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CONTENTS OF UVOLUME XVI.

No. 1.— November, 1899.

PAGES
I. Wiciiam PatTreN AND WILLIAM A. REDEN-
BAUGH.
Studies on Limulus. TI. The Endocrania
of Limulus, Apus, and Mygale. . . 1-26

eG. CARL) FIUBER.
A Contribution on the Minute Anatomy
of the Sympathetic Gangla of the
Different Classes of Vertebrates. . . 27-90

III. Witiiam PatTeNn AND WILLIAM A. REDEN-
BAUGH.
Studies on Limulus. IT. The Nervous
System of Limulus Polyphemus, with
Observations upon the General Anatomy 91-200

IV. EstHer FussELt Byrnes.
The Maturation and Fertilization of the
Ege of Limax Agrestis (Linné) . . 201-236

V. James PERRIN SMITH.
Larval Stages of Schloenbachia . . . 237-268

No. 2.— February, 1900.

I. P. Carvin MEnscu.
Stolonization in Autolytus Varians . . 269-322

II. Henry L. Bruner, Pu.D.
On the Heart of Lungless Salamanders . 323-336 .

III.

IV.

II.

1

LV,

CONTENTS.

Harris M. BENEDICT.
On the Siructure of Two Fish Tapeworms
from the Genus Proteocephalus Wetn-
land 1858

SAMUEL J. HOLMEs.
The Early Development of Planorbts

No. 3.— August, Igoo.

PAGES

337-368

369-458

WILLIAM PATTEN AND ANNAH PUTNAM HAZEN.

The Development of the Coxal Gland,
Branchial Cartilages, and Genital Ducts
of Limulus Polyphemus

Henry McELDERRY KNOWER.
The Embryology of a Termite .

T. H. MorGan AND ANNAH PUTNAM HAZEN.
The Gastrulation of Amphioxus

KATHARINE Foot AND ELLA CHURCH STRO-
BELL.
Photographs of the Egg of Allolobophora
Foetida RSS AMBER E, ie ct UT hi Sa

Che Atheneum Press.
GINN & COMPANY, BOSTON, U.S.A.

459-502

505-568

569-600

601-618

Volume X V1, November, I&Q9. Number I.

JOURNAL

OF

NOR P OMe ey.

STUDIES ON LIMULUS.

I. THE ENDOCRANIA OF LIMULUS, APUS, AND MYGALE,
BY
WILLIAM PATTEN anp WILLIAM A. REDENBAUGH.

DARTMOUTH COLLEGE, Hanover, N. H.

INTRODUCTION.

In 1888 I began a detailed study of the anatomy and
development of Limulus, for the purpose of determining
whether such a study would justify the conclusion that
Limulus and other arachnids are closely related to ancestral
vertebrates. That they are so related seemed probable in view
of certain resemblances between the mode of development of
the brain and eyes of Lzmulus and scorpion and those of
vertebrates.

An elaborate attack on the problem was planned. The struc-
ture, development, and physiology of all the organs of Lzmalus
were to be worked out, and a comparison made between them
and the corresponding organs in vertebrates. But it was soon
discovered that an inquiry of this nature, made from a special
point of view, opens many side problems that necessitate

frequent digressions in order to discuss tentative homologies
I

2 PATTEN AND REDENBAUGH. [VoL. XVI.

and other questions of a purely theoretical nature. This mode
of treatment, therefore, is open to the constant danger of con-
fusing fact and theory, of destroying the simplicity and direct-
ness of the purely descriptive parts, and of detracting somewhat
from their permanent value. Moreover, comparisons instituted
for the purpose of indicating broad homologies fail to carry
conviction when standing alone, as they would have to do if
each system of organs were treated separately. In such com-
plicated problems as the origin of vertebrates, resemblances
between several systems of organs and the corresponding parts
in the hypothetical ancestors must be treated together, in order
to show how one comparison harmonizes with and supports the
other. It seemed best, therefore, to publish each descriptive
part as soon as completed, with only an occasional attempt
to point out the relations of certain organs in Lzmulus to the
corresponding structures in vertebrates. At some later period
we hope to combine our results into an organic whole.

While there may be differences of opinion as to the probable
value of such an undertaking, there can be no doubt that if
it leads to a fairly complete account of one species, that alone
will be a sufficient return for the labor, and will give the work
a value that cannot be derived from fragmentary accounts of
different, even though closely related, forms. Moreover, this
mode of treatment has obvious advantages, in that long famil-
iarity with a given form enables the investigator to avoid
the repetition of much preliminary work, and thus obtain his
results more rapidly.

The senior author has for a long time regarded the endo-
cranium, the branchial cartilages, and the segmental cartilages
of the spinal cord of arachnids as forerunners of the corre-
sponding cartilages in vertebrates, and he began to study their
anatomy, histology, and development some ten years ago. A
preliminary statement as to the structure of the endocranium
in scorpions and Lzmu/us was given in his paper on the “ Origin
of Vertebrates from Arachnids”’ ('g9). Since then, from time
to time, many suggestive details have been gathered; but as no
immediate opportunity was likely to present itself to get this
material into proper shape for publication, it was decided to turn

No. 1.] STUDIES ON LIMULUS. 3

over a part of it in the form of some rough sketches and notes
to Mr. Redenbaugh, then a student in the biological laboratory
at Dartmouth. Mr. Redenbaugh began the work anew, and
with great care and skill has brought it to completion. His
work on the endosternite of Lzmzlus coincides very closely
with my own. The drawings that accompany this paper have
been made along the lines of my original sketches, some of
which were intended to emphasize the relations of the cranial
cartilages to the nervous system, and to show their resem-
blance to the corresponding parts in vertebrates. But every
drawing has been made from the dissections, and with the
utmost fidelity to details of size and position.

The descriptions of the endosternite of Apuws and Mygale,
with some unimportant exceptions, are the work of Mr.
Redenbaugh.

Another paper, belonging to this series, on “‘ The Structure
and Development of the Nephridia, Branchial Cartilages, and
Genital Ducts of Limulus,’ by Miss Hazen and myself, is
completed and in the hands of the publishers, and further
work on the histology and development of the endosternite
and branchial cartilages is in progress.

The second paper of this series, that on “The Peripheral
Nervous System of Limulus,” was begun some years ago, and
considerable progress was made on it. But the work was
laid aside for a time, owing to the pressure of other duties;
the notes and drawings were finally given to Mr. Redenbaugh,
who went over the entire subject again, receiving from
Dartmouth College, in recognition of his work, the degree
Of Ph.D:

A description of the “Structure and Development of the
Central Nervous System of Limulus”’ is being prepared, also
papers on the “ Physiology of the Brain and Spinal Cord.”

W. PATTEN.

The structure, which has been called by various names, vzz.,
prosomatic endochondrite, or endosternite, cartilaginous ster-
num, endocranium, and plastron, has been found in many of
the Avachnida and in a few Crustacea. In all cases it is a

4 PATTEN AND REDENBAUGH. [VoL. XVI.

cartilaginous body of varied form lying dorsal to the cephalic
ganglionic mass and ventral to the intestine. It functions as
a centrum for the attachment of various muscles.

Schimkewitsch in ‘94 gave a very complete summary of all
the literature upon the subject. Therefore it will only be neces-
sary to call to mind those works which have an immediate
bearing upon the forms herein described.

In ’g1 Lankester figured the plastrons of Limulus, Mygale,
and Scorpio, and briefly compared them with each other in their
general characteristics.

In 'g4 he gave a more complete account of the plastron of
Scorpio. He figured and described those of Apus, Mygale, and
Limulus, and gave something concerning the histology of all
four of them.

In ’g5, with the aid of his pupils, Mr. Benham and Miss Beck,
he compared the plastrons of Lzmzulus and Scorpio with refer-
ence to their muscular attachments.

In 'g9 Patten figured and described the plastron of a different
species of scorpion, and laid special stress upon the existence
of the subneural arch, or “ occipital ring.” His figure differed
from Lankester’s principally in the absence of a diaphragm.

In 92, 93, and ’94 came a series of papers by Schimkewitsch
and Bernard, in which Bernard sought to homologize the apo-
demes of Galeodes with the endosternites of other arachnids, and
to show that the endosternites of arachnids are apodematous
structures due to fusion and compression of the cephalotho-
racic segments and later specialized for muscular attachments.
He regarded the endosternite of Zzmulus as a derivation of the
ventral muscle bands and believed it to be homologous with that
of Agus and not with that of arachnids. Schimkewitsch, on
the other hand, maintained that the apodemes of Galeodes were
entirely represented by less developed apodemes in Scorgzo, and
that the endosternite was a morphologically different structure.
He also stated that the arachnid endosternite was composed of
two parts: (1) of a transversal muscle corresponding to the
adductor muscle of Crustacea; and (2) of a pair, or perhaps
several pairs, of mesodermic tendons connected with the trans-
verse muscle strands.

No. I.] STUDIES ON LIMULUS. 5

METHODS.

In the work upon Zzmulus both fresh and alcoholic material
was employed, but in that upon A/yga/e and Apus only alcoholic
specimens were used. The plastron of Lzmulus, with adjoin-
ing parts, was excised and allowed to soften in water until the
muscles could be easily picked away. Dissection was then car-
ried on under water or weak alcohol with fine pointed forceps.
In other specimens the muscles were allowed to macerate
completely, and were then brushed off, leaving the cartilage
intact.

Serial sections of Apus, both transverse and longitudinal,
were made, and a model of the plastron magnified seventy times
was reconstructed from the longitudinal sections.

As Mygale material was scanty, only cross-sections of this
animal were made. The viscera were cut from the cephalothorax
and imbedded in celloidin. Sections were cut and mounted
serially, stained in borax carmine, and a reconstruction of the
cartilage made with an amplification of twenty times. With
these models as guides, drawings were made and minute details
added by careful study of the sections under the microscope.

I. PLASTRON OF LIMULUS.
(See Pl. I, Figs. 1-4.)

1. General Description.—In the following descriptions, for
reasons which will appear later, the terms “haemal” and
“neural” will be substituted for dorsal and ventral, respec-
tively, and the terminology of Lankester will be used in desig-
nating the parts of the endosternite.

In Limulus the plastron lies in about the center of the
cephalothorax, with the anterior margin about opposite the
chelicerae, and the posterior extremity on a level with the chi-
laria. The anterior processes extend some distance beyond the
bases of the chelicerae. The mouth of the animal is located a
little anterior to the center of the plastron, and from it the
oesophagus passes forward between the anterior processes to
form the <-shaped proventriculus, the haemal arm of which

6 PATTEN AND REDENBAUGH. [VoL. XVI.

communicates with the intestine. The intestine begins a short
distance in front of the plastron and proceeds straight back-
ward, close to the haemal side of the plastron. The oesophag-
eal collar surrounds the entrance to the oesophagus, and lies
entirely on the neural side of the plastron. The ventral cord
passes posteriorly along the same side.

The plastron itself is roughly rectangular, with the long axis
parallel to the long axis of theanimal. It is produced anteriorly
into two stout processes (anterior cornua, a.c., Pl. I, Figs. 1 and
2), posteriorly into a cleft median process (posterior process,
ppr., Figs. 1-4) and a pair of short bars (capsuliginous bars,
cap.b., Figs. 1-4).

Laterally, near the anterior margin, are two pairs of long rod-
like tendons (lateral cornua, /.c., Figs. 1 and 2), and near the
posterior border is a pair of short stout processes (latero-poste-
rior processes, /.p.f7., Figs. 1-4).

Neurally a pair of processes arise from near the bases of the
latero-posterior processes, pass around the ventral cord, and
unite on the neural side of it to form the occipital ring (0c.r,,
Figs. 1, 3,and 4). This ring is connected posteriorly with the
capsuliginous bars (caf.0.).

Haemally a pair of short stout processes (dorsal or haemal
processes, /.pr., Fig. 2) arise midway along the lateral margins
of the plastron.

The body of the plastron is a thick plate of fibroid cartilage,
flat or slightly concave on the neural side, with the lateral mar-
gins (m.r., Figs. 1 and 3) sharply elevated, particularly towards
the anterior end. The haemal side is concave in transverse
section. The lateral edges are much thickened anteriorly, and
produced beyond the body of the plastron to form the anterior
cornua.

2. The anterior cornua (a.c., Figs. 1 and 2) are a pair of
stout transversely flattened processes. To each process are
attached three muscles, which go to the haemal side of the
carapace; one directly forward from the extremity, one perpen-
dicularly from the inner surface of the process, and one obliquely
forward from the haemal margin.

The neural margins of the processes and the entire lateral

No. I.] STUDIES ON LIMULUS. 7

portions of the plastron, including the latero-posterior processes,
give attachment to the plastro-coxal muscles of the thoracic
appendages from the second to the sixth pairs.

Anteriorly the muscles do not cover the neural surface of the
plastron, but posteriorly, as the muscles increase in size with
the increase in size of the appendages, they encroach upon the
neural surface even to the median line. There is, therefore, on
the anterior neural surface of the plastron a triangular space
which, except for a few loose strands (plastro-buccal muscles)
going to the oesophagus, is free from muscles and comparatively
smooth.

3. The lateral cornua (lc., Figs. 1 and 2) spring from the
anterior haemal side of the plastron. They are long and slen-
der, and each is attached by a short muscle to the haemal side
of the carapace, close to the origins of the tergo-coxal muscles.
The first pair of cornua pass between the tergo-coxal muscles of
the third and fourth pairs of appendages, the second pair between
the tergo-coxals of the fourth and fifth pairs of appendages.

The bases of these lateral cornua give to the plastron a
greater thickness at the anterior border than in the middle.

4. The haemal processes (h.pr., Fig. 2) are very stout, and
spring from the haemal side near the lateral edge of the plastron,
about halfway between the latero-posterior processes (/.p.f7.)
and the lateral cornua (/c.). Their bases anteriorly are close
to the edge of the plastron, but posteriorly, owing to the widen-
ing of the plastron towards the latero-posterior processes, they
lie about halfway between the middle and the edges of the
plastron. These processes incline slightly outwards, and each
gives attachment to two muscles: one going haemally and a
little laterally from the extremity of the process to the cara-
pace, and the other posteriorly and slightly haemally from the
posterior margin of the process to the first entapophysis.

5. The latero-posterior processes (lp.pr., Figs. 1-4) are lateral
expansions of the posterior portion of the plastron. They are
flattened haemo-neurally, and rapidly taper to a point. Along
the posterior margin of each, on the neural side, is a sharp
ridge, which towards the median line is continuous with the base
of the occipital ring. The latero-posterior processes give attach-

8 PATTEN AND REDENBAUGA. [VoL. XVI.

ment to some of the plastro-coxal muscles of the sixth pair of legs,
which are the most powerful appendages of the animal.

A marginal ridge is formed (m.7., Fig. 1) on the neural side
of the plastron, which becomes quite prominent anteriorly ; pos-
teriorly it dwindles to a low rounded shoulder at the base of the
latero-posterior process, then rises a little, to form the base of the
occipital ring (oc.7., Figs. 1, 3, and 4). There is also a shoulder
(s., Fig. 4) running across the body of the plastron, connecting
the posterior borders of the latero-posterior processes.

6. The posterior process (ppr., Figs. 1-4) begins as a thick-
ening on the haemal side of the plastron between the haemal
processes. It increases in thickness posteriorly, and ends in a
bifid process, each division of which is deeply grooved on the
haemal side. Along the whole haemal side of the process are at-
tached two large muscles going to the first pair of entapophyses.

On the body of the plastron, on both sides of the base of the
posterior process, are attached longitudinal abdominal muscles,
passing backward between the last described muscles and the
plastron. The attachments of these muscles extend a little an-
terior to the haemal processes. In front of this the body of
the plastron is destitute of muscles on the haemal side.

A pair of small chilarial muscles are attached to the haemal
side of the extremity of the posterior process.

7. The capsuliginous bars (cap.b., Figs. 1-4) are a pair of
processes of peculiar structure arising from the posterior mar-
gin of the plastron, alongside the posterior process. The
latero-posterior process on each side is extended backwards as
a much thinner layer of cartilaginous tissue. It is flush with
the neural surface of the posterior process, but where it joins
the latero-posterior process there is a very abrupt shoulder
upon the neural side and aslight one upon the haemal side.
To the posterior margins of these thin portions are attached
the rod-like bars. The latter bend neurally and slightly
toward the median line, enter the bases of the chilaria, and are
attached to their posterior sides.

A small transverse muscle joins the distal ends of the two
bars. Two other small muscles run to the chilaria from the
thin portions of the plastron near the bases of the bars.

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No. I.] STODIES ON LIMCLES. 9

It is especially noteworthy that the capsuliginous bars differ
histologically from the rest of the plastron, and that they are
united with the thinner portions of the plastron by what appears
to be a true joint. The body of the plastron is composed of
fibroid cartilage, while the bars are of capsuliginous cartilage,
exactly like that found in the abdominal appendages, the histol-
ogy of which has been described by Gegenbaur (’58) and Lan-
kester ('84) and more recently by Gaskell (’97).

8. Lhe Occipital Ring (oc.r., Figs. 1, 3, and 4).— At the
points where the marginal ridges (m.7., Fig. 1) meet the bases
of the latero-posterior processes, two outgrowths are formed
which are united with each other distally on the neural side of
the ventral cord by a connective tissue membrane. At their
bases the processes are slender, but distally they enlarge and
thicken, and are joined to the capsuliginous bars (cap.6.) by
strands of connective tissue.

That the occipital ring or subneural arch thus formed is a
true cartilaginous ring is beyond doubt, for serial sections of
a young animal through this region show the continuity of the
cartilaginous tissue entirely around the cord (Fig. 4). Upon
the neural side of the ring are two depressions (ch.m., Figs. 1,
3, and 4), to which are attached a pair of muscles going to the
insides of the chilaria. From the anterior side of the ring,
muscle strands pass forward to the integument immediately
behind the mouth.

9. Accessory Structures. — Besides the processes and muscle
attachments above mentioned, there are found along the late-
ral edges of the plastron on the haemal side, and attached to
it by connective tissue fibers, tough membranes (mem., Fig.
2), to which are attached the ‘‘veno-pericardiac muscles”’ of
Lankester, or the ‘brides transparentes”’ of Milne-Edwards
(v.p.m.1-2, Fig. 2). Anteriorly this membrane springs from
the side of the plastron just back of the lateral cornua. About
midway between the lateral cornua and the haemal process it
affords attachment to the anterior or first veno-pericardiac mus-
cle (v.f.m.1, Fig. 2). Just anterior to the latero-posterior proc-
ess it gives attachment to the second veno-pericardiac muscle
(v.p.m.2, Fig. 2), and at the same point sends a bundle of con-

IO PATTEN AND REDENBAUGEH. [VoL. XVI.

densed connective tissue neurally to the integument between
the fifth and sixth pairs of legs. A similar bundle unites it
with the integument just outside of the chilaria, and from
here on it is attached by a double base all along the integu-
ment of the abdomen, between the ventral longitudinal and
the “‘branchio-thoracic’’ muscles. It furnishes attachment for
all the veno-pericardiac muscles.

Benham, in his paper “On the Muscular and Endoskeletal
Systems of Limulus” ('85), has described the veno-pericardiac
muscles as attached to the dorsal side of the longitudinal venous
sinus. The venous sinuses of Lzmulus, asa rule, have no walls
substantial enough for a muscle attachment ; but posterior to
the second veno-pericardiac muscle the above-described mem-
brane is double and spans the venous sinus. Anterior to
this point the venous sinuses have no connection with the
membrane.

10. Foramina.—Two pairs of foramina for the passage of
nerves have been found. One pair (f!, Figs. 1-3) may be
seen at the bases of the latero-posterior processes, just out-
side the neural marginal ridge and appearing on the haemal
side of the plastron, a little posterior to the haemal processes.
These two foramina furnish passage for the intestinal branches
(in.n.6, Fig. 3) of the haemal nerves belonging to the sixth
thoracic neuromere. :

The second pair of foramina (f2, Figs. 1-3) are located
in the posterior thinner portion of the plastron, near the bases
of the processes which form the occipital ring. Through these
pass the intestinal branches (2.7.7, Fig. 3) of the haemal nerves
of the chilarial neuromere.

11. Relation of the Brain to the Plastron.— Following the
nomenclature adopted by Patten (89, ’93), the term “brain”’
will be applied to the entire circumoesophageal collar; the
term ‘fore-brain’’ to the supraoesophageal portion, or the
part derived from the preoral lobes of the embryo; “hind-
brain” to the part formed by the fusion of the six thoracic
neuromeres; and “accessory brain” to that portion formed
from the two abdominal neuromeres which’ fuse secondarily
with the thoracic neuromeres.

No. I.] STUDIES ON LIMULUS. IE

The brain forms a close-fitting collar around the oesophagus
and lies a little in front of the center of the plastron. Six
pairs of large pedal nerves radiate from the neural side of the
collar and innervate the six pairs of thoracic appendages. Six
pairs of integumentary nerves belonging to the same neuro-
meres radiate from the haemal side and innervate the skin
and other organs on the haemal and neural sides of the
carapace.

In Pl. I, Fig. 3, only the posterior half of the collar is repre-
sented ; 7.7.4, z.2.5, and 2.7.6 are the three posterior pedal or
neural nerves of the thoracic neuromeres; /.7.4, 4.2.5, and
h.m.© are the three integumentary or haemal nerves of the
same neuromeres. Of these, the haemal nerves (4.72.6), which
belong to the same metamere as the sixth pair of legs, give off
small branches (27.7.©) which pass through foramina (/.!) in the
plastron and communicate with a sympathetic system supplying
the intestine and the longitudinal abdominal muscles. The
main portions of the nerves give off branches to the region
of the heart, and then ramify over the skin of the haemal and
neural surfaces of the cephalothorax.

The ventral cord (v.c.), the chilarial (7.2.7), and the opercular
(z.2.8) nerves, and two pairs of integumentary nerves (4.7.7 and
h.n.8) belonging to the chilarial and opercular neuromeres, pass
out from the posterior side of the brain through the occipital
ring.

The chilarial nerves (#.7.7, Figs. 3 and 4) arise on the neural
side of the posterior end of the oesophageal collar and pass
directly backwards through the occipital ring close to its neural
portion and innervate the chilaria.

On the haemal side of the collar arise a pair of integumentary
or haemal nerves (4.7.7, Figs. 3 and 4) belonging to the chilarial
neuromere. They diverge laterally and pass through the occip-
ital ring, sending branches (zv.7.7) through foramina (/.2) to
supply the intestine and the longitudinal abdominal muscles.
The main portions of the nerves bend sharply outwards after
leaving the occipital ring, and, after giving off cardiac branches,
supply the skin on the haemal and neural surfaces of the ante-
rior portion of the abdomen. The chilaria, though apparently

EZ PATTEN AND REDENBAUGH. [VoL. XVI.

in the thoracic region, belong primarily to the category of
abdominal appendages, as shown (1) by the development of
the chilarial neuromere ; (2) by the distribution of the haemal
nerves of the neuromere; and (3) by the possession of a gill
bar of capsuliginous cartilage closely resembling those found
in the other abdominal appendages.

The opercular nerves (7.7.8, Figs. 3 and 4) arise a little poste-
rior to the chilarial nerves and pass backward alongside the
ventral cord through the occipital ring and innervate the genital
operculum. The haemal nerves (4.7.8, Figs. 3 and 4) belonging
to this neuromere arise slightly posterior to the haemal nerves
of the chilarial neuromere, and, diverging at a less angle than
the preceding, pass through the occipital ring and out towards
the sides of the body immediately posterior to the capsuligi-
nous bars (cap.d.). As they turn outwards each sends a branch
(tz.n.8, Figs. 3 and 4) haemally to the intestine and longitudinal
abdominal muscles. The main part of the nerve finally sends
a branch to the heart, and then distributes itself over the
surface of the abdomen. The ventral cord (v.c., Figs. 3 and 4)
passes straight back from the oesophageal collar through the
occipital ring and does not branch until it reaches the ganglion
of the first gill metamere.

II. ABDOMINAL ENDOCHONDRITES OF LIMULUS.

In the abdominal region of Lzmulus is a series of cartilages
spanning the ventral cord on the neural side. There are six in
all, one at the base of each of the abdominal appendages, from
the operculum to the fifth gill. Like the plastron, they are
composed of fibroid cartilage and serve as centra for the
attachment of muscles, but they differ from the plastron in
being placed on the neural side of the central nervous system
instead of upon the haemal side. These cartilages vary consid-
erably in different individuals and in different metameres of the
same individual, but the one represented in Fig. 3 (ad.en.) is
typical. It consists of an irregularly shaped body with a flat
neural surface, which is in contact with the integument except
across the middle portion. This middle part lies directly under

Not] STUDIES ON LIMULUS. 13

the base of the operculum and is indented by two pits (of.m.),
which represent the origin of a pair of muscles inserted on the
inside of the appendage.

The posterior and anterior prolongations of the endochondrite
(pp. and a.p., Fig. 3) serve partly for the attachment of muscle
strands of the longitudinal muscles of the abdomen, but in
many cases the anterior and posterior processes of successive
endochondrites are continuous with each other. On the haemal
side of the endochondrite is a pair of haemal processes (4.f.,
Fig. 3), one on each side of the ventral cord. These project
haemally, and a little outward and backward, and furnish
attachment for a pair of haemo-neural muscles inserted on the
haemal side of the carapace just median to the entapophyses.

III. THe ENbDOSTERNITE oF APUS.
(Pl. II, Figs. 5-10.)

This structure, like that of Limulus, is located in the cephalo-
thorax, between the central nervous system and the intestine.
It lies behind the mouth and opposite the mandibles. The body
of the plastron is elongated in a direction transverse to the long
axis of the animal, and its flaring ends (m., Figs. 5, 6, and
10) give attachment to the powerful adductor muscles of the
mandibles.

On the posterior side a pair of chitinous apodemes (afo.,
Figs. 5, 6, 8-10) project into the plastron. These are formed
by the invagination of the chitin between the bases of the first
and second appendages behind the mandibles. These append-
ages have been called the first and second maxillae.!_ Lankester
regards them as one appendage consisting of two parts and
calls it a maxilla. Bernard calls the anterior portion a cleft
underlip, and the posterior portion the first maxilla.

From the inner ends of the apodemes a pair of tendonous
cords run forward directly through the body of the plastron at
right angles to its fibers, and emerge on the anterior side as a
pair of anterior cornua (a.c., Figs. 5, 6, 8, and 9). These proc-

1 A. Gerstaecker, Die Klassen und Ordnungen der Arthropoden, Ba. v. Crustacea.

14 PATTEN AND REDENBAUGH. [VoL. XVI.

esses are each split into three divisions. The neural portion
(z., Figs. 5, 6, and g) is in contact with the integument at the
side of the mouth, and furnishes attachment for a few muscle
strands going to the integument anterior to the labrum. The
middle (m., Figs. 5, 6, and 9) and haemal divisions (Z., Figs.
5-9) both furnish attachment for muscles going to the posterior
side of the flexure of the oesophagus. The haemal division also
continues as a thin strand around the side of the oesophagus to
the haemal side of the carapace, anterior to the eyes. The
neural and middle divisions are rather stout, while the haemal
one is thin and joined at the base to its fellow upon the oppo-
site side. Thus in median longitudinal section the plastron
appears to terminate in a knife edge (Z., Fig. 7).

Posteriorly the plastron terminates, in the median portion, in
a thin membrane (z., Figs. 5-8), which runs backward and is
attached to the integument between the longitudinal commis-
sures of the ventral cord. It is continued laterally onto the
posterior sides of the apodemes (z., Fig. 8).

The ventral longitudinal muscles of the abdomen are attached
to the posterior sides of the apodemes and to the plastron itself
on each side of the median line. A process (x, Figs. 5 and 8)
projecting neurally from each apodeme sends muscle strands to
the inside of the second pair of maxillae.

Haemo-neural muscles are attached to the haemal sides of
the apodemes. There are also a pair of muscles inserted on
the posterior neural portion of the plastron (y., Figs. 5, 7, and
IO), passing to the integument between the longitudinal com-
missures, just back of the first cross-commissures.

IV. ENDOSTERNITE OF MYGALE.
(Pl. II, Figs. 11 and 12.)

This plastron, like those heretofore described, lies between
the alimentary canal and the central nervous system. Its gen-
eral contour from the neural aspect is oval, with the longer axis
parallel to the long axis of the animal.

The anterior edge is deeply indented by a bay running to the

No. I.] STUDIES ON LIMULUS. 15

middle of the plastron, thus forming a pair of very large ante-
rior cornua (a.c., Figs. 11 and 12).

The body of the plastron may be considered as a plate of
cartilage with crenate margins and concave on the neural side.
Radiating from a common center on the neural side are four
paired, plate-shaped processes (z.p/.1-4, Fig. 11), and one poste-
rior unpaired one (7.7.5, Fig. 11). The posterior one is thinner
than the others and gradually tapers out, ending in three low
ridges. The anterior pair borders the inner neural margins of
the anterior cornua (a.c.). From about the middle of the an-
terior cornua spring a pair of processes (z.pr., Figs. 11 and 12),
which bend around the brain and attach themselves to the
integument close together on the neural side.

On the haemal side of the plastron two high ridges converge
from the distal ends of the anterior cornua to the posterior end
of the plastron, forming a deep gully between them, in which
lies the alimentary tract. These ridges are split up into five
paired haemal processes (4.f7.1-5, Fig. 12) of unequal length,
those in the middle being longer than those at either end.
The plastron ends posteriorly in a short median process (f.p7.,
Figs. 11 and 12).

The muscles arising from the plastron are too numerous and
complicated to allow of a full description in this paper. From
nearly the whole of the neural side muscles go to the legs;
haemo-neural muscles are attached to the haemal processes
(Z.pr.1-5), and longitudinal muscles to the posterior process
(ppr).

The brain lies just haemal to the neural processes (w.g7.),
which are in contact with the integument. The oesophagus
passes through the brain and between the anterior cornua to
the sucking stomach, which lies in the groove on the haemal
side of the plastron. Muscle strands run from the stomach to
the walls of the groove. TW. 2. ape ee.

There are, therefore, four distinct structures in arachnids
that may serve as parts of a true endoskeleton :

I. The endosternite or endocrantum, a broad flat plate of
fibro-cartilage lying on the dorsal side of the brain, and serving

16 PATTEN AND REDENBAUGH. [VoL. XVI.

mainly for the attachment of the muscles that move the thoracic
appendages and the flexor muscles of the thorax and abdomen.
In scorpions and Limzlus a complete cartilaginous occipital
ring is formed about the spinal cord, near its union with the
brain. The plate is a continuous structure, and there is no
indication whatever that it consists of two separate lateral
bars, as maintained by Gaskell.

We recognize three parts to the endosternite of arachnids,
namely, two lateral bars, a broad plate of cartilage on the
dorsal side of the nervous system uniting the posterior ends
of the bars, and a bridge of cartilage on the ventral side, which,
together with the above-mentioned parts, forms a continuous
ring about the anterior end of the spinal cord.

This whole structure, after the extensive reduction of the tho-
racic appendages and the muscles that move them, is retained,
apparently, in the ancestral vertebrates, and becomes, as was
first pointed out by Patten in ’gg, the primordial cranium. The
lateral bars, the transverse plate, and the ventral arch corre-
spond respectively to the trabeculae, the basilar plate, or para-
chordals, and the occipital ring. If we add the olfactory and
auditory capsules and roof over the remaining dorsal surface,
we obtain a complete cartilaginous cranium like that of higher
forms. As we shall show elsewhere (American Naturalist),
Gaskell’s attempt to utilize the endosternite in his version of
the origin of vertebrates is a complete failure, since under the
conditions of his theory it must be torn apart and put together
again upside down, with the old occipital ring replaced by a new
one on the opposite side of the endosternite from the one on
which it actually occurs. The endosternite has a true carti-
laginous consistency, and is composed of a mass of interwoven
fibers containing variously formed stellate lacunae, which may
be very clearly seen after macerating out the cell contents.
The lacunae are united by numerous anastomosing canaliculi,
which in the living cartilage contain minute branching proc-
esses of the cartilage cells situated in the lacunae.

II. The six segmentally arranged cartilages of the spinal
cord (Pl. VI, Fig. 1) contain the same kind of cartilage as the
endosternite. They are the forerunners, we believe, of the

ING ST) STUDIES ON LIMULUS. Ny

segmental cartilages found in corresponding regions in primi-
tive vertebrates, and which give rise to the vertebral column.
The cartilages serve evidently as points of attachment for seg-
mentally arranged muscles that meet at those points. But we
must not conclude that that is the only reason why the carti-
lages are there, because cartilages are not always formed at
points where muscles are attached to one another. It is obvi-
ously impossible to carry the comparison with vertebrate carti-
lages any farther. It is enough for our purpose to show that
the conditions in Zzmulus are such as to produce a series of
segmentally arranged cartilages about the spinal cord, similar
to those in primitive vertebrates.

III. There are seven pairs of branchial cartilages composed
of an entirely different tissue histologically (and chemically,
also, according to Gaskell) from that in the endosternite and
the segmental cartilages.

The first pair arise from the inner surface of the chilaria, and
are attached to the posterior margin of the endocranium so that
they appear to form a part of it. The remaining six pairs arise
from the base of the abdominal appendages and go to the cor-
responding entopophyses. They develop, as will be shown in
one of the papers of this series, at a very early embryonic period
as clearly defined outgrowths of the walls of the mesoblastic
somites. Their union with the epidermis is secondary, and
they are in no wise derived from the modification of chitinous
ingrowths from the epidermis, as maintained by Gaskell.

These branchial bars of the mesothorax correspond, we
believe, to the cartilaginous bars of the visceral clefts of verte-
brates, as we first indicated in ’g9. In ’93 we called attention
to the surprising histological resemblance between these carti-
lages and those of Petromyzon, and still later (96) we showed
that there was a certain number of embryos in which one or
more pairs of appendages were invaginated instead of evaginated.
Transverse slits were thus formed along the sides of the head,
resembling vertebrate gill slits and recalling to mind the lung
books of scorpions and spiders. In Lzmudlus the appendages
most frequently invaginated, the thoracic ones, are not pro-
vided with gills. This again is suggestive since in vertebrates

18 PATTEN AND REDENBAUGH. (VoL. XVI.

the most anterior visceral arches are likewise devoid of gill
lamellae. On the neural side of each thoracic appendage of
Limulus, except the first and last, is a large group of sense
organs supplied by a special ganglionated tegumentary nerve,
i.e. the gustatory organs of the coxal spines (Patten, '93) and
the sense organs on the endopodite of the abdominal append-
ages. These sense organs and nerves correspond in position
and in mode of development respectively to the suprabranchial
sense organs and the rami dorsali of the cranial nerves of
vertebrates.

IV. The Dermal Skeleton.— Limulus is the only invertebrate
where the chitinous exoskeleton has begun to form a system
of true dermal bones (Patten, '94). They arise as innumerable
ingrowths of the ectoderm that unite to form a mass of anasto-
mosing chitinous trabeculae. The tissue thus formed resem-
bles coarse cancellated bone, but more especially the coarse bony
networks that form the inner layers of the cephalic shields of
the Cephalaspidae. Within these trabeculae are numerous cavi-
ties, which, after the shell is macerated and dried, become filled
with air, and then bear a strong resemblance to true bone
lacunae. They are spindle-shaped lacunae, with two or more
very fine canaliculi leading off from them, which appear to unite
in some cases with the canaliculi of neighboring lacunae. In
living tissue the lacunae are filled with a substance resembling
protoplasm, and the larger ones appear to be nucleated. Thus,
an entirely new dermal structure is forming here unlike that
known in any other invertebrate; namely, local ingrowths of
the ectoderm, forming a network of chitinous trabeculae,
into which numerous cells migrate to form true bone cor-
puscles.

As the trabeculae cannot be shed periodically, like the rest
of the exoskeleton, they are retained permanently within the
body. Since, as we now know, some forms of chitin are very
closely allied to chondrin, perhaps this condition may be the
means of ultimately completing the chemical metamorphosis
of chitin into chondrin. But there is no evidence that other
chitinous ingrowths, such as the entopophyses or the apodemes
of many arthropods, are invaded by cells, or that they have

No. I.] STUDIES ON LIMULUS. 19

made any decided approach in chemical composition towards
true bony or cartilaginous structures.

We can readily understand how such a sub-dermal framework
as we see in Lzmulus might lose its connection with the epi-
dermis, and the latter form a continuous layer above it. Then
the hard superficial chitinous armor would no longer be needed,
either for the attachment of muscles or for protection, and
might disappear altogether, thus obviating the dangers of peri-
odically shedding the old chitinous exoskeleton.

Thus Lzmulus appears to have laid the foundation for an
elaborate system of internal supports; namely, the primordial
cranium, the branchial cartilages, the cartilages of the spinal
cord, and the sub-ectodermal structures which resemble dermal
bones. These parts have different modes of origin, and differ
widely histologically from one another, yet they agree in all
essential features with the corresponding parts of the vertebrate
skeleton. In vertebrates we so frequently see these structures
welded together into a common framework for the whole body,
that we underestimate the importance of certain facts of verte-
brate ontogeny, which indicate more and more clearly that
the vertebrate skeleton is also composed of several parts quite

different in structure and origin.
W. PATTEN.

20 PATTEN AND REDENBAUGH. [VoL. XVI.

BIBLIOGRAPHY.

’58 GEGENBAUR, C. Anatomische Untersuchung eines Limulus. df. d.
Nat. Ges. zu Halle. Bd.iv. Halle 4°.

'66—79 GERSTAECKER, A. Die Klassen und Ordnungen der Arthropoden.
Crustacea. Bd. v.

'73, MILNE-EDWARDS, ALPH. Recherches sur l’Anatomie des Limules.
Ann. Sciences Naturelles. Sér.5. Tome xvii.

’'8la LANKESTER, E. RAy. Limulus an Arachnid. Q./7. M.S. Vol. xxi.

’'81lb LANKESTER, E. Ray. Observations and Reflections on the Append-
ages and on the Nervous System of Apus cancriformis. Q./. M7. S.
Vol. xxi.

'84 LANKESTER, E. Ray. On the Skeleto-Trophic Tissues and Coxal
Glands of Limulus, Scorpio, and Mygale. Q./. M@. S. Vol. xxiv.

’'85 BENHAM, W. B.S. On the Muscular and Endoskeletal Systems of
Limulus. TZvaus. Zool. Soc. Vol. xi.

’85 Beck, Miss E. J. On the Muscular and Endoskeletal Systems of
Scorpio. TZyvrans. Zool. Soc. Vol. xi.

’85 LANKESTER, E. Ray. Comparison of the Muscular and Endoskeletal
Systems of Limulus and Scorpio. TZvans. Zool. Soc. Vol. xi.

’89 PATTEN, WM. On the Origin of Vertebrates from Arachnids. @Q./.
M.S. | Vol. xxxi, Part:3, Nas:

'92a BERNARD, H. M. The Apodidae, a Morphological Study. London.

'92b BERNARD, H. M. The Apodemes of Apus and the Endophragmal
System of Astacus. Ann. and Mag. of N. H. Vol. x.

’93 SCHIMKEWITSCH, W. Endosternite of Arachnida. Zool. Anz. Jhg. 16,
No. 425, pp. 300-308.

’'93 KINGSLEY, J. S. Embryology of Limulus. Part II. /Journ. of Morph.
Vol. viii.

'93 PATTEN, WM. On the Morphology and Physiology of the Brain and
Sense Organs of Limulus. Q. /. 7..S. Vol. xxxv, No. 137. July, 1893.

'94 PATTEN, WM. On Structures resembling Dermal Bones in Limulus.
Anat. Anz. Bd. x, No. 14. May 5.

'94 BERNARD, H. M. The Endosternite of Scorpio compared with Homol-
ogous Structures in other Arachnida. Ann. and Mag. of VN. H. (6).
Vol. xiii.

'94a SCHIMKEWITSCH, W. Sur la Signification de l’Endosternite des
Arachnides. II. Zool. Anz. Jhg.17, No. 444.

'94b SCHIMKEWITSCH, W. Ueber Bau und Entwicklung des Endosternits
der Arachniden. Zool. Jahrb. Abth. f. Anat. Bad. viii, Heft 2.

’'96 PATTEN, W. Variations in the Development of Limulus. /ourn. of
Morph. Vol. xii.

'97 GASKELL, W. H. On the Origin of Vertebrates deduced from the
Study of Ammocoetes. Journ. of Anat. and Phys. Vol. xxxii.

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STUDIES ON LIMULUS. 21

REFERENCE LETTERS.

abdominal endochondrite.

anterior cornua.

anterior process of abdomi-
nal endochondrite.

apodeme.

capsuliginous bars.

chilaria.

origin of chilarial muscles.

cross-commissures.

foramina.

haemal division of anterior
cornu of plastron of Apus.

haemal nerves.

haemal processes of ab-
dominal endochondrite.,

haemal processes of plas-
tron.

intestinal nerves.

integument.

lateral cornua.

latero-posterior processes.

middle division of anterior
cornu of plastron of Apus.

mLem.

membrane to which are
attached the veno-peri-
cardiac muscles.

marginal ridge.

neural division of anterior
cornu of plastron of Apus.

neural nerves.

neural plates.

neural processes.

occipital ring.

oesophageal collar.

origin of opercular muscles.

posterior processes of ab-
dominal endochondrite.

posterior process.

shoulder joining latero-pos-
terior processes.

veno-pericardiac muscles.

. points of muscular attach-

ment on plastron of Apus.

membrane attached to pos-
terior side of plastron of
Apus.

eh

PESTER mK Ma

a‘

rate

STUDIES ON LIMULUS. 23

EXPLANATION OF PLATE I.

Fic. 1. X 2. Endosternite of Zzmu/us from neural side, anterior extremity
towards top of plate. Only the bases of the lateral cornua (/. c.) are represented.
The anterior cornua (a. c.) are prolongations of the thickened sides of the plas-
tron. The sides of the plastron are roughened by muscular attachment. Inside
the marginal ridge (#. 7.) the floor of the plastron anteriorly is smooth and
marked only by the slight transverse furrows indicating the course of the fibers
of the cartilage, for there are no muscles attached here except a few strands
going to the oesophagus. Posterior to this space the irregular markings indicate
an area of muscular attachment, the anterior limit of which is marked by a V-
shaped outline. The marginal ridge (m. rv.) runs along each side of the plas-
tron, from the anterior cornua to the occipital ring (oc. 7). Anteriorly the ridge
rises sharply above the floor of the plastron, but posteriorly it is only a low,
rounded shoulder.

The occipital ring (oc. .), shaded lighter than the rest of the plastron, is marked
on the anterior margin by the attachments of muscle fibers going to the integu-
ment behind the mouth. On the top are two shallow pits (cz. m.) in which a
pair of muscles going to the chilaria take their origin. From the posterior side
of the ring thin strands of connective tissue go to the capsuliginous bars (caf. é.).
The capsuliginous bars (caf. 6.) arise from the thinner portion of the plastron, in
the angle between the posterior process (/. Zr.) and the latero-posterior processes
(2. p. pr.). The posterior process (/. 7.) is unpaired and is divided into two
divisions.

The first pair of foramina (/*) are represented by black dots at the bases of the
latero-posterior processes in the thick portion of the cartilage; the second pair
(7”) by the light circles in the thin portion back of the occipital ring.

Fic. 2. X 2. Endosternite of Zzmulus from haemal side. The distal ends of
the lateral cornua upon the left side are disconnected from the proximal portions.
These cornua are fully one-third longer than here represented. The drawing
shows their mode of attachment to the haemal side of the plastron and the
courses of the fibers in and about their bases. This portion of the haemal sur-
face is free from muscles. The area of muscular attachment begins with the
‘splintery appearance just anterior to the haemal processes (#. fr.). The edges of
the plastron between the lateral cornua and the haemal processes are slightly
‘ elevated. The haemal processes (2. Zr.) are stout and rise a considerable distance
above the body of the plastron.

The first pair of foramina (7-") appear as dark slits a short distance behind the
haemal processes.

The posterior process (7. #7.) begins as a low ridge in the median line opposite
the haemal processes. Each division of its forked end is deeply grooved. The
capsuliginous bar (caf. 6.) on the left is shown entering the base of one of the
chilaria (c#.), to the posterior side of which it is attached. Running along the left
side of the plastron is a connective tissue membrane (mem.) to which the veno-
pericardiac muscles are attached. The bases of the two anterior of these muscles
(v. ~. m.‘—*) are represented. Neural to the base of the second muscle (vz. /. m.?)
the membrane is attached to the integument (z77¢.) by a bundle of connective tissue

24 PATTEN AND REDENBAUGH.

fibers. Alongside the chilaria it is attached to the integument by a similar bundle
of connective tissue.

Fic. 3. X 2%. Posterior portion of plastron of Zimu/us and first abdominal
endochondrite, with posterior half of oesophageal collar (oe. col.) and nerves,
neural side. Four of the cross-commissures (cr. com.) are shown. In each neu-
romere are two pairs of nerves—a neural pair (7. #.4~*) and a haemal pair (4. 7.4—*).
Of the neural nerves, x. 7.4, 2. 2.5, and x. 2.° supply the fourth, fifth, and sixth
pairs of legs respectively; 7. 7.’, the chilaria; and xz. 7.8, the operculum. All the
haemal nerves supply the skin of the carapace and other organs; 4. 7.° and 4. 2.7
give off small nerves (cz. .° and zz. 2.7) through the first and second pairs of
foramina (/7 and /?) respectively; 4. .° also gives off a small nerve (. .°) just
posterior to the plastron. This is seen through the semi-transparent connective
tissue attached to the capsuliginous bars. These small nerves (zz. 2.°-*) com-
municate with the sympathetic system supplying the intestine and longitudinal
abdominal muscles. The nerves x. 2.7—*, 4. 2.7’, and the ventral cord (v. c.) pass
through the occipital ring. The ventral cord passes haemal to the abdominal
endochondrites, the first of which (ad. ez.) lies at the base of the operculum. The
shallow pits (of. m.) on the surface of this endochondrite, similar to those on the
occipital ring, represent the attachments of a pair of muscles entering the opercu-
lum. Posterior and anterior to these pits the endochondrite is in contact with
the integument. In some cases the anterior and posterior processes (a. Z. and £. .)
of successive endochondrites are continuous with each other as thin strands of
connective tissue. The haemal processes (Z. .) which straddle the ventral cord
give attachment to haemo-neural muscles.

Fic. 4. X 2. Endosternite of Zzmu/us from posterior side, showing the occi-
pital ring (oc. ~.) and its relation to the nerves. The portion of the plastron
anterior to the occipital ring is not represented.

S.is a thickened shoulder uniting the bases of the latero-posterior processes
(2. p. pr.). Other reference letters as in Fig. 3.

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26 PATTEN AND REDENBAUGH.

EXPLANATION OF PLATE II.

Fic. 5. X 45. Endosternite of Agus, neural side, anterior extremity towards
top of plate. The jagged ends (m.) furnish attachment for the muscles of the
mandibles. The anterior cornua (a. c.) are each divided into three parts (4., m.,
and z.). A pair of chitinous apodemes (afo.) project into the endosternite on
the posterior side. Muscles going to the maxillae are attached to the small
processes at x. A few strands going to the integument are attached at y. The
process z is a thin membrane attached to the integument between the longitudi-
nal commissures of the ventral cord.

Fic. 6. X 45. Endosternite of Apfus, haemal side. Longitudinal abdominal
muscles are attached to the jagged processes (w.) on the posterior side. Other
reference letters as in Fig. 5.

Fic. 7. X 60. Sagittal section of endosternite of Afws near median line;
anterior end towards the right of figure. It shows the neighboring integument
(¢nt.) with the muscles going to it from y, the relations of the membrane z to the
integument, and the positions of the cross-commissures (cv. com.*~3) with reference
to the endosternite.

Fic. 8. X 60. A section of the plastron of Afus a little farther from the
median line than the preceding. It shows the apodeme (a/o.) and the tendinous
cord running frem it, through the plastron, to the anterior cornu (a. c.). The
process x, the membrane z, and the projections due to attachment of abdominal
muscles to posterior side of apodeme are cut by this section.

Fic. 9. X 60. A section of the plastron of Afws still farther from the median
line. The apodeme (aZo.) is approaching the exterior. All three divisions (4., m.,
and 7.) of the anterior cornu (a. c.) are cut through.

Fic. 10. X60. A cross-section through posterior part of plastron of Apus.
It cuts the inner ends of the apodemes (afo.), the muscles at y, and a pair of
ganglia of the ventral cord (v. c.). .

Fic. 11. X10. Endosternite of A/yga/e, neural side. The anterior cornua
(a. c.) are very large. The neural processes (#. Zr.) are in contact with the integu-
ment. The neural plates (z. 4.'~5) project vertically from the body of the plastron.
The ends of the five haemal processes (4. f7.'-5) protrude beyond the edges of the
plastron. The posterior process (/. fv.) is not cleft, as in Limulus.

Fic. 12. X10. Endosternite of A/yga/e, haemal side. Five pairs of haemal
processes (#. fr.'—5) rise from the sides of the hollow in which the intestine lies.
Reference letters as in Fig, 11.

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A CONTRIBUTION ON THE MINUTE ANATOMY
OF THE SYMPATHETIC; GANGEDA OF THE: DIF-
FERENT CLASSES OF VERTEBRATES.

G. CARL HUBER.

INTRODUCTION.

EVEN a cursory review of our knowledge of the minute anat-
omy of any tissue or organ teaches us that the various steps in
the perfection of our knowledge are in a large measure synchro-
nous with advances made in microscopical technic. This is,
perhaps, to no one more clearly shown than to him engaged
in neurological work. The introduction of solutions of osmic
acid and gold chloride, the Weigert’s haematoxylin method, the
methods of Golgiand Ehrlich, and the numerous other special
methods, have, each in its turn, directed the attention of investi-
gators to results hitherto unattained. This is especially true
of the chrome silver and the methylene blue method; the results
obtainable by these two methods, variously modified (embracing,
as they do, a large portion of our more exact knowledge of the
shape of neurons, and especially of their relation to each other
and to the myriads of cells under their influence), have led
many workers to devote much of their time to the investigation of
various portions of the central and peripheral nervous system
of vertebrates, and to some extent, also, of the still larger group
of invertebrates. Of this number many, no doubt, encour-
aged by the earlier results of Kolliker and Ehrlich, have used
these methods for the investigation of the sympathetic or gan-
glionic nervous system. And it is gratifying to reflect that the
researches of Kolliker, Ehrlich, Aronson, Arnstein, Smirnow,
Cajal, Van Gehuchten, V. Lenhossék, Sala, Retzius, d’Erchia,
Dogiel, and others have very much broadened our knowledge con-
cerning this system, many points having been observed so often
that they are beginning to be accepted as demonstrated facts.

27

28 HUBER. [VoL. XVI.

My reason for presenting the following research may, if such
reason be required, be briefly stated as follows:

It seemed to me desirable to confirm some of the observa-
tions previously made, with what seemed to me somewhat im-
proved histological methods, hoping at the same time to throw
new light on some of the still disputed questions, and especially
to broaden our knowledge of these structures by systematically
investigating the sympathetic ganglia of types from the several
classes of vertebrates, using in each case the same methods.

During the investigation, material was obtained from the
following vertebrates:

FISHES.

Ambloplites rupestris (Raf.), rock bass.
Micropterus dolomieu (Raf.), small-mouth black bass.
Perca flavescens (Mitch.), perch.

AMPHIBIA.
Rana Catesbiana. Rana Hal.
REPTILIA.
Chrysemys picta. Chelydra serpentina.
Emys meleagaris.
BIRDS.
Gallus domesticus — chicken.
MAMMALIA.

Dog, cat, rabbit, and guinea pig.

The subject-matter to be discussed in this research will be
taken up as follows:

1) A brief discussion of the methods used.

2) A descriptive account of the results obtained in the prep-
arations made from each of the above classes of vertebrates,
in the order above named, with the literature bearing on the
subject.

3) The general conclusions which may be drawn.

Method.

In the earlier portion of this work both the chrome silver
and the methylene blue method were used; the former was,
however, soon discarded, owing to its greater uncertainty ;

No. 1.] SYMPATHETIC GANGLIA OF VERTEBRATES. 29

largely, however, because in my hands more definite results
were obtained with the methylene blue method. For the past
two years the latter method alone has been used, and the
results here presented are based exclusively on observations
made with it.

Ehrlich’s methylene blue method has so often been modified
in one way or another, that, in order to discuss it at all, it
seems quite imperative to take it up historically. Riese (1)
has, however, collected all the literature bearing on this method,
appearing before 1891; for the earlier modifications, therefore,
the reader is referred to this summary. Since that time the
most important modification of the method has been suggested
by Bethe (2), who, in recommending the use of ammonium molyb-
date to convert the unstable methylene blue compound, as formed
in fresh tissues, into one practically insoluble in water, alcohol,
and the imbedding media, has given us a method by which
methylene blue stained tissues may be imbedded in paraffin, sec-
tioned and counterstained at will. Too much cannot be said
for this important addition to the method suggested by Ehrlich.

The fixative suggested by Bethe has the following compo-
sition :

AmmMoniumimolybdate . . < .« =) » /.aese ieee
PN CMARGESE re pega) Yeh) «<6, o> 1a) 5 Se Oe Lem:
Elydrocensperoxide 3 9: « 2. -2i evan G.em:
Etydrochloric acid 4) . .)-| . os eee See

Into this solution, cooled to + 2° C., the stained tissues are
placed for four to five hours; are then washed in water, dehy-
drated in alcohol, and imbedded in paraffin. Meyer (4) suggests
the omission of the hydrogen peroxide from the above formula,
because, as he believes, it has a bleaching action — “etwas
bleichend gewirkt hat.’ Bethe (3) in a more recent communi-
cation has, however, drawn attention to the fact that this strong
oxidizer is not, as such, present in the fixing fluid, because it at
once unites with the molybddnsauren ammonium, and this has
in the main lost the properties of the hydrogen peroxide. For .
a further discussion of this question the reader is referred to
Bethe’s (3) recent article. I may, however, add that before
Meyer’s paper came into my hands similar conclusions had

S320 HUBER. [Vor. XVI.

been reached by me; namely, that better results were obtained
when the hydrogen peroxide was omitted from Bethe’s formula.
It may be stated that it has always been my plan to expose
the ganglia, or other tissues to be stained, to the air, either in
the animal or on the slide, until all the elements which it was
thought would stain were clearly seen under the microscope;
or, in other words, until the leukobase, the tetramethyldiamido-
thiodiphenylamin, which is formed by the living tissues when
methylene blue comes in contact with them, is again oxi-
dized, as it is in the presence of air. Hydrogen peroxide,
or, as used in the formula, hypermolybddnsauren ammonium,
would in such cases be unnecessary, and does, to some extent
at least, as Meyer suggests, have a bleaching effect. I wish
further to call attention to other modifications which Bethe
(3) himself has suggested. He has tried to obviate the neces-
sity of placing the tissues into an “ice-cool mixture,” as his
earlier method requires, by changing the unstable methylene
blue stain, as found in fresh tissues, into one insoluble in water.
To this end he places the fresh tissue in a saturated aqueous
solution of ammonium picrate (suggested by Smirnow and
Dogiel), and, at the expiration of fifteen to twenty minutes,
transfers them to a solution prepared after one of several form-
ulas, one of which is the following :

Ammonium ‘molybdate’ <=) ees ens) ak EM:
Aqgiia.dest. 2.0) 2 Samay alti neenee te nl tee PO VC- CE
Hydrochloric! acid i oen is meets can em EL ene:

Tissues two to three millimeters in thickness remain in this
second solution three-fourths to one hour.

My experience with this modification of Bethe’s earlier method
has not been extended; as far as it goes, I have been led to
conclude that the prefixation in ammonium picrate is open to
a number of objections.

In the first place, picrate of ammonium seems, even in the
short time the tissues are exposed to it, to act more or less as
a macerating fluid, which is obviously an objection where the
relation of end fibrillae to cells is to be investigated. Secondly,
tissues prefixed in the picrate solution do not stain so readily
as when the molybdate alone is used.

No. 1.] SYMPATHETIC GANGLIA OF VERTEBRATES. 31

The methylene blue method, as used by me in this research,
may be briefly described as follows: A 1%, 2%, or 4% solution
of methylene blue (Griibler’s rectificiert nach Ehrlich), made in
a normal salt solution, was injected into a vein, easily accessible.
In frogs, the large lateral cutaneous vein; in Reptilia, the jugu-
lar; in birds, the humeral (Owen); in Mammalia, the jugular
or femoral, usually the former, were used. As to the percent-
age of the solution used, no definite statement can be made;
the above solutions were used with equally good results in some
experiments, and equally unsatisfactory results in others. Ina
general way it may be said that the stronger solutions stained
more readily the cell body and branches of the sympathetic
neurons, while the weaker solutions brought to view more
clearly the pericellular plexuses; these statements are, how-
ever, open to many exceptions.

The quantity injected varied with the size of the animal
used ; from 2 to 4 c.cm. for a frog, to 60 to 80 c.cm. for a
dog. The solution was allowed to flow into the circulation
until the animal became blue, or until the heart’s action was
stopped.

Forty-five minutes to one hour after the injection, the gan-
glia or tissues to be examined were exposed ; the larger ganglia,
with the afferent and efferent nerves, were freed from the sur-
rounding tissues, but not removed until they assumed a blue
color, when they were excised and placed on a slide, and, if
the staining seemed satisfactory, were placed in the fixative.
Smaller ganglia were at once removed to a slide moistened with
normal salt, and examined from time to time, until the stain
was developed, when they were also placed in the fixative. The
fixative used by me had the following composition :

Ammonium molybdate - . = \29\7 0) s esem:
WGN atdestos ks, 2) so 1 he) ONG CON:
Hydrochloric acid, 2. a. 2 ere ott:

The molybdate is ground in a mortar and the water added ;
‘the solution is then removed to a flask and heated until per-
fectly clear; the hydrochloric acid is then added. The solution
so made is placed in small glass jars, and these are surrounded

32 HUBER. [VoL. XVI.

with snow or ice. The solution is best made some time before
the injection, so that it may be properly cooled before using.
In the fixative the tissues remain three to five hours. They
are then washed in distilled water for about one hour and de-
hydrated and hardened in absolute alcohol, placed in xylol and
imbedded in paraffin. As a rule, the ganglia were cut into
serial sections, fixed to slide with the albumen fixative, and
mounted in Canada balsam ; other series were double-stained
in alum carmine before mounting.

My experience as to the durability of preparations made in
this way has not been the most satisfactory, although, as a rule,
they have kept without fading for several months. Some of
the sections (ganglia of Amphibia) have been well preserved for
over two years, and are only now beginning to show signs of
fading ; others (sections of sympathetic ganglia of the chicken)
began to fade at the end of several weeks. My experience
would go to show that sections double-stained in alum car-
mine keep better than such as are stained only in the meth-
ylene blue. Why some sections should seem to fade more
readily than others I am unable to say; it would seem, how-
ever, that sections thoroughly dehydrated and thoroughly
washed in oil of bergamot and xylol fade less quickly than
those where these precautions are not taken. Sections exposed
to light fade more readily than those kept in the dark. Bethe
(2) in discussing this phase of the matter says: “Die Halt-
barkeit der Praparate, die mit der einfachen Fixation hergestellt
sind, ist keine unbedingte. Sehr dicke Praparate zeigen oft
schon nach zwei bis drei Monaten zuerst ein Dunkelwerden des
Canadabalsams, auf den Triibung des Protoplasmas und Diffus-
werden der Farbung folgt.” <‘Schnitte halten sich besser,
vielleicht deswegen, weil hier der Ueberschuss von Ammonium-
molybdat, von dem in Verkindung mit Resten von Alkohol die
Zerstorung auszugehen scheint, besser ausgewaschen wird.”

In attempting to stain the sympathetic ganglia of fishes by
injecting methylene blue into the circulation (through the cau-
dal vein), it was found that the loose adipose tissue in the gill ’
region, in front of the vertebral column, by the removal of
which a number of small ganglia were brought to view, stained

Nowt.j SPATE TIC GANGLIA OF VERTEBRATES. 33
so deeply that after an injection a search for them was usually
fruitless. In fishes, therefore, it was found advisable to re-
move the ganglia, unstained, to a slide or watch crystal, and,
following Dogiel’s suggestion, to stain them ina 4% solution
of methylene blue in normal salt. A portion of a ganglion thus
treated was usually stained in forty-five minutes to one hour,
the staining being controlled under the microscope. The tis-
sues so stained were then fixed in ammonium molybdate, the
further treatment being as above described.

In attempting to stain the tissues on the slide, I have tried a
number of methods, which, in the hands of other investigators,
have given good results. I may especially mention the meth-
ods recommended by Lawdowsky (5). He suggests the dilution
of the methylene blue with one of the following solutions:
Blood serum, egg albumen, a 7,—4-—}% solution of ammonium
chlorate, or ferrum ammonium chloratum. The successful
staining obtained by Lawdowsky has not been realized by me;
on the contrary, results obtained with methylene blue diluted
in normal salt have been much more satisfactory.

In closing the discussion of the methylene blue method, I
need hardly add that, even with the greatest care, negative re-
sults are only too frequent. Why, when this method is used
over and over again in exactly the same manner, in some in-
stances successful staining is obtained, in others only failure,
I am unable to say; but such is the case. Then, of course, it
must be remembered that the investigator is always at a loss to
know whether the preparation before him tells the whole story,
or only a portion of it; whether, in other words, the structure
before him is completely or only partially brought to view by
the methylene blue.

SyMPATHETIC GANGLIA OF FISHES.

The sympathetic system of teleosts, to which subclass my
investigations were confined, consists of a cephalic, a trunkal,
and a caudal or post-anal portion, made up of a series of gan-
glia, united by intervening nerves into two cords lying close to
the vertebral column. The cephalic portion extends forward

HUBER. Vou. XVI.
34

under the place of exit of the vagus, glosso-pharyngeal, and
facial nerves, and sends branches to the trigeminus; at the
point of union of the sympathetic with the cranial nerves men-
tioned, small sympathetic ganglia are usually found. At the
anterior end of the trunk the two chains approach each other
and present a relatively large ganglion — the splanchnic gan-
glion. In the trunk the two chains are found immediately
under the. vertebral column above the kidney, in which they
may in part be imbedded. The caudal portion is found within
the haemal arch, accompanying the aorta. The trunkal and
caudal portions receive rami communicantes from the ante-
rior roots of the spinal nérves. At the point of junction of
the rami with the sympathetic cords small ganglia are found,
which may be of microscopic size, or large enough to be recog-
nized with the naked eye. This brief description of the sym-
pathetic system of fishes is taken from Stanius’s Handbuch der
Zootomte, Zweiter Theil, Die Wirbelthiere, Zweite Auflage,
pp. 143-146. The ganglia especially studied by me were the
splanchnic and several small ganglia found in connection with
the cardiac and intestinal branches of the vagus and the cells
of Auerbach’s plexus.

Literature. — The literature bearing on this portion of the
subject is very meager. In the literature at my disposal I
have found no observations on the structure of the larger sym-
pathetic ganglia of fishes, made with the Golgi or methylene
blue method. Monti (6) has used the Golgi method in study-
ing the intestinal canal of teleosts with reference to its nerve
supply, and has described small ganglia, composed of multipo-
lar cells, possessing a single, unbranched axis-cylinder process.
These ganglia take part in the formation of a plexus found in
the submucosa, and from it fibers are given off which form a
plexus in the muscularis mucosa. This latter plexus is con-
tinuous with a periglandular plexus, in which nerve cells, also
with a single axis-cylinder process and surrounded with a very
complicated network of fine fibrillae, are found. Sakusseff (7),
a pupil of Dogiel, has stained the nerve plexus in the intestine
of fishes with methylene blue. I was, however, not able to
obtain the original article. Dogiel, in his brief reference to

No. 1.] SYMPATHETIC GANGLIA OF VERTEBRATES. 35

this work, draws attention only to the fine fibers which
leave the plexuses, passing through the mucosa to reach the
epithelium.

The ganglia studied by me varied in size from such as were
just recognizable with the naked eye to others about } mm. in
their longest diameter. They were most often recognized as
spindle-shaped swellings in the course of one of the vagus
branches. They were removed to a slide and stained in 55%
methylene blue solution in normal salt ; fixed in ammonium
molybdate and teased or sectioned, some of the sections being
further stained in alum carmine. The ganglia are surrounded
by a fibrous capsule, which is continuous with the perineural
sheath of the nerves connected with the ganglion.

In sections, sympathetic nerve cells, medullated fibers, very
small medullated fibers, and Remak’s fibers may be seen.

Shape and Structure of the Ganglion Cells. — The shape of
the nerve cells in the sympathetic ganglia of fishes varies.
The cell body may be more or less regularly round or oval, and
from it one or several processes may have their origin. The
cells may therefore be unipolar or multipolar. In cells not too
deeply stained in methylene blue the protoplasm appears gran-
ular, the granules staining more deeply than the remaining
portion of the cell. The granules are very small, and evenly
distributed through the protoplasm. The granules are prob-
ably the chromophile granules described for other nerve cells.
In case the cell is deeply stained, it assumes a diffuse blue
color, the granules showing only very indistinctly. In my prep-
arations the nucleus was sometimes stained more deeply, again
less deeply than the protoplasm, usually showing no distinct
structure, although in preparations double-stained with alum
carmine a nucleolus may now and then be made out. The cell
body is invested in a nucleated capsule.

The neuraxis arises from a cone-shaped extension of the cell
body. (See Pl. III, Fig. 1, ae.) In sections it is sometimes
difficult to make out with any degree of certainty which of
the several processes is the neuraxis; for instance, e, of the
above figure. In the neighborhood of the cell the neuraxis
is non-medullated; whether at some distance from the cell it

36 HUBER. [VoL. XVI.

becomes surrounded with a sheath of myelin, I have been unable
to determine.

The dendrites vary in number ; four to six are usually seen.
They are quite short, and in my preparations do not undergo
much branching; this of course may be due to imperfect stain-
ing. The dendrites terminate between the ganglion cells, are
extra-capsular, and form a loose network, the arrangement of
which depends in part on the number and relative position of
the adjacent neurons.

Medullated Fibers of the Ganglion. —In sections a large num-
ber of medullated fibers are seen in the sympathetic ganglia of
fishes. Many of these pass through the ganglia without giving
off any branches. This is especially the case in the smaller
ganglia, which are recognized as spindle-shaped enlargements in
the course of a nerve trunk. Other medullated fibers, which
give off one or several branches in the respective ganglion, are
frequently met with. These branches, which are usually smaller
than the parent fiber, end in a network on the cell bodies of the
sympathetic cells. This network varies much in complexity ;
and in double-stained preparations it may easily be seen that
this network is zztra-capsular. It may be quite simple, as may
be seen in Pl. III, Figs. 2 and 3, where only the network is
shown. In Fig. 2, a, the portion of the neuraxis shown (a
collateral branch of a medullated fiber), divides into four smaller
branches, which anastomose to form a pericellular plexus; in
Pl. III, Fig. 3, both the neuraxis and the terminal fibrillae are
very small and beset with varicose enlargements. This pericel-
lular plexus enclosed a long oval cell, lying between the nerve
fibers of a ganglion; the cell was only faintly perceptible and
is outlined in black; the processes were not made out. In
Fig. 4 is shown a more complicated ending. The large axis
cylinder, a, which was surrounded by a medullary sheath (not
shown in the figure), breaks up within the capsule into a num-
ber of branches, some of which pass over the cell, others ending
between the cell and the capsule in a number of branching,
twisted, or coiled fibrillae. In Pl. III, Fig. 5, the same cell is
again represented, after staining in alum carmine (the prepa-
ration from which Fig. 4 of Pl. III was sketched was broken

No.1.] SYMPATHETIC GANGLIA OF VERTEBRATES. Sy

down, stained, and remounted). The latter figure may serve to
show that the end fibrillae shown in Pl. III, Fig. 4, terminate
between nuclei found within the capsule. In all instances (see
also Pl. III, Figs. 6 and 7) where this more complicated end-
ing was observed, the cell body of the sympathetic cell seemed
imbedded in these nuclei; some of which, no doubt, belong to
the cells of the capsule; others, if my observations are to be
relied on, are within the capsule and seem to be grouped more
particularly about that portion of the sympathetic cell from
which the neuraxis arises. The number of the nuclei varies ;
in Pl. III, Fig. 6, sketched from a cell obtained by teasing a
small ganglion, and which was completely isolated from the
surrounding structures, I estimated that ten to twelve nuclei
are within the capsule. The capsule enclosing this cell (c) was
very clearly made out. The branches of the nerve fiber ending
within the capsule are clearly shown in the figure, they alone
being stained in methylene blue, the other structures taking
the alum carmine. In PI. III, Fig. 5, above referred to, and
in Pl. III, Fig. 7, the number of these nuclei is far greater.
In the latter figure, which was also sketched from a teased
preparation, may be seen the neuraxis of a large medullated
fiber, from which two non-medullated collateral branches (a! and
a'') are given off, these terminating within the capsule of the
sympathetic ganglion cells (A and £#) in a system of varicose
end branches, the majority of which end between the intra-
capsular nuclei.

It may be of interest to note that Arndt (8) has diagrammed
a cell which resembles very closely the one shown in PI. III,
Fig. 6. The cell referred to is reproduced by him in Pl. XIV,
Fig. 38. Arndt, in his account, describes it as coming from
a sympathetic ganglion of Perca, macerated in a 35% solution
of acetic acid.

I have not been able to formulate any definite conclusions
as to the nature of the nuclei above mentioned. I beg, how-
ever, to be allowed to give expression to a hypothesis which
has often suggested itself in studying the preparations made
from the sympathetic ganglia of fishes, but more particularly
. from similar preparations made from Reptilia ; namely, that these

38 HUBER. [VoL. XVI.

nuclei may belong to very much branched neuroglia cells, only
the nuclei staining in the preparations at my disposal. This
hypothesis is here only mentioned, and will be further discussed
subsequently.

The medullated fibers ending in the pericellular plexuses may
now and then be traced into some nerve root coming to the
ganglion; their further course has not been ascertained.

A few very small medullated fibers are occasionally stained
in sections of the sympathetic ganglia of fishes. My observa-
tions on these do not allow of any definite statement concerning
them.

The non-medullated fibers seen in the ganglia are no doubt,
to a large extent, the neuraxes of the sympathetic cells, con-
stituting the ganglia, although in sections it is an exceedingly
difficult task to trace such non-medullated fibers to a ganglion
cell; I was able to do so only a few times. Small bundles of
them may be traced into the nerves leaving the ganglia.

SYMPATHETIC GANGLIA OF AMPHIBIA.

The sympathetic system of the frog comprises a series of
ganglia, lying on each side of the vertebral column, united
by intervening nerves to form the ganglionated chains. The
number of sympathetic ganglia usually corresponds to that of
the spinal nerves, ten pairs of ganglia being found. Numerous
smaller ganglia are found in the walls of the various organs —
Bidder’s and other ganglia in the heart; ganglia in the lungs,
the pharynx, the intestinal canal, and the bladder. Sections
were usually made of the larger ganglia — the first and second
(this being the largest), and seventh, eighth, and ninth. The
last three, on account of their size, the length of their rami,
and their exposed position, are very easily found.

The methylene blue method was used to the exclusion of
other methods. The stain was injected into the circulation
of the living frog. The larger ganglia were fixed in ammo-
nium molybdate, sectioned, and some of the sections double-
stained in alum carmine. The smaller ganglia were fixed in
ammonium picrate and cleared in glycerine.

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No.1.] SYMPATHETIC GANGLIA OF VERTEBRATES. 39

Literature. —The ganglion cells of the frog’s sympathetic
were first described by Beale and Arnold. In Beale’s (g)
account the following statement may be found: “In the
fully formed cell a fiber comes from the center of the cell
(straight fiber), and one or more fibers (spiral fibers) proceed
from its surface.’ ‘These are wound spirally around the
first fiber.”’ Arnold (10) described similar bodies in connection
with the nerves in the frog’s lung, believing them to be peculiar
to this organ. In Arnold’s (11, 12) later publications he states
that these cells were found generally in the sympathetic system
of the frog, and gives a much more detailed account of the
straight and the spiral processes. The straight fiber he was
able to trace through the protoplasm of the cell to the nucleolus,
while the spiral fiber had its origin in a network of fine fibrillae
connected with the branches of the nucleolus. (See Pl. I, Figs.
3, 4, and 6, Virchow’s Archive, Vol. XXXII.) Courvoisier (13)
corroborated many of the observations made by Arnold. His
results may be summed up as follows: The straight fiber
begins or ends in the nucleus, while the spiral fiber has its
origin in a network which is connected to the nucleolus by
“root fibers” — Wurzelfaden. This network gives origin also
to “commissural fibers,’’ which connect the network of adjacent
cells. This network was also described by Kollman and Arn-
stein (14). On the other hand, Schwalbe (15) stated that the
sympathetic ganglia of frogs contained two kinds of cells —
bipolar cells, with straight and spiral fibers, the latter arising
from the cell body and making only a few turns around the
straight fiber, and unipolar cells, with only the straight process,
with a prominent capsule, the thickenings or folds of which
may simulate a spiral fiber. This latter form Schwalbe believed
to be the more common.

Arndt (16), while not denying the nervous nature of the
spiral fiber, suggests that the fibrillar structure of the capsule
may in some instances be mistaken for a spiral fiber, inasmuch
as the fibrillae of the capsule are now and then arranged trans-
versely to the axis of the straight fiber as it leaves the cell.

Axel, Key, and Retzius (17) made further contributions by
showing that the spiral fiber became invested with a medullary

40 HUBER. [Vou. XVI.

sheath at some distance from the cell body; thus for the first
time pointing out conclusively its nervous nature.

The true nature of the spiral fiber and the network, which
had been described by some of the earlier investigators, and
their relation to the protoplasm and the straight process of the
cell, were, however, not understood until Ehrlich’s methylene
blue method became known. Ehrlich (18), in his first communi-
cation on the reaction of methylene blue on living nerve tissues,
gives a very clear description of the spiral fiber and network.
I take the following brief account from this article: ‘The
spiral fiber, which alone was stained blue, divides into fine
fibrillae, which unite to form a network, which network may
enclose the entire cell or only a portion of the cell. From this
network fine fibrillae are given off, which end on the cell in
small terminal swellings. This mode of ending of the spiral
fiber was observed in all the larger ganglia examined; also
in the smaller ganglia found in the heart, bladder, lung, and
pharynx.” The fact that the spiral fiber was medullated led
Ehrlich to believe that it was a cerebro-spinal fiber, and that
its course was centralward, and to express the opinion that it
conveyed impulses from the nerve centers to the sympathetic
ganglia through the network about the cell. Ehrlich’s obser-
vations were soon corroborated by Aronson (19), and by Arn-
stein and his pupil Smirnow. In the account given by Arnstein
(20) the network formed by the end branches of the spiral fiber
is described as a closed one; the terminal fibrillae mentioned
by Ehrlich and Aronson were only occasionally seen, and then
only when the network was imperfectly stained. Arnstein
states that they found it extremely difficult to determine which
direction —centralward or toward the periphery —the spiral
fiber takes on leaving the cell; often it was impossible to
decide. In the sympathetic cells found in the pharynx of the
frog, the spiral fiber went to the periphery, often branching in
its course. Retzius (21), in a short but very comprehensive
account of his observations on methylene blue stained sympa-
thetic ganglia of the frog, adds a number of details to obser-
vations previously made. He here points out that the terminal
network of the spiral fiber is always within the capsule of the

No.1.] SYMPATHETIC GANGLIA OF VERTEBRATES. I

ganglion cell. The network is described as a closed one with
varicose fibrillae. At the nodal points of the network, nodular
enlargements are often seen; these vary in shape and number.
He further observed that the spiral fiber became myelated at
some distance from the cell, and that it often divided into two
branches, — “tubes en T,’’ —the two arms being often traced
in divergent directions. (See Fig. 3 of his article.) Retzius
looks upon the spiral fiber as of cerebro-spinal origin, and
reaches the following conclusions as to its function: “ Nach-
dem die Spiralenfasern sich getheilt haben, schicken sie den
einen Arm an eine sympathische Ganglienzelle, um in dieser
Weise eine Verbindung mit ihr einzugehen.”

Lawdowsky (22) (whose account I have not been able to
obtain) and Feist (23) have also written on the frog’s sympa-
thetic. The latter’s observations corroborate in the main those
made before him; he finds, however, a different explanation for
the network and spiral fiber. Feist, while admitting that the
spiral and network often stain alike, does not regard this as
sufficient reason for regarding them as identical in structure.
The conditions, he says, may just as well be the following:
«The ganglion cell is united to the capsule through a layer of
protoplasm, arranged in the form of a network. The spiral
fiber, as it approaches the cell, pierces the protoplasmic net-
work at some nodal point and then enters the substance of
the ‘cell

Feist (23) also finds, as his Figs. 20, 21, and especially 22,
may show, that the straight process undergoes division at some
distance from the cell. As a result of his investigations he
reaches the following conclusion, which is so at variance
with results previously obtained that it will be given in full:
«Nach dieser Beobachtung riickt die bipolare Ganglienzelle des
Froschsympathicus in das Schema der multipolaren Hirn- und
Riickenmarksganglienzellen der Sauger ein, nur mit dem Un-
terschiede, dass bei jener die verastelten Fortsatze nahe bei
einander von einer weit vom Centrum der Zelle entfernten
Stelle, der Theilungsstelle der geraden Faser, abgehen, welch
letztere hiernach nur eine lang ausgezogene Partie der Zellsub-
stanz darstellt.’’ If I understand Feist correctly, the spiral

42 HUBER. [VoL. XVI.

process would thus become the neuraxis of the cell, the straight
process, with its branches, the dendrites; a conviction entirely
at variance with the facts in the case, as will be subsequently
shown.

Before concluding the review of the literature bearing on the
structure of the sympathetic ganglia of Amphibia, mention must
be made of Smirnow’s (24) later publications on the subject.
I wish, however, at this place to draw attention only to that
portion of his writings in which he discusses the relation of
the network to the spiral fiber and its further course after
leaving the cell.

According to Smirnow, the spiral fibers extend toward the
periphery, and are, according to the location of the nerve cell,
distributed to various peripheral tissues. ‘They form anasto-
moses with other cells, in so far as they may join the processes
of other cells; or may become continuous with the network
enclosing other cells; or they may be distributed to heart
muscle or form vasomotor fibers to the blood vessels. The
distribution of the spiral fibers is such that, as a rule, a portion
of it serves to form anastomoses with other cells, while the
remaining portion goes to muscle tissue or to blood vessels.”

Nearly all writers who, after Ehrlich, have used the methylene
blue method in their investigation of the sympathetic ganglia
of Amphibia, are agreed that the spiral fibers and the network
enclosing the cell body are continuous, and that they stain more
readily than do the other structures of the ganglion. There
exists, however, a difference of opinion as to the nature of the
spiral fibers; Ehrlich (18), Aronson (19), and Retzius (21) look-
ing upon them as cerebro-spinal fibers, while Arnstein (20),
Smirnow (24), and Feist (23) ascribe to them other properties.

The results obtained by the writer will be taken up as
follows :

1) Sympathetic ganglion cell of Amphibia, its structure, the
straight process and the capsule of the cell.

2) The spiral fibers and the pericellular network and the
course of the spiral fiber after it leaves the straight process.

Sympathetic Ganglion Cell of Amphibia. — It may be stated
at the outstart that the writer regards the sympathetic neuron

No.1.] SYYPATHETIC GANGLIA OF VERTEBRATES. 43

of Amphibia as a unipolar cell, adopting in this respect Kolli-
ker’s (25) view. In methylene blue stained preparations the
cell body of such neurons remains, as a rule, wholly unstained.
In such sections, double-stained in alum carmine, its outline
may, however, be clearly made out, as Pl. III, Figs. 9 and Io,
may show.

In such sections it may be seen that the cell body is more
or less regularly round or oval, becoming attenuated at one
end, from which portion arises the straight process, a, of Pl.
III, Figs. 9 and 10 respectively. The straight process often
remains unstained ; in some cells, however, it is stained faintly
blue, and in such cases it gradually loses its blue color as it
approaches the cell body, becoming continuous with the drawn-
out portion of the cell. Smirnow (24) states that the straight
process may in some cases take on a medullary sheath (see
Fig. 16 of Smirnow’s article). This I have not seen. In sec-
tions only relatively short, straight processes are seen; they
are not, as far as my observations go, medullated near the cell
body. In sections of the nerve roots coming from the sympa-
thetic ganglia, very small medullated fibers may now and then
be seen, no doubt medullated sympathetic fibers such as Smir-
now describes, and as were seen many years ago by Kolliker
and others in the sympathetic nerves of the frog. The great
majority of the sympathetic fibers of the frog are, however,
non-medullated. Schwalbe, Feist, and Smirnow state that the
straight process may undergo branching at a variable distance
from the cell body. The former (Schwalbe and Feist) regard
the straight process as a dendrite, if I understand Feist correctly,
as has been stated in the review of the literature. Smirnow
(24) likens the branching seen by him (Fig. 11 of his article)
to a “T-shaped process, as seen in the spinal ganglia. He
leaves it an open question whether all straight processes divide
in this way.

In sections of larger ganglia such a division was never seen
by me. The straight process is here, as a rule, so indistinctly
stained that it could not be traced for any distance. In surface
preparations of the bladder and pharynx, stained, zztra vitam,
in methylene blue, fixed in ammonium picrate, and cleared in

44 HUBER. [VoL. XVI.

glycerine, I was, however, able in a number of instances to
trace the straight process of a sympathetic cell for some dis-
tance from the cell body, and in one preparation of the pharyn-
geal mucous membrane and the oesophagus of a frog prepared
as above stated, a straight process coming from a sympathetic
cell, which divided into two branches, was clearly made out.
These branches could be traced toward a small gland situated
in the upper part of the oesophagus, where both underwent a
second division; from here one of the resulting branches could
be traced on to one of the alveoli of the gland, where it was
lost to view. The appearance here presented seemed to indi-
cate that the straight process extended toward the periphery,
and in this case innervated the small gland above mentioned,
although the ultimate endings on the gland cells could not be
made out. In the few cases seen by me, where the straight
process branched, this did not simulate a “‘T’’-shaped branching
as described by Smirnow; the branching was usually at an acute
angle. The arrangement seen by him I should regard as acci-
dental. The observations made by me led to the conclusion
that the straight process is the neuraxis of the unipolar sym-
pathetic cell of Amphibia, innervating gland and non-striped
muscle tissue, as my observations on the bladder of the frog
would show. This conclusion is, therefore, wholly in accord
with the following statement made by Kolliker (25): “Die
geraden Fasern sind nicht Dendriten oder Protoplasmafortsat-
zen zu vergleichen, sondern einfach Achsencylinderfortsatze der
betreffenden Zellen, die zu Muskelfasern treten und hierbei
Verdstelungen zeigen.”

The statement has been made that the sympathetic neurons
of Amphibia were unipolar cells. As an exception to this, I
may say that the sympathetic cells found in the wall of the
intestinal canal (stomach, small and large intestine) of frogs are
distinctly multipolar, as the two cells reproduced in Pl. III,
Fig. 11, may show. These cells were sketched from a prepara-
tion of the large intestine of a frog, stained, zztra vitam, in
methylene blue, and fixed in ammonium picrate; in the figure
the purplish color assumed by methylene blue stained tissue
when fixed in the picrate is not reproduced. The cell body of

No.1.] SYMPATHETIC GANGLIA OF VERTEBRATES. 45

these neurons is very irregular; the neuraxis, a, which in the
cells sketched could be traced for a long distance as an exceed-
ingly fine fiber, was lost in the network of Auerbach’s plexus.
The dendrites, 4, have a very irregular, jagged appearance,
perhaps better shown in the figure than can be described.
Such multipolar cells were seen only in the intestinal canal; in
all other sympathetic ganglia of the frog known to me the cells
are unipolar.

Pericellular Network and Spiral Fiber.—In well-stained meth-
ylene blue preparations of any sympathetic ganglion of a frog it
may be seen that the cell body of each neuron constituting said
ganglion is enclosed in a pericellular network or plexus. In
such preparations, double-stained in alum carmine, it may
readily be seen that this network is intra-capsular (see Pl. III,
Figs. 9 and 10, ¢, capsule of cells), and is in contiguity with the
enclosed cell body, but never continuous with it. The arrange-
ment and structure of the fibrillae constituting the network are
very variable, as may be seen from PI. III, Figs. 8-10. The
fibrillae may be very loosely or more densely woven; their
arrangement seems largely accidental. The fibrillae may be
smooth or varicose. This apparent difference in structure
seems to depend somewhat on the length of time the tissues
are exposed to the air before fixing them. It will be remem-
bered that with this method the nerve tissues are usually almost
colorless about one hour after the injection, the interval of time
allowed to elapse before the ganglia are exposed ; if, on exposing
the ganglion to the air, they become blue in a few moments, as
they often do, the fibrillae of the network have a much smoother
appearance than when it is necessary to expose them ten, fifteen,
or even twenty minutes before the stain has developed, in which
case the fibrillae usually present a very varicose appearance.
Now and then quite large nodular masses, stained deeply blue,
may be seen at some nodal points in the pericellular network,
d, of Pl. III, Figs. 8 and 9; Retzius (21) has also described
such nodular enlargements. The question as to whether the
network is an open one, as described by Ehrlich (18), Aronson
(19), and Lawdowsky (22), or a closed one, — Arnstein (20),
Smirnow (24), and Feist (23), — has, I believe, been most cor-

46 HUBER. [VoL. XVI.

rectly answered by Retzius (21), who states that, as a rule, the
fibrillae form a closed network, but that now and then free-
ending fibrillae may be observed. In Pl. III, Fig. 8, which
represents a portion of a section of the sympathetic ganglion
on the second spinal nerve of a frog, in which the structures
were very well stained in methylene blue, I have indicated by
the letter ¢ a number of instances where fibrillae seem to end
free, usually in a small nodular enlargement. That the net-
work is continuous with the spiral fiber seems evident to all
investigators, with the exception of Feist (23), who have studied
the sympathetic ganglia of Amphibia with the methylene blue
stain. Most often the network and spiral are alone stained,
and in sections of 20 w to 40 mw thickness the spiral fiber may
often be traced with the utmost clearness through the “ gran-
ular mass” into the fibrillae of the network ; the granular mass,
g, of Pl. III, Figs. 8-10, forming a more densely woven portion
of the network, as has been pointed out by Retzius (21).

My own observations on the course of the spiral fiber after
it leaves the straight process lead me to conclude that its course
is centralward; or, to express it in another form, the pericellular
plexus and spiral fiber are parts of a neuron, the cell body of
which is situated more centrally than the respective sympathetic
ganglia, in which the structures are found. These conclusions
are based on observations made on ganglia prepared in the
following way: a small quantity (5 to 1 c.cm.) of a 1% solu-
tion of methylene blue was injected into the circulation of a
small frog. At the expiration of about an hour the entire sym-
pathetic system was exposed. The entire sympathetic chain
on one side, with the rami communicantes and segments of the
spinal nerves from which they had their origin, was then
removed and placed on a slide moistened with normal salt.
As soon as the stain seemed developed as much as it would,
the preparation was fixed in ammonium picrate and cleared in
glycerine. When properly fixed and cleared, the entire chain
was placed on a slide, where all the surrounding tissues were
removed with teasing needles, care being taken not to tear any
of the rami from the ganglia. The entire chain, thus freed
from all connective tissue, was then mounted in ammonium

No.1.] SYMPATHETIC GANGLIA OF VERTEBRATES. 47

picrate and glycerine. By injecting only a small quantity of
the methylene blue stain, as above stated, now and then only
a few nerve fibers were stained in some of the ganglia of the
chain, the ganglia clearing in the glycerine to such an extent
that such fibers could be followed for long distances, even under
the oil immersion. In such preparations it was often possible
to trace a medullated fiber from a white ramus into a ganglion,
or from the interganglionic nerve trunk into the respective
sympathetic ganglion, and through several branchings to its
ending in a spiral and pericellular network.

Some of my best examples are reproduced in Fig. I. I
may say that the fibers here sketched were drawn under a
zs in. oil immersion lens, with the camera lucida, and drawings
reduced 5 or 25 times, as stated in the description of the
figure. In the above figure, x by the side of a fiber indicates
a node of Ranvier, and y the change of a medullated to a non-
medullated fiber. In fiber A is shown the neuraxis of a large
medullated fiber, which entered the first sympathetic ganglion
through a nerve uniting the ganglion with the vagus ganglion;
the fiber was traced for a distance much greater than shown in
the figure. Soon after entering the ganglion this nerve divided
into two branches, both of which were medullated. The right
branch was traced through three nodes of Ranvier, when it
became non-medullated. After a short distance this non-
medullated fiber divided into two branches, one of which was
traced into a spiral fiber and pericellular basket; the other
terminated abruptly. The left branch of fiber A was traced
through four nodes of Ranvier, where a small collateral branch
was given off (6 in the figure), which could be followed for only
a short distance. At the succeeding node the fiber became
non-medullated and divided into three branches, c, d, and e.
Branch ¢ was traced to a pericellular network; branch d, re-
flected upon itself at d’, divided into two branches, one of
which ended in a spiral and network ; branch e was followed a
long distance, when it also terminated in a spiral and pericel-
lular network. Fibers B, C, and D are sketched from the eighth
sympathetic ganglion. In each case the fiber sketched shows
only a short segment of the neuraxis before branching; these

48 ; HUBER. [Vot. XVI.

Fic. I. — Spiral fibers from sympathetic ganglia of frog. (See text for description.)

No.1.] SYMPATHETIC GANGLIA OF VERTEBRATES. 49

were traced in every instance into a white ramus of the
ganglion.

Fiber & may be explained somewhat more fully, as it ex-
plains, I believe, some observations made by Retzius (21) and
Smirnow (24). This fiber entered the eighth sympathetic gan-
glion through a white ramus, and could be traced through a
number of nodes of Ranvier, only two of which are shown. At
the second node shown in the figure there was given off a non-
medullated collateral branch, which terminated in a spiral and
pericellular plexus ; two nodes further on the fiber became non-
medullated, ultimately branching into two branches, each of
which terminated in a spiral and plexus. In a teased prepara-
tion, showing only the upper portion of the fiber, it may easily
be seen how one might be led to say that the spiral fiber
branched “ex 7,” as Retzius (21) has done, and diagrammed in
Fig. 3 of his article. Arnstein (20) and Smirnow (24) have
also described a branching of the spiral fibers; they, it will be
remembered, assume that the spiral fiber has a peripheral course.
Such observations were made, I believe, on only partially stained
fibers. Whenever I was able to trace a spiral fiber to a medul-
lated fiber which had a peripheral course, such medullated fiber
always terminated in a spiral in some more distal portion of the
ganglion.

The anastomosis between pericellular baskets of adjacent
ganglion cells, described by Smirnow (24) and long ago men-
tioned by Courvoisier (13) and others, I have never seen in
sections. In methylene blue preparations of whole ganglia,
cleared in glycerine, I have now and then seen what might
be regarded as an anastomosis between two baskets. It would
not, it seems to me, deserve the importance given it by
Smirnow (24).

The observations above mentioned seem to me to present
very strong evidence in favor of the hypothesis long ago
expressed by Ehrlich (18) and Retzius (21), namely, that the
spiral fiber was the ending of a cerebro-spihal fiber. They seem
to me to show also that the course of the spiral fiber, after it
leaves the straight process, is centralward. I have attempted
by the degeneration method to determine this more clearly by

50 HUBER. [VoL. XVI.

experiments of the following nature: the spinal cord of frogs
was destroyed by inserting a hot needle through the peripheral
end of the spinal canal; anaesthetized frogs stand this very
well. In the frogs so operated upon, the posterior extremities
seemed entirely paralyzed. At the end of five, ten, and twenty-
five days a certain number of frogs were injected with meth-
ylene blue, with a view of staining the sympathetic ganglia. It
was found, however, that even in frogs in which a portion of
the cord had been destroyed twenty-five days previous to the
injection, the nerves were not as yet degenerated; even the
motor endings, in the sartorius, for instance, could be stained
very easily. The sacral sympathetic ganglia showed, in all but
one instance, the spiral fibers. This experiment (of ten days’
duration) seemed at first conclusive, as only a few spiral fibers
and baskets were stained; the unipolar ganglion cells stained
much more readily than usual. Later experiments, where a
longer time elapsed between the primary operation and the
injection, did not, however, corroborate the above result. It
may be stated that not much reliance can be placed on these
experiments, as they were made on “ winter frogs”’ in the early
spring, at a time when it is well known that their metabolic
changes are at their lowest. This, I think, may account for
the fact that no degeneration of nerves was observed, dissection
showing that the posterior portion of the spinal cord was com-
pletely destroyed. It is my aim to repeat such experiments on
more suitable frogs. |

The following brief summary of the results obtained in the
study of the sympathetic ganglia of Amphibia may here be in
place :

1) The sympathetic neurons of the frog are unipolar cells,
the neuraxes of which (straight processes) innervate involuntary
muscle and gland tissue (Kolliker). .

2) Entering the sympathetic ganglia through the white
rami, or through the interganglionic nerves, are seen medul-
lated fibers, probably of cerebro-spinal origin, which fibers divide
in the ganglion into two, possibly three, fibers, also medullated ;
at various places in the course of these fibers non-medullated,
collateral branches are given off, which, with or without further

No.1.] SYMPATHETIC GANGLIA OF VERTEBRATES. 51

branching, end in spiral fibers and pericellular plexuses. After
a longer or shorter course in the ganglion, the medullated
branches lose their medullary sheath, and, as non-medullated
fibers, also end in spiral and pericellular plexuses.

3) The pericellular plexuses surround the cell bodies of the
sympathetic neurons, and are always intra-capsular. They
represent the ending of cerebro-spinal fibers in the ganglion.

SYMPATHETIC GANGLIA OF REPTILIA.

In the tortoise the sympathetic chain begins in the superior
cervical ganglion. The nerve connecting this ganglion with
the succeeding one runs in close relation to the pneumogastric
to about the middle of the neck, where it leaves the nerve, and,
passing outwards and backwards, reaches the middle cervical
ganglion; in Chelydra serpentina, a ganglion nearly a centi-
meter in length. From this ganglion several branches are
given off, the largest of which passes toward the axilla and
ends in the inferior cervical ganglion. Crossing the brachial
plexus, the nerve which forms the continuation of the cord
ends in the stellate ganglion. The relative size of these three
ganglia differs in the several species studied; also, to some
extent, in the same species. They are all, however, relatively
large, and for this reason were used almost exclusively. They
may be readily exposed by removing the plastron and pushing
aside the tissues of the neck. From the stellate ganglion two
filaments run back, one on each side of the subclavian artery,
to a small ganglion which lies on the first dorsal nerve. In
the succeeding dorsal segments are found small sympathetic
ganglia, resting on the spinal ganglia. The sympathetic cord
then runs back and becomes connected with sacral nerves.
The two chains may then be traced as a single nerve into the
tail, a number of small ganglia being found here. The account
of the sympathetic system of the tortoise has been taken, with
almost no alteration, from Martin and Moal’s Handbook of
Vertebrate Dissection, Part I: How to Dissect a Chelonian.
Their description has reference more especially to the red-
bellied slider terrapin (Pseudemys rugosa); their description

52 HUBER. [VoL. XVI.

has been followed, as a number of dissections of the species
worked on by me seem to agree with it in all essentials.

The sympathetic ganglia in the pharynx, oesophagus, in the
heart (see Gaskall, Journal of Physiology, Vol. V), in the wall
of the stomach and small intestine, were also studied. The
intra vitam methylene blue method was alone used. The gan-
glia were fixed in ammonium molybdate, sectioned, and some
of the sections mounted at once in balsam; others were double-
stained in alum carmine before mounting in balsam.

In the literature at my disposal I have not seen arty reference
to observations made on the sympathetic ganglia of Reptilia
with either the methylene blue or the chrome silver method.
The older literature was not searched carefully enough to say
whether these structures have been examined at all; this did
not seem necessary, as such references would have no direct
bearing on the observations here recorded.

Sympathetic Neurons of Reptilia: Tortotse. — The sympa-
thetic neurons of the Reptilia studied vary greatly in shape and
size. Their shape may be multipolar, bipolar, or unipolar. Multi-
polar cells are found in all ganglia, and in well-stained cells the
dendrites and neuraxes can usually be differentiated. Only
one neuraxis is found; this may arise from the cell body, as
is the usual way, or from a dendrite at some distance from the
cell body.

Bipolar cells are not numerous, and are usually found near
one of the poles of a ganglion, between the efferent or afferent
nerve fibers. Such cells are now and then seen in peripheral
nerves some distance ‘from a ganglion; they are now and then
seen in the nerves found in the oesophageal mucosa. Cells
which I have classed as unipolar belong to the largest cells
seen; their peculiarity calls for a somewhat more extended
description. These cells belong to the largest cells found;
they are much more numerous in the larger ganglia of the
chain, although they have often been recognized in many of
the smaller ganglia. The cell body of such cells (see Fig. IT,
and Pl. IV, Fig. 12) is usually more or less regularly round or
oval, and very often presents a depression at one side of the
cell body, from which arises one large process. This process

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Nost.) SYMPATHETIC GANGLIA OF VERTEBRATES. 53

varies greatly in shape and in the course it assumes after leav-
ing the cell body. It may be quite straight (Fig. II, A, and
Pl. IV, Fig. 12, A and #), and break up at a variable distance
from the cell into secondary branches; or it may assume a
tortuous course (Fig. II, &, CG, and £), and may (see figure)
be partly wound around the cell or twisted upon itself. This

Fic. I1.— Sympathetic neurons of Reptilia. (See text for description.)

process may again be quite short (Fig. II, D), and break up at
once into a number of large secondary branches. In such
cells the neuraxis may arise directly from this large process,
Ol Cel Ns. Wl) TV i Pics 12) and ca, oulcellsms, oc, and of
Fig. II, in which case the large process becomes gradually
attenuated and stains more deeply; or it may arise from one
of the dendritic branches at some distance from the cell body,

54 ILO BET [VoL. XVI.

as in cell A, Fig. 12 (in which case the neuraxis was recog-
nized by a nucleus of the sheath of Schwann, 4, of figure), or
again, cell D of Fig. II, where the neuraxis was recognized
by its greater affinity to the methylene blue. It should be
stated that the figures here reproduced were sketched from
sections of sympathetic ganglia of Chrysemys picta and Chel-
ydra serpentina, which accounts for the abrupt ending of some
of the processes. The facts here given have been abundantly
verified on fresh tissues.

In methylene blue stained preparations of sympathetic gan-
glia of Reptilia, examined fresh or in sections of such ganglia
fixed in ammonium molybdate, the chromophile granules of the
protoplasm are clearly seen. These granules vary in shape and
size, and are distributed more or less evenly through the cell
protoplasm, leaving free, however, a narrow zone immediately
surrounding the nucleus, as may be seen in PI. IV, Fig. 12.

The cell body of the sympathetic neuron of Reptilia is sur-
rounded by a nucleated capsule, which seems continuous with
the neurolemma of the neuraxis of said cell; although my
observations on this point are not conclusive.

Within the capsule of many of the sympathetic cells (and
this is especially true of the large unipolar cells above described)
are found a varying number of nuclei, the nature of which has
not been clearly determined. They are always recognized in
sections of ganglia stained in methylene blue and double-stained
in alum carmine; they may, however, also be seen in sections of
ganglia hardened and stained in the more usual methods. In
discussing similar nuclei found within the capsule surrounding
the sympathetic ganglion cells of fishes, it was mentioned that
these nuclei were looked upon as nuclei of neuroglia cells.
Cajal has described cells which he regards as neuroglia cells in
the superior cervical ganglion, and Dogiel (40) has drawn atten-
tion to spindle-shaped or star-shaped cells with long branch-
ing processes found in the sympathetic ganglia of Mammalia.
Such cells, stained in methylene blue, are shown by Dogiel in
Pl. XIV, Fig. 15. They are described as forming a network
outside of the capsule of the ganglion cells. By reason of
the fact that neuroglia cells are not stained in methylene blue,

No.1.) SYMPATHETIC GANGLIA OF VERTEBRATES. 55

Dogiel is inclined to think that the cells described by him do
not belong to neuroglia tissue. He likens them to the branched
connective tissue elements, which are found on the membrana
propria of gland alveoli, which, as he states, are also occasion-
ally stained in methylene blue. The cells in question have
never been stained by me in methylene blue. This may be
due to the fact that in all my work the methylene blue solu-
tion was injected into the living animal, in which case, as is
well known, many elements do not stain as readily as when the
methylene blue solution is added directly to the freshly removed
tissues. My only reason for suggesting that the nuclei found
within the capsule of many of the sympathetic cells of Reptilia
are nuclei of neuroglia cells is based on some observations
made on ganglia hardened in Miiller’s fluid, imbedded in par-
affin and sectioned, the sections being then stained in haema-
toxylin and double-stained in the acid fuchsin —picric acid
solution suggested by Van Gieson. In such sections are seen
a large number of fine fibrillae, which seem to be more or less
intimately connected with the nuclei above mentioned. These
fibrillae are not, I believe, nerve fibrillae, belonging to the
pericellular plexus presently to be described ; their smooth and
regular contour and their apparent connection with the nuclei
would exclude such a supposition. Time has not been at my
disposal to consider these nuclei more fully. I mention the
above hypothesis in the hope that it may stimulate further
inquiry into their structure.

In the sympathetic ganglia of Reptilia, stained in methylene
blue, medullated nerve fibers, which enter the ganglia through
their rami, are abundantly found. Many of these medullated
fibers have been traced into intra-capsular, pericellular plex-
uses. Such pericellular plexuses may be described under two
heads:

@) Simple pericellular plexuses, without spiral fibers, sur-
rounding, as a rule, the cell body of multipolar or bipolar
ganglion cells.

6) Complex pericellular plexuses, with spiral fibers, more
often found surrounding the cell body of the large unipolar
cells above mentioned.

56 HUBER. [VoL. XVI.

The pericellular plexuses of the simpler type resemble very
closely those to be described for birds and mammals. They
are found in all sympathetic ganglia, but are relatively more
numerous in the smaller ganglia. When the shape of the
enclosed cell can be ascertained, it will, as a rule, be found to
be multipolar. But as the sympathetic cells are generally only
imperfectly stained, when the pericellular plexuses are well
stained, the enclosed cells are usually not clearly made out.
The above statement may, therefore, be open to exceptions.
In Pl. IV, Fig. 13, three of the simpler pericellular plexuses
are sketched. In each case the non-medullated axis-cylinder
(a collateral branch or end branch of a medullated fiber, as
one may occasionally trace such a branch to a medullated fiber)
breaks up, just before or after it pierces the capsule (not shown
in the figures), into several secondary branches, which, after
further division, are woven into a network varying greatly in
complexity and arrangement. The fibrillae of the network are
often very varicose, showing quite large nodular enlargements.
This network or plexus is generally seen as a closed one; only
here and there some of the fibrillae seem to end free. (See d
of Fig. 13.) These plexuses are always intra-capsular, and sur-
round the cell body of a sympathetic cell; the capsule, though
often not stained, may be recognized if much of the light is
cut off.

The more complex pericellular plexuses vary greatly in struc-
ture; they are usually found, as above stated, surrounding the
large unipolar cells. Their structure may best be made clear
by reference to a number of figures showing some of the dif-
ferent types found. In Pl. IV, Fig. 16, the relation of such
an end plexus to the enclosed ganglion cell and capsule is
well shown. The axis-cylinder of the nerve fiber (2) ending
in the plexus breaks up into several branches, which are wound
around the single process of the sympathetic cell enclosed in
its end plexus. From these spirally arranged branches second-
ary branches are given off, which, on approaching the cell body
of the sympathetic cell, are woven into a loose basket-like net-
work. Very often the axis-cylinder ending in such pericellular
plexuses is spirally wound around the process of the sympa-

No.1.] SYMPATHETIC GANGLIA OF VERTEBRATES. 57

thetic cell before breaking up into secondary branches. The
number of such spirals and their arrangement are subject to
much variation. In Fig. 14 an axis-cylinder with four spiral
turns is shown. In Fig. 15 is shown a long spiral, which sur-
rounds a long and straight process of a unipolar sympathetic
ganglion cell. In Pl. IV, Fig. 17, we may see a portion of four
pericellular plexuses; special attention is, however, drawn to
the one in connection with the axis-cylinder showing the spiral
turns. The spiral fiber, a, here shown is a collateral branch
of a medullated fiber, a’; the collateral branch, after making
many turns around the process of the enclosed sympathetic
cell (the process and cell body of the sympathetic cell are not
shown in the figure; they can, however, be quite clearly made
out in the section), breaks up into a number of fine varicose
fibrillae, which form the pericellular plexus. Attention needs
further to be drawn to the network of fine fibrillae seen within
the spiral fiber. Many of these fine fibers branch from the
spiral fiber. Their arrangement is, however, so complex that
they can be followed for only a short distance. Such intra-
spiral fibers were seen only a very few times, when the prepara-
tions seemed very well stained. They form a network which
is in very close proximity to the straight process of the enclosed
sympathetic cell, and suggest the possibility that the process
may be stimulated directly through the intra-spiral fibrillae.
This figure shows further some very small varicose fibers (c),
which enter the spiral from without. Where these fibers come
from, and the nature of their ending, I have not been able to
ascertain. I should like to suggest that such fibers may be
sympathetic fibers ending on the sympathetic cell or on one of
its large processes. I possess, however, very few observations
that would tend to strengthen such a view. My reason for
suggesting the hypothesis is based on the fact that, in birds
and Mammalia, end branches of sympathetic fibers would in
some cases seem to end on dendritic branches of other sym-
pathetic cells, as will be stated in the following pages. In
Pl. IV, Figs. 14-16, only the spiral fiber and the pericellular
plexuses are shown; their arrangement and structure are
more easily seen when the other parts of the ganglion are

58 HUBER. [Vox. XVI.

not sketched. In Pl. IV, Fig. 18, I have reproduced a sym-
pathetic cell with pericellular plexus, taken from one of my
most successful preparations, where the structures other than
the spiral fiber and pericellular plexus may be seen. This
sketch is made from a section of an inferior cervical ganglion
of Chelydra serpentina, stained in methylene blue and double-
stained in alum carmine. In this sketch the cell body of the
sympathetic cell came out more deeply colored than in the
section; in all other respects the coloring and arrangement
of nerve fibers and nuclei were followed as accurately as pos-
sible. In this figure may be seen B, the cell body, C and D,
the nucleus and nucleolus, and A, the process of a large uni-
polar sympathetic cell. The capsule, c, was clearly made out
and could be followed for some distance along the process,
when it became unobservable. Three medullated fibers, a, a’,
and a’, take part in the formation of the spiral and pericellular
plexus. Their interlacement is, however, such that they could
be followed for only a short distance. At their distal end these
spiral fibers break up into secondary branches, which become
very varicose and, after further branching, form a basket-like
network which surrounds the cell body of the sympathetic cell,
the arrangement of which is better shown in the figure than I
can describe it. The figure shows most clearly that this net-
work is intra-capsular. The figure may further serve to give an
idea of the number and the arrangement of the intra-capsular
nuclei. These, as has been previously observed, are regarded
as nuclei of neuroglia cells. Fig. 19 has been added to give
the general appearance of a methylene blue preparation of
a sympathetic ganglion of a tortoise, double-stained in alum
carmine. In this section the processes of the cells were only
partly stained, and the fact that this sketch is from a section will
explain the abrupt ending of some of the branches. The figure
shows three of the more complex pericellular plexuses, A, with
spiral fibers, and one simpler pericellular plexus, 4; shows
also the arrangement of the medullated and the non-medullated
(varicose fibers in the figure) fibers and of the dendrites as seen
in sections.

My reasons for assuming that the intra-capsular, pericellular

No.1.] SYMPATHETIC GANGLIA OF VERTEBRATES. 59

plexuses above described are the endings in the sympathetic
ganglia of Reptilia of cerebro-spinal fibers may briefly be stated
as follows :

a) The medullated fibers in the sympathetic ganglia may,
from their size and structure, be regarded as fibers of cerebro-
spinal origin.

6) In serial sections of well-stained, sympathetic ganglia,
single medullated fibers, or small bundles of such, may often
be traced from the white rami into the ganglia, where they
undergo division, or give off collateral branches, and may now
and then, as shown in PI. IV, Fig. 17, be traced into a spiral
fiber and pericellular plexus.

c) The small dorsal sympathetic ganglia of the Reptilia inves-
tigated are in close apposition to the spinal ganglia. By remov-
ing the bodies of the vertebrae, the spinal cord, with the roots
of the spinal nerves, the spinal and sympathetic ganglia may
easily be exposed. After exposing the structures as above indi-
cated, I have, in a large number of experiments, divided the
roots of the spinal nerves close to the spinal cord, and removed
these with the spinal and sympathetic ganglia, with short seg-
ments of the nerves uniting the sympathetic ganglion to the
sympathetic chain, and with a short portion of the spinal nerve.
The tissues so removed were then fixed in ammonium molyb-
date, imbedded in paraffin, and cut in serial sections. As a
rule, in the sections so obtained only a few of the nerve fibers
and nerve cells were well stained in methylene blue. In some
series, however, the staining was such that small bundles of
medullated fibers could be traced from the anterior root into
the sympathetic ganglion. The writer (26) has already drawn
attention to the fact that in the dorsal spinal ganglia of Reptilia,
stained in methylene blue, a small group of multipolar cells was
found within the connective tissue capsule of the spinal ganglion.
This group of multipolar cells is found on the ventral side of
the ganglion, immediately under the capsule of the ganglion,
and separated from the sensory cells by a connective tissue
septum. Into this group of cells, which, by reason of their
shape and structure, are looked upon as sympathetic cells, I
have several times been able to trace medullated fibers coming

60 HUBER. [Vou. XVI.

from the anterior root ; and now and then such a fiber could be
traced into a pericellular plexus of the simpler variety.

Such observations, it seems to me, point conclusively to the
cerebro-spinal origin of the medullated fibers ending in the
ganglion in intra-capsular, pericellular plexuses.

The non-medullated fibers seen in sections of a ganglion
wind their course through the ganglion between its constituent
cells. Many of these are no doubt the neuraxes of the sympa-
thetic neurons of the ganglion, although it is very seldom that
in section such fibers can be traced to a ganglion cell. This
may, however, now and then be done, as may be seen from
Pl. IV, Fig. 12. Serial sections show further that some non-
medullated fibers enter the ganglia through the interganglionic
cords. Their mode of ending in the ganglia has not been defi-
nitely ascertained, unless possibly that shown in Pl. IV, Fig. 17
(fibers c), and described with that figure, may be their mode of
ending.

A brief summary of the observations made on the sympa-
thetic ganglia of Reptilia above described may here be given:

1) The sympathetic neurons of Reptilia may be multipolar,
bipolar, or unipolar. The first two types need no further men-
tion. The unipolar cells, very characteristic for the sympathetic
ganglia of Reptilia, are relatively large, possessing one large
process, varying in length and its relation to the cell body of
the respective neuron. All these cells have one neuraxis, which
near the cell is non-medullated; whether at some distance from
the cell it becomes invested witha sheath of myelin I am unable
to say. The neuraxis arises from the cell body or from some
one of the dendrites. The number of dendrites varies. The
cell body of all sympathetic cells of Reptilia is surrounded by
a nucleated capsule.

2) Medullated fibers, no doubt of cerebro-spinal origin, end
in the ganglia, either in simple pericellular plexuses surround-
ing the multipolar or bipolar cells, or in more complex pericel-
lular plexuses with spiral fibers surrounding the cell body of the
unipolar cells. These pericellular plexuses are intra-capsular.
The medullated fibers branch in the ganglia, and may thus
influence a number of sympathetic cells through their several

No.1.] SYMPATHETIC GANGLIA OF VERTEBRATES. O61

pericellular plexuses; each branch probably having such an
ending.

3) The ending of the non-medullated fibers in the ganglion
has not been determined definitely.

SYMPATHETIC GANGLIA OF BIRDS.

The observations here recorded were made on half-grown
chickens. A 1% to 4% solution of methylene blue in normal
salt was injected into the humeral vein (Owen), just above its
division over the distal end of the humerus. The methylene
blue was allowed to flow into the circulation as quickly as pos-
sible; as in birds, a small quantity of the solution causes a
stoppage of the heart. At the expiration of thirty to forty
minutes the sternum with the attached muscles was removed,
and the heart and lungs displaced to one side. In this way the
dorsal sympathetic ganglia are readily exposed. In the chicken
the dorsal sympathetic ganglia rest on the spinal ganglia, so
that a white ramus as such is wanting. The sympathetic gan-
glia may, therefore, be best removed with the spinal ganglia;
this was usually done. The tissues so removed were fixed in
ammonium molybdate, the further treatment being as previously
indicated. It should be stated at the outstart that the results
obtained on the sympathetic ganglia of birds has been far from
what might be desired. And this for two reasons:

1) I have found it very difficult to stain these structures at
all well in methylene blue. Very generally, all the structures
of the ganglion stain a diffuse blue; in which case, sections
show scarcely more than may be seen in sections of these
ganglia stained with the ordinary stains.

2) The few sections that were well stained would fade in a
few days or weeks, no matter what care was taken. In some
few instances, however, results which may be compared with
those obtained on other vertebrates have been at my disposal.

In the literature I have been able to consult I find no refer-
ence to observations made on the sympathetic ganglia of birds
with the methylene blue method, although these ganglia have
been stained by Cajal (27), Retzius (28), and Lenhossék (29).

62 HOBER. [VoL. XVI.

Ramon y Cajal’s studies on the sympathetic system were pub-
lished in three papers, which followed each other in close succes-
sion. In his second communication, to which I wish here to
refer more particularly, he discusses observations made on dove
embryos of the fourteenth and sixteenth day, and chick embryos
of the seventeenth and eighteenth day. He here states that
the sympathetic cells are multipolar, and that all the processes
have the character of nervous processes. He describes long
and short processes; the latter end free within the ganglion,
the former pass beyond the bounds of the ganglion. In his
third communication Cajal retracts in part these statements,
and states that while the cells are multipolar, the great majority
possess one axis-cylinder branch, the others being dendrites.
In a brief account of his observations made on the sympathetic
ganglia of vertebrates, Retzius refers in a few lines to those
made on chick embryos of the fifteenth to the eighteenth day,
in which he merely confirms the conclusions ultimately reached
by Cajal as above given.

Lenhossék has studied the sympathetic ganglia of embryo
chicks of the tenth and fifteenth day; the best results were
obtained with the older embryos studied.

In cross-sections of chick embryos of the above age, stained
after the double Golgi method, Lenhossék made the following
observations :— Both in the cervical and thoracic regions a ramus
communicans is wanting ; the sympathetic ganglion lies directly
on the median side of the ventral spinal nerve. In the thoracic
region the spinal nerve divides into a smaller dorsal branch and
a relatively larger ramus ventralis, on which the sympathetic
ganglion is found. These observations I cam to some extent
corroborate on the grown chicken, although, as above stated, I
find the sympathetic ganglion, or a portion of it, resting on the
spinal ganglion. Lenhossék describes multipolar cells, with
one neuraxis and several dendrites, five to ten in number. The
dendrites are described as quite thick and relatively short, some
ending free without any branching.

The neuraxes are described as taking one of three paths:

1) Rarely into one of the peripheral branches of the ganglion
going to the viscera.

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No.1.] SYMPATHETIC GANGLIA OF VERTEBRATES. 63

2) In frontal sections of chick embryos of the fifteenth day
it was seen that some of the neuraxes passed up or down in the
ramus internodalis, and entered one of the contiguous ganglia.

3) Many of the neuraxes take a peripheral course in the ven-
tral branch of the spinal nerve of the segment.

Both Cajal and Lenhossék were able to trace cerebro-
spinal nerve fibers into the sympathetic ganglia of the chick.
The latter describes these as coming from the ventral ramus
of the spinal nerve, as branching in the ganglia, and as termi-
nating in a “free ending.’ Cajal was able to trace such fibers
to the anterior root, while Lenhossék is inclined to regard them
as coming from the posterior root. Neither of these investi-
gators describes pericellular plexuses in the sympathetic ganglia
of birds.

The results obtained by me with the methylene blue method
confirm in many particulars the observations made by Cajal
and Lenhossék with the Golgi method. I also find the sympa-
thetic neurons multipolar, with numerous dendrites and one
neuraxis. The body of such neurons may be irregularly round
or oval, or of a triangular shape. In ganglia stained in meth-
ylene blue, and in those so stained and fixed in ammonium
molybdate and sectioned, chromophile granules may be seen
in the protoplasm of the ganglion cell, if the staining is not
too intense or diffuse, as is often the case. These granules are
very fine and evenly distributed through the protoplasm, as
(Pl. V, Fig. 21) a portion of a section of one of the ganglia of
the dorsal sympathetic chain may show.

The dendrites, as Lenhossék has correctly stated, are short
and thick and not prone to much branching. They form an
interlacing network between the cell bodies of the ganglion
cells. As the cell bodies of such cells are surrounded with
a nucleated capsule (see c of Fig. 22), this dendritic plexus is
extra-capsular.

Cajal mentions this arrangement of the dendrites, formed by
the “short process,” and describes it as pericellular nests —
“‘nido pericellular.” He further suggests that through such
pericellular nests ganglion cells may be physiologically asso-
ciated.

64 HUBER. [VoL. XVI.

Van Gehuchten, Sala, and Dogiel have observed a similar
arrangement of the dendrites of the sympathetic cells in the
ganglia of Mammalia; they, however, regard the nest-like
arrangement as accidental. A study of Pl. V, Fig. 21, may
show that the latter interpretation seems the more plausible
one, and especially if we take into consideration that such
dendrites are extra-capsular. It is further to be remembered
that if these dendrites have the power to conduct nervous
impulses, as they no doubt may have, they would, reasoning
from analogy, conduct toward the cell body —be “ cellulipetal”’ ;
and I am at present not aware of any instance where a dendrite
is stimulated by the cell body or dendrites of another neuron.
We may thus assume that the arrangement of the dendritic
branches of the sympathetic neurons in the sympathetic ganglia
of birds is mainly accidental, depending in a great measure on
the relative position of contiguous ganglion cells. Participating
in this network between the ganglion cells as above described,
are found non-medullated and medullated nerve fibers. The
former are, no doubt, in part the neuraxes of the sympathetic
cells of the ganglion. In serial sections, however, it may be
seen that some of the non-medullated fibers enter the ganglia
from without; this, both Cajal and Lenhossék have described.
The latter pictures in his article (Fig. 13) a bundle of such
fibers entering a ganglion. This figure, as the descriptive text
shows, was sketched from a dorsal sympathetic ganglion of a 14-
day chick. Lenhossék has this to say concerning these fibers:
«Wir haben es hier offenbar mit den Fortsatzen von ander-
weitig, etwa in den visceralen Ganglien gelegenen sympa-
thischen Zellen zu thun.” He goes on to say that the ending
of these fibers is by a simple end brush and not by an end basket;
and, to quote again: “Wobei Endaste manchmal auffallende
Verdickungen zeigten, an den Stellen, wo sie sich an die Zellen
anlegten.” An ending such as here described has not been
seen by me. The non-medullated fibers, as far as I have been
able to determine, are always extra-capsular, and do not there-
fore end on the cell body of the sympathetic cells. A few
times, however, an ending such as shown in Pl. V, Fig. 20, has
been seen by me. In this figure, which is a portion of a section,

Nesta) SVUPATHE TIC GANGEIA Of VERTEBRATES. 65

of 20 mw thickness, of one of the dorsal sympathetic ganglia,
is shown a sympathetic nerve cell with several dendrites. On
one of the branches, d, is shown the ending of a non-medullated
fiber, 2; the non-medullated fiber being stained somewhat more
deeply than the dendrite. In a careful search of many prepa-
rations, only a few such endings have been found; yet in rela-
tively thin sections, studied under ,/,-inch oil immersion, they
have now and then been quite clearly made out.

Concerning the medullated fibers found in the sympathetic
ganglia of birds, the following observations have been made:

It will be remembered that the sympathetic ganglia of the
bird lie on the ventral branch of the spinal nerve and partly
on the ventral side of the spinal ganglia, and are removed with
these structures. In such preparations, stained in methylene
blue and examined before fixing, even under a low power, the
multipolar-sympathetic cells may be made out with certainty,
and axis-cylinders of medullated fibers may now and then be
traced between such multipolar cells. Cajal, it will be remem-
bered, describes such fibers as coming from the anterior roots,
while Lenhossék regards them as coming from the posterior
roots, as may be gathered from the following statement made
by him: “In zwei Fallen, schien es mir, als handelte es sich
gerade umgekehrt um Fasern die aus dem Spinalganglion
kommen, also um sensible Fasern, doch kann ich dies nicht
mit voller Bestimmtheit vertreten.” In ganglia removed as
above stated, and examined before fixing, and especially in those
where only a few nerve fibers were stained, I have several times
been able to trace axis-cylinders of medullated fibers ending in
the sympathetic ganglia toward the anterior root; this may
also be seen in serial sections of the sympathetic ganglia and
the structures in connection with them. My observations, as
far as they go, are therefore in accord with those given by Cajal.

There is no doubt that many of the medullated fibers end-
ing in the sympathetic ganglia of birds, do so in pericellular
plexuses, although Cajal, Retzius, and Lenhossék were unable
to see them in Golgi preparations of these structures. The
latter says in this connection: ‘Von einer faserkorbartigen
Anordnung im Inneren des sympathischen Ganglion ver-

66 HUBER. (VoL. XVI.

mochte ich beim Hiihnchen nichts wahrzunehmen; es handelte
sich immer um einfache Aufzweigungen.” The pericellular
plexuses seen by me are the endings of collateral branches,
or ultimate endings of medullated fibers. A number of such
endings are reproduced in Figs. 20-22, In Fig. 20, from the
medullated fiber A, is given off at the node of Ranvier, x, a
non-medullated collateral branch, a, which may be traced into
two pericellular plexuses. Fig. 22 was sketched from a section
of a methylene blue stained, dorsal sympathetic ganglion of
a chicken, fixed in ammonium molybdate, the sections being
further stained in alum carmine. Here the relation of the
pericellular plexus to the cell body of the enclosed cell and its
capsule is clearly shown.

The pericellular plexuses observed in birds are of a relatively
simple structure. The end branch, or collateral branch destined
to form such an ending, divides, just before or after it reaches
the capsule of a sympathetic cell, into a number of fine varicose
fibers which are woven into a very loose plexus and often end
free on the cell.

My observations on the sympathetic ganglia of birds may be
briefly summarized as follows:

1) The sympathetic neurons are multipolar, with one neu-
raxis and several dendrites. The cell body of such a neuron is
enclosed in a nucleated capsule.

2) The non-medullated fibers entering the ganglia from with-
out, end, after branching, on the dendritic branches of the sym-
pathetic neurons.

3) The medullated fibers of cerebro-spinal origin, which enter
the ganglia, most probably from the anterior roots, end, after
branching in the ganglia, in pericellular plexuses of a relatively
simple structure. These plexuses surround the cell bodies of
the sympathetic neurons and are intra-capsular.

SYMPATHETIC GANGLIA OF MAMMALIA.

The sympathetic ganglia of Mammalia have been investigated
with the Golgi method by Kolliker (30-33), Ramon y Cajal
(34-36), Retzius (28), Van Gehuchten (37), Sala (38), d’ Erchia

Noms KVvPATHETIC GANGLIA OF VERTEBRATES. 67

(39), and Lenhossék (29); and with the methylene blue method
by Aronson (19) and Dogiel (40, 41). Each writer has in turn
reviewed the work of those who have preceded him, to such
an extent that a special review of the literature seems here
uncalled for. The observations made by others will thus be
considered in giving my own results.

The observations here to be recorded cover a period of now
nearly three years; in the earlier portion of this work the
Golgi method was to some extent used, but in the last two
years the zztva-vitam methylene method alone was used, and
the results to be recorded pertain exclusively to observations
made with it.

The animals investigated varied in age from such as were
two or three weeks old to such as were full grown. The meth-
ylene blue solution was injected through the jugular or femoral
vein, usually the former; the quantity varying with the size of
the animal.

The ganglia studied were the superior and inferior cervical,
the stellate ganglion, the smaller ganglia of the chain, and
many of the peripheral ganglia. These were exposed forty-five
minutes to an hour after the injection; were fixed in ammo-
nium molybdate and studied in sections, either stained only
in the methylene blue or double-stained in this dye and alum
carmine.

Some of the results here to be given were known to me before
Dogiel’s (40) article (from which I shall quote freely and to
some extent follow) reached me. It seems, nevertheless, advis-
able to give them, for, notwithstanding the fact that we have
both used the methylene blue method in our investigations,
Dogiel’s observations were made on tissues stained on the slide
and fixed in ammonium picrate; and, furthermore, his observa-
tions pertain more particularly to the smaller peripheral ganglia
(wall of the gall bladder and so forth), where it is possible to
study the gangliaasa whole. He was in this way able to obtain
preparations of sympathetic neurons, which for completeness
of staining, judging from his pictures, seem not to have been
equaled. My own observations, as above stated, were made
on sections, usually double-stained in alum carmine; and while

68 HUBER. [Vou. XVI.

in such sections the sympathetic cells are not so clearly shown,
by reason of the fact that many of the processes are cut from
the cells, yet the relation of the dendrites and pericellular
plexuses to the cell body and capsule of the sympathetic cells
of the ganglion is more clearly shown than in ganglia studied
as a whole.

SYMPATHETIC NEURONS OF MAMMALIA.

All writers who have reported observations made with the
Golgi or methylene blue method are agreed that the great
majority of the sympathetic neurons are multipolar. Dogiel
(40) states that near the poles of the sympathetic ganglia bipolar
and unipolar cells are to be found. Their number is relatively
small. My own observations lead to the conclusion that such
cells are usually found between the afferent and efferent nerve
fibers of the ganglion. In the protoplasm of all these cells
chromophile granules are seen ; in this respect my observations
are in accord with those made by Dogiel (40).

The nucleus is usually only imperfectly stained in methylene
blue, more often of a diffuse blue, which may be of a darker or
a lighter hue than the cell body. Nucleoli are only rarely seen
with this stain, although they are readily found in preparations
stained in alum carmine.

In some Mammalia, namely, the rabbit, hare, and guinea pig,
sympathetic cells with two or even three nuclei are found, as
has been shown by Remak (44), Guye (42), Schwalbe (15), and
more recently Apolant (43). Schwalbe and Apolant explain this
curious phenomenon by stating that it is the result of an incom-
plete cell division, the nucleus dividing but not the protoplasm,
and the latter has shown that the multiplication of the nuclei
takes place by an amitotic cell division, which may be recog-
nized in embryo rabbits as early as the third week. Apolant
reaches the following conclusions concerning this point: ‘Ich
glaube daher, dass die Bildung der beiden Kerne in einem
innigen Zusammenhange mit den Wachsthumsverhiltnissen der
Zelle steht, der Art, dass die anfangliche, iiberwiegende Aus-
bildung des Kernes zu einer Theilung desselben fiihrt, welche

No.1.] SYMPATHETIC GANGLIA OF VERTEBRATES. 69

ihrerseits die Veranlassung zu einem starkeren Wachsthum
der Zelle abgiebt. Ich vindicire also dem Process keine func-
tionelle, sondern lediglich eine biologische Bedeutung fiir die
Zelle.” My own observations on sympathetic ganglion cells
with more than one nucleus were made on ganglia of the guinea
pig stained in methylene blue, a method which, in the hands of
Apolant, gave only unsatisfactory results ; they may, therefore,

Fic. III. — Sympathetic neurons of guinea pig. (For description see text.)

receive this brief mention. The majority of my preparations
were from the solar ganglion. The larger number of the sympa-
thetic neurons in this ganglion are multipolar, with two and
sometimes three nuclei. Such cells are reproduced in A and B
of Fig. III; as may be seen from this figure, these cells differ
from sympathetic cells found in other Mammalia only in having
more than one nucleus. The number of dendrites varies;
only one neuraxis is made out (a, in figure), if the cells are well
stained.

70 HOBIER. [VoL. XVI.

In D of Fig. III is shown a multipolar cell with two nuclei,
where the portions of the cell body containing the nuclei are
united by a band of protoplasm, giving the appearance of a
cell in the later stages of cell division. This condition was
seen only a few times, and is very much like that shown by
Apolant in Figs. 7 and 8 of his article. Not all the multipolar
cells have two nuclei, as may be seen in cell C of the above
figure. Mononuclear multipolar cells are, however, rarely seen.
Bipolar cells are proportionally not more numerous in the
rodents above mentioned than in other Mammalia. They are
found near the poles of the ganglia, between the afferent and
efferent nerves, and may be mononuclear or possess two nuclei.
In order to close the discussion of these cells I may be allowed
to anticipate somewhat, and state that I have often found the
multipolar cells, with two or more nuclei, surrounded by a peri-
cellular plexus; one such is shown in & of Fig. ITI.

I must, therefore, agree with Apolant when he states that the
presence of two or more nuclei in the sympathetic cells of the
rodents under discussion is not to be looked upon as expressing
a degenerative process. In all other respects the structure of
these cells, —the presence of chromophile granules, —as also
the structure of the ganglia taken as a whole, is identical with
the structure of the sympathetic cells and ganglia of other
mammals, in which the sympathetic neurons have only one
nucleus.

The Dendrites. — In the sympathetic cell near the center of
the ganglion, the dendrites, the number of which varies, may
arise from any part of the cell body. In the peripheral cells,
as Dogiel (40) has correctly stated, the dendrites are usually
given off from that portion of the cell body pointing toward the
center of the ganglion. The dendrites branch and rebranch
and form between the ganglion cells an intercellular plexus.
This plexus is well shown in Fig. 26—a portion of a section
of the solar ganglion of a cat. It may here be seen that the
dendrites intertwine in such a way as to leave open spaces, in
which the cell body of one, two, or perhaps three sympathetic
cells are found. This basket-like arrangement is what Ramon y
Cajal has described as pericellular nests. The dendrites do not,

No.1.] SYMPATHETIC GANGLIA OF VERTEBRATES. 71

however, come in contact with the cell bodies of the sympa-
thetic cells, but are separated from them by their capsule.

Dogiel (40) has further described a “general peripheral
plexus,” situated under the fibrous capsule of the ganglion. In
the formation of this kind of plexus, dendrites from nearly all
the cells of the smaller ganglia and many of the cells of the
larger ganglia take part, the dendrites from the cells situated
more centrally in the ganglia winding their way out until the
plexus is reached.

Neuraxis.—It is now very generally conceded that the sympa-
thetic neurons of Mammalia possess one neuraxis — axis-cylin-
der. This may arise from the cell body or from a dendrite at
a variable distance from the cell body. In making the latter
statement I have been guided largely by observations made
by others, as in sections where, as a rule, only relatively short
segments of the axis-cylinder are met with, it is often exceed-
ingly difficult to classify the processes of any particular sympa-
thetic cell. In most preparations the axis-cylinder branches
are not stained in any way characteristically. Sometimes,
however, as may be seen in Pl. V, Fig. 26, they stain a deeper
and more purple shade of blue, which color differentiation may
aid in making out which one of the several processes of a sym-
pathetic cell is the axis-cylinder. In such sections I have been
able to confirm the statements made above. Near the cell
body the neuraxes of the sympathetic cells of Mammalia have
a very regular contour and maintain about the same size; and
Dogiel (40) has described a very delicate longitudinal striation,
caused by a deeper staining of the ultimate fibrillae of the
axis-cylinder.

Kolliker (32), who has for many years paid especial attention
to the structure of the neuraxes of sympathetic cells, has quite
recently summarized his observations as follows:

1) The sympathetic nerve fibers (axis-cylinder processes of
sympathetic cells) are in many cases surrounded by a very deli-
cate sheath of myelin.

2) In some of these fibers the sheath of myelin accompa-
nies the neuraxis to its periphery — nerve fibers from the ciliary
ganglion and the pilo-motor nerves of the cat.

72 HUBER. [Vor. XVI.

3) In other instances the medullary sheath is sooner or later
lost, the neuraxes continuing as Remak’s fibers — fibers going
to the intestine, liver, and spleen.

4) In many cases the neuraxes of sympathetic neurons are
non-medullated throughout. This, it would seem to me, is the
structure of the neuraxes of sympathetic cells in the peripheral
ganglia —those of the heart, salivary glands, intestine, blad-
der, etc.

Lenhossék (29) and Dogiel (40) have described the giving off
of collateral branches from the axis-cylinders of the sympathetic
cells of mammalia; such branches have been traced into the
intercellular plexus; their mode of ending has not, however,
been determined.

Before closing the discussion of the sympathetic neurons in
Mammalia, it is necessary to mention some cells found in the
sympathetic ganglia, which Dogiel (41) has recently described
as sensory sympathetic cells. These cells are said to have the fol-
lowing structural peculiarities : The cells are multipolar, with one
to sixteen dendrites and one neuraxis; they are characterized
by the structure of the dendrites, which are longer and more slen-
der than the dendrites of the other sympathetic cells. These
dendrites ramify in the ganglion and may often be traced into
one of the nerve trunks connected with the ganglion, in which
they may often be followed for long distances. In the ganglia
of Auerbach’s plexus such dendritic processes could be followed
from a ganglion into one of its nerve roots, and then some into
the mucosa, others into the submucosa, and soon. The neu-
raxes of such cells are described as coming from the cell body
or from some dendrite; in the smaller ganglia such cells have
non-medullated axis-cylinders; in the larger ganglia of the
chain this process becomes surrounded with a medullary sheath
some distance from the cell body of the sympathetic cell from
which it arises. In the plexuses of the intestine Dogiel was
able to trace the neuraxes of such sensory sympathetic cells
through several ganglia. In the ganglia through which they
pass, or in which they end, collateral branches are given off
which terminate in the intercellular plexus. Dogiel suggests
that such cells may form the anatomical basis for certain phe-

No.1.] SYMPATHETIC GANGLIA OF VERTEBRATES. 73

nomena, such as peripheral reflexes, etc., which have been
observed in the sympathetic system. My own observations,
based almost entirely on sections, do not give me sufficient
evidence on which to judge these interesting and, if corrobo-
rated, most important observations which Dogiel has given
us. Cells with long, slender processes, such as he has described,
have now and then been seen by me, but I have not been able
to trace these processes for any distance. I may, therefore,
dispense with any further discussion of these so-called sen-
sory sympathetic cells.

Non-Medullated and Medullated Nerve Fibers in the Sympa-
thetic Ganglia. — In sections of sympathetic ganglia stained in
methylene blue, smaller and larger medullated fibers and non-
medullated fibers may be seen between the ganglion cells.
These have been observed by Kolliker, Ramon y Cajal, Len-
hossék, Sala, Van Gehuchten, and others, in Golgi preparations
of the sympathetic ganglia of Mammalia, and by Aronson and
Dogiel in methylene blue stained preparations of these struc-
tures. In serial sections of the chain ganglia removed with
the white rami, stained in methylene blue, bundles of medul-
lated fibers may readily be traced from the white rami into the
ganglia, although it is not always easy to trace individual fibers
for any long distance. In the ganglia such medullated fibers
are seen branching into two or three branches, which may or
may not be medullated, and in a number of instances such
branches were traced to a sympathetic cell, where, after further
branching, they terminated in a pericellular plexus which sur-
rounded the cell body of the sympathetic cell. In well-stained
preparations such pericellular plexuses are easily found, although
in sections it is not always easy to connect such plexuses with
any particular nerve fiber. These pericellular plexuses vary
much in complexity and in the arrangement of fibrils which
form them. They may be very loosely woven, or, again, made
up of alarge number of fibrils. The fibrils may be quite smooth,
or show numerous and large varicose enlargements. The ones
shown in Pl. V, Fig. 23, a small portion of a section of the
stellate ganglion of a dog, stained in methylene blue and alum
carmine, may be looked upon as presenting the general appear-

74 HUBER. [VoL. XVI.

ance of the pericellular plexuses in the sympathetic ganglia as
seen by me. In double-stained sections there can be no doubt
that the pericellular plexuses are in contact with the cell body
of the sympathetic cell—are intra-capsular. This agrees with
the following statement found in Dogiel’s (40) account: ‘ Das-
selbe”’ (speaking of the pericellular plexuses) “liegt unmittelbar
der Oberflache der Ganglienzellen an und befindet sich, wie
mir scheint, zum Unterschiede von dem intercellularen Geflecht
nicht iiber, sondern unter der Zellenhiille.’”’ The fibers termi-
nating in the pericellular plexuses are usually non-medullated
for some distance from the cells around which they end (a, PI.
V, Fig. 23), although in a few instances they are medullated
to the point where they pierce the capsule, 4, of the same
figure. In some few instances much more complicated peri-
cellular plexuses were seen, in which the fiber or fibers termi-
nating in such plexuses were spirally wound around some
process, probably the axis-cylinder branch of the cell enclosed
by the pericellular plexus. One such ending is shown in Pl. V,
Fig. 24, taken from the stellate ganglion of adog. In the few
instances seen by me the network resulting from the division
and redivision of such spiral fibers is much more complicated
than the pericellular plexuses usually observed. The fibrils are.
very varicose, often presenting quite large nodular enlargements.
Aronson (19) has described spiral fibers in the sympathetic gan-
glia of the rabbit. His description leads one to infer that they
are quite common. A comparison of the figures given by
Aronson (especially Fig. 1) with my Pl. V, Fig. 24, may suf-
fice to show that very dissimilar structures are spoken of in
the two accounts. Spiral fibers, such as he describes, formed
by one, two, or three varicose fibers, twisted once, twice, or
three times around the neuraxis of the sympathetic cell and
terminating in an ordinary pericellular plexus, have now and
then been seen by me. In this account, however, the term
‘‘ spiral fiber”’ is confined to such as are diagrammed in Pl. V,
Fig. 24, ending in a more complex pericellular plexus. These
were only rarely seen by me. They resemble, to some extent,
the more complicated pericellular plexuses, with spiral fibers,
described for Reptilia.

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No.1.] SYMPATHETIC GANGLIA OF VERTEBRATES. 75

Pericellular plexuses, enclosing the cell body of the sympa-
thetic neurons of Mammalia, have been described for nearly all
sympathetic ganglia in all parts of the sympathetic system; in
the cranial sympathetic ganglia as follows: Ciliary ganglia by
Kolliker (32) and Michel; spheno-palatine by Lenhossék (29) ;
and submaxillary and sublingual by myself (45); in the larger
ganglia of the chain by a number of investigators, beginning
with Aronson (19); in the respiratory passages by Arnstein (46) ;
in the heart of the rabbit by Aronson (19); intestinal canal
and other peripheral ganglia by Dogiel; in the suprarenal by
Dogiel (47); in the epididymis by Timofeew (48); and have
further been seen by me in the bladder and oesophagus of the
cat. In all the Mammalia studied, and in all the ganglia, they
have essentially the same structure, and so far as my observa-
tions go, in sections of the ganglia of the sympathetic chain and
various peripheral ganglia of the dog, cat, rabbit, and guinea pig,
the pericellular plexuses are always intra-capsular. Sometimes
only one fiber may end in a pericellular plexus; sometimes two,
three, or even more fibers may take part in the formation of
one plexus ; these fibers are always, so far as may be gathered
from the investigations on the sympathetic ganglia of Mammalia,
collateral or end branches of medullated fibers.

So far as concerns the small medullated fibers, which may
be traced from this or that nerve root of a ganglion into said
ganglion, my own observations confirm wholly the account
given by Dogiel (40). Such fibers may readily be seen in
sections of methylene blue stained ganglia, where they are
found branching and rebranching, and forming, with the den-
dritic processes of the ganglion cells, what Dogiel has described
as the intercellular plexus; this plexus is always extra-capsular
and may be seen in Pl. V, Fig. 26—a portion of a section
about 20 uw in thickness. In sections one-third or one-half that
thickness I have now and then observed what I have looked
upon as the ending of the fibers under discussion. In Pl. V,
Fig. 25, is reproduced a cell from a section about 10 wp in thick-
ness of the solar ganglion of a cat stained in methylene blue.
The cell body of this cell was deeply stained, its neuraxis, a,
and dendrite, 4, not so deeply ; these could, however, be clearly

76 HUBER. (Vor. XVI.

made out. A very small non-medullated fiber, ¢c, could be
traced with the utmost clearness to its ending on one of the
protoplasmic branches of the cell in question; this non-medul-
lated fiber terminated in two very small nodular enlargements,
which were in contact with this dendrite, as shown in c’ of this
figure. In a number of instances such endings were made out,
so that I feel justified in concluding that at least some of the
non-medullated fibers in the ganglia, possibly also the termi-
nal branches of the ssza// medullated fibers, terminate in the
ganglia after this manner.

In giving my own conclusions as to the nature of the fibers
ending in the sympathetic ganglia of Mammalia, I can do no
better than to quote the conclusion reached by Dogiel (40)
concerning this point, which is as follows: ‘‘ Die feinen Fasern,
welche in den Ganglien mit intercellularem Geflechte endigen,
zu den sympathischen, augenscheinlich vorzugsweise markhalti-
gen Fasern gehoren, die dicken Fasern aber, deren Endverzwei-
gungen in den Ganglien pericellulare Geflechte bilden, zu den
markhaltigen Fasern zu rechnen sind, welche aus dem Cere-
brospinalsystem entspringen.” These pericellular plexuses, as
already stated, are always intra-capsular.

GENERAL CONCLUSIONS.

In these general conclusions my aim is to be as brief as
possible. Two reasons may here be given in justification of
this: (1) the results obtained in each of the vertebrate classes
studied have to some extent been summarized in the foregoing
pages; (2) the writer has, in a series of ‘Special Lectures on
the Sympathetic Nervous System,” given before the medical
students of Michigan University, and published in the Journal
of Comparative Neurology, Vol. VII, No. 2, September, 1897,
dwelt more fully on many of the questions which will here be
touched upon.

From a study of the sympathetic ganglia of vertebrates the
following facts concerning the shape and structure of the sym-
pathetic neurons, and the nerves ending in the ganglia, may be
deduced :

No.1.] SYMPATHETIC GANGLIA OF VERTEBRATES. 77

1) In all vertebrates, excepting the Amphibia, the great
majority of the sympathetic neurons are multipolar, possessing
a varying number of dendrites; but each cell has only one
neuraxis. In Amphibia the sympathetic neurons, with the
exception of those found in the coats of the intestinal canal
and stomach, which are multipolar, are unipolar cells, as
Kolliker has previously stated. In vertebrates other than
Amphibia, some few unipolar and bipolar sympathetic neurons
are to be found.

2) In all the vertebrates examined chromophile granules
were found. One relatively large nucleus is the very general
rule ; the sympathetic cells of rabbits, hares, and guinea pigs
forming an exception; many sympathetic cells with two or,
occasionally, even three nuclei being here found.

3) The neuraxes of sympathetic cells may be medullated
throughout, medullated for only a portion of the process, or
non-medullated throughout; the medullary sheath, if present,
forms a relatively thin layer, thinner than in the small medul-
lated cerebro-spinal fibers. (The above statements are taken
from Kolliker’s writings.)

The neuraxes of sympathetic neurons have one or the other
of the following distributions:

a) To involuntary smooth muscle or heart muscle.

6) To glandular tissues.

c) To other sympathetic ganglia (?).

a2) To the spinal ganglia.

In the special lectures above alluded to, the writer has dis-
cussed, somewhat at length, each of the above possible modes
of termination of the neuraxes of sympathetic neurons, consid-
ering also the literature bearing on this subject. It may here
suffice to add that Arnstein (46) has recently traced, and pic-
tured the neuraxis of a sympathetic neuron ending in smooth
muscle tissue; the writer has traced small branches of non-
medullated fibers from some of the small ganglia found in the
cat’s auricle to their ending on heart muscle, and also the
neuraxes of sympathetic neurons of the sublingual ganglion
(Langley) to the epilamellar plexus surrounding alveoli of the
gland of the same name; from Dogiel’s (40) work, some of

78 HUBER. [VoL. XVI.

which I have corroborated, the conclusion may be drawn that
neuraxes of sympathetic neurons (especially those surrounded
by a thin layer of myelin) end in the intercellular plexuses of
sympathetic ganglia, probably on the dendrites of sympathetic
cells; and, finally, Ramon y Cajal’s (36) and Dogiel’s (49)
observations on the ending of sympathetic nerve fibers in the
spinal ganglia of Mammalia support the view that the neuraxes
of sympathetic neurons end in the spinal ganglia.

4) The cell bodies of sympathetic neurons are surrounded by
a nucleated capsule, which apparently has the same structure
in all vertebrates.

5) In the sympathetic ganglia of all vertebrates studied by
others and myself (speaking here of results obtained with either
the Golgi or the methylene blue method), medullated fibers
ending in pericellular intra-capsular plexuses have been found.
These are, in all instances described, of essentially the same
structure. I am well aware, as the accompanying plates may
show, that the relation of the pericellular plexuses to the cell
bodies of the enclosed sympathetic neurons varies somewhat in
the different vertebrates, as do also the course, structure, and
relation to other processes of the nerve fibers ending in such
pericellular plexuses; yet these differences are not essential
and important. The question now arises, What is the origin
of the medullated fibers thus ending in the pericellular plexuses?
“Bei der Ermittelung der schwierigen, hier zur Sprache kom-
menden Verhaltnisse haben sich”’ (as Kolliker aptly states) ‘die
Physiologie und die feine Anatomie briiderlich die Hand ge-
reicht.”” The anatomical side of the question has been repeat-
edly touched in the preceding pages, where it may have been
seen that abundant observations have been made, both with the
Golgi and methylene blue method, to show that such medullated
fibers enter the ganglion either through the white rami (chain
ganglia) or through some nerve root of the ganglion. Once in
the ganglion, the medullated fibers have been shown to branch
and even rebranch before ending in the pericellular plexus.

Physiologists have long known that all sympathetic effects,
which may be produced by stimulating a sympathetic nerve in
any region, may also be produced by stimulating some spinal

No.1.] SYMPATHETIC GANGLIA OF VERTEBRATES. ‘79

nerve within the vertebral canal. This, as is now well known, and
first clearly shown by Gaskell (50), is explained by the fact that
certain small medullated fibers, which leave the cord through
the anterior roots of certain spinal nerves — first dorsal to the
third or fourth lumbar inclusive—reach the sympathetic ganglia
through the white rami communicantes. These fibers end, not
only in the ganglia of the chain, but also may be traced into
the pre-vertebral and even the peripheral ganglia, as the follow-
ing statement taken from Gaskell’s account may show:

“The white rami communicantes are formed by an outflow
of medullated fibers from both the anterior and posterior roots
of the spinal nerves between the second thoracic and second
lumbar inclusive, which medullated fibers pass not alone into
their metameric sympathetic iateral ganglia, but also form three
main streams, upwards into the cervical ganglia, downwards into
the lumbar and sacral ganglia, and outwards into the collateral
(pre-vertebral) ganglia. The white rami communicantes alone
constitute the rami viscerales of the morphologist. The outflow
of visceral nerves from the central nervous system into the so-
called sympathetic system takes place by their means alone.”

That these small medullated fibers are the fibers which, in
all vertebrates studied, end in the sympathetic ganglia in peri-
cellular plexuses, may now be assumed with much certainty; and
this for two main reasons:

a) Medullated fibers have been traced from the white rami
into the sympathetic ganglia, or from some nerve root into the
peripheral ganglia, and have been seen to end in pericellular
plexuses, as repeatedly stated.

6) The researches of Langley (51), Anderson, and Dickinson
have taught us that in nicotin we have a drug which shows
most clearly that the action of such fibers is interrupted in the
sympathetic ganglia; that the sympathetic effects which may
be obtained on stimulating a cerebro-spinal nerve, containing
the small medullated fibers first described by Gaskell, or a
white ramus made up of such fibers, cannot be obtained if a
solution of nicotin of proper strength be injected into the circu-
lation of the animal or be applied to the sympathetic ganglia
with which such fibers are connected.

80 HUBER. [VoL. XVI.

Such observations led Langley to conclude that the nerve
fibers, issuing from the cord and reaching the sympathetic
ganglia through the white rami, had each a sympathetic nerve
cell in its course, which sympathetic cell was paralyzed by
nicotin. Langley has suggested the term pre-ganglionic fibers
or pre-cellular fibers to designate the efferent medullated fibers
before they reach the nerve cells, and post-ganglionic or post-
cellular fibers after they have traversed the nerve cells. Taking
these statements into consideration, it would seem reasonable,
to say the least, that the pre-ganglionic fibers are the medul-
lated fibers which end in the sympathetic ganglia in pericellular
plexuses, and the post-ganglionic fibers, the neuraxes of the
sympathetic neurons of said ganglia.

It is not, however, to be supposed that such pre-ganglionic
fibers end only in the chain ganglia in pericellular plexuses.
Small medullated fibers may, as we have quoted from Gaskell,
be traced into the peripheral ganglia, and the mere fact that in
such ganglia pericellular plexuses, similar in structure to those
found in the chain ganglia, have been described as the endings
of medullated fibers, would warrant the conclusion that many
of the pre-ganglionic fibers reach also the peripheral ganglia.
Kolliker (33) has stated the fact so tersely that I may be
allowed to quote again from him: “ Hierbei ist der Verlauf
derselben”’ (meaning here the pre-ganglionic fibers) “ein langerer
oder kiirzerer. Die einen enden an den nachstgelegenen Gang-
lienzellen, andere durchlaufen mehrere Ganglien, bevor sie zu
ihren Endigungen gelangen und konnen hierbei durch Colla-
teralen auf eine Mehrheit von Zellen einwirken. Noch andere
endlich finden erst an den am meist peripherisch gelegenen
Ganglien ihr Ende, wobei es unentschieden bleibt, ob sie in
ihrem Verlaufe auf zwischengelegene Zellen einwirken.”

These observations warrant, it seems to the writer, the fol-
lowing statements: the sympathetic neurons form the periph-
eral links in a nerve chain, of which the second link is formed
by a neuron, the cell body of which is situated in the cerebro-
spinal axis, the neuraxes of which form the pre-ganglionic fibers
ending in pericellular plexuses, which unite the two links
physiologically.

No.1.] SYMPATHETIC GANGLIA OF VERTEBRATES. 81

It is probable, then, that in all vertebrates this statement
holds good: an impulse, leaving the cerebro-spinal system, and
having sympathetic effects, is transferred from a pre-ganglionic
fiber to one or several sympathetic cells which convey it along
their neuraxes to the periphery.

The writer would here again offer the suggestion first made
in the lectures above referred to, that nicotin does not primarily
paralyze the sympathetic cells of the sympathetic ganglia, but
the pericellular plexuses, the endings of the pre-ganglionic fibers
in the sympathetic ganglia. This theory, for it is but a theory,
is based on the analogy which exists between the physiological
action of nicotin and certain other drugs, notably curare. The
latter drug, as is well known, paralyzes the motor endings in
striped muscle, and has an action very similar to nicotin on the
sympathetic ganglia; on the other hand, nicotin paralyzes also
the motor endings in striated muscle, not quite so readily as
curare, but in a similar manner; its action on the sympathetic
ganglia has already been explained. It would seem reasonable,
therefore, to suppose that, in both cases above alluded to, curare
and nicotin paralyze the ending of the cerebro-spinal fiber; in
the one case, the motor ending in striped muscle; in the other
case, the pericellular plexuses in the sympathetic ganglia.

The question as to whether the neuraxes of sympathetic
cells may end in other sympathetic ganglia, and may in this
way influence other sympathetic cells, seems as yet open to
discussion. Kolliker and Langley are of the opinion that the
neuraxes of sympathetic neurons end always in the periphery,
in involuntary muscle, gland tissue, etc.; while Dogiel, with
whom my own observations on this point lead me to concur,
believes that the fine fibers, which end in, and help to form the
intercellular plexus of the sympathetic ganglia, are the neuraxes
of sympathetic neurons, more especially the myelinate ones.
They end, I believe, on the dendrites of the sympathetic neurons
of the ganglion.

HIsTOLOGICAL LABORATORY, UNIVERSITY OF MICHIGAN,
July, 1897.

1 Tn his later writings, Professor Langley gives this explanation of the action of
nicotin on the sympathetic ganglia.

82

HUBER. [VoL. XVI.

LITERATURE CONSULTED.

NoTE.—In a number of instances the reference number is repeated, as the same thought

1. ‘91

2. '95

3. ‘96

fg ee)

4. '96

5.) oS

6. '95

7. '95

8. '74

9. '63

Io. 63

II. ’65

is expressed in two or more articles by the writers mentioned.

METHOD.

RiESE. Zusammenfassendes Referat iiber die vital Methylen-
blaufarbung des Nervengewebes. Centralblatt f. allgemeine
Path. u. path. Anatomie. Vol. ii, Nos. 20, 21.

BETHE. Studien tiber das Centralnervensystem von Carcinus
Maenas nebst Angaben iiber ein neues Verfahren der Methylen-
blaufixation. Archiv f. mikr. Anat. Vol. xliv.

BETHE. Eine neue Methode der Methylenblaufixation. Avxat.
Anzeiger. Vol. xii, No. 18.

MeyYER. Die subcutane Methylenblauinjection, ein Mittel zur
Darstellung der Elemente des Centralnervensystems von Sauge-
thieren. Archiv f. mikr. Anat. Vol. xlvi.

MEYER. Ueber eine Verbindungsweise der Neuronen. Nebst
Mittheilungen tiber die Technik und die Erfolge der subcutanen
Methylenblauinjection. Archiv f. mikr. Anat. Vol. xlvii.

Lawpowsky. Zur Methodik der Methylenblaufarbung und uber
einige neue Erscheinungen des Chemotropismus. Zez¢. f. wzss.
Mikr. Vol. xii.

FISHES.

Mont!. Contributo alla conoscenza dei nervi del tubo digerente
dei pesci. Rendiconti del r. Instituto lombardo di sc. e lettere.
Serie ii, Vol. xxviii.

SAKUSSEFF. Comptes rendus des séances de la société impériale
des natur. de Saint-Petersburg. December, 1895. Taken from
Dogiel, Zwei Arten sympathischer Nervenzellen. Anat. Anzez-
ger. Vol. xi, No. 22. 18096.

ARNDT. Untersuchungen iiber die Ganglienzellen des Nervus
sympathicus. Archiv f. mikr. Anat. Vol. x.

AMPHIBIA.

BEALE. On the Structure of the So-called Apolar, Unipolar, and
Bipolar Nerve-Cells of the Frog. Quart. Journ. of Micr. Sct.
New Series. Vol. iii.

ARNOLD. Zur Histologie der Lunge. Virchows Archiv. Vol.
XXVili.

ARNOLD. Ueber die feineren histologischen Verhaltnisse der
Ganglienzellen in dem Sympathicus des Frosches. Virchows
Archiv. Vol. xxxii.

No. I.]
12. '67
13. '66
14. '66
15. '68
16, '74
FZ. 2G
18. '86
19. 86
20. ’87
21. '89
22. ’89
23. 90
24. 90
25. ’96
26. ’96
Pig ehh
28. '92
29. ‘94

SYMPATHETIC GANGLIA OF VERTEBRATES. 83

ARNOLD. Beitraége zu der feineren Structur der Ganglienzellen.
Virchows Archiv. Vol. xli.

CouURVOISIER. Beobachtungen tiber den sympathischen Granz-
strang. Archiv f. mikr. Anat. Vol. ii.

KOLLMAN and ARNSTEIN. Zezt. f. Biologie. Vol. ii.

SCHWALBE. Ueber den Bau der Spinalganglien nebst Bemerk-
ungen uber die sympathischen Ganglienzellen. Archiv f. mtkr.
Anat. Vol. iv.

ARNDT. Untersuchungen iiber die Ganglienkérper des Nervus
sympathicus. Archiv f. mikr. Anat. Vol. x.

AXEL Key and Rerzius. Studien in der Anatomie des Ner-
vensystems und Bindegewebes. Vol. ii.

EHRLICH. Ueber die Methylenblaureaction der lebenden Nerven-
substanz. Deutsche med. Wochenschrift. Vol. xii, No. 4.

ARONSON. Beitrage zur Kenntniss der centralen und peripheren
Nervenendigungen. /uzaug. Dis. Berlin, Aug. 14. j

ARNSTEIN. Die Methylenblaufarbung als histologische Methode.
Anat. Anzeiger. Vol. ii.

Rerzius. Zur Kenntniss der Ganglienzellen des Sympathicus.
Verhand. d. Biol. Vereins in Stockholm. Vol. ii, Nos. 1, 2.
November.

LAwbowsky. Weitere Beobachtung tiber Nervenendigungen, etc.
Beilage zum lxi. Bande der Denkschriften der Kaiserlichen
Akad. d. Wissensch. zu St. Petersburg. 1889. (Russian.)
Taken from Smirnow.

FeIst. Beitrage zur Kenntniss der vitalen Methylenblaufarbung
des Nervengewebes. Archiv f. Anat. u. Physiol. Anat.
Abtheil.

SMIRNOW. Die Structur der Nervenzellen im Sympathicus der
Amphibien. Archiv f. mikr. Anat. Vol. xxxv.

KO6OLLIKER. Handbuch der Gewebelehre des Menschen. Sechste
umgearbeitete Auflage, zweiter Band, zweite Halfte, p. 864.

Huper. The Spinal Ganglia of Amphibia. Anat. Anzeiger.
Vol. xii.

BIRDS.

Ramon y CajAL. Algunos detalles sobre las cellulas simpaticas.
Pequenas contribuciones al conocimento del sistemo nervioso.
Vol. vi, pp. 56-58. Barcelona. 1891. Taken from Ergedbnisse
der Anat. u. Entwicklungsgeschichte. Vol.ii. 1892.

REtTzIus. Ueber den Typus der sympathischen Ganglienzellen der
héheren Wirbelthiere. Bzo/. Unters. Neue Folge. Vol. iii.

v. LENHOSSEK. Ueber das Ganglion Sphenopalatinum und den
Bau der sympathischen Ganglien. Seztrdge zur Histologie des
Nervensystems und der Sinnesorgane. Wiesbaden.

30.

Si:

32:

33°

34.

35.

36.

37:

38.

39:

4o.

4l.
42.
43.

44.

ks

96

’91

"91

'93

92

92

93

94
194,

95

96

66

'96

HUBER. [VoL. XVI.

MAMMALIA.

ARONSON. (19)

KOLLIKER. Histologische Mitteilungen. Sztzungsber. der phys.-
med. Ges. zu Wiirzburg. 1889.

KOLLIKER. Sympathische Zellen aus dem Ganglion cervicale
supremum und aus dem Ganglion solare des Kalbes. Demon-
strated and described at the sixth meeting of the “ Anatomische
Gesellschaft” in Wien, June 7-9. Verhandlungen. 1892.

KOLLIKER. Ueber die feinere Anatomie und die physiologische
Bedeutung des sympathischen Nervensystems. ‘ Gesellschaft
Deutscher Naturforscher und Aerzte.” Verhandlungen. 1894.
Allgemeiner Theil.

K6LLIKER. Handbuch der Gewebelehre des Menschen. Sechste
umgearbeitete Auflage, zweiter Band, zweite Halfte, pp. 850—
864.

RAMON y CAJAL. Estrutura y conexiones de los ganglios sim-
paticos. Peguenas contribuctones al conocimento del sistemo
nervioso. Barcelona. 1891.

Ramon y Caja. Estructura del gran simpdtico de los mami-
feros. Gaceta sanitaria del 10 Décembre, 1891. Taken from
Ergebnisse der Anat. u. Entwicklungsgeschichte. Vol. ii.
1892.

Ramon y CajaAL. Neue Darstellungen vom histologischen Baue
des Centralnervensystems. Archiv f. Anat. u. Physiol. Anat.
Abtheil.

VAN GEHUCHTEN. Les cellules nerveuses du sympathique chez
quelques mammiféres et chez homme. La Ced/ule. Vol. viii.
1892.

RETZIUS. (28)

SALA. Sur la fine anat. des ganglions sympathiques. Arch.
Ital. de Biol.

v. LENHOSSEK. (29)

'95 p’ERCHIA. Struttura del ganglio ciliare. Monztore Zoologica
Italiano. Nos. 9, to, 1894, and No. 7, 1895.

DociEL. Zur Frage iiber den feineren Bau des sympathischen
Nervensystems bei den Sadugethieren. Archiv f. mtkr. Anat.
Vol. xlvi.

DociEeL. Zwei Arten sympathischer Nervenzellen. Axat. Anzez-
ger. Vol, xt

Guye. Die Ganglienzellen des Sympathicus beim Kaninchen.
Med. Centralblatt.

APOLANT. Ueber die sympathischen Ganglienzellen der Nager.
Archiv f. mikr. Anat. Vol. xlvii.

REMAK. Quoted by Schwalbe. (15)

No. I.]
45. '96
46. '97
47. ’95
48. '94
49. '96
50.
51. ’89
90
90
92
92
95

SYMPATHETIC GANGLIA OF VERTEBRATES. 85

Huser. Observations on the Innervations of the Sublingual and
Submaxillary Glands. Journ. of Experimental Medicine.
Wolk h INO,

ARNSTEIN (ADAM PLOSCHKO). Die Nervenendigungen und
Ganglien der Respirationsorgane. Anat. Anzeiger. Vol. xiii.
DoGIEL. Zur Frage tiber die Darmgeflechte der Sdugetiere.

Anat. Anzeiger. Vol. xi.

TIMOFEEW. Zur Kenntnis der Nervenendigungen in den mann-
lichen Geschlechtsorganen der Sduger. Anat. Anzeiger.
Vol. ix.

Dociet. Der Bau der Spinalganglien bei den Sdugetieren.
Anat. Anzeiger. Vol. xii.

GASKELL. On the Structure, Distribution, and Function of the
Nerves which innervate the Visceral and Vascular Systems.
Journ. of Physiology. Vol. vii.

LANGLEY and LEE DICKINSON. On the Local Paralysis of Pe-
ripheral Ganglia, and on the Connection of Different Classes of
Nerve Fibers with them. Proceed. of the Royal Society of
London. Vol. xlvi, p. 423.

LANGLEY and LEE DICKINSON. On the Progressive Paralysis of
Different Classes of Nerve Cells in the Superior Cervical Gan-
glion. Proceed. of the Royal Society of London. Vol. x\vii,
P. 379-

LANGLEY. On the Physiology of Salivary Secretion. Journ. of
Physiology. Vol. xi.

LANGLEY. On the Origin from the Spinal Cord of the Cervical
and Upper Thoracic Sympathetic Fibers, with Some Observa-
tions on White and Gray Rami Communicantes. PAz/. Trans.
of the Royal Society of London. Vol. clxxxiii.

LANGLEY and ANDERSON. The Action of Nicotin on the Ciliary
Ganglion, and on the Endings of the Third Cranial Nerve.
Journ. of Physiology. Vol. xiii.

LANGLEY. A Short Account of the Sympathetic System. Physi-
ological Congress, Berne. Further references to Langley’s work
may be found in Journ. of Physiology. Vols. xiv, xv, 1893;
Xvi, Xvil, 1894 ; xviii, xix, 1895; xx, 1896.

The literature which has appeared since this article was sent to the editor will receive
consideration in a future publication. — G. C. H.

86 HUBER.

EXPLANATION OF PLATE III.

All figures, with the exception of Nos. 1, 19, and 26, were sketched with the
aid of camera lucida under a ;;-inch oil immersion and No. I eyepiece, the image
being reflected to the table, this giving a magnification of 890 diameters. Figs. 1,
19, and 26 were sketched under a No. 7 objective and No. 1 eyepiece, with about
400 diameters amplification.

Fics. 1-7. Sympathetic Ganglia of Fishes.

Fic. 1. Sympathetic neurons from sections of sympathetic ganglia, stained in
methylene blue, of small-mouth black bass (M/icropterus dolomieu Raf.); a, 6, and
c, unipolar cells; ¢ and e, multipolar cells.

Fics. 2, 3. Pericellular plexuses found in sections of sympathetic ganglia of
black bass. Methylene blue stain. a, neuraxis, ending in pericellular plexus. In
Fig. 3 the enclosed ganglion cell is faintly indicated in black.

Fic. 4. From teased preparation of sympathetic ganglion of black bass,
stained in methylene blue, fixed in ammonium molybdate and hardened in alcohol.
e, sympathetic cell; 7, nucleus; and _¢, its capsule ; a, medullated fiber terminating
within the capsule in pericellular plexus.

Fic. 5. The same cell, double-stained in alum carmine. Some of the end
branches of pericellular plexus terminate between the intra-capsular nuclei.

Fic. 6. Completely isolated cell, obtained by teasing a sympathetic ganglion
of black bass, stained in methylene blue and alum carmine. a, cell body of a
sympathetic neuron ; 4, medullated fiber ending within capsule c.

Fic. 7. From the same ganglion from which Fig. 6 was taken. 4, large
medullated fiber giving off two collateral branches, a’ and a”, ending within the
capsules of cells 4 and B, respectively.

Fics. 8-11. Sympathetic Ganglia of Amphibia.

Fic. 8. Small portion of a section of sympathetic ganglion of Rana Catesbiana,
stained in methylene blue; only spiral fibers and pericellular plexuses stained.
Shows the connection of spiral fiber, a, with pericellular plexus, the structure of
which is well shown in this figure. In d@ we may see one of the large nodular
swellings now and then seen; e, free ending of fibril of the pericellular plexus.

Fics. 9, 10. Two ganglion cells from section of sympathetic ganglion of Rana
C., stained in methylene blue and alum carmine. a, neuraxis of unipolar cell;
4, sheath nuclei of the neuraxis; c, capsule; d@, nodular swellings in pericellular
plexus ; e, free endings of fibrils of plexus; s. /, spiral fibers.

Fic. 11. Sympathetic neurons of Auerbach’s plexus of large intestine of

Rana C., stained in methylene blue and fixed in ammonium picrate. a, neuraxis;
6, dendrites.

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88 HUBER.

EXPLANATION OF PLATE IV.

Fics. 12-19. Sympathetic Cells of Reptilia.

Fic. 12. Two sympathetic cells from the inferior cervical ganglion of Chrys-
emys picta; methylene blue stain. a, neuraxis; 4, sheath nucleus of neuraxis ;
c, clear zone of protoplasm surrounding nucleus; protoplasm shows chromophile
granules.

Fic. 13. From section of dorsal ganglion of Chelydra serpentina, stained in
methylene blue. Simple pericellular plexuses. a, neuraxis of fiber ending in
pericellular plexus ; 4, free endings of fibrils.

Fics. 14-17. More complicated pericellular plexuses, with spiral fiber. All
sketched from sympathetic ganglia, Chelydra serpentina, stained in methylene
blue.

Fic. 18. From section of sympathetic ganglion of Chelydra serpentina, stained
in methylene blue and alum carmine. 4, neuraxis; Z, cell body; C, nucleus;
and ZY, nucleolus of large unipolar sympathetic neuron. a, a’, a”, three neuraxes
of medullated nerve fibers, forming a spiral and ending in pericellular plexus;
c, capsule.

Fic. 19. A portion of a section of an inferior cervical ganglion of Chelydra
serpentina, stained in methylene blue and alum carmine; showing a number of
more complicated pericellular plexuses with spiral fibers, 4, and one simpler
plexus without spiral fiber, 8. Also the network of medullated and non-medul-
lated fibers between ganglion cells.

PLIV.

Journal of Morphology Vol.Xxv1.

go HUBER.

EXPLANATION OF PLATE V.

Fics. 20-22. Sympathetic Ganglia of Birds.

Fic. 20. From methylene blue stained section of one of the dorsal sympathetic
ganglia of chicken. A, medullated fiber, with node of Ranvier at x, at which
place is given off a non-medullated collateral branch, a, which terminates in two
pericellular plexuses ; 7, a non-medullated, probably a sympathetic nerve, ending
on d, the dendrite of the sympathetic cell.

Fic. 21. A portion of a section of a dorsal sympathetic ganglion of chicken,
given to show the arrangement of the multipolar cells and the “intercellular
plexus,” formed by the dendrites, medullated and non-medullated fibers of the
ganglion; 4, pericellular plexus. The capsules of the sympathetic cells were not
stained, and therefore not shown in the figure.

Fic. 22. Three sympathetic cells, with pericellular plexuses from dorsal gan-
glion of chicken. Methylene blue and alum carmine. a, neuraxis of nerve fiber
ending in pericellular plexus; ¢, capsules of sympathetic cells.

FIGs. 23-26. Sympathetic Ganglia of Mammalia.

Fic. 23. A portion of the stellate ganglion of dog. Methylene blue and
alum carmine. a@, non-medullated fiber, collateral branch of pre-ganglionic fiber
ending in pericellular plexus; 4, fiber ending in pericellular plexus which is medul-
lated to within a short distance of the capsule.

Fic. 24. Sympathetic cell from stellate ganglion of dog (methylene blue and
alum carmine), showing a more complex pericellular plexus with spiral fibers.

Fic. 25. Sympathetic neuron from semilunar ganglion of the cat, methylene
blue staining. a, neuraxis; d, dendrite ; c, non-medullated, probably sympathetic
fiber ending on dendrite at ¢’.

Fic. 26. A portion of a section of semilunar ganglion of cat, methylene blue
staining, showing arrangement of sympathetic cells and “ intercellular network ”
formed by the dendrites of the sympathetic cells and the medullated and non-
medullated fibers of the ganglion.

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STUDIES ON LIMUEUS:

Il. THE NERVOUS SYSTEM OF LIMULUS POLVYPHEMUS, WITH
OBSERVATIONS UPON THE GENERAL ANATOMY.

BY

WILLIAM PATTEN anpd WILLIAM A. REDENBAUGH.

DartTMouTH CoLiecE, Hanover, N.H.

CONTENTS.
INR OD UCTUON scot scnast cess occeccet sont cutse2astagsshoeis cscs te caenutegee ree eee ee ee 98

1. Historical.
Researches of Van der Hoeven, Gegenbaur, Dohrn, Lockwood, Packard,
Owen, Milne-Edwards, Lankester, Benham, Patten, Viallanes, and Hyde.... 102
2. Methods.

Maceration in water and weak alcohol previous to dissection; sectioning

MCS CAIN IN Sees ae wce ste setseseodecccchnsetcnteissthecesnsrstaecnassecSbeceee de oar ea ee ee ee 103
GOLAE EV OT LACIE Oe ee ere, ee i oee cones ttactade caaeeee ee EE 104
NICU Y LCT RUILERIICL ROD oaks. cache acted siaesesaatedueatce giant eee eR 104

I. PRELIMINARY DESCRIPTION OF THE ANATOMY OF LIMULUS.

1. External Form.

Cephalothorax : eyes, olfactory organs, appendages, and nephridial openings 105

A DLOILET > APPCDGALES CAIUS occcstsscnce scouvsetuacssteuseectens ere eee ee 106
COUM OLAS PIPER Pre essa) lh eas eaS SA cath Stes Soe ectne Snes ete a ate ae cee eR 106
PEATLDP OP TAY SE Ss Rene Dc a basa oes ann tok is Goa Pe a 106
LL EVLLEIEOU SS (LOUIE OB erence rs eh cas sna Bahia oan saat eet ce eee aoe 106
CADDIE LOE SMR ree ae ao 2 2a Sea SLE aca g uc cece es kaa a oe 106
CLG ANS ale oar a et ee eee reed ae ae ie a) Ae eae le 107

Thoracic appendages from the second to the fifth; modification of chelae
of second pair in the male; entocoxites, sensory knobs, mandibles, inner

mandibles, or “ epicoxites,” apodeme of third joint... eceeeeeee 107
Sixth pair of appendages; flabellum, modification of distal joints for

|SUEER GSN PELs ECE eh NEL Ad ME SSN ONE Hh 1 i Sic Peer eee 108
(C) TIE CIP ae Aare er ae Rael eee eee Besa cen Te ce OY Eee Ee 108
@perculum.+ ‘xenital: papillae!) 2) 2.2! csc os ee teed teeta evap a rosaete ee tceeen sen, ZOD
AGN GARD OOK oo veets ae eed ceed BRL ea eer sae re Re oh ere eee tae eee ee 109

Q2 PATTEN AND REDENBAUGH. [VoL. XVI.

2. The Endoskeletal System.

a. The Endocranium. PAGE
IMmuscles) attachedito end Granites srecetens seers eee easton ene eee eee 109
Processés; ‘occipital ‘rim gp) se 22225 os eee secrete te ceaat ce cease tenn aera ee ae 109
Branchial: (Days) si. ccicccccdesssasceh-stccescooacccessodaceneceserseoet cee eeee eae sae ae eee a ee 109
MPO TAMU A sb sncc dsdesecdacd cto soe Soe eae ee ee ne a es 1@ fe)

b. Zhe Abdominal Endochondrites.

Location, muscles attached, and Processes ..............-2.-:c-.-ecsnsececsecosseceaseserarece IIO

c. The Branchial Cartilages.

Tuankester’sCescription <hr casio rec ct ccto cs semen eons anea eee ee 110
Location amd fume mses coe claece seers arse aaa ee IIo
Resemblance to capsuliginous bars of endocranium....................:::00e1eee00-- III

3. The Muscular System.
Reference ‘to, Benham’ sspapeticsscceesccsee: oes tants ree eeeee nce ee een ser can cso eern eee III
Muscles attached to endocranium ; plastro-coxals, tergo-proplastrals, lat-
eral proplastro-tergals, plastro-tergals, plastro-entapophysials, longi-

tudinal sabdomninal mS CL es sees eee ee eee eee eee eee III
Inter-tergal mussel eS c.s50csp sie cacsssnesesn tac teaten atts cstececvectend datanersncbcnearecaeteeemeees 113
Haemo-neural muscles: se crsscficsoccteececeessteecneletcepeces tes Sacecedaserstnae cons ataets Snares eee 113
Veno-pericardiac muscles; their neural attachment to the membrane

spanning: the) venous-collectimsy Sims lessees. tee sree ree eee eee ee 113
Muscles of caudal spine; flexors and extensOYrs ..........2...:2.22:ceeececeeeeeeeeeeeeeee II4
Anal usel esis ee oes Se eerste cect ence eer eas eeceeeree eeettcs cane tives seeceeenname 114

The Cheliceral Muscles.
Tergo-coxal muscles (of Lankester), or muscles of first joint..................... 115
Muscles: of ‘second Ou t.c:4550- oot. 2 sighs caece, scene deco seeteses cave ces cecseeesas evien dase eee 116
Muscles: of ‘third oints ices sesleecrcnscecscstten cece cea tees ear sapcbecsll eckson eee wee eae 117
The Muscles of the Second, Third, Fourth, and Fifth Appendages.
Plastro-coxals and tergo-coxals, or muscles of coxopodite ...................-+-.--- 117
Muscles:of basippo dite ere ies are see a eee eee raya eee eee 117
Muscles\Gf ischiopodite so es eee ae ea eee ere TT
Musclesof mero-canpopo dite jews eee te eects sees eee ae ee 118
Muscles of propodite rs .i ein eee os a een eee se ene esas Serene eee 118
Muscles: of dactylopo dite xii oe ooo sec ba Sess aoc cas oss ecma soe canes eee 118
Muscles. of inner main cil ef oere case o eee sea eae aca nee eee ee ee sn 120

The Muscles of the Sixth Appendage.
Comparison of the muscles of other thoracic appendages; muscles of
Gistall joints: <4 eee ee ne a ce ae TO
The Muscles of the Chilaria.
Muscles attached to occipital ring, posterior portion of endocranium, and
haemal side ‘of (carapace 2 ache cce teenie see eee ees cere ee eee eee 122
The Muscles of the Operculum.
Reference to Lankester. Extensors or adductors, flexors or abductors,
internal branchials, and branchio-thoracic muscles..........-.....-.--:--:ee-eeee0++ 124
Lhe Muscles af the Gills \ cece eee ee ee ae 124

4. The Digestive System.

The mouth, mandibles, oesophagus, proventriculus, intestine, rectum,
anus, and liver........ Jl Sak ee aes SEN OES ee Oe ek ae ee 124

No. 1.] STUDIES ON LIMULUS. 93

5. Zhe Circulatory System.

a. The Heart. PAGE
Location, general appearance, and size; functional and rudimentary
OStiat; pericardial sinus) and |pericax dimmers reece eee ena 126
“ Alary muscles” and other supports for the heart; branchio-cardiac
CO a ESV eat eR OR RR ee eR ee te A Fe tee a at eee te 127
Arteries arising from the heart; aortic arches and frontal artery, aortic
Valve ,lateral/arteries’s valves of the ostia eee 128

fistology of the Heart.
Longitudinal connective-tissue fibers, basement membrane, internal layer

of anastomosing muscle fibers, and longitudinal nerves ...............2.200++-+- 129
b. Arterial System.
The frontal and marginal arteries; the aortic archeS.............:.:ssessssecsseseeees 129
Masculannne enclosing, brain ......0c00ce ene ee eee eee 130
Ventral artery and branches anastomosing with the superior abdominal
BIE 6 ee oc cece eee ae soe bos epee EE EES SESE CeO OnE gcoe oseceesateroncteonceeseeecereetenensseace 130
Lateral arteries, variation, and occasional occurrence of a fifth lateral
ERC TY oisee ceca nce nde nena cand ceaycctnnsssceazacseudicctcozcesgare de: Beee eeeeee aoe ee ee een See 130
The collateral arteries; hepatic and marginal arteries; superior abdomi-
Rl RAC EN Yeats ae Sarmcccee ce cocina ve nawectnnsnecstscousectsdetpet sare eat -teeak ee Renee eee 131
c. Venous System.
Nena us-collectitigy SINGSOS) -223.15.-::-.c0c0cate cies cnccicuceeee eee ee 131
Branehio-cargiaGrCAMals: t2..!o.5. sentaccosececstence-cotesaesecseeecitna eee ee eee 131
RO nIG ar cli all $S1 miu Sips ees cakes ates ee cccsc seco ct cecesdsascastaveteeseesesaeee reece ee 131

6. The Excretory System.
Nephridiay or brick-red. (lands ......::.22cstsscasctscos ceaacensustet pcos eeeeee eee 132

7. The Reproductive System.
Modification of second pair of chelae in male; ovaries and testes;
eeprniea ePoaAUAG ee cc cnsnzesaszcenssateedeates nis occu: <tiecctemecees renee 132

II. THE NERVOUS SYSTEM.

The work of Owen, Milne-Edwards, Packard, Viallanes, and Patten
HpOn' EVE NERVOUS! SYStOM cic: aca. cash c carotene het eee ee 132

1. Zhe Central Nervous System.
a. The Brain.

Circum-oesophageal collar; general appearance................csssccceeeeeceeeeeoe ees 133
(1) The Fore-Brain.

General Pious ene EVeS jatdS iii Oy fC TL tee eee eee ee ee 134
(2) The Mid-Brain, or Tween-Brain.

Consistsiof chelicerall MeurOomene oeesereee erected eee eee ee 134

Elements ofa typical neuromere.2...2 2-7... pcan enema ates eee es cs AGA

iNeuralforzchelicerall Nerves !2.:-0. ss e ne ee ee e 134

taemaltorlateral nerves: cccco asec cease eee cea ea ee ee ae ess 134

Preoralicommissure- ang rostral Nenves ese 135
SICOMIOR Vea MELVES <<. «scenes vase cuec obscene cata eee eee pe ee eames tan en BR

94 PATTEN AND REDENBAUGAH. [VoL. XVI.

(3) Zhe Hind-Brain. PAGE
Second to sixth thoracic neuromeres; ganglia, cross-commissures,
neural nerves with mandibular and ento-coxal branches, haemal

nerves with intestinal and cardiac branches ...............22....2::21cce1-e ee 135
(4) Zhe “Accessory Brain.
Neural nerves, haemal nerves, with intestinal and cardiac branches...... 135
Fusion of cardiac branches and connection with lateral sympathetic
and pericardial nerves; the vagus regiOM...........-..-----:---ssceseeeseereeeeeeeeeeees 136
b. Zhe Ventral Cord.
Abdominal eangliavanidyth etry Oca:tl 0 reese eee ee eae eee nearer 136
Five branchial neuromeres; neural nerves, haemal nerves with intes-
tinal-and)cardiare iran cles yess sess ee aes ea 137
Two post-branchial neuromeres without neural nerves ; haemal nerves
with intestinal and post-cardiac branches.............2...--2:ceecceeeeceeeeeeeeeeeeees 137

2. Peripheral Nervous System.
a. Nerves from the Fore-Brain.
(1). Olfactory Nerves.

Median and lateral; Patten’s description of them ...........-......----1s-ee 137
(2) Median Eye Nerves.

AP wodescrib ed) tty wie tases reese ee ene 138

Origin: ands tori ution see ee eee sees e rcs peepee sane Fence 138

Patten’s description of the distal end of the median eye nerve...... ........ 139
(3) Lateral Eye Nerves.

Origin, course, pigmental body, distribution, blood vessel...................... 139

Description of a Typical Neuromere.
(1) Abdominal Neuromere.
A pair of ganglia and cross-commissures; neural nerve; haemal nerve
with double intestinal branch, cardiac branch communicating with
the lateral sympathetic and pericardial nerves, and integumentary
Loy 24 00) o Yeeaie Wee nV aN te yg Uae enon aE Ey ERR EY I eran EPR eco 139
(2) Cephalic or Cranial Neuromere.
A pair of ganglia with cross-commissure ; neural-nerve with mandibu-
lar, ento-coxal, and pedal branches; haemal nerve with intestinal
cardiac and! integumentany,|bramChesyie sense eeseeee cen seneseeeree eee aereaenee 141
b. Werves from the Mid-Brain.
(1) Weural Nerves.
Origin ; external and internal pedal nerves and their distribution to
Cheli@erae issih ee Fs ET aia oe eee ead aoe eet 142
(2) Haemal Nerves or Lateral Nerves.
Origin ; fusion with second haemal nerve; distribution upon neural side

of: cephiallothorxaxanel ailoc me ma ese ee eae eee es aoe 143
Problematical sense organ opposite flabellum ..........2.200.-..--------eseeeee 143
Comparison with other haemal nerves .........-------2.-..s-00s0sccee-ceeseees itt A eee 144
Mentioned by Owen, Viallanes, and Pattem............-----.---c--:-seeeseeeseeeeeeee 144

(3) The Stomodaeal Nerves.
Origin and distribution to oesophagus, proventriculus, and pyloric
Val Vee isccchescicile ses dst te ees a Td 0 oe ee ee see 144
Ganglion upon proventriculus and cardiac branches described by Milne-
Bicwarcsiees sree sss ieossuoesestedes a? eet ose SO Lae ees 144

No. 1.] STUDIES ON LIMULUS. 95

(4) Rostral Nerves. PAGE
Two found by Milne-Edwards ; three by Patten; origin from anterior
commissure and distribution to the rostrum eect 145
c. Nerves from the Hind-Brain.
S CCONGRLONSIRE: TEU TOME T Ca eater anes meee ema nee Neen aL eee nD A 145

(1) Meural Nerves.
General distribution; division into mandibular, ento-coxal, and pedal

|} eH YS) 2) 2 SS en MERI Aer nie Ses ic} Fh AE cae) Ce ee 145
(a) Mandibular Branches.
Distribution to mandibles of third pair of appendages..............2..2..... 145
Comparison of these with branches in second, fourth, fifth, and sixth
APPEM GAGES Fe ass he aback cass she oe Re eee ER RE 147

(b) Lxto-coxal Branches.
Anterior ento-coxal nerve; origin and distribution to anterior coxal

muscles and anterior sensory knobs we ee ee 147
Posterior ento-coxal nerve ; distribution to posterior coxal muscles

and posterior sensory knob.....................-... : achaveeaee ts) LAO
Median ento-coxal nerve ; diseabauon to medians sensory set See 149
Absence of median one in second appendage..................2...-ssessee coon 149

In sixth appendage median ento-coxal nerve becomes flabellar nerve 150
(c) Pedal Branches.
External and internal pedal nerves ; distribution to distal joints
Ol AP PENG Pesca. o esc sae Soe cone ce ccs anes eee ee 150
Modificationsiin sixth leg... oe. Ss jacttiuis Aecoes never seen ea ecm 150

(2) Haemal Nerves.
General description of haemal nerves ; division into intestinal, cardiac,
aga anterumentany ibTanches...... 02.222 cen nee ee eee 150
(a) Lntestinal Branches.
Origin of one in sixth neuromere and passage through foramen in en-
docranium to longitudinal abdominal muscles and intestine............ ISI
iintestinalinervesnysecond meurome4nres se I51
(b) Cardiac Branches.
Origin, course, and distribution to inter-tergal muscle and haemal

(5) 1G GI 2 1k I ae tan nn EMER Dee ease ATC 8a I51
Absence in third, fourth, and fifth neuromeres; doubtful cardiac
[pa ROVEAS SST DaT SE) A pe nae ee roee eS SBA enced nN cert ego
(c) Lutegumentary Branches.
Branches to the neural and to the haemal surface... eects 153
Relations of the haemal nerves to each other and to the median eye
AMGMLALS HAL MOE VCE. 8 2 ac csv csl ay. tn coc oe 154

d. Nerves from the Accessory Brain.
Kreme ra KEMMATIES fen 8 8 2 bata ace carl Ue he en Ra nic!
(1) Meural Nerves.
(a) Chilarial Nerve.
Origin, passage through occipital ring, and distribution to chilarium 154
(b) Opercular Nerve.
Origin and distribution to operculum; external, median, and internal
OperculariMerves' 2225 ocs.c.c:f fasts: Ue coc estar eee een SMe 154

96 PATTEN AND REDENBAUGAH. [VoL. XVI.

(2) Haemal Nerves. PAGE
General’ course of the nerves! :-.5.2.2.5 25.-ice aero ee eee eee 156
(a) Zutestinal Branches.
Of chilarial neuromere; passes through foramen of endocranium to
longitudinal abdominal muscles and intestine ....... 2.0... teens 156
Of opercular neuromere 22) ee schstaeluesassenadereeoze 156
(b) Cardiac Branches.
Fusion of the seventh and eighth; relations to lateral sympathetic
and pericardial nerve ; distribution to integument and inter-tergal

PAUSCIES 2.5 czdehecazzaes kee aceh sate das erasers atest arene ee oe er 156
(c) LZutegumentary Branches.
Distribution to lateral expansions of the carapace.............0.::ec+eee 158
e. Werves from the Five Branchial Neuromeres.
General remarks; reference to typical neuromere................--.----1:0----+- 158
(1) Meural or Gill Nerves.
Distribution to gill; external, median, and internal branches.............. 160
(2) Haemal Nerves.
General! Course) 2tisaes ea Fare c rater ea eae ee sett otra oe OO
(a) LZutestinal Branches.
Double nature; anterior branch; posterior branch..........2.2-...---:-.---+-- 160
Irresularity my pOStenOn MEWUrOMeNeS jee sese ere ceneen a cneencr ee ceaesesaeen ences aeons 161

(b) Cardiac Branches.
Connection with lateral sympathetic and pericardial nerves, and

communication with the median cardiac nerve..............::0ce1eee 161
(c) Lntegumentary Branches.
Innervation of the five anterior abdominal spines.............-....2.2.-220--+ 162
f. Merves from the Post-Branchial Neuromeres.
General remarks 25.25.2252 oto e tates oh soca ce va canecasscseusy eaeest ete ee 163
(1) Lerves from the First Post-Branchial Neuromere.
General course ......... das togckasrcubetct spec betestoucsu saad sued wiontuys sontucdssceneeea Sooners 163
(a) Intestinal Branches.
Drregularity 22s2505.0.t 2. scccnescstbesssscsetecesnctsstceet sasattsosncosesceacs cosines coneeetemeeareee 163

(b) Post-Cardiac Branch.
Relation to the lateral sympathetic; branch to extensor of telson .... 163
(c) Lntegumentary Branches.

Innervation of last abdominal spine .............. iid, Helwan wemeeee eR OA
(2) Merves from the Second Post- Brana Nene
Origin: size; and) general (Cours eae ee ee eae eee ee TOT

(a) LZntestinal Branches.
Supply intestine and anastomose with corresponding branch from

last MeUROMEeT esac seis cecte soe ce see cece e ee ce cek See ee een See 164
(b) Post-Cardiac Branch.
Distribution to muscles of telson and epidermiis.................--2.210--.0----- 164
(c) LZntegumentary Branches.
Innervates posterior angles of abdomen and muscles of telson.......... 165

(3) Werves from Third Post-Branchial Neuromere.
Two main branches; one to muscles of telson, the other to telson... 165
Intestinal nerve to anal muscles and rectum.................-..-.--:00-eeseeee-0-e- 165
Milne-Edwards describes a rectal ganglion....................s.sesseseoeeeseseeeseeeees 165

No. 1.] STUDIES ON LIMULUS.

3. Sympathetic Systems.
a. Lateral Sympathetic.

Description by Milne-Edwards... See ;
Location, extent, relations to branchio: thoracic auaeleee aad the seg-

97

PAGE

. 166

mentalvcandiactand haemalinenviess sess ee eres ee 166
Description of lateral sympathetic nerve as found in one specimen 166
Ganglionic enlargements mentioned by Milne-Edwards............................ 169

b. Nerves of Heart.
Milne-Edwards’s description of the cardiac nerves..............:0--s:eeeeeeseeees 169
(1) Zhe Cardiac Plexus.
Medianysaneilionated in er vierces cena te eens erence ee TC.
leateralucardiacmenves rand) ple xus):cc ss eee eae ee 170
(2) Segmental Cardiac Nerves.

Gemeralideseriyy tion eee ose ck ccsnee ce coke eee ter 170

Fused branches of seventh and eighth neuromeres, and their distribu-
tion) wantenon branches, pericardial meiycl sss aaa eee 171

Post-cardiac nerves........... wboeueadh edesdeciccceoundidasnedetbe Mevae oeaee os eee aee ee 172

Caxdiacibranchyfrom\ the: sixth! mewromene see eee ee 172

Relation of segmental cardiac nerves to ostia of heart «0.00.2... 172

c. Nerves of Alimentary Tract.
(1) Zhe Rostral Nerves.
(2) Stomodaeal Nerves.

Milne-Edwards’s description of the ganglion upon the side of the pro-
ventriculus, and branches of the stomodaeal nerves going to the
intestine anGdstont he Weare aoe se ceec sees see 173

(3) Zntestinal Nerves.

Innervation of posterior part of intestine, and the rectal ganglion of
IMU GSTS Clwaiecl Sueeeee eee area: sco. 2 fate eet ee eee : os 19721

Typical arrangement of ees tnall nerve in region Ue fet ree heer
GhialneurOMIEKeS et see et 88 oe Se ease es 175

AAT TANS Menta MyPOSterOlgnie MOM CLES) anes aaa aaene ee aeE 175

Suggestions of elaborate plexus surrounding intestine....... Bs eee 176

Intestinal nenvesjof antemor NeuUrOmMenes iss een ee 176

SUMMARY.

I. Sixteen neuromeres besides fore-brain ................ tbacten ceecease precast xe) LF
2. Elementary part of each neuromere acl excepronit to Seas Fae eee ees 0 177
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4. Brain divisible into four regions
5: keculiarities)/of accessory brain) or <“vasusi region: seen ee ee 178
6. The typical cranial and abdominal neuromeres.....................00cceceseseeeceeeeeess 178
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Sai@auptae TOLVES cs. 2 26 Ro. nabs Ak, cece ee eee ee rea cet ee 179

98 PATTEN AND REDENBAUGH. [VoL. XVI.

PAGE

g. Fusion of muscles of different neuromeres accompanied by fusion of

nerves! Supplying Muscles:. £..22c5:52.s5.cccu8s eer eret eee eee ee 179
10. Evidence that chilaria are not only distinct appendages, but resemble the
abdominal rather than the thoracic appendages.........220.22.-22:0::cceeseeeeeeeeee 179

11. Innervation of liver, nephridia, and generative organs as yet unobserved 180

INTRODUCTION.

THE senior author first began work on the nervous system
of Limulus in 1889, and after various interruptions the distribu-
tion of the more important nerves was worked out and recorded
in the shape of rough sketches and notes. Especial atten-
tion was given to the distribution of the haemal, cardiac, and
sympathetic nerves, with the hope of finding facts that might
point toward conditions similar to those in vertebrates. In
some respects these hopes were not realized, or at least
not in the particular ways looked for, while in others, con-
ditions were found that more than exceeded our expectations.
For example, a very careful search was made for lateral
line nerves similar to those in vertebrates. Various embryo-
logical data ‘seemed to indicate that one might be found run-
ning parallel with and close to the free edge of the carapace
and abdomen, because in the embryos this area is early marked
out by a thickened band of ectoderm along which are scattered
a series of minute sense organs. Every attempt to find sucha
nerve in that region both in the embryos and in the adult
failed. But a nerve was finally found on the neural surface
nearer the median line that fills some of the requirements of
a lateral line nerve (Pl. VI, 2x), for it is a purely sensory
cranial nerve extending at right angles to the course of the
other cranial and spinal nerves, nearly, if not quite, the whole
length of the head and trunk. It branches more freely in the
abdominal region and supplies the skin along its course, but the
exact nature of the sense organs to which its branches are dis-
tributed, and whether this nerve is split off originally from the
sensory thickening that runs round the embryo, and which
corresponds in position with the edge of the future carapace
and abdomen, could not be determined. The fact that it proba-
bly does not supply the line of marginal sense organs in the

No. 1.] STUDIES ON LIMULUS. 99

adult is against such a view of its origin. In another paper
we shall describe the central terminations of this nerve and
we will then discuss its probable relations to corresponding
cranial nerves in vertebrates.

I had hoped to find some points of comparison between
vertebrates and Limulus in the mode of union of the cardiac
nerves with the central nervous system, especially in the rela-
tions of the cardiac branches to those cranial nerves arising
from what I have called the vagus region of the brain. These
inquiries at once led to the discovery of the three great lon-
gitudinal cardiac nerves, the nerve plexuses uniting them with
one another, and the segmentally arranged nerves ‘by which
the cardiac plexus is united with the central nervous system.
When we came to study these nerves more carefully we found
that we had obtained data showing in Limulus an approach
toward the well-known conditions in vertebrates. This resem-
blance is shown: (1) by the absence of cardiac nerves in front
of the sixth thoracic neuromere ; (2) by the increased size, and
(3) by the fusion of the branches arising from the nerves of the
vagus region. In the transition from invertebrates to verte-
brates, we may assume that as the elongated arachnid heart was
forced forward into the head region it gradually lost its connec-
tion with the spinal nerves and retained its union with the
nerves of the vagus region only. In Limulus the fusion with
each other of at least two cardiac nerves in the vagus region,
and their increased size, indicate the adaptability of these
nerves for this purpose.

The main sympathetic system of Limulus also presents strik-
ing resemblances to the condition found in primitive vertebrates.
Instead of the single trunk described by Milne-Edwards, we find
two longitudinal trunks, suggesting the condition found in
some Amphibia. One trunk is mainly related to the cardiac
nerves (Pl. IX, Fig. 6), the other is deeper and nearer the
median line and more intimately related to the intestine
(Pl. VIII, Fig. 4). Both sets of nerves form anastomosing
plexuses. They are united by regularly arranged rami com-
municantes with the haemal nerves, give off segmental
branches to the heart and intestine, and in addition supply

100 PATTEN AND REDENBAUGH. [VoL. XVI.

adjacent trunk muscles. The segmental intestinal nerves, like
the cardiac nerves, are absent from the anterior five thoracic
segments ; therefore the intestinal and the cardiac branches of
the sixth, seventh, and eighth nerves having a greater territory
to cover are larger and better developed than the corresponding
nerves in the trunk region. These enlarged intestinal nerves
supply that part of the alimentary canal confined to the thorax,
exclusive of the oesophagus and proventriculus, into which the
liver ducts open. This part corresponds roughly to the gastric
region of vertebrates, and the nerves that supply it may thus
be fairly regarded as comparable with the intestinal branches
of the vagus, especially since they have their origin in spinal
nerves which for other reasons we had regarded as comparable
with the system of vagus nerves in vertebrates.

We thus have in Limulus a complex condition that clearly
indicates the initial differentiation of sympathetic and vagus
systems similar to those found in vertebrates. This is shown
(1) by the presence in Limulus of double longitudinal nerve
trunks that form anastomosing plexuses, united on one side
by segmental rami communicantes with the spinal nerves, and
on the other by segmental cardiac and intestinal nerves with
the heart and intestine; (2) by the absence or great reduction
of segmental cardiac and intestinal nerves anterior to the
sixth thoracic neuromere; (3) by the consequent increase in
size, and in the territory covered by the intestinal and cardiac
nerves of the sixth, seventh, and eighth neuromeres ; (4) by the
origin of the enlarged cardiac and intestinal nerves of the
seventh and eighth thoracic segments from those spinal nerves
which on entirely independent grounds we had long ago regarded
as comparable with the vagus group in vertebrates.

The sympathetic system of Limulus differs from that of
vertebrates in the absence of segmental ganglia, although iso-
lated ganglion cells are scattered here and there along the
main intestinal and cardiac nerves and over the surface of the
intestine.

The division of labor between Mr. Redenbaugh and myself
in the preparation of this paper was rather unusual. I began

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INGF 1.) STUDIES ON LIMULUS. IOI

work on the peripheral nervous system of Limulus more than
ten years ago, and have continued my observations on it from
time to time ever since.

I had thus worked out the semi-maceration method of dissec-
tion, the structure of the large, median cardiac valve, the
cardiac plexuses, and the distribution of the main peripheral
nerves, neural and haemal. A great deal of time was devoted
to the distribution of the cardiac nerves, in order to determine
their mode of union with the central nervous system; also
much time was given to studying out the relations of the
longitudinal, sympathetic, and intestinal nerves. Many of the
results thus obtained were recorded in the shape of notes and
rough drawings, and the entire work was well in hand when it
was turned over to Mr. Redenbaugh for completion. By his
very careful work Mr. Redenbaugh was able to add many new
and important details, especially in regard to those relations of
the cardiac and intestinal branches that require such careful
dissection. All these points have been verified by both of us,
in some cases several times.

The drawings were made in most instances by Mr. Reden-
baugh along the lines of my original plans and sketches, but I
have added some details of color and finish where it seemed
advisable to make them more distinct or more intelligible.

The descriptive parts were written entirely by Mr. Reden-
baugh and are presented in very nearly the form accepted by
the Biological Department of Dartmouth College as a thesis for

the degree of Doctor of Philosophy. W. Patten.

Most of the work in this paper has been done in the Bio-
logical Laboratory of Dartmouth College. We wish to acknowl-
edge the kindness of Commissioner J. J. Brice, and of the late
Col. Marshall MacDonald, for facilities afforded by the United
States Fish Commission Laboratory at Woods Holl, Mass.,
during the summers of 1894 and 1896.

102 PATTEN AND REDENBAUGH. [VoL. XVI.

1. HISTORICAL.

Before the year 1872 we find little mention of the nervous
system of Limulus, although a number of papers had appeared
upon the natural history, histology, and systematic position of
the King Crab. Van der Hoeven, in 1838, published a mono-
graph upon the anatomy of Limulus, in which he gave a very
good account of the external form, appendages, and grosser
internal anatomy. In the same year Milne-Edwards made
the first observations upon the development. Gegenbaur, in
'58, described the histology of some of the tissues of Limulus.
Lockwood, in ‘70, Dohrn, in '71, and Packard in '72, con-
tributed considerable to our knowledge of the embryology of
the animal.

The first important description of the nervous system ap-
peared in a paper by Owen in’72. He figured the brain and
ventral cord, and the principal nerves arising therefrom.

Milne-Edwards, in '73, carried the investigation of the
nervous system much further, and also gave a very complete
account of the circulatory system.

In ’80 Packard described the histology of the digestive sys-
tem, structure of the liver, nephridia and eyes, and gave
some observations upon the brain, particularly its internal
structure and development. In '93 he published further
observations upon the brain with notes upon its embry-
ology. In this paper he deals almost entirely with the
internal structure.

Lankester, in ’84, described the skeleto-trophic tissues and
coxal glands, and in ’85, with the assistance of W. B. S. Benham,
the muscular and endoskeletal systems.

In '89 Patten gave a short account of the development of
the brain, and in '93, treated of it in greater detail, tracing the
later modifications to practically the adult stage.

In the year ’93 Viallanes, also, published a paper upon the
brain of Limulus; and Miss Ida H. Hyde investigated the
nervous mechanism of the respiratory movements of Limulus,
and maintained that the respiratory centers were located in the
ventral cord.

No. 1.] STUDIES ON LIMULUS. 103

2. METHODS.

The results obtained in our work upon Limulus have been
obtained largely by careful dissection. In order to accom-
plish much by this method, however, it has been necessary to
prepare the material in a special way. In fresh specimens the
nerves were found to be so transparent, and the other tissues
so tough, that it was impossible to trace out the smaller nerves
with any degree of accuracy, and in most alcoholic material the
clotted blood and organic precipitates upon the tissues rendered
it difficult to distinguish the smaller nerves from arteries.

Specimens which had been for a long time (two or three
years) in alcohol of from 50 per cent to 70 per cent were found
to be in remarkably good condition for dissection. The nerves
were white, and easily traced in the partially macerated tissues
when dissection was carried on under water or weak alcohol.

Equally good material was procured by taking large female
Limuli at the end of the spawning season, when all the
ova had been shed, and treating them in the following man-
ner. They were first allowed to bleed freely, then cut in halves
along the median line, and the parts macerated for several
days in water. Finally, they were transferred to 70 per cent
alcohol until ready for use. In this way many of the organic
substances, which would have been precipitated by the alcohol,
were dissolved out. The alcohol whitened the nerves and made
them stand out in contrast with the other tissues. . It was diffi-
cult to determine the proper length of time to continue the
maceration, as it varied with different specimens and with the
temperature. A number were tried and the best selected.

Dissection was carried on under water or weak alcohol with
the aid of a lens, fine pointed forceps, and needles. When-
ever any doubt arose in regard to the character of the tissues,
the doubtful portions were excised, stained, and examined under
a compound microscope.

Injected specimens were used in tracing out the arteries.
A great deal of the work of dissection was verified by exami-
nation of Dr. Patten’s serial sections, both longitudinal and
transverse, of young crabs, from 1 to 1} inches long exclusive

104 PATTEN AND REDENBAUGH. [VoL. XVI.

of the caudal spine. These crabs had been imbedded in cel-
loidin, sectioned, and stained in borax carmine, or in haema-
toxylin and picro-acid-fuchsin.

The histology of the heart, pericardium, alary muscles, etc.,
was studied by means of sections and pieces, excised, stained,
and mounted. The heart with the neighboring tissues was cut
out and hardened in Flemming’s strong solution. Heidenhain’s
iron haematoxylin and eosin gave in most cases the best stain
for sections; Kleinenberg’s haematoxylin and eosin were, also,
found very satisfactory.

The nerves upon the heart were made out by means of
Lowit’s gold chloride method, and the results verified by the
methylen blue method.

Gold Chloride Method.—The hearts of young Limuli,
about 5 inches in length, were dissected out, slit open along
the ventral side, spread out, and treated with formic acid (one
volume of water to one of the acid) until the tissues became
transparent. They were then put for fifteen minutes or half an
hour in 1 per cent gold chloride solution, and afterwards left
in the dark for twenty-four hours in dilute formic acid (one
part acid to three of water). Finally, they were put into strong
formic acid, and left for three or four days in the dark, until the
muscles on the insideof the heart had macerated to such an extent
that they could be washed away by careful manipulations of
the pipette, leaving the nerve plexus intact. The hearts
were then spread out on slides and mounted in glycerine, acidu-
lated with formic acid.

Methylen Blue Method. — Methylen blue was used in various
ways. Some very good stains of the nerves in the appendages
and upon the heart were obtained by injecting small crabs with
a I per cent aqueous solution.

The injection method proved successful in only a small per-
centage of cases. More uniform results were obtained by
immersing portions of the crabs in a solution of methylen blue
in serum, a method first used by Dr. Patten. The serum was
obtained from the clotted blood of large Limuli. Enough of the
stain was dissolved in the serum to give it a clear blue color,
and the solution was kept well oxygenated by forcing air into

Now1-] STUDIES ON LIMULUS. 105

it with a pipette. The tissues to be stained were kept exposed
to the air and moistened from time to time with the solution.
This method almost invariably gave a stain of the nerves in
from ten minutes to one hour.

The nerves upon the heart, pericardium, oesophagus, pro-
ventriculus, rectum, gills and sides of the carapace (after
stripping off the chitin) were easily stained by this method.

As yet all attempts to obtain a stain of the nerves upon the
intestine have failed.

I. PRELIMINARY DESCRIPTION OF THE ANATOMY OF
LIMULUS.

I. ExTERNAL Form.

The carapace of Limulus has been divided by Lankester into
three regions: (1) the prosomatic carapace or cephalothorax
(Pls. VI and VIII, Figs. 1 and 3, pros.) ; (2) the meso-metaso-
matic or abdominal carapace (Pls. VI and VIII, Figs. 1 and
3, mes.) ; and (3) the postanal spine, caudal spine or telson
(Pls. VI-IX, Figs. 1-5, ¢e/.). The terms “haemal” and
“neural’’ will be substituted for dorsal and ventral, in the
following descriptions.

The cephalothorax bears upon its haemal surface two large,
lateral, compound eyes (Pls.VI and VII, Figs. 1 and 2, Ze.) and
tiree (simple, median eyes (Pl. VIII, Pig 32722). Barly
observers found only two ocelli, but Patten (Quar. Jour. Micr.
Scz., 1893) described two ectoparietal eyes, and an endoparietal
eye formed of two retinas fused together.

The neural surface of the cephalothorax bears the olfactory
organs, mouth, nephridial openings, and seven pairs of append-
ages ; vzz., the chelicerae, five pairs of ambulatory legs, and
the chilaria.

The olfactory organs (Pls. VII and VIII, Figs. 2 and 3,
ol.or.) lie in the median line anterior to the chelicerae, and
about one-third the distance between the bases of the chelicerae

1 For a more detailed description, see Lankester, “ Limulus an Arachnid,”

Quar. Jour. Micr. Sci.. and Benham, “ Muscular and Endoskeletal Systems of
Limulus,” Zraxs. Zoél. Soc., London, 1885.

106 PATTEN AND REDENBAUGH. [VoL. XVI. ,

and the anterior edge of the carapace (see Patten, Quar. Journ.
Micr. Sci., 1893). The seven pairs of appendages surround
the mouth (Pl. VIII, Fig. 3, #.), which lies nearly in the center
of the cephalothorax. The nephridial openings (PI. VII, Fig. 2,
7.0.) may be seen just back of the fifth pair of legs.

The abdomen (Pls. VI and VIII, Figs. 1 and 3, mes.) is
attached to the cephalothorax by a transverse hinged joint on
the haemal side of the animal, and is capable of movement in a
haemo-neural direction only. It bears on its lateral edges six
pairs of spines (Pl. VI, Fig.1, @.s.9"4), the first pair belonging
to the first branchial metamere, and the last pair to the first
post-branchial metamere.

On the neural surface of the abdomen are six pairs of lamellar
appendages (Pl. VIII, Fig. 3, af.°”%); the genital openings
are on the posterior side of the first pair of abdominal append-
ages, and the anus (Pls. VI and VIII, Figs. 1 and 3, a.) is at the
base of the caudal spine.

The caudal spine (Pls. VI, VIII, and IX, Figs. 1, 3-5, eZ), or
telson, is a long, sword-shaped, terminal segment capable of
movement upon the abdomen in any direction.

Entapophyses.— Seven pairs of entapophyses, or chitinous
infoldings of the haemal side of the carapace (Pls. VI, VIII,
and IX, Figs. 1, 4-6, ewta.7"4), serve for the attachment of
muscles. These infoldings may be seen from the exterior; one
pair (ezta.7-*) on the cephalothorax, just anterior to the hinge,
and the other six pairs (extfa.7"#) upon the abdomen. The
first pair (exta.7*), which are much larger than the others,
are probably formed by the fusion of the two pairs belonging
to the chilarial and opercular metameres; the next five pairs
(enta.73) upon the abdomen belong to the five gill metameres ;
the last pair (ex¢a.74) belong to the first post-branchial metamere.

Tendinous Stigmata.— Six pairs of chitinous infoldings of
the neural surface of the abdomen, close behind the bases of
the six pairs of abdominal appendages, serve for the attachment
of the branchio-thoracic muscles. They have received the name
of ¢endinous stigmata (Pls. VI and IX, Figs. 1 and 6, 7.5.43).

The Appendages.—In describing the appendages it seemed
desirable to consider them all as turned outward so as to lie at

No. I.] STUDIES ON LIMULUS. 107

right angles to the median line and parallel to each other. The
homologies can then be more readily made out. The first pair,
the chelicerae (Text-fig. 1; Pl. VIII, Fig. 3, a.’), lies almost
parallel to the median line, so that the morphologically anterior
side faces the median line. They are small chelate appendages,
anterior to the mouth, consisting of but three segments, and
are regarded as homologous with the antennae of insects and
myriapods.

The next four pairs of appendages (Text-fig. 2), from the
second to the fifth, serve as ambulatory legs and also as masti-
catory organs. In the female they are all chelate. In the male
the chelae of the second pair of appendages are modified to serve
as clasping organs. The propodite is thickened, and its terminal
process, which is ordinarily the anterior blade of the chela, is
aborted ; the dactylopodite, or posterior blade of the chela,
is curved anteriorly so as to be opposed to the aborted end of
the propodite.

Each of these appendages is composed of six joints, of which
the fourth is double, being formed by the fusion of the meropo-
dite and the carpopodite. The proximal margin of the coxopo-
dite (Text-fig. 2, 7-cox.) is much thickened, and forms a structure
of complicated outline which has been called by Lankester the
“entocoxite.” If the coxopodite be examined from the proximal
side, the entocoxite may be clearly seen (Pls. VI and VII, Figs.
I and 2, ezt.2°). The outer portion is divided by chitinous bars
into three spaces filled with areolar tissue containing numerous
nerve endings. These spaces appear as slight swellings or knobs,
probably highly sensory, upon the outer extremity of the base of
the coxopodite. The inner, or median, portion of the coxopodite
is modified to form a mandible (Text-fig. 2, #zaxz.) which projects
over the mouth and bears numerous, inwardly projecting spines
provided with gustatory buds. The mandibles of the third,
fourth, and fifth pairs of appendages bear an inner detached por-
tion (Text-fig. 2, z.man.) furnished with a small flexor muscle.
These inner mandibles have been called by Lankester the “epicox-
ites.” They are also supplied with spines and gustatory buds.!

1 For the structure of the gustatory buds of the mandibles, and also of similar

buds in the chelae, see Patten, “‘ Morphology and Physiology of the Brain and Sense
Organs of Limulus,” Quar. Journ. Micr. Sci., 1893.

108 PATTEN AND REDENBAUGH. [VoL. XVI.

A chitinous infolding, or apodeme (Text-fig. 2, apfo.), arises
from the arthroidal membrane, between the third and fourth
joints, and projects into the cavity of the third joint. It fur-
nishes attachment for a large flexor muscle arising from the
anterior side of the second joint.

The sixth pair of appendages (Text-fig. 3) are the powerful
legs used for burrowing and pushing the animal along through
the sand. The inner mandibular portion (Text-fig. 3, man.)
lacks gustatory spines, is very massive, and serves as a crush-
ing jaw. Two of the sensory knobs of the outer portion of
the coxopodite are like the corresponding parts of the other
ambulatory legs, but the third or median one is replaced by
a spatulate organ, the flabellum (Text-fig. 3, fad., and Pls. VI
and VII, Figs. 1 and 2, emz.°).

The homologies of these sensory knobs will be discussed later,
under the nervous system.

The fifth joint (Text-fig. 3, 5-pvo.), instead of forming with
the sixth a chela, as in the other legs, is oblong in longitudinal
section, and bears upon its distal end a rosette of four shorter
spatulate organs, and the sixth joint. The latter (Text-fig.
3, 6-dac.) bears at its distal end two small terminal joints,
which are opposed to each other and function as a small chela.

The chilaria (Text-fig. 4), which lie posterior to the mouth,
are a pair of small appendages consisting of a single segment.
Owen regarded them as detached portions of the sixth pair of
appendages, but they undoubtedly represent true appendages
and belong to a distinct metamere.

The first pair of appendages upon the abdomen (Text-fig. 5)
are almost completely fused in the median line, and form a large
operculum, which overlaps the five pairs of gills. Each half
consists of a large proximal portion marked off by radiating
creases into triangular areas, and a small distal portion
divided into two lobes—an inner lobe, or endopodite (Text-fig. 5,
z./.), consisting of two joints, and an outer lobe, or exopodite
(0.2.), consisting of one joint. Two genital papillae upon the
posterior side of the proximal portion mark the external open-
ings of the oviducts (Text-fig. 5, ov.). There is no gill book
upon this appendage.

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No. 1.] STUDIES ON LIMULUS. 109

The remaining five pairs of abdominal appendages (Text-fig.
6) are alike in form, but decrease in size towards the posterior
end of the body. A deep median cleft divides them into right
and left halves, between which in the median line is inserted
a membranous tongue or median lobe (Text-fig. 6, 7.7). The
basal portion of each half bears a gill book (Text-fig. 17, ¢.d.)
consisting of numerous overlapping leaflets. The endopodite
(z.2.) is slender, and produced a short distance beyond the broad
exopodite or outer lobe (o./.).

2. THE ENDOSKELETAL SYSTEM.

In Limulus there are a number of cartilaginous bodies which
serve for the attachment of muscles. These are the plastron, or
endocranium (Text-figs. 2-5; Pls. VI and VIII, Figs. 1, 3, and
4, endo.); six small abdominal endochondrites (Text-figs. 5
and 6; Pls. VI, VIII, and IX, Figs. 1, 3, 4, and 6, a.e.4%), one
at the base of each pair of abdominal appendages; and six pairs
of branchial bars (Text-figs. 5 and 6; Pls. VI and IX, Figs. 1
and 6, 4.c.3"%) supporting the operculum and gills. Another
pair of branchial bars (d.c.”7) supporting the chilaria are fused
with the endocranium.

a. The Endocranium.

The endocranium has been fully described in the first paper
of this series. Here we shall merely state that this piece of
cartilage serves as a centrum for the attachment of the longitu-
dinal abdominal muscles (Text-figs. 5 and 6; Pls. VI, VIII, and
IX, Figs. 1, 4, and 6, /.a.m.), tergo-plastrals (Pls. VIII and IX,
Figs. 4 and §, @./p.z.), tergo-proplastrals (Text-fig. 8; Pls. VI,
VIII, and IX, Figs. 1, 4, and 5, ¢..m.7“), veno-pericardiac
muscles (Text-fig. 4; Pl. VIII, Fig. 4, v.f.m.%7), and numerous
muscles inserted upon the coxopodites of the appendages, from
second to the seventh, inclusive.

A pair of bars (Text-figs. 4 and 5; Pls. VI, VII, and VIII,
Figs. 1-4, 0.c.”) of capsuliginous cartilage, identical with that of
the branchial bars of the abdominal appendages, are fused,

110 PATTEN AND REDENBAUGH. [VoL. XVI.

proximally, with the posterior border of the endocranium and
are attached distally to the posterior sides of the chilaria.

Near the posterior extremity of the endocranium are two pairs
of foramina (Text-figs. 3-5; Pls. VI and VIII, Figs. 1, 3, and 4,
f.°7) which afford a passage for intestinal nerves (2.7.97).

b. Zhe Abdominal Endochondrites.

These are six in number (Text-figs. 5 and 6; Pls. VI, VIII,
and IX, Figs. 1, 3, 4, and 6, a.e.43), metamerically arranged
in the median line neural to the ventral cord, one at the base
of each pair of abdominal appendages (ap.23). It will be
noticed that they lie upon the side of the central nervous sys-
tem opposite to that on which the endocranium lies. They are
fibroid in structure, like the endocranium, and act as centra for
the attachment of the internal branchial muscles (Text-figs. 5
and 6; Pl. IX, Fig. 6, 2.d.m. °#3), longitudinal abdominal muscles
(Text-figs. 5 and 6; Pls. VI, VIII, and IX, Figs. 1, 4, and 6,
.a.m.), and haemo-neural muscles (Pls. VI, VIII, and IX, Figs.
I, 4-6, h.n.m 73),

c. The Branchial Cartilages.

These structures were described by Gegenbaur in 1858.
Lankester (Quar. Journ. Micr. Scz., 1884) described a “ pair
of ligamentous bands, the extapophysial ligaments, which pass
from one to another of the dorsal ingrowths of the integu-
ment known as the dorsal entapophyses.” ‘The ligamentous
band is not of equal dimensions throughout, but where it is
attached to an entapophysis it gives off at right angles a
conical, knob-like protuberance.”’

This ligament is continuous only between the first and sec-
ond pairs of entapophyses. From the outer side of every enta-
pophysis except the seventh, a cartilaginous bar (Text-figs. 5
and 6; Pls. VI and IX, Figs. 1 and 6, 6.c.4) passes neurally
to the inside of the appendage of the same metamere af.*%.
It acts as a skeletal support for the appendage and also for
the attachment of numerous muscles. These bars consist of a
highly characteristic core of capsuliginous cartilage enveloped

No. I.] STUDIES ON LIMULUS. Tor

in a tough fibroid cortical layer. The same kind of cartilage
is found in the structures arising from the posterior portion of
the endocranium and attached to the insides of the chilaria.

3. THE Muscurar System.

Lankester (Zvaus. Zool. Soc., London, 1885), with the assist-
ance of Mr. Benham, gave a detailed account of the muscular
system of Limulus. He, however, omitted the chilarial and the
anal muscles, as well as the muscles of the thoracic append-
ages, except the coxal muscles. The anal muscles were figured
by A. Milne-Edwards (Aun. Sct. Nat., 1873).

In the cephalothorax the edges and neural surface of the
endocranium afford attachment for a great many muscles which
radiate to the bases of the ambulatory legs and are inserted
upon the inner proximal portions of the coxopodites from
the second to the sixth, inclusive. Lankester calls them the
plastro-coxal muscles (Text-figs. 2 and 3; Pl. VII, Fig. 2,
Prands,candz, 62,4, /,andg, and corresponding muscles in the
second to the sixth appendages). They assist in the compli-
cated chewing movements of the mandibles.

Three pairs of muscles, the texgo-proplastrals (Text-fig. 13 ;
Pls. VI, VIII, and IX, Figs. 1, 4, and 5, ¢.4.m.*°), suspend the
anterior cornua of the plastron from the haemal portion of the
carapace.

A small muscle, lateral proplastro-tergal, fastens each of the
lateral cornua to the haemal side of the carapace.

A pair of plastro-tergals (Pls. VIII and IX, Figs. 4 and 5,
@.l.p.t.) attach the haemal processes of the endocranium to the
carapace, and a pair of plastro-entapophysials (Pl. VIII, Fig. 4,
d.lp.e.) pass from the same processes to the first pair of
entapophyses.

From the posterior portion of the haemal surface of the
endocranium large meso-plastro-entapophystals (P|. VIII, Fig. 4,
m.p.e.) also go to the first pair of entapophyses. All of these
muscles serve to suspend the endocranium in a firm position in
the cephalothorax and to counteract the contractions of the
coxal muscles and longitudinal abdominal muscles, which would

PE2 PATTEN AND REDENBAUGH. [VoL. XVI.

tend to draw the endocranium out of place. They may also
serve to compress the cephalothorax and thus aid in the expul-
sion of the genital products.

A few muscle strands, plastro-buccal muscles, go from the
anterior neural side of the plastron to the oesophagus, and a few
more, beneath the skin behind the mouth, go from the occipital
ring to the oesophagus.

A mass of longitudinal abdominal muscles (Text-figs. 5 and 6;
Pls. VI and VIII, Figs. 1 and 4, /.a.m.) arises from the posterior
haemal side of the endocranium and passes backward, giving off
slips to each pair of abdominal entapophyses and to the abdom-
inal endochondrites. It terminates upon the integument just
posterior to the last pair of gills. Portions of this mass join
together the successive endochondrites. A pair of slips are
inserted upon the integument posterior to each of the four gills
and just median to the infoldings of the tendinous stigmata.
They unite with the terminal portion of the mass of abdominal
muscles, pass backward, and are attached to the integument
posterior to the last pair of gills.

Four zuter-entapophysial muscles (Pl. VIII, Fig. 4, z.e..) pass
from the first pair of entapophyses to the next four pairs.

The tendinous stigmata (¢.s.273) of the six pairs of abdomi-
nal appendages furnish attachment for a bundle of drvanchio-
thoracic muscles (Text-figs. 5 and 6; Pls. VI and IX, Figs. 1, 5,
and 6, 8.¢.%.) which run forward just neural and median to the
row of entapophyses, and external to the large bundle of abdomi-
nal muscles proceeding from the endocranium. After passing
median to the tergo-coxal muscles, external to the tergo-plastrals,
and haemal to the plastro-coxals, the branchio-thoracic muscles
attach themselves by two slips (Pl. IX, Fig. 5, 0.2.2.7 744) to
the haemal side of the carapace, external to the pericardial
sinus (/.S.).

Between the branchio-thoracic muscles and the longitudinal
abdominal muscles is a double membrane closely investing
these muscles like a perimysium, and affording attachment for
the veno-pericardiac muscles (see Pl. IX, Fig. 6, v.f..9).

All of these longitudinal muscles act together as flexors of
the abdomen.

No. 1.] STUDIES ON LIMULUS. 113

The abdomen is extended by two pairs of powerful zzter-tergal
muscles, Pls. VIII and IX, Figs. 4 and 5, z.#. (only the external
ones are represented) in the median line on the haemal side of
the animal. The external pair of muscles arise from the median
faces of the first pair of entapophyses, and the internal pair
from an extended area upon the haemal, median portion of the
carapace. Both pairs are inserted in the median line on the
anterior, haemal border of the abdominal carapace.

Seven pairs of haemo-neural muscles (P\s. VI, VIII, and IX,
Figs. 1, 4-6, 4.”.m.5"4) arise from the haemal side of the
abdominal carapace. The first six pairs (4.2.m.'3) are inserted
upon the six abdominal endochondrites, and hence belong to
the opercular and five branchial metameres, respectively. The
seventh pair (4.2.m./4) are inserted upon the neural side of the
carapace posterior to the last appendage. The first pair
(i.n.m.°), which belong to the opercular metamere, arise upon
the anterior border of the abdominal carapace, from the median
side of a pair of small protuberances which may possibly rep-
resent the remnants of a pair of entapophyses, though it is
more likely that the entapophyses of the chilarial and opercular —
metameres have fused with each other. As will be seen later,
the fact that there is a fusion of other structures in this region
serves to support this hypothesis. The remaining six pairs of
haemo-neural muscles (/.7.#.9"4) arise from the carapace on the
median side of the six pairs of abdominal entapophyses.

The haemo-neural muscles aid in holding the endochondrites
in place by counteracting the contractions of the internal bran-
chial muscles (2.6.%.) which arise from the endochondrites.
They also serve to compress the abdomen.

Eight pairs of veno-peritcardiac muscles (Text-fig. 4; Pls.
VIII and IX, Figs. 4 and 6, v.p.m.°), ‘‘ brides transparentes,”’
of Milne-Edwards, are attached to the neural side of the peri-
cardium opposite the eight pairs of ostia (0s.°”3) of the heart.
These muscles pass neurally on each side of the intestine to the
integument upon the neural side of the body. Instead of being
attached directly to the integument, however, the bases of the
muscles expand and become continuous with a tough connective-
tissue membrane running longitudinally between the branchio-

114 PATTEN AND REDENBAUGAH. [VoL. XVI.

thoracic muscles and the longitudinal abdominal muscles. In
the abdominal region this membrane is double, the two portions
partially investing the branchio-thoracic muscles on the one
hand, and the longitudinal abdominal muscles upon the other.
Between the successive appendages it is attached to the integu-
ment. The space between the two portions is the ventral col-
lecting venous sinus (Pl. IX, Fig. 6, v.c.s.), which, with its
fellow upon the opposite side of the body, carries the blood to the
gills to be aérated. In the thoracic region the membrane does
not enclose a venous sinus. It is single and attached to the sides
of the endocranium, and, in places, to the integument between
the plastro-coxal muscles.

The caudal spine is moved by various muscles which arise
by a large number of slips, each of which Lankester has desig-
nated by a special name. For our purpose, however, it will be
better to divide them into three groups and consider each
group as one pair of muscles, thus making one pair of flexors
and two pairs of extensors. The pair of flexors (Pl. VI, Fig. 1,
t.f.m.) arise by numerous slips from the neural portion of the
abdominal carapace posterior to the appendages, and from the
outer and posterior sides of the last three pairs of entapophyses,
and are inserted upon the outer neural portions of the arthroi-
dal membrane which attaches the caudal spine to the abdomen.

One pair of extensors (Pl. IX, Fig. 5, ¢.e.m.*) arise by numer-
ous slips from the haemal side of the abdomen posterior to the
heart, and from the inner sides of the last three pairs of enta-
pophyses, and are inserted upon the haemal portion of the
above-mentioned arthroidal membrane, near the median line.
The second pair of extensors (Pls. VI and IX, Pigs andes;
t.e.m.°) arise upon both the haemal and neural sides of the cara-
pace, and are inserted upon the arthroidal membrane, external
to the first pair of extensors.

Coordinate action of the muscles on either side of the median
line moves the caudal spine in a lateral direction.

A sphincter muscle (Pls. VI and VIII, Figs. 1, 3 and 4, s.a.)
closesthe anus. A pair of slender muscles (Pl. VI, Fig. 1, 0.a.),
which may be called the occ/udor ant, arising on the neural side
of the carapace, just posterior to the last appendage, are inserted

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No. 1.]

STUDIES ON LIMULUS.

ries

upon the anterior side of the proctodaeum, close to the anus.
These serve to draw the anus forward, and by elongating the
anal slit possibly assist the sphincter ani in its function. A
pair of band-shaped muscles (Pls. VI, VIII, and IX, Figs. 1,

3-5, /.a.), or levator ant, are in-
serted upon the sides of the anus,
and pass haemally and outward
to the haemal side of the cara-
pace. They serve to open the
anus.

The Cheliceral Muscles (Text-
fig. 1).—In the chelicerae the
first joint is moved by four mus-
eles (Text-fie./1,.e7, 7.7; and 2.2.7).
Lankester regarded these as one
muscle and called it the tergo-
coxal muscle of the chelicera.
They have their origin on the
haemal side of the carapace, de-
scend vertically on the median
side of the anterior cornua of the
endocranium, and are _ inserted
upon the base of the first joint
of the appendage. The flexor
(77) is inserted upon the inner
margin, the extensor (e.’) upon
the outer margin, and a small
muscle (/7.7) upon the _ sides
midway between the flexor and
extensor muscles. In these de-
scriptions the appendages are
supposed to be revolved out-
ward, so as to lie at right angles

to the median line and parallel to
flexor and extensor muscles act
and give to the whole appendage

the other appendages.
in opposition to each other
a movement toward or away

h c/ ¥

Fic. 1.— Diagram showing muscles and dis-

tribution of nerves in chelicera of adult
Limulus, from the anterior side (natural
size). 7,2, and 3, first, second, and third
joints of chelicera; ¢.!-3, extensors of first,
second, and third joints, respectively ; 713,
flexors of first, second, and third joints, re-
spectively; 72.7.1, lateral muscles of first
joint; e.!, £1, and 2.7.1 constitute the tergo-
coxal muscles; .c., haemal side of the
carapace; 47., fore-brain; z.z.!, neural
nerve or cheliceral nerve; z.4.7z., internal
pedal nerve; ¢.4.7%., external pedal nerve ;
f..2x.1, haemal nerve of cheliceral neuromere
or lateral nerve.

The

from the mouth. The small lateral muscles give it a slight

lateral movement.

116 PATTEN AND REDENBAUGH. [VoL. XVI.

The second joint is moved by two muscles. A large flexor
(7) arises from the anterior side of the proximal joint, and
is inserted upon the inner proximal margin of the second.
A smaller extensor muscle (e.?) arises from the outer and pos-
terior sides of the proximal joint, and is inserted upon the outer

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NG

Al NG

Fic. 2.— Diagram showing muscles and distribution of nerves in the third leg of Limulus, from the
anterior side (34 natural size).
I-cox., coxopodite, or first joint; 2-das., basipodite, or second joint; 3-zsc., ischiopodite, or

ea fused carpopodite and meropodite, or fourth joint ; 5-470., propodite, or
C+ }

third joint; ¢- {
fifth joint; 6-dac., dactylopodite, or sixth joint; a@fo., apodeme; %.c., haemal side of carapace ;
endo., endocranium; ’¢., heart; zzz., intestine; z.#zaz., internal mandible; #.,mouth; »az.,
mandible; Z., pericardium; #.s., pericardial sinus.

Muscies : 32 and b, plastro-coxal muscles inserted upon anterior side of entocoxite;
3¢and 4, tergo-coxal muscles inserted upon anterior side of entocoxite; ¢.2-6, extensors of
second to sixth joints; 42-6, flexors of second to sixth joints; 7.™, flexor of inner mandible.

NERVES: @.e.., anterior ento-coxal nerve; 4r., brain; e.f.7., external pedal nerve, %.,
haemal branch of integumentary nerve; 4.7.3, haemal nerve ; 7.2.3, integumentary branch ;
z.p.m., internal pedal nerve; .e.7%., median ento-coxal nerve; 7.72., mandibular nerves; 7.,
neural branch of integumentary nerve ; 7.7.3, neural nerve ; 4.e.7., posterior ento-coxal nerve.

No. 1.] STUDIES ON LIMULUS. 117

proximal margin of the second. These muscles move the
chela toward and away from the mouth.

The third joint, or movable blade of the chela, is moved by
two muscles, —a large flexor (f) arising from the anterior and
inner sides of the second joint and inserted upon the ante-
rior proximal margin of the third, and a small extensor (e.3)
arising from the posterior side of the second joint and inserted
upon the posterior proximal margin of the third. By these
muscles the third joint is moved at right angles to the move-
ments of the other joints of the appendages, and the chela
opened and closed.

The Muscles of the Second, Third, Fourth, and Fifth Append-
ages (Text-fig. 2).— As these appendages are very similar in
their musculature, the third one may be taken as a type, and
the others compared with it. The muscles which move the
coxopodite are nine in number. Four plastro-coxals (Text-fig. 2,
peande Pl WIL, Pig. 2,7%%* 42) arise from the side of the plas-
tron and are inserted upon the median half of the entocoxite,
two on the anterior side and two on the posterior. The re-
iadining tive (Dext-fig..2, 7°*79¢;,.Pl. VIL, Pig. 2, 7447) anise
from the haemal side of the carapace and are inserted upon the
outer portion of the entocoxite. Two of these, # and 4%, or
3° and 3%, are inserted upon the anterior side, and three, 4%, 4%,
and 47, upon the posterior.

In the second appendage the muscle corresponding to ¢ is
absent or fused with the one corresponding to 4%. In the sixth
appendage there is an extra plastro-coxal muscle, 6%, on the pos-
terior side, arising from a point on the plastron much farther
forward than the origins of the other plastro-coxals of that
appendage. The tergo-plastral muscle corresponding to 4’ is
apparently fused with 6%. All of these muscles assist in perform-
ing the complicated chewing movements of the coxopodites
as well as the forward and backward movements of the legs in
walking and swimming.

The second joint, or basipodite (Text-fig. 2, 2-das.), is moved
by two muscles: a large flexor (f”) arising from the posterior
side of the coxopodite (/—coxr.) and inserted on the inner proxi-
mal margin of the basipodite ; a smaller extensor (e.?) arising

118 PATTEN AND REDENBAUGA. [VoL. XVI.

from the anterior side of the coxopodite and inserted upon the
outer proximal margin of the basipodite.

The third joint, or ischiopodite (3-zsc.), is moved by a more
complicated set of muscles. A large flexor muscle (3) arises
from the posterior side of the basipodite, and is inserted upon
the inner proximal margin of the ischiopodite. Another large
muscle (7.4), arising from the anterior side of the basipodite, is
inserted upon a chitinous infolding of the arthroidal membrane
(apo.) on the median side of the appendage between the ischi-
opodite and the mero-carpopodite. This muscle acts as a flexor
of the mero-carpopodite, and also as an extensor of the ischi-
opodite. Three small muscles (e.3), arising from the outer side
of the ischiopodite, and inserted upon the outer margin of
the distal portion of the basipodite, act as extensors of the
ischiopodite.

Besides the large flexor of the mero-carpopodite, above men-
tioned, a pair of small flexors (4) of the same joint arise from
the outer side of the ischiopodite, and are inserted upon the
inner margin of the proximal portion of the mero-carpopodite.
A pair of slender muscles (e.4) arising from the outer side of
the ischiopodite, near the distal end, and inserted upon the
inner margin of the proximal portion of the propodite, act as
extensors of the mero-carpopodite, and also as flexors of the
propodite.

Another pair of flexors (5) of the propodite arise from the
outer side of the mero-carpopodite, and are inserted upon the
inner proximal margin of the propodite. The extensors (e.5) of
the propodite are a pair of muscles arising from the outer side
of the mero-carpopodite, and inserted upon the outer proximal
margin of the propodite. The propodite is capable of move-
ment upon the mero-carpopodite in any direction. Movement
in an anterior direction is effected by the codrdinate contraction
of the anterior extensors and flexors, and movement in a pos-
terior direction, by contraction of the posterior extensors and
flexors.

The dactylopodite (6-dac.) is moved by a large flexor (/-)
arising from the anterior, inner, and outer sides of the propodite,
and inserted on the anterior proximal margin of the dactylopo-

No. 1.] STUDIES ON LIMULUS. 119

he’ im!

Fic. 3.— Diagram showing the muscles and distribution of the nerves in the sixth leg of Limulus,
from the anterior side (24 natural size).

I-cox., coxopodite, or first joint; 2-das., basipodite, or second joint; 3-zsc., ischiopodite, or
third joint; ¢- fea fused carpopodite and meropodite, or fourth joint ; 5-4ro0., propodite, or

”
fifth joint; 6-dac., dactylopodite, or sixth joint; @fo., apodeme; dr., brain; c.c., cross-commis-
sure; ezdo., endocranium; jéaé., flabellum ; /.c., haemal side of carapace; Az., heart; zzz.,
intestine ; 7zaz., mandible ; Z.s., pericardial sinus.

MuscLss: 64 and b, plastro-coxal muscles inserted upon anterior side of entocoxite;
6cand 4d, tergo-coxal muscles inserted upon anterior side of entocoxite; e.2-7, extensors of
second to seventh joints ;_ 42-7, flexors of second to seventh joints; z.7., inter-tergal muscle.

NERVES: a@.e.z., anterior ento-coxal nerve ; ¢.f.7., external pedal nerve ; 4., haemal branch
of integumentary nerve; 4.7.3, haemal nerve; 2.7.6, intestinal nerve; 2.7.6, integumentary
branch of haemal nerve; 7.4.7., internal pedal nerve; Z.c.7., lateral cardiac nerve; 7.c..,
median cardiac nerve; 7.e.2., median ento-coxal nerve or flabellar nerve ; 7.2., mandibular
herve; #., neural branch of integumentary nerve; .7.6, neural nerve; Z., pericardium;
?.e.2., posterior ento-coxal nerve ; s.c.7.6, segmental cardiac nerves.

120 PATTEN AND REDENBAUGH. [VoL. XVI.

dite, and by a small extensor (e.°) arising from the posterior side
of the propodite and inserted upon the posterior proximal margin
of the dactylopodite. These muscles close and open the chela.

A small flexor muscle (f.”) is found at the base of the inner
mandible. This muscle is absent in the second appendage.

Fic. 4.— Diagram showing the muscles and nerves of the chilaria of Limulus, from anterior side.
The appendages are revolved outward about 45° (magnified nearly 14% diameters).
é.c.7, capsuliginous bar or branchial cartilage; ezdo., endocranium; #.c., haemal side of
carapace; At., heart; zzz., intestine; oc.r., occipital ring; Z.s., pericardial sinus.
Muscies: 7 a7-€, plastro-coxal muscles ; 7fand g, tergo-coxal muscles; 2.7., inter-tergal
muscles; v.f.7.7, veno-pericardiac muscles.

Nerves: 4.2.7 and 8, haemal nerves of chilarial and opercular neuromeres ; 7.7.7, intestinal
nerve ; 22.2.7 and 8, integumentary branches of haemal nerves of chilarial and opercular seg-
ments; /.c.z., lateral cardiac nerve; Z.s.2., lateral sympathetic nerve; 7z.c.2., median cardiac
nerve ; 7.72.7 and 8, neural nerves of chilarial and opercular neuromeres; Z., pericardium ;
s.c.2.7 and 8, fused segmental cardiac nerves of chilarial and opercular neuromeres.

The Muscles of the Sixth Appendage (Text-fig. 3).— These
are similar to the muscles of the second, third, fourth, and fifth
appendages, except those which move the joints beyond the
propodite. A large muscle (f°), arising from the posterior,

No. I.] STUDIES ON LIMULUS. I2I
ul.

Fic. 5. — Diagram showing the muscles and distribution of the nerves in the operculum. The oper-

culum is flexed upon the abdomen, and is seen from the neural side (about 1% natural size).

a.e., abdominal endochondrite of opercular segment; 4.c.7, capsuliginous bar or branchial
cartilage of chilarium; 4.c.8, branchial cartilage of operculum ; ezdo., endocranium ; z./., inner
lobe of operculum; oc.v., occipital ring; a./., outer lobe of operculum ; ow., oviduct.

Musctes: a.d..8, abductor muscle of operculum; 4.¢.7z., branchio-thoracic muscles;
e.6.m.8, external branchial muscle; z4.7z., internal branchial muscle; 7./.7z., muscle of inner
lobe ;_ 0.2.72., muscle of outer lobe. a

NERVES : ¢.0.7., external branch of opercular nerve ; 7.7.7 and 8, haemal nerves of chilarial
and opercular segments; 2.7.7 and 8, intestinal nerves of chilarial and opercular neuromeres ;
72.2.7 and 8, integumentary branches of haemal nerves of chilarial and opercular neuromeres ;
z.0.7%., internal branch of opercular nerve; Z.s.2., lateral sympathetic nerve; 7z.0.7., median
branch of opercular nerve; 7.7.8, neural or opercular nerve; s.c.7.7 and 8, fused segmental
cardiac nerves of the seventh and eighth neuromeres ; v.c., ventral cord.

122 PATTEN AND REDENBAUGA. [VoL. XVI.

median, and anterior sides of the propodite, is inserted upon the
arthroidal membrane between the bases of the whorl of spatu-
late organs upon the distal extremity of the propodite, and acts
as a flexor for them all, as well as for the slender dactylopodite
(6-dac.). A smaller muscle arising from the outer side of the
propodite, and inserted upon the outer proximal margin of the
dactylopodite, acts as the extensor of this joint, and also of
the spatulate organs. The inner proximal margin of the dac-
tylopodite extends like a spur toward the bases of the spatu-
late organs, and upon the contraction of the extensor muscle
acts as a lever and lifts up the arthroidal membrane in the
middle of the whorl of spatulate organs. This action tips
them outward.

At the distal end of the longer joint are two smaller append-
ages opposed to each other like a chela, and opened and closed
by small flexor (77) and extensor (e.7) muscles.

In the sixth appendage, as in the second, the inner mandible
with its flexor muscle is absent.

The Muscles of the Chilaria (Text-fig. 4).—These muscles
are comparable with those of the abdominal rather than with
those of the thoracic appendages. A muscle (Text-fig. 4, Pls.
VII and WIM Fies: 27andte,.7>) sarises, from the rToottot
the occipital ring, and is inserted by two slips to the posterior
and anterior sides of the chilarium. This muscle draws the
appendage forward. A few transverse strands of muscle
fibers (7%) are attached to the posterior margins of the bases
of the two chilaria, and draw the two appendages toward
the median line. A muscle (7%) arises on the neural side
of the posterior process of the endocranium, and is inserted
upon the inner margin of the base of the chilarium.

Another small muscle (7°) arises on the inner side of the
capsuliginous bar, and is inserted upon the base of the chilarium,
close to the insertion of the last-described muscle. These two
muscles aid in drawing the appendage backward and toward the
median line. Still another small muscle (7) arises from the
posterior border of the endocranium, outside of the capsuligi-
nous bar, and is inserted upon the outer margin of the base of
the chilarium. A long muscle (7) arises from the haemal side

No. 1.] STODIES ON LIMES: 123

Hh ple
y

Fic. 6. — Diagram showing the muscles and distribution of nerves in the first gill. The appendage
is flexed upon the abdomen, and is seen from the neural side (about 1% natural size).

a.e.9, abdominal endochondrite; 4.c.8 and 9, branchial cartilages of operculum and first gill ;
2.2., inner lobe of gill; 7.Z., median lobe of gill; 0./., outer lobe of gill.

MuscieEs : @.6.72.9, abductor muscle of gill; 4.¢.2%2., branchio-thoracic muscles; ¢.4.7.9,
external branchial muscle; 7.4.12.9, internal branchial muscle; 7./.7., inner lobe muscles ;
2.a.m., longitudinal abdominal muscles; 0.2.7., outer lobe muscles.

NERVES : a.g., first abdominal ganglion; e.4.z., external branch of neural nerve; g.z.,
branch of neural nerve supplying gill book; 4.7.9, haemal nerves; 7.6.7., internal branch cf
neural nerve ; 2.7.9, intestinal nerve (two branches are shown, a posterior and an anterior one) ;
iz.2.9, integumentary branch of haemal nerve; /.s.7., lateral sympathetic nerve ; 7z.4.7., median
branch of neural nerve; 7.7.9, neural nerve; s.c.z.9, segmental cardiac nerve ; v.c., ventral cord.

124 PATTEN AND REDENBAUGH. [VoL. XVI.

of the carapace, just anterior to the hinge, and passes haemally
outside of the branchio-thoracic and longitudinal abdominal
muscles to its insertion, close to that of the last-described
muscle. These two muscles draw the appendage outward and
backward.

Lastly, a muscle (7%) arising from the haemal side of the
carapace passes neurally, and is inserted upon the integument
a short distance from the base of the chilarium.

The Muscles of the Operculum (Text-fig. 5).— These have
been minutely described by Lankester, so it will not be neces-
sary to give them in detail. The appendages naturally lie
flexed backward upon the abdomen. Large extensor, or abduc-
tor, muscles (a.d.m.*), arising from the haemal side of the
cephalothorax and from the first entapophysis, are inserted
upon the anterior lamella and upon the anterior side of the
branchial bar (4.c.2) of the operculum. A large flexor, or
adductor, muscle (Text-fig. 5; Pls. VI and IX, Figs. 1 and 5,
e.6.m.*), arising from the haemal side of the abdominal carapace
just posterior to the hinge, is inserted upon the posterior lamella
and the posterior side of the branchial bar. A small internal
branchial muscle (Text-fig. 5, 2.0.7.) arises from the neural side
of the first abdominal endochondrite (a.¢.) and is inserted upon
the inner side of the branchial bar (0.c.%). The branchio-thoracic
muscles have already been described. A few strands of mus-
cles (o./.4. and 2z./.m.) flex and extend the inner and outer lobes
of the appendage.

The Muscles of the Gills (Text-fig. 6).— These are similar to
those of the operculum, except that the extensors or abductors
(a.6.m.?) arise from the entapophyses of the preceding metamere
instead of from the haemal side of the carapace ; and the mus-
cles which serve to flex and extend the terminal portions of the
appendage are more numerous and better developed.

4. DIGESTIVE SYSTEM.

The mouth (Text-fig, 2; Pl. VIEL, Pigs 33-7.) ismsituared
nearly in the center of the neural side of the cephalothorax, and
is surrounded by the chelicerae, five pairs of mandibles, and the

No. 1.]} STUDIES ON LIMULUS. 125

chilaria. The mandibles and chelae of the second, third,
fourth, and fifth pairs of legs, aided by the chelicerae, tear
the food to pieces and cram it into the mouth. The mandi-
bles of the sixth pair of legs crush the hard portions, and the
chilaria serve to push the food forward, within reach of the
mandibles.

The oesophagus (Pls. VI and VIII, Figs. 1, 3, and 4, oe.)
passes through the circum-oesophageal collar, turns anteriorly
neural to the endocranium, and runs forward to the muscular
stomach or proventriculus (Pls. VI, VIII, and IX, Figs. 1, 3-5,
prov.), which lies at the anterior extremity of the cephalothorax.

The proventriculus (Pls. VI, VIII, and IX, Figs. 1, 3-5, prov.)
is V-shaped, and the haemal arm communicates with the intes-
tine by a pyloric valve, which appears as a large muscular
papilla at the anterior end of the intestine. The walls of the
oesophagus, proventriculus, and pyloric valve are very muscular,
and are lined with chitin, which is thrown into longitudinal
ridges or rugae.

The zztestine (Text-figs. 2-4; Pls. VI, VIII, and IX, Figs.
I, 3, 4, and 6, zwz.) is a straight tube running posteriorly from
the proventriculus, haemal to the endocranium and ventral cord,
and neural to the heart. Its posterior extremity passes into a
short rectum or proctodaeum (Pl. VIII, Figs. 3 and 4, proc.).
Anteriorly the intestine is large, but decreases in size posteriorly.
The larger anterior portion receives two pairs of hepatic ducts
(Pl. VIII, Fig. 3, %.¢.*), which enter at the sides of the intestine
nearly opposite the mouth. The walls of the intestine are sup-
plied with both longitudinal and circular muscle fibers, but they
are much thinner than the walls of the other portions of the
alimentary canal.

The rectum or proctodacum (P\. VIII, Figs. 3 and 4, proc.) isa
short tube passing from the intestine to the anus. It is lined
like the oesophagus with chitinous rugae, and its walls are
supplied with well-developed muscles for the ejection of faeces.

The anus (Pls. VI and VIII, Figs. 1 and 3, a.) is a longitudinal
slit capable of being opened and closed by the anal muscles
already described.

The /ver consists of a great mass of tubules ramifying over

120 PATTEN AND REDENBAUGH. [Vox. XVI.

a large portion of the cephalothorax and abdomen. These com-
municate with ducts which are collected into the two pairs of
‘large hepatic ducts (4.2.7) entering the intestine.

5. THE CIRCULATORY SYSTEM.

The circulatory system has been worked out very accurately
by A. Milne-Edwards, but some structures have been over-
looked in connection with the heart. This organ will, therefore,
be taken up in some detail.

a. Lhe Heart (Text-figs. 2-4; Pls. VIII and IX,
Figs. 3, 5-8, 4z.).

' The heart, which is very large in Limulus in comparison
with the size of the body, lies on the haemal side of the
animal, directly beneath the carapace and haemal to the
intestine. It extends from a point midway between the lateral
eyes back to about the middle of the abdomen, being fully one-
half as long as the body exclusive of the caudal spine. It has
the general appearance of a jointed tube, and attains a length
of about five inches in the adult male and about six inches
in the female, with a diameter of from half to three-quarters
of an inch. Longitudinal strands of connective tissue give it
a striated appearance, and a large median ganglionated nerve
(Text-figs. 3 and 4; Pls. VIII-X, Figs. 3, 5, 6, 8-10, m.c.m.)
is very conspicuous upon the haemal side. In cross-section it
is somewhat triangular in the middle portion, but is flattened
haemo-neurally toward the extremities. The largest portion is
just back of the middle, and from this point it tapers in both
directions.

There are eight pairs of transverse slit-like ostia (Pls. VIII-
X, Figs. 3, 5, 6, 8, and 9, os.) upon the haemal side of the
heart, partially concealed by a grating of longitudinal connec-
tive-tissue strands lying across the openings. A ninth pair of
rudimentary ostia (Pl. IX, Fig. 8, ~os.) are discernible at the
anterior extremity. They appear as two shallow pits on the
inner surface of the haemal wall of the heart, just behind
the aortic arches’ (ao.a.) and in front of the aortic valve (a.v.).

No..1.] STUDIES ON LIMULUS. 127

Around the heart is a large pericardial sinus (Text-figs.
2-4; Pls. VIII and IX, Figs. 3, 5, and 6, .s.), enclosed by a
membranous pericardium (f.)._ Upon the neural side this mem-*
brane is well defined, stretching across the body, between the
heart and the intestine. At the sides it is attached, in the abdom-
inal region, to the entapophyses, to the bases of the branchial
cartilages, and to the haemal carapace between the successive
entapophyses. In the cephalothorax it is attached to the first
pair of entapophyses, and to the carapace outside of the origins
of the inter-tergal muscles. Posteriorly and anteriorly the peri-
cardium is continuous with the neural walls of the heart.

Upon the haemal side of the pericardial space the pericardium,
as such, does not exist, or, at least, is indistinguishable from the
epidermis. A considerable amount of areolar tissue fills the
haemal side, and many of the interstices of the pericardial
sinus. .

Eight pairs of rather broad bands of connective tissue, the
alary muscles (Pl. IX, Figs. § and 6, a.4.m.°3) of Van der
Hoeven, spring from the lateral edges of the heart, opposite
the ostia, and fuse at their distal ends with the pericardium,
forming a strong lateral support for the heart. Those in the
abdominal region enter the venous canals opening into the
pericardial sinus.

Neurally the heart is attached to the pericardium throughout
its entire length by numerous connective-tissue fibers. Hae-
mally it is suspended from the carapace, opposite each pair of
ostia, by small strands of connective tissue which are continuous
with the longitudinal fibers of the heart. From the anterior
extremity of the heart, opposite the rudimentary ostia, a pair
of tendinous bands (a/.m.5), comparable to a pair of alary mus-
cles, run forward and upward a short distance beyond the limits
of the pericardium, and attach themselves to the carapace close
to the insertions of the tergo-proplastral muscles (Pl. IX, Fig.
5, 2p.m.°). At the posterior end a sheet of connective tissue
attaches the extremity of the heart to the carapace.

The pericardial sinus (Text-figs. 2-4; Pls. VIII and IX,
Figs. 3, 5, and 6, #.s.) surrounds the heart from the extreme
posterior end to a point about opposite the rudimentary ostia.

128 PATTEN AND REDENBAUGH. (VoL. XVI.

The posterior portion receives the five pairs of canals (4.c.c.7)
from the gills.

In normal specimens there are eleven arteries given off from
the heart, three from the anterior extremity, and four pairs
from the sides, opposite the four anterior pairs of functional
ostia. The two large anterior arteries, aortic arches (Pls. VIII
and IX, Figs. 3, 5, and 8, ao.a.), curve downward, one on each
side of the proventriculus, to the circum-oesophageal collar.
The median artery, arteria frontalis (f.ar.), goes directly
forward over the haemal surface of the proventriculus. A
large pocket-shaped valve (Pl. IX, Fig. 8, a.v.), much like a
vertebrate semilunar valve, lies upon the haemal wall of the
heart at the base of the aortic arches, and just behind the rudi-
mentary ostia. It prevents a backward flow of the blood from
the aortic arches and from the arteria frontalis.

The lateral arteries, arteriae laterales (Pls. VIII-X, Figs.
3, 5, 8, and 9, /.ar.°9), arise from the lateral, neural corners of
the heart, directly beneath the four anterior pairs of functional
ostia (os.°9), and pass downward into the pericardium, and out-
ward to a pair of longitudinal collecting arteries, the arteriae
collaterales (c.ar.). The arteriae col-
laterales (Pl. IX, Figs. 5, 6, and 8,
c.ar.) pass backward, giving off nu-
merous branches, and unite behind
the posterior end of the heart to form
a median artery, the arteria abdomt-
nalis superior (s.a.ar.), which appears

STLW.

Fic. 7. — Diagram showing the mechan- :
ism of the valves of the ostia. .w., to proceed from the posterior ex-

haemal wall of heart; 2.w., neural
wall of heart; os., ostium of heart;

tremity of the heart.
£7., grating of elastic Abers upon the | The arzeviac daterales anes supplied
outside of the heart, bridging over ' i p
the ostium, and preventing the lips with paired semilunar valves (Pl. IX,
of the ostium from spreading apart; . 6 6 °
m.s., muscle fibers attached to the lips Fig. 8, s.v.°9%) at their points of ori-
of the ostia at the outer corners. :

gin from the heart. These valves

are upon the posterior and anterior walls of the arteries.

Each of the ostia is also provided with a valve, the action
of which is best seen in young Limuli. The lips of the
ostia (Text-fig. 7, os.) turn inward toward the lumen of the
heart, and this invagination is greatest at the outer corners,

No. I.] STUDIES ON LIMULUS. 129

where a few muscle fibers (m.s.) are inserted upon the invagi-
nated lips and attached to the neural wall of the heart. Upon
the outside of the heart a grating (gr.) of elastic fibers stretches
across the ostia and prevents the lips from separating too
much. The valves are thus kept from being evaginated by the
pressure of the blood when the heart contracts.

flistology of the Heart.— A cross-section of the heart
shows the cardiac walls to be composed of three layers: (1)
a median, dense, connective-tissue, basement membrane (PI. IX,
Fig. 7, 6.mem.); (2) an outer longitudinal layer of thick elastic
fibers (/.c.s.); and (3) an inner muscular layer (a.m.f.). The
muscle fibers, which are distinctly cross-striated, are inserted
upon the basement membrane and extend across the lumen of
the heart, branching, and anastomosing with each other. They
are loosely arranged around a central lumen, and form a circu-
lar layer thickest in the lateral angles of the heart. There is
no endothelium, and the blood circulates freely around the indi-
vidual muscle fibers. This muscular layer extends from the
extreme posterior limit of the heart to a point on the neural
side, opposite the aortic valve. On the haemal side (PI. IX,
Fig 5) its anterior boundary curves around the posterior end of
the aortic valve.

In each angle of the heart is a longitudinal nerve (Text-
figs. 2-4; Pls. VIII-X, Figs. 3, 5-10, m.c.m. and /.c.z.) lying
between the elastic fibers, outside of the basement membrane,
but enclosed by a sheath which is continuous with the mem-
brane.

b. Arterial System.

The origins of the arteries from the heart in the normal con-
dition have been described above. There is, however, often
considerable variation. The arterta frontalis (Pls. VIII and
IX, Figs. 3-5 and 8, fa.v.) may arise directly between the
two aortic arches, or from either one, or it may be absent
entirely. When present it gives off a small branch, which
goes posteriorly, haemal to the heart, and supplies the inter-
tergal muscles, areolar tissue, and epidermis in the haemal
median line. The main trunk runs forward in the median

130 PATTEN AND REDENBAUGA. [VoL. XVI.

line, supplying the tissues on the haemal side of the body in
the neighborhood of the proventriculus, and divides at the
anterior margin of the carapace to form two anterior marginal
arteries. These follow the edges of the carapace around to the
posterior angles of the cephalothorax, where they are joined
by branches from the arteriae collaterales.

The aortic arches (Pls.VIII and IX, Figs. 3-5 and 8, ao.2.)
curve downward upon each side of the proventriculus, supply
this organ and the oesophagus, and then follow the oesophagus
backward to the vascular ring, which encloses the nerve collar.
The vascular ring gives off arteries anteriorly to the oesopha-
gus, to the tissues in the median line neural to the oesophagus,
and to the median and lateral eyes. The chelicerae, five pairs
of ambulatory appendages, chilaria, and operculum also receive
large arteries from the vascular ring. The ventral artery (PI.
IX, Fig. 6, v.av.), which sheathes the ventral cord, is given off
from the posterior side of the ring. The longitudinal abdominal
muscles, the neural side of the intestine, and the five pairs of
gills receive their blood supply from the ventral artery. Poste-
riorly this artery divides into a number of branches, some sup-
plying the muscles of the caudal spine, but the main branches
pass on either side of the rectum to the interior of the caudal
spine. A little in front of the anus these arteries give off
branches which, after supplying the rectum, pass haemally on
each side of the rectum and anastomose with the arteria abdomt-
nalis superior (S.a.ar.). -

The origins of the four pairs of lateral arteries (/.ar.°9) and
their union with the arteriae collaterales (c.ar.) have already
been described. An interesting variation, however, sometimes
occurs. A fifth lateral artery (Pl. IX, Fig..5, 4.275) has been
found arising from the base of the left aortic arch, anterior to
the aortic valve, and opposite the rudimentary ostia (7.0s.). This
artery has no semilunar valves, but it joins the collateral artery
just as do the other lateral arteries. In the same specimen in
which this lateral artery was found, a small artery was found in
the corresponding position upon the opposite side, but this did
not connect with the collateral artery of that side. It supplied
the tergo-proplastral muscles.

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No. 1.] STUDIES ON LIMULUS. 131

Anteriorly the collateral arteries give off branches to the
tergo-proplastral and tergo-coxal muscles, and to the intestine.
Opposite the second pair of ostia a large pair of arteries are
given off, which pass outward toward the posterior angles of
the cephalothorax and anastomose with the anterior marginal
arteries. Midway between the median line and the edge of the
carapace a large branch, the hepatic artery, is given off ante-
riorly, supplying the liver and anastomosing with the lateral eye
arteries. At the same point a fosterior marginal artery is
given off. This passes posteriorly along the margin of the
abdominal carapace and anastomoses with the superior abdom-
inal artery at the base of the caudal spine.

The posterior portions of the collateral arteries send branches
to the intestine, to the external branchial muscles, and to the
muscles of the caudal spine. The arterta abdominalis superior
(s.a.ar.) gives off branches to the haemal side of the intestine,
anastomoses with branches from the ventral artery and poste-
rior marginal artery, and terminates in the caudal spine. All
the arteries divide ultimately into very fine arterioles, which
open into venous spaces.

c. Venous System.

There are no veins with definite walls lined with epithelium.
The venous system consists, for the most part, of tubular spaces
in the areolar tissue, or irregular spaces between the various
organs. The blood is collected from these spaces into a pair
of longitudinal sinuses (Pl. IX, Fig. 6, v.c.s.) upon the neural
side of the body. These sinuses have already been described
as spaces, between the branchio-thoracic muscles and the
longitudinal abdominal muscles, roofed in on the haemal side
by a membrane which furnishes attachment for the veno-peri-
cardiac muscles. From these venous sinuses the blood passes
into the operculum and the lamellae of the five pairs of gills.
From each gill a large branchio-cardiac canal (PI. IX, Figs. 5
and 6, 0.c.c.73) carries the blood to the pericardial sinus (/-s.).
A canal (0.c.c.8) from the operculum unites with the branchio-
cardiac canal (4.c.c.”) of the first gill. There are, therefore,

132 PATTEN AND REDENBAUGA. [VoL. XVI.

only five pairs of these canals entering the pericardial sinus,
although Milne-Edwards describes six.

From the pericardial sinus the blood enters the heart through
eight pairs of functional ostia.

6. THE ExcrRETORY SYSTEM.

The nephridia, brick-red glands, or coxal glands (Pl. VII,
Fig. 2, 2.75), as they have been variously called, consist of a mass
of tubules in the cephalothorax on each side of the endocranium.
An elongated portion lies on the haemal side of the plastro-
coxal muscles, and four lobes. (z.?%) descend from this into
the bases of the second, third, fourth, and fifth appendages,
respectively. The lobes lie between the slips of the superior
plastro-coxal muscles and communicate with each other by
ducts on the neural sides of these muscles. A duct leads
to the exterior from the lobe in the fifth appendage. The
external opening (z.0.) is upon the posterior side of the base
of the fifth appendage.

7. THE REPRODUCTIVE SYSTEM.

Limulus is dioecious, and the male can be distinguished by
the thicker and subchelate character of the second pair of
appendages which are used, during the breeding season, in
clinging to the posterior margin of the abdominal carapace
of the female. Both the ovary and testes are retiform, the
network of tubules, which compose these glands, extending
through the cephalothorax and a large part of the abdomen.
The paired oviducts and vasa deferentia have muscular walls,
and open to the exterior by apertures at the summits of two
genital papillae upon the posterior surface of the base of the
operculum.

II. THE NERVOUS SYSTEM.

As has already been said, we are indebted for out knqwledge _
of the nervous system of Limulus chiefly to Owen and Milne-
Edwards, although Packard, Viallanes, and Patten have more

No. 1.] STUDIES ON LIMULUS. 133

recently devoted some attention to the development and mor-
phology of the brain.

Owen and Milne-Edwards gave a general plan of the distri-
bution of the larger nerves arising from the brain and ventral
cord, but did not agree upon the number of these nerves, and
left practically untouched the innervation of the heart, intes-
tine, and appendages. Packard and Viallanes confined their
investigations principally to the supra-oesophageal ganglion and
the nerves arising therefrom, and restricted the term “brain”
to this portion of the nervous system.

Patten figured and described the entirecircum-oesophageal col-
lar and ventral cord and applied the term “brain”’ to the whole
nerve ring. He furthermore divided the brain into four regions :
(1) the fore-brain, which comprises the cerebral lobes or supra-
oesophageal ganglion; (2) the mid-brain, or cheliceral neuro-
mere; (3) the hind-brain, or six thoracic neuromeres; and (4)
the accessory brain, or chilarial and opercular neuromeres. In
the following description of the brain the nomenclature of
Dr. Patten will be followed.

1. THE CENTRAL Nervous SYSTEM.

The central nervous system may be divided into brain and
ventral cord, the former consisting of the fused ganglia of the
circum-oesophageal collar, and the latter of the abdominal
ganglia and their longitudinal connectives.

As the primary object of this paper is to give a clear presen-
tation of the distribution of the peripheral nerves, the internal
structures of the central nervous system will not be discussed
further than is necessary in defining the origins of the various
nerves.

a. The Brain.

In the adult Limulus the brain (Pls. VI-VIII, and X,
Figs. 1-3, 11, and 12) is nearly circular and fits snugly around
the oesophagus close to the mouth. It is included within a
vascular ring and is thus completely bathed in blood. At the
sides of the oesophagus there is a slight flexure, in a neural

134 PATTEN AND REDENBAUGH. [VoL. XVI.

direction, which throws the cerebral lobes a little downward.
One cross-commissure (a.c.) anterior to the mouth and four
(~.0.c.25) posterior to the mouth can be seen, from the exterior.
Numerous nerves radiate from the sides of the brain. Those
going to the appendages bend neurally, giving to the brain a
concave appearance upon the neural side and a convex appear-
ance upon the haemal side.

(1) The Fore-Brain. — The fore-brain (f.47.) lies entirely in
front of the mouth, and, when the arterial sheath is removed,
appears as two convoluted lobes, the cerebral lobes, separated
upon the neural side by a deep longitudinal fissure. Upon the
haemal side the fore-brain is depressed in the middle line be-
tween the enlarged bases of the lateral eye nerves (/.e.7.).

From the semicircular lobes lying in this depression the
median eye nerve (#.ey.z.) arises by four roots.

The median olfactory nerves (o/.z.) arise from the anterior
extremity of the fore-brain, the lateral ones from the middle
lobes of the optic ganglia.

(2) The Mid-Brain or Tween-Brain.— This is represented
by the cheliceral or first thoracic neuromere. A typical neuro-
mere (Text-fig. 9) in Limulus consists, according to Patten,
of a pair of ganglia united across the median line by several
cross-commissures (c¢.c.); two pairs of nerves, a neural pair
(x.2.) supplying the appendages, and a haemal pair (/.7.) sup-
plying the internal organs and the lateral expansions of the
carapace.

The neural nerves (Text-figs. 1 and 13; Pls. VI-VIII and
X, Figs. 1-3, 11, and 12, 2.2.) arise from the neural side of
the brain posterior to the cerebral lobes and pass neurally to
the chelicerae. Some small nerves (Text-fig. 1, ¢..7.), arising
near the bases of the neural nerves, supply the tergo-coxal
muscles of the chelicerae.

The haemal nerves (Pl. X, Figs. 11 and 12, /.7.*) arise from
the haemal side of the brain just posterior to the origins of the
lateral eye nerves (/.e.7.), and present a very exceptional distri-
bution. After fusing with the haemal nerves of the second
neuromere they separate again, and, curving around posteriorly,
innervate the epidermis upon the neural side of the body out-

No. 1.] STUDIES ON LIMULUS. 135

side the bases of the appendages, and extend far back upon the
abdomen.

The pre-oral cross-commissure (a.c.), which belongs to this
neuromere, is more neural in position than the post-oral ones,
and is also characterized by the fact that it gives off three
nerves (/a.z.) to the rostrum, or upper lip.

In addition to these peculiarities the cheliceral neuromere
bears upon its inner side a pair of stomodaeal ganglia and a
pair of stomodaeal nerves (s¢.z.) which supply the oesophagus
and proventriculus.

(3) Lhe Hind-Brain.— The hind-brain consists of five thoracic
neuromeres, the second to the sixth inclusive. Each neuromere
(Text-fig. 9) consists of a pair of ganglia united by cross-com-
missures, and two pairs of nerves, a neural pair (#..) supplying
the appendages, and a haemal pair (4.z.) supplying internal or-
gans and the lateral expansions of the carapace. The mandibular
nerves (#.7.) arise from the bases of the neural nerves upon the
neural side, and the ento-coxal nerves, three in number (a.e.z.,
p.e.n., and m.e.n.), upon the haemal side of the same nerves.

AS pair or nerves (Text-fig. 13; Pl. X, Figs. 11 and) 125 2772);
which resemble in their distribution the intestinal nerves, arise
from the haemal sides of the bases of the haemal nerves (4.7.7)
of the second thoracic neuromere. In the third, fourth, and
fifth neuromeres the intestinal branches are absent; in the
sixth they are again present (z.7.°), but at some distance from the
bases of the haemal nerves (/.7.°).

In the sixth neuromere a cardiac branch (Text-figs. 3 and 9;
Pls. VI-IX, Figs. 1-3 and 5, s.c.z.°) arises from the haemal
nerve still farther out than the origin of the intestinal nerve.
A small nerve (Pls. VI and VII, Figs. 1 and 2, x.), which could
not be traced out, was found arising from the haemal nerve
(4.2.5) of the fifth neuromere, and apparently corresponding to
the cardiac nerves of other segments.

(4) Zhe Accessory Lrain,— This contains two neuromeres,
the chilarial and opercular neuromeres, which are fused with
each other and form the posterior side of the circum-oeso-
phageal collar. Each contains the usual number of elements
of the typical neuromere.

I 36 PATTEN AND REDENBAUGH. [VoL. XVI.

The chilarial and opercular nerves (Pls. VI-VIII and X, Figs.
I-3, I1, and 12, 2.7.7 and z.z.*) arise from the neural side of
this portion of the brain and pass backward through the occipital
ring to their respective appendages. Mandibular nerves are
absent, and the ento-coxal nerves are so modified as not to be
recognizable as such.

The haemal nerves (4.2.7 and .z.°) arise from the haemal
side of the brain and pass backward through the occipital ring
and outward to the sides of the carapace. Intestinal branches
(2.2.7 and 2.7.8) arise at some distance from the bases of the
haemal nerves, and cardiac branches (s.c.z.7 and s.c.z.5) arise
still farther out.

It is worthy of notice that the cardiac branches of these two
neuromeres fuse with each other, give a recurrent branch to
the lateral sympathetic, and send a pericardial branch, poste-
riorly, in the pericardium ; the latter branch also gives recurrent
branches to the cardiac nerves of the five branchial neuro-
meres. The fusion of sympathetic nerves in this region is of
the utmost importance, as it supports in a most satisfactory
manner the suggestion of Dr. Patten that we have here, in
both scorpions and Limulus, the beginnings of a vagus region.

The neural and haemal nerves of the accessory brain, together
with the ventral cord, pass through the occipital ring.

b. Zhe Ventral Cord.

This portion of the central nervous system consists of five
paired branchial ganglia and three paired post-branchial gan-
glia (Text-figs. 6 and 18, a.g.%°), united by two longitudinal
connectives. The double nature of the ventral cord is not
apparent unless the ensheathing artery is removed.

The first five branchial ganglia (a.g¢.7’3) are separate, but the
three post-branchial ganglia (a.g.#”°) are intimately united with
one another. The first three abdominal ganglia lie just in
front of the corresponding abdominal endochondrites (Text-fig.
6, a.e.9; Pls. VI and VIII, Figs. 1, 3, and 4, a.e.%"") ; the fourth
and fifth (a.g.’?4) lie in front of the next endochondrite (a.e.”) ;
the three fused terminal ganglia (a.g.’+”) lie considerably in

Noe1.] STUDIES ON LIMULUS. 137

front of the last endochondrite (a.e.7), which belongs to the fifth
branchial metamere.

The five branchial neuromeres are typical neuromeres, each
with its double ganglion and cross-commissure, and neural and
haemal pairs of nerves (Text-fig. 8). The neural nerves (Text-
Has. 6,5, and» 12; Pls. VI, VILL, and ix iitiess i032, 45 and .6,
n.n.?"3) arise from the posterior end of the ganglion and give off
intestinal branches (2.7.93) close to their origins. The cardiac
branches (s.c.z.7“3) communicate with the lateral sympathetic
(Z.s.2.) and with the pericardial nerve (f.z.) by recurrent
branches.

The post-branchial neuromeres are not well defined. The
ganglia are closely fused together, and the neural nerves are
absent.

The haemal nerves (4.7.74) of all three neuromeres give
off intestinal branches (7.z.’*7°), and post-cardiac branches
(s.c.2.7#75), which have a distribution similar to the cardiac
branches, are given off from the first two post-branchial nerves.
However, as the heart does not extend into the post-branchial
metameres, the post-cardiac nerves do not communicate with
this organ.

2. PERIPHERAL NERVOUS SYSTEM.

a. Werves from the Fore-Brain.

Six nerves have been found arising from the fore-brain, three
olfactory, one median eye, and two lateral eye nerves. These
are all accompanied by blood vessels, but there are other blood
vessels given off from the same region, which do not accompany
nerves and which might easily be mistaken for nerves in a
hasty dissection. This fact probably accounts for the varied
number of nerves found by different investigators.

(1) Olfactory Nerves. — Owen, Milne-Edwards, and Packard
have described two integumentary nerves arising from the
anterior side of the fore-brain. Patten described three nerves,
and showed that they had a most remarkable development and
that they supplied an aggregation of sense buds on the
under surface of the carapace in the median line, anterior to

I 38 PATTEN AND REDENBAUGH. [VoL. XVI.

the chelicerae. He regarded these buds as probably olfactory
in function and called the nerves supplying them the olfactory
nerves.

The median olfactory nerve (Pls. VI-VIII, Figs. 1-3,
m.ol.n., ol.n.) arises from the anterior extremity of the fore-brain
in the median line, by two roots, one from each of the cerebral
lobes, and passes directly forward beneath the skin to the olfac-
tory organs (o/.0r.). The proximal end is composed of a mixture
of nerve fibers and small ganglion cells which arise, according
to Patten, as early outgrowths of the cerebral hemispheres.
«‘The distal end divides into many diverging branches, which
can be followed by means of a hand lens to the posterior edge
of the olfactory organ; they there begin to anastomose, and
form a dense plexus underlying the olfactory region, but a
considerable number of fibers extend beyond the olfactory
region to the neighboring ectoderm. The lateral olfactory
nerves (Pls. VI-VIII, Figs. 1-3, olm., rv.ol.n., l.ol.n.) arise
apparently from the anterior part of the brain, but in sections
one can follow their roots to the ventral surface into the middle
lobe of the optic ganglia. In the adult the proximal ends
of the nerves consist of coarse transparent nerve tubes
and masses of very large ganglion cells. Their distal extremi-
ties also contain many clusters of large ganglion cells. The
nerve terminates abruptly just beneath the cuticle on the
lateral edge of the olfactory organ. The lateral olfactory nerve
is accompanied by a large blood vessel that divides into numer-
ous branches, supplying the tissues in front of the olfactory
organ; small nerve filaments accompany some of these blood
vessels, and probably supply the ectoderm in the same region.
Some larger nerve branches leave the median border of the
lateral nerves a little distance back of the olfactory organ, and
mingle with the plexus formed by the median nerve”’ (Patten).

(2) Median Eye Nerves.— Owen described two median eye
nerves, but other authorities have found but one. The median
eye nerve (Pls. VI-IX, Figs. 1-3 and 5, m.ey.x.) apparently
arises from the anterior border of the fore-brain, but examina-
tion of sections shows that it arises by four roots from the
semicircular lobes upon the haemal side of the fore-brain. It

No. 1.] STUDIES ON LIMULUS. 139

passes forward neural to the oesophagus and a little to the right
of the median line; turns haemally to the right of the proven-
triculus and passes to the median eye upon the haemal surface
of the carapace in the median line.

According to Dr. Patten the median eye consists of two
ectoparietal and one endoparietal eye, and the endoparietal eye
is formed by the fusion of a pair of retinas. He finds that ‘the
distal end (of the median eye nerve) splits up into four branches,
two of which plunge directly into the median diverticulum or
endoparietal eye, and the other two pass to the paired retinas
of the ectoparietal eyes.”

(3) Lateral Eye Nerves. — The lateral eye nerves (Pls. VI
and VIII, Figs. 1-3, 7.x.) arise from the large optic ganglia
upon the haemal side of the cerebral lobes, pass forward
median to the tergo-coxal muscles of the chelicerae, turn out-
ward around the anterior outer corners of the entocoxites of
the second pair of appendages, and then pass backward to the
lateral eyes. The distal extremity breaks up into two small
branches and a large one. The large one passes directly to the
retina of the lateral eye; the two smaller ones pass farther
backward to a pigmented body beneath the retina.

A large blood vessel encloses the lateral eye nerve for the
greater part of its course, and is continued beyond the lateral
eye where it anastomoses with the hepatic artery.

DESCRIPTION OF A TYPICAL NEUROMERE.

The nerves of all the neuromeres posterior to the fore-brain
conform more or less closely in their distribution to a common
plan, which it will be well to keep in mind as we take up the
successive neuromeres.

(1) Abdominal Neuromere. — The most primitive, and hence
the most typical neuromeres are those in the abdominal
region, vzz., those from the ninth to the thirteenth. In these
neuromeres (Text-fig. 8) we have a pair of ganglia (a.g.)
united by cross-commissures, and also partially fused in the
median line. Posteriorly, a pair of neural nerves arise, and,
anteriorly, a pair of haemal ones. The neural nerves (x.x.)

I40 PATTEN AND REDENBAUGH. [VoL. XVI.

supply the appendages, and the haemal nerves (4..) supply
the body portion of the metamere.

Close to the ganglion each haemal nerve gives off two small
nerves; one of these joins a plexus supplying the longitudinal
abdominal muscles, the other divides into two portions, the first
going to a haemo-neural muscle, and the second to the intestine.

Fic. 8. — Diagram of typical abdominal neuromere.

a.g., abdominal ganglion; ¢.4.7., external branchial nerve; g.., gill nerve; %.7., haemal
nerve; 2.4.2., internal branchial nerve; z.z., intestinal nerve; zz.z., integumentary nerve;
Z.s.m., lateral sympathetic nerve; #z.c.7., median cardiac nerve; z.z., neural nerve; /.z.,
pericardial nerve; s.c.z., segmental cardiac nerve; /.%.m., nerve to haemo-neural muscle ;
int., nerve to intestine; Z.a.7., nerve to longitudinal abdominal muscles; 4/., nerve to plexus
in tissues surrounding the intestine,

The intestinal portion generally communicates, outside of the
intestine, with a plexus which unites the corresponding nerves
of successive neuromeres.

At some distance from the ganglion the haemal nerve sends
haemally a cardiac nerve (s.c.z.). This communicates by a
recurrent branch with the lateral sympathetic (/.s.2.) which
supplies the branchio-thoracic muscles. Another recurrent
branch of the cardiac nerve joins the pericardial nerve (7.z.),

No. I.] STUDIES ON LIMULUS. 141

which runs longitudinally in the areolar tissue alongside the
heart. The distal end of the cardiac nerve communicates
with the median nerve (m.c.z.) of the heart.

The main or integumentary branch (27.z.) of the haemal nerve
passes outward into the lateral expansions of the carapace.

(2) The Cephalic or Cranial Neuromere.— In the cephalotho-
rax the typical neuromere (Fig. 9) is somewhat modified. The

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Fic. 9. — Diagram of a typical cranial neuromere.
ér.g., ganglion forming part of the brain; c.c., cross-commissures; ¢.7., ento-coxal nerves ;

e.p.n., external pedal nerve; 4., haemal branch of integumentary nerve ; 4.7., haemal nerve ;
i.m., intestinal nerve ; z#.7., integumentary nerve; z.f.7., internal pedal nerve ; .7., man-
dibular nerves; .c.z., median cardiac nerve; %., neural branch of integumentary nerve;
2.m., neural nerve; #.7., pericardial nerve; s.c.z., segmental cardiac nerve; zzt., nerve to
intestine ; /.¢.7., nerve to longitudinal abdominal muscles.

two ganglia are more or less separated, and the cross-commis-

sures are in some cases very long.

The ganglia of the successive neuromeres are crowded
together, so that the neural and haemal nerves appear to arise
from the neural and haemal sides, respectively, of the ganglia,
instead of from the posterior and anterior sides.

The neural nerve (z..) arises from an enormous ganglion,
which in the adult is much obscured by the thick membranes

142 PATTEN AND REDENBAUGH. [VoL. XVI.

that surround it. It divides near the ganglion into the ento-
coxal (e.z.), mandibular (#.z.), and pedal branches. The ento-
coxal branches, three in number, supply the muscles inserted
upon the entocoxite, and numerous sense organs in the ento-
coxite itself; the mandibular branch supplies the mandibles ;
and the pedal branches the remainder of the appendage. In
the coxopodite the main pedal branch divides into an external
(e.p.2.) and an internal portion (2.f.z.).

The thoracic haemal nerve, like the abdominal one, has an
intestinal, a cardiac, and an integumentary branch. The intes-
tinal branch (z.z.) arises at some distance from the brain;
the cardiac branch (s.c.z.) does not communicate with the
lateral sympathetic, and its communication with the heart is
doubtful; the integumentary nerve divides into a haemal and
a neural portion (4. and z.).

b. Merves from the Mid-Brain (Text-fig. 10).

The mid-brain region contains in addition to the nerves and
neuromere of the cheliceral segment a pair of stomodaeal nerves
and ganglia and three rostral nerves, arising from the pre-stomo-
daeal commissure.

(1) Zhe Neural Nerves.—The neural nerves (Text-fig. 10;
Pls, VI-VIII and X, Figs. 1-3, 11, and 12, 2.m.’) arise from
ganglionic swellings situated just back of the cerebral lobes,
and pass directly to the inside of the chelicerae.

At the base of the neural nerve arises one or more external
pedal nerves (e.p.z.). They innervate the tergo-coxal muscles
(e.7, £7, /.m.*), and then pass into the chelicera outside of the
main nerve (2.f.7.), giving branches to the extensor muscle (e.?)
of the second joint, and ending in the epidermis upon the outer
side of the joint. The branches supplying the tergo-coxal
muscles represent the ento-coxal branches of a typical neuromere.

The main cheliceral nerve (2.f.7.) innervates the epidermis in
the first, and the flexor muscle (7) of the second joint. Here
it gives branches to the skin and to the muscles (e3 and 3)
which move the third joint, and then divides into two large
sensory branches which break up in the chelae to supply the

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No. 1.]

gustatory organs.

STUDIES ON LIMULUS.

143

A large blood vessel accompanies the

cheliceral nerve and supplies the appendage with blood.
(2) Zhe Haemal Nerves.— The non-ganglionated haemal
nerves (/.z., 4.n.7) arise from the haemal side of the neuro-

mere. Each nerve passes for-
ward, dorsal to the lateral eye
nerve, upon the median side of
the tergo-coxal muscles of the
chelicerae, and there fuses with
the haemal nerve (%.z.) of the
second thoracic neuromere. It
soon leaves it, however, and,
passing haemal to this nerve,
turns posteriorly around the base
of the second appendage, keeping
close to the median eye nerve.
The rest of its course lies in the
epidermis upon the neural sur-
face of the carapace, outside the
bases of the appendages. It does
not branch much, if any, until it
reaches the skin, just posterior
to the sixth leg, beneath a sclerite
which lies opposite the flabellum.
Here it gives off a number of
branches which ramify over the
skin. The main nerve is con-
tinued onto the abdomen, where
it branches at regular intervals,
sending a small fiber toward the
bases of the first five abdominal
appendages. Opposite the last
appendage it breaks up into nu-
merous filaments, ramifying over
that region.

Fic. 10, — Diagram showing muscles and dis-

tribution of nerves in chelicera of adult
Limulus, from the anterior side (natural
size). 7,2, and 3, first, second, and third
joints of chelicera; ¢.1-3, extensors of first,
second, and third joints, respectively ; £3,
flexors of first, second, and third joints, re-
spectively ; 72.7.1, lateral muscles of first
joint ; ¢.1, 7.1, and 2.7.1 constitute the tergo-
coxal muscles; 4.c., haemal side of the
carapace; $r., fore-brain; 2.7.1, neural
nerve or cheliceral nerve; 7.4.7., internal
pedal nerve ; ¢.f.7., external pedal nerve ;
hk.n.t, haemal nerve of cheliceral neuro-
mere or lateral nerve.

a large portion of the skin in

It is a noteworthy fact that this nerve branches profusely in
the skin beneath a sclerite similar to that in the olfactory region,
and also that this sclerite and the flabellum, which is highly

144 PATTEN AND KEDENBAUGH. [VoL. XVI.

sensory, lie opposite each other in a channel through which a
continuous current of water passes when the gills are being
aérated.

This nerve differs so much in character from the other haemal
nerves that it might be considered as an entirely different nerve.
Its origin seems to correspond with the origins of the haemal
nerve of the typical cranial neuromere, and it passes anterior
to the tergo-coxal muscles of the chelicera, just as the haemal
nerves of the other neuromeres pass anterior to the tergo-coxal
muscles of their own metamere. Its fusion with the second
haemal nerve, and its intrusions into all the succeeding meta-
meres without communicating with the haemal nerves, are
peculiarities which differentiate it from the other haemal
nerves.

It was first described by Owen as a branch of the second
haemal nerve, but he did not trace it into the abdominal region.
Milne-Edwards overlooked it entirely. Viallanes, in his figure
of the brain, figures the root of it and designates it as the
recurrent nerve. Patten did not describe its distribution, but
in his figure of the brain represented its proximal end as the
third pair of haemal nerves.

(3) Zhe Stomodaeal Nerves. — The stomodaeal nerves (Pls.
VIII and X, Figs. 3, 11, and 12, s¢.z.) were described by
Milne-Edwards and by Owen, and called the stomato-gastric
nerves. They arise from ganglionic swellings of the nerve
collar and extend along each side of the oesophagus, to
the proventriculus. Numerous branches are given off to the
oesophagus, and sometimes small branches are found arising
from the ganglionated bases of the nerves. At the sides of the
proventriculus the nerve breaks up into several branches which
ramify over the proventriculus, and on to the pyloric valve and
the beginning of the intestine. No branches could be traced
beyond the pyloric valve.

Milne-Edwards described a ganglion upon each side of the
proventriculus, and two very fine branches of the stomato-gastric
nerves communicating with the median nerve of the heart, but
I have been unable to find either the ganglia or the cardiac
branches.

No. 1.] STUDIES ON LIMULUS. 145

A blood vessel accompanies the stomodaeal nerve and com-
municates with the aortic arch at the side of the proventriculus,
and small vessels from the aortic arch accompany the branches
of the stomodaeal nerve over the proventriculus.

(4) Rostral Nerves. — Milne-Edwards described two rostral or
labral nerves arising from the cerebral lobes. Patten found
three, a median and two lateral nerves (Pls. VII, VIII, and X,
Figs. 2, 3, 11, and 12, /.a.z.), and correctly described them as
arising, not from the cerebral lobes, but from the pre-oral com-
missure. They innervate the rostrum or upper lip.

c. Nerves from the Hind-Brain (Text-fig. 11).

The second, third, fourth, fifth, and sixth thoracic neuromeres,
which make up the hind-brain, are so much alike that a descrip-
tion of one will, with a few modifications, suffice for all.
The third neuromere (Text-fig. 11) is most characteristic and
contains the usual elements, a pair of ganglia united by cross-
commissures, a pair of neural and a pair of haemal nerves.

(1) Meural Nerves.— The neural nerves of the hind-brain
arise from ganglionated bases and radiate from the nerve collar
to the five pairs of appendages. Owing to the increasing dis-
tance from the brain to the base of the appendage, the basal
portions of the more posterior nerves are elongated, and the
entocoxal and mandibular nerves, which in the anterior neuro-
meres arise close to the brain, in the sixth neuromere arise
at a considerable distance from it (Pl. X, Figs. 11 and 12).

The typical neural nerve (Text-figs. 11, 12, and 8; Pl. X, Figs.
11 and 12, .2.7°) divides, soon after leaving the brain, into
three portions, a mandibular portion (#.7.) supplying the gusta-
tory organs of the mandibles, a pedal portion (¢.4.~. and e.p..)
supplying the main portion of the appendage, and three ento-
coxal branches (e.7.) supplying the tergo-coxal muscles and the
sensory knobs of the coxopodite.

(a) Mandibular Branches.— These nerves were first described
by Patten (93). In the third neuromere there are three man-
dibular branches (Text-fig. 11, #..) which arise close together
from the neural side of the nerve, not far from the brain. If

146 PATTEN AND REDENBAUGH. [VoL. XVI.

the ensheathing artery be removed, the three nerves may be
seen arising from a common trunk, which may be traced to the
margin of the nerve collar. Each of the three mandibular
branches bears at its proximal end a small ganglionic swelling.
The first branch gives off a small nerve to the inner mandible

Fic. 11. — Diagram showing muscles and distribution of nerves in the third leg of Limulus, from the
anterior side (34 natural size).

I-cox., coxopodite, or first joint; 2-4as., basipodite, or second joint; 3-zsc., ischiopodite, or

third joint; ¢ { eh, fused carpopodite and meropodite, or fourth joint ; 5-4v0., propodite, or
Sf

fifth joint ; 6-dac., dactylopodite, or sixth joint; 2/o., apodeme ; /.c., haemal side of carapace ;
endo., endocranium ; /z., heart; z¢., intestine ; z.7zaz., internal mandible ; 7., mouth; szaz.,
mandible ; Z., pericardium ; /.s., pericardial sinus.

Muscies: 32 and b, plastro-coxal muscles inserted upon anterior side of entocoxite ;
3¢and 4, tergo-coxal muscles inserted upon anterior side of entocoxite; ¢.?, extensors of
second to sixth joints; 42-6, flexors of second to sixth joints ; 7m, flexor of inner mandible.

NERVES: a@.e.z., anterior ento-coxal nerve; 4y., brain; e.f.7., external pedal nerve; %.,
haemal branch of integumentary nerve; 4.7.3, haemal nerve; 7.2.3, integumentary branch ;
z.p.n., internal pedal nerve; 77.e.2., median ento-coxal nerve ; 7z.7., mandibular nerves; z.,
neural branch of integumentary nerve ; 7.7.3, neural nerve ; f.e.7., posterior ento-coxal nerve.

No. 1.] STUDIES ON LIMULUS. 147

(z.man.) and its flexor muscle (f.”), and then breaks up into fine
branches, which ramify over the posterior surface of the man-
dible (#zan.) and innervate some of the gustatory spines. The
second branch supplies the anterior surface of the mandible and
the more anterior of the gustatory spines. The third branch
innervates the outer portion of the mandible and sends some
fine branches to the skin of the inner proximal portion of the
basipodite (2—-das.).

In the second appendage the first mandibular branch is much
reduced in size and innervates only the inner portion of the
mandible. There is no inner mandible and, consequently, no
corresponding nerve. The second mandibular branch is enlarged
and innervates the greater part of the mandible upon both the
anterior and posterior sides. The third branch is about as in
the third appendage.

In the fourth and fifth appendages there are only two man-
dibular branches, each with a ganglion near the base. The
first one, however, divides into three branches, one to the inner
mandible and its flexor muscle, one to the posterior, and one to
the anterior side of the mandible. The second ganglionated
mandibular branch has a distribution similar to that of the third
branch in the third appendage. It is evident that the ultimate
distribution of the mandibular branches of these appendages is
the same as in the third, but the mode of branching at their
bases is a little different.

In the sixth appendage (Text-fig. 12) there are only two man-
dibular branches (m.z.). The first is not ganglionated and is
much reduced. It divides into three branches, which are dis-
tributed to the inner portion of the mandible, where there are
no gustatory spines. The second mandibular branch is gan-
glionated and corresponds to the third branch in the third
appendage. It divides into two portions, one supplying the
outer part of the mandible upon which there are a few spines,
and the other supplying the inner proximal portion of the
basipodite.

(b) Exto-coxal Branches.—In the third appendage (Text-figs.
g and 11; Pl. VII, Fig. 2) there are three ento-coxal branches, an
anterior (a.¢.7.), a posterior (f.e.7.), and a median one (m.¢.u.).

148 PATTEN AND REDENBAUGA. [VoL. XVI.

1m!

Fic. 12. — Diagram showing the muscles and distribution of the nerves in the sixth leg of Limulus,
from the anterior side (% natural size).
I-cox., coxopodite, or first joint ; 2-das., basipodite, or second joint ; 3-zsc., ischiopodite, or
e os car.
third joint; g- POD)
fifth joint ; 6-dac., dactylopodite, or sixth joint ; a@fo., apodeme; 4+., brain; c.c., cross-commis-
sure ; ezdo., endocranium; //ad., flabellum; %.c., haemal side of carapace ; #z., heart; zzz.,
intestine; 7zaz., mandible; /.s., pericardial sinus.

Musc.es: 64 and », plastro-coxal muscles inserted upon anterior side of entocoxite;
6c and 4, tergo-coxal muscles inserted upon anterior side of entocoxite; e.2-7, extensors of
second to seventh joints ; 42-7, flexors of second to seventh joints; z.7z., inter-tergal muscle.

NERVES : a.e.2., anterior ento-coxal nerve ; ¢.f.2., external pedal nerve; #., haemal branch
of integumentary nerve ; 4.7.3, haemal nerve; z.2.6, intestinal nerve; z.7., integumentary
branch of haemal nerve; 7.4.7., internal pedal nerve; Z.c.7., lateral cardiac nerve; 7.c.7.
median cardiac nerve; .e.2., median ento-coxal nerve or flabellar nerve; .2., mandibular
nerve; z., neural branch of integumentary nerve; 2.7.6, neural nerve; %., pericardium ;
p.e.m., posterior ento-coxal nerve ; s.c.7.°, segmental cardiac nerves.

fused carpopodite'and meropodite, or fourth joint ; 5-470., propodite, or
’

No: 7.] STUDIES ON LIMULUS. I49

They arise upon the haemal side of the neural nerve, the two
former very near the brain, the latter or median one a little farther
out. Sometimes there are two additional nerves supplying the
inner plastro-coxal muscles, but these may be regarded as
branches of the main ento-coxal nerves, for in many specimens
these muscles are supplied by nerves which are undoubted
branches of the main ento-coxal nerves.

The anterior ento-coxal nerve (a.¢.z.) arises from the anterior
haemal side of the neural nerve quite close to the brain, gives off
a small branch to the innermost, anterior, plastro-coxal muscle
(37), then passes down through the substance of the nephrid-
ium (Pl. VII, Fig. 2, 2.2) and innervates all the muscles (3*%),
which are inserted upon the anterior border of the entocoxite,
but apparently gives no branches to the nephridium. It termi-
nates in delicate filaments in the areolar tissue at the outer
extremity of the entocoxite, and sends some fibers to the ante-
rior sensory knob.

The posterior ento-coxal nerve (/.¢.z.) arises from the poste-
rior, haemal side of the neural nerve, and innervates all the
muscles which are inserted upon the posterior border of the
entocoxite. It also passes through the substance of the ne-
phridium, but does not give off any nerves to it. It terminates
in areolar tissue at the outer extremity of the entocoxite and
innervates the posterior sensory knob.

The median ento-coxal nerve (7.¢.z.) arises from the haemal
side of the neural nerves some distance farther from the brain
than the other ento-coxal nerves. It is much smaller than these
nerves, and passes out over the surface of the nephridium accom-
panied by a blood vessel. It is entirely sensory in function and ter-
minates in the median sensory knob and the surrounding tissues.

In the second appendage (Pl. VII, Fig. 2) the anterior and
posterior ento-coxal nerves (a.e..? and f.¢.z.2) are similar to
those in the third appendage, except that separate nerves supply-
ing the inner plastro-coxal muscles are often present. The
median nerve has not yet been found, but it probably exists, and
has been overlooked on account of its extreme tenuity. All the
median ento-coxal nerves, except the sixth, are very small and
difficult to find.

150 PATTEN AND REDENBAUGH. [VoL. XVI.

In the fourth and fifth appendages the ento-coxal nerves are
essentially the same as in the third appendage.

In the sixth appendage (Text-fig. 12; Pl. VII, Fig. 2) there is
an interesting variation. The anterior and posterior ento-coxal
nerves (a.e.z. and p.e.z.) are similar to those in the third append-
age, except that they arise at some distance from the brain; but
the median ento-coxal nerve (m.e.z.) is much enlarged and be-
comes the flabellar nerve, which breaks up into many filaments
inside the flabellum (/7ad.) and supplies the numerous sense
buds of this organ. It also gives off a few branches to the
epidermis around the base of the flabellum. A large blood
vessel accompanies it into the flabellum.

(c) Pedal Branches. (See Text-fig. 11). — In the coxopodite
(1-cox.) the main pedal nerve gives small branches to the flexor
(7?) and extensor muscles (e.?) of the basipodite (2-0as.). A
larger branch, the external pedal nerve (e.p.7.), leaves the
outer side of the pedal nerve and runs parallel to it along
the outer side of the leg, through all the joints as far as
the distal end of the propodite (5-pvo.)._ This branch is found
in all the thoracic appendages, not excepting the chelicerae,
and seems to be mainly sensory in function, though it does
supply a few muscles.

The main pedal branch, or internal pedal nerve (z.f..), lies
for the most part toward the inner side of the leg between
the muscles of the anterior and those of the posterior sides. It
gives off both sensory and motor branches all along its course,
and supplies all the muscles not supplied by the external pedal
nerve. In the second, third, fourth, and fifth legs it terminates
in four branches, two to each blade of the chelae.

In the propodite of the sixth leg (Text-fig. 12) it gives off
numerous branches to the spatulate organs. The main branch
continues into the slender outer joint (6—dac.), and, after sup-
plying the extensor and flexor muscles (e¢.7 and f.7) of the termi-
nal chelate portions, divides into two branches which distribute
themselves in the two terminal joints.

(2) Haemal Nerves. — The typical haemal nerve (Text-fig. 8)
consists of three branches, an intestinal (z.7.), a cardiac (s.c.7.),
and an integumentary branch (¢z.7.). In most of the thoracic

No. 1.] STUDIES ON LIMULUS. en

neuromeres the intestinal and cardiac branches are absent.
The sixth neuromere, however, contains the typical number of
branches.

In all cases the haemal nerve (Text-figs. 11 and 12; Pls. VI-
VIII, and X, Figs. 1-3, 11, and 12, 4.7.) arises from the haemal
side of the brain slightly anterior to the neural nerve of the
same neuromere. It is about half as large as the neural nerve,
does not have a ganglionated base, and is not accompanied by
an artery except for a short distance from its origin. It is pe-
culiar in having at a greater or less distance from its origin a
ganglion-like swelling. This swelling, which has been described
by Milne-Edwards, contains, however, no ganglion cells, but the
fibers at these points undergo a complicated interlacing, and
numerous nuclei are present.

(a) Lntestinal Branches. — The intestinal branch is absent in
the third, fourth, and fifth neuromeres. In the sixth (Text-fig.
12); Pls. VI-VILI ‘and: X, Figs. 1-4; ni wand123 a7 yait
arises from the haemal side of the haemal nerve and passes
between the plastro-coxal muscles of the fifth and sixth legs,
through a foramen (PI. VIII, Fig. 4, 7°) in the endochondrite,
into the longitudinal abdominal muscles attached to the endo-
chondrite. Here it communicates witha plexus which supplies
these muscles. Some of the branches, however, pass on to the
intestine. In the third, fourth, and fifth metameres the intestine
is supplied by nerves which pass forward from the plexus in the
longitudinal abdominal muscles. Some of the nerves have also
been traced from this plexus to the tergo-plastral and veno-peri-
cardiac muscles in this region.

In the second metamere a nerve (Text-fig. 13; Pls. VIII and X,
Figs. 3, 11, and 12, z.7.”) is given off from the haemal nerve
(4.2.2) close to its base. It passes haemally upon the median
side of the anterior cornu (a.cor.), and supplies the tergo-proplas-
tral muscles (4.7.74), No branch has been observed going
to the intestine, but as it supplies muscles similar to those sup-
plied by the other intestinal branches, and also has an origin
similar to that of the other intestinal branches, it has been
included in the same category.

(b) Cardiac Branch.— The cardiac branch (Text-figs. 12

152 PATTEN AND REDENBAUGH. [VoL. XVI.

and g; Pls. VI-X, Figs. 1-3, 5, 11, and 12, s.c.z.°) is present
in the sixth neuromere only. It arises from the haemal
side of the haemal nerve (4%.2.°), about midway between
the brain and the outer edge of the entocoxite. Where it
separates from the haemal nerve a small recurrent branch is
sometimes seen passing backward from the cardiac nerve to
the integumentary portion (zz.7.) of the main nerve. It passes

Fic. 13. — Diagram showing the distribution of the intestinal nerve of the second neuromere. The
left anterior cornu of the endocranium with attached muscles, one of the aortic arches, and
the anterior portion of the brain are represented as seen from the median side. The fore-brain
and endocranium are cut in two in the middle line.

@.coy., anterior cornu of the endocranium ; ao.a., aortic arch; ezdo., endocranium ; %.c.,
haemal side of carapace.

Musctes: 22 and b, plastro-coxal muscles inserted upon the anterior border of the ento-
coxite of the second appendage ; #.4.7.4-c, tergo-proplastral muscles.

NERVES: @.c., anterior commissure; 4~., fore-brain; 4.2.2, haemal nerve of second neuro-
mere; 2.7.2, intestinal nerve of second neuromere; Z.e.7., lateral eye nerve; 7.0/.”., median
olfactory nerve; 7.2.1, cheliceral nerve; o.c., portion of circum-oesophageal collar.

haemally, outside of the branchio-thoracic muscles (4.¢.m.) and,
bending toward the anterior side, passes through the pericardium
to the inter-tergal muscle. Within this muscle its branches
anastomose with the cardiac branches of the next posterior
neuromere.

Some of the branches supply the epidermis in the haemal
median line over the first pair of ostia (os.°) of the heart, and,
although no connection has yet been found between these

No. 1.] STUDIES ON LIMULUS. 153

branches and the median nerve (w.e.7.) of the heart, it is very
probable that such a connection exists. The connective-tissue
strands supporting the heart in this region are numerous,
rendering it very difficult to distinguish nerve fibers and trace
them through the mass of other fibers.

In the second, third, and fourth thoracic neuromeres no car-
diac branches have been found; in the fifth a small nerve was
found which corresponded in origin to the cardiac branches
of the other haemal nerves, but its distribution could not be
traced out.

(c) Lnutegumentary Branches.— The haemal nerve becomes
enlarged and flattened near the outer margin of the entocoxite
and forms the integumentary nerve (Text-figs. 9, 11, and 12; Pls.
Vi and VII, Figs. 1 and 2, zz.z.). It then divides into two
main branches, one (z.) going to the neural surface of the cara-
pace, and the other (/.) to the haemal surface.

The neural branch soon divides into two more branches, and
these break up into numerous fibers, which ramify over the
neural surface of the carapace and supply the skin, and prob-
ably the numerous muscle strands passing between the two
surfaces of the lateral expansions of the carapace.

The haemal branch gives off near its origin a small nerve,
which turns haemally toward the median line, and supplies
the epidermis of the haemal side between the pericardium and
the outer edges of the entocoxites. In some cases these
branches anastomose in the epidermis with the correspond-
ing branches of the other haemal nerves. The main haemal
branch breaks up into small branches which innervate the skin
upon the haemal side of the lateral expansions of the carapace.

The second haemal nerve (4.7.?) is somewhat larger than the
others, and its integumentary branches have a larger area of
distribution, for they supply the thicker anterior portion of the
cephalic shield. It lies almost parallel to the median line, while
the sixth one (4.7.°) lies at right angles to it. The interme-
diate haemal nerves (4.7.3), owing to the rounded form of the
cephalothorax and the central position of the brain, necessarily
diverge from each other like the radii of a circle.

Another noticeable feature about the haemal nerves is a

154 PATTEN AND REDENBAUGH. [VoL. XVI.

bend near their proximal ends; the proximal end of the
second haemal nerve is straight ; the third nerve has a slight
flexure; the fourth has a greater one ; and the fifth and sixth
have very marked flexures.

The median eye nerve (m.e.z.) passes between the haemal
and the neural integumentary branches of the second, third,
fourth, and fifth haemal nerves. The first haemal nerve or
lateral nerve (/.z.) passes haemal to the second haemal nerve
and neural to all others.

d. Nerves from the Accessory Brain (Text-figs. 14 and 15).

The accessory brain consists of two neuromeres fused
together, the seventh, or chilarial neuromere and the eighth, or
opercular neuromere. Both neuromeres have all the typical
elements, but they resemble the abdominal rather than the
cranial type. The haemal and neural nerves all arise from the
posterior side of the brain, and together with the ventral cord
pass through the occipital ring.

(1) Meural Nerves. —The neural nerves of the two neuro-
meres differ so much from each other that it will be necessary
to describe them separately.

(a) Chilarial Nerve.— The paired chilarial nerve (Text-fig.
14; Pls. VI-VIII, and X, Figs. 1-3, 11, and 12, 7.7.7) arises
from the posterior side of the brain near the median line and
neural to the origin of the ventral cord (v.c.). It passes poste-
riorly near the median line close beneath the roof of the occipital
ring (0c.r.) into the chilarium. As it enters the base of the
appendage, it gives off branches to all the muscles of the
chilarium. The main nerve breaks up inside the appendage,
supplies the epidermis, and sends a large fiber into each of the
gustatory spines which fringe its inner margin.

(b) Opercular Nerve. — The opercular nerve (Text-figs. 14
and 15; Pls. VI-VIII, and X, Figs. 1-3, 11, and 12, .7.°) arises
just posterior to the chilarial nerve and passes backwards through
the occipital ring near the median line neural to the ventral cord.
At the base of the operculum (a/.) it gives off a small branch
to the internal branchial muscle (Text-fig. 12, 2.6.1.) and then

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No. 1.] STUDIES ON LIMULUS. 155

divides into three main branches, the external, median, and
internal, opercular nerves (e.0.7., m.0.2., and 7.0.n.).

The first of these (2.0.z.) again divides into a motor
and a sensory branch; the motor branch innervates the large
abductor muscles (ad.7.5) upon the anterior face of the append-

Fic. 14. — Diagram showing the muscles and nerves of the chilaria of Limulus, from anterior side.
The appendages are revolved outward about 45° (magnified nearly 1% diameters).

6.c.7, capsuliginous bar or branchial cartilage ; exdo., endocranium; /.c., haemal side of
carapace; A¢., heart ; z¢., intestine ; oc.7., occipital ring; .s., pericardial sinus.

Musciss : 7 4-e, plastro-coxal muscles; 7 f and g, tergo-coxal muscles ; 7.#., inter-tergal
muscles; v.f.7.7, veno-pericardiac muscles.

NervEs : 4.7.7 and 8, haemal nerves of chilarial and opercular neuromeres ; 2.7.7, intestinal
nerve ; 2.2.7 and 8, integumentary branches of haemal nerves of chilarial and opercular seg-
ments ; é.c.., lateral cardiac nerve ; Z.s.7., lateral sympathetic nerve ; 7.c.2., median cardiac
nerve ; 7.7.7 and 8, neural nerves of chilarial and opercular neuromeres; ¢., pericardium ;
s.c.z.7 and 8, fused segmented cardiac nerves of chilarial and opercular neuromeres.

age, and the sensory branch supplies the epidermis of the

anterior face and outer margin of the base of the appendage.
The second branch (m.0.z.) also divides into a motor anda

sensory branch; the motor branch supplies the external bran-

I 50 PATTEN AND REDENBAUGH. [VoL. XVI.

chial or abductor muscle (e.4.7.*) upon the posterior face of the
appendage; the sensory branch innervates the epidermis of the
middle portion of the appendage.

The third branch (2.0.z.) is mainly sensory and innervates the
distal portions of the appendage. It also contains some motor
elements which supply the muscle strands (0./.m. and 2./.m.),
moving the distal portions (0./. and z./.) of the appendage.

(2) Haemal Nerves. — The haemal nerves (Text-figs. 14 and
15; Pls. VI-VIII and X, Figs. 1-3, 11, and 12, 4.2.7 and 4.2.)
arise from haemal side of the brain and pass back through the
occipital ring and outward to the sides of the body, between
the sixth pair of legs and the operculum. The one belonging
to the chilarial neuromere arises outside of the opercular one
and turns outward anterior to the capsuliginous bar, while the
other one turns outward posterior to the bar. They are typical
haemal nerves and have the usual intestinal, cardiac, and integu-
mentary branches.

(a) Intestinal Branches. — The intestinal branch (2.7.7) of the
chilarial haemal nerve is given off at the bend in the nerve
between the capsuliginous bar (0.c.”) and the base of the occip-
ital ring, and passes through a foramen (f7) in the endo-
cranium to the longitudinal abdominal (/.a.#.) muscles and, like
the intestinal nerve of the sixth neuromere, communicates with
a plexus supplying these muscles and sends a branch to the
intestine.

The intestinal branch (z.z.°) of the opercular haemal nerve is
also given off at the bend of the nerve posterior to the capsu-
liginous bar (0.c.7). It does not pass through the endocranium
but plunges directly into the abdominal muscles. Its distribu-
tion is similar to that of the preceding. It is a notable fact
that in many cases these intestinal branches have been seen to
arise from the haemal nerve by two roots.

(b) Cardiac Branches. —The cardiac branches (s.c.7.7 745)
of these two neuromeres arise from the haemal nerves at some
distance beyond the origins of the intestinal nerves, and fuse
together into one large nerve. The opercular root gives off a
branch to the lateral sympathetic (/.s.z.) which innervates the
branchio-thoracic muscles (0.¢..). The fused cardiac nerve

No. 1] STUDIES ON LIMULUS. 157
ul.

Fic. 15.— Diagram showing the muscles and distribution of the nerves in the operculum. The
operculum is flexed upon the abdomen, and is seen from the neural side (about 1% natural size).

a.e., abdominal endochondrite of opercular segment ; 4.c.7, capsuliginous bar or branchial
cartilage of chilarium ; 4.c.®, branchial cartilage of operculum ; ezdo., endocranium ; 2.é., inner
lobe of operculum ; oc.v., occipital ring ; 9./., outer lobe of operculum ; 0.v., oviduct.

Musctes : a.d.1m.8, abductor muscle of operculum; 4.¢.%., branchio-thoracic muscles;
e.6.m.8, external branchial muscle ; 2.d.7., internal branchial muscle ; z./.7z., muscle of inner
lobe ; 0.2.7z., muscle of outer lobe.

NERVES : ¢.0.., external branch of opercular nerve; 4.7.7 and 8, haemal nerves of chilarial
and opercular segments ; 7.7.7 and 8, intestinal nerves of chilarial and opercular neuromeres ;
in.n.7 and 8, integumentary branches of haemal nerves of chilarial and opercular neuromeres ;
Z.0.#., internal branch of opercular nerve; Z.s.7., lateral sympathetic nerve; .0.7., median
branch of opercular nerve ; 7.7.8, neural or opercular nerve; s.c.%.7 and 8, fused segmental
cardiac nerves of the seventh and eighth neuromeres; v.c., ventral cord.

I 55 PATTEN AND REDENBAUGH. [VoL. XVI.

passes haemally outside of the branchio-thoracic muscles, turns
toward the median line anterior to the base of the large enta-
pophysis (eta.7**4%), and enters the large inter-tergal muscles
haemal to the heart. Here it breaks up into anastomosing
branches, running forward inside the muscle, and into small
fibers which pass toward the median line in the epidermis over
the second and third pairs of ostia (0s.7*°45) of the heart. A
large branch, the pericardial nerve (/.z.), passes posteriorly in
the epidermis haemal to the heart and gives recurrent branches
to the cardiac nerves of the gill region.

(c) Lutegumentary Branches. —The integumentary portion
of the chilarial nerve (Pl. VI, Fig. 1) supplies a large area of the
epidermis on the posterior portion of the cephalothorax, includ-
_ing the posterior angles. A few branches are also distributed
to the anterior border of the abdomen. As the lateral expan-
sions of the carapace are thin in this region, haemal and neural
branches cannot easily be distinguished, but near the median
line we find the usual small haemal branch running in the
epidermis haemally and toward the median line.

The integumentary branch of the opercular nerve has a limited
area of distribution in the anterior portion of the abdomen. It
confines itself to the opercular segment, which is conspicuously
marked off by the auricular-shaped processes of the abdominal
carapace just posterior to the hinge. A small nerve is given
off to the epidermis outside the base of the operculum.

e. Lerves from the Five Branchial Neuromeres (Text-figs.
16 and 17).

The nerves from the five branchial neuromeres are very
similar in their distribution. These neuromeres are the most
typical and primitive, and all the others may be considered as
derived from neuromeres similar to them. Each branchial
neuromere (Text-fig. 8) contains a pair of fused ganglia (a.g.)
united by cross-commissures, and a pair of haemal and
neural nerves. The neural nerves (z.z.) arise from the pos-
terior side of the ganglion and innervate the appendage, and
the haemal nerves (/.z.) arise from the anterior side and inner-

No: 1.] STUDIES ON LIMULUS. 159

ul.

Ne a4
i i ep

Fic. 16. — Diagram showing the muscles and distribution of nerves in the first gill. The appendage
is flexed upon the abdomen, and is seen from the neural side (about 1% natural size).

a.e.9, abdominal endochondrite ; 4.c.8 and 9, branchial cartilages of operculum and first gill;
z.2., inner lobe of gill; #z.2., median lobe of gill; 0.2., outer lobe of gill.

MusctsEs : @2.6.m.9, abductor muscle of gill; 4.¢.%z., branchio-thoracic muscles; ¢.4.72.9,
external branchial muscle; 7.4.7.9, internal branchial muscle ; 7./.#z., inner lobe muscles ;
Z.a.m., longitudinal abdominal muscles ; 0./.7z., outer lobe muscles.

NERVES: a@.g., first abdominal ganglion ; ¢.4.7., external branch of neural nerve; g.7.,
branch of neural nerve supplying gill book; 4.7.9, haemal nerves; 7.4.7., internal branch of
neural nerve; 2.7.9, intestinal nerve (two branches are shown, a posterior and an anterior one);
in.n.9, integumentary branchof haemal nerve; Z.s.7., lateral sympathetic nerve ; #z.6.7., median
branch of neural nerve ; 7.77.9, neural nerve ; s.c.7.9, segmental cardiac nerve ; v.c., ventral cord.

160 PATTEN AND REDENBAUGH. [VoL. XVI.

vate the body portion of the metamere. The haemal nerve is
divisible into intestinal (¢..), cardiac (s.c.z.), and integumen-
tary branches (zz.z.), and of these the intestinal and cardiac
branches communicate with corresponding branches of other
neuromeres by longitudinal connectives.

(1) Meural or Gill Nerves. — The neural nerve (Text-figs.
16 and 17, 2.2.9) enters the base of the gill and immediately
divides into three branches, the external (¢.6.7.), median (#7.0.2.),
and internal (2.d.2.) branchial nerves.

The external branchial nerve gives a motor branch to the
abductor muscles (a.m.%) of the anterior face of the gill and
a sensory branch to the epidermis of the same region and to
the outer portion of the base of the gill.

The median branchial nerve (m.0.7.) gives a motor branch to
the external branchial or abductor muscle (e.d.m.%) upon the
posterior face of the appendage, and a sensory branch (g.7.) to
the gill book (Text-fig. 17, .4.)._ This passes outward posterior
to the branchial cartilage (0.c.%) to the inner edge of the gill
book, where it divides into two bundles of fibers which go in
opposite directions along the edge of the gill book, and give off
a fine nerve fiber to each gill leaf. These fibers follow the
margins of the leaves and break up into fine filaments which
supply the numerous sense buds in the epidermis.

The internal branchial nerve supplies the muscles (0./.m, and
2./.m.) and epidermis of the distal portions (0.7. and 2./) of the
appendage, and corresponds very closely to the third branch
(Z.o.2.) in the operculum. The median lobe (./.) of the ap-
pendage is also supplied by a branch from the internal branchial
nerve.

(2) Haemal Nerves. —The haemal nerve (Text-fig. 16; Pls.
Vi and VIII, Figs. 1 and 3, 4.7.93) of the branchial neuromere
arises from the anterior end of the abdominal ganglion and
passes out over the neural surfaces of the longitudinal abdomi-
nal muscles, anterior to the appendage of its own metamere.
It divides into three principal branches — (a) intestinal, (6)
cardiac, (c) integumentary branches.

(a) Intestinal Branches.—In all the gill neuromeres the intes-
tinal branch (2.2.93) arises from the proximal end of the haemal

No. 1.] STUDIES ON LIMULUS. 161

nerve very close to the ganglion and is either double at its
origin or divides, not far from its origin, into two branches. One
branch joins the plexus in the longitudinal muscles; the other
goes to the intestine and in the majority of cases sends also a
branch to the haemo-neural muscle (4.2.m.%) of its own meta-

ral

Fic. 17.— Diagram of the first gill, from the posterior side, showing the distribution of the gill
nerve to the gill book (about natural size).
g-6., gill book; z./., inner lobe of the appendage; #./., median lobe of appendage ;
o.l., outer lobe of appendage.
Nerves: e¢.6.7., external branchial nerve; g.z., gill nerve; 7.4.7., internal branchial
nerve; 7.6.2z., median branchial nerve; 7.7.9, neural nerve of the ninth neuromere.

mere. In the last gill neuromere the double nature of the
intestinal branch was not found, but as these nerves are all
very delicate some of the branches might easily be overlooked
in dissection. Sometimes the branch which supplies the in-
testine goes some distance in the connective tissue, and divides
before entering the intestine ; and it has been seen to anastomose
with the intestinal branches from the other neuromeres.

(b) Zhe Cardiac Branches. — The cardiac branch in the

162 PATTEN AND REDENBAUGH. [VoL. XVI.

branchial neuromeres is given off (Text-figs. 16 and 18; Pls.
VI, VIL, and IX, Figs, 1,:3,\5, and 6,s.c:72.9-) outside of the
branchio-thoracic muscles (6.¢.1.) or tendinous stigmata (¢.5.7),
and, after giving a recurrent branch to the lateral sympathetic
(2.s.2.), passes haemally, anterior to the entapophysis and poste-
rior to the branchio-cardiac canal (4.c.c.7%) of its own neuromere.
Having reached the haemal side of the animal, it turns toward the
median line, gives a recurrent branch anteriorly to the peri-
cardial nerve (f.z.), and breaks up into small branches which
ramify through the epidermis, haemal to the heart. Over each
pair of ostia (os.73) of the heart a pair of these branches pass
down from the epidermis to the median nerve (m.c.v.) of the
heart.

The cardiac nerve (s.c.z.7%) of the fifth branchial neuromere
is a little different from the others. A portion of it some-
times separates from the haemal nerve far back near the base
of the latter, and follows it along to the lateral sympathetic,
where it receives another branch from the haemal nerve. This
latter branch, and not the main cardiac branch, gives the recur-
rent branch to the lateral sympathetic (Text-fig. 12). Near
the entapophysis (ez¢a.7) it gives a branch to the slip of the
extensor muscle (¢.¢.7.%) of the caudal spine, which is attached
to the last three entapophyses (enxza?4)._ In this muscle it
anastomoses with branches from the post-cardiac nerves which
supply the same muscle. The connection of the cardiac branch
of this neuromere with the pericardiac nerve (/.v.) is also irregu-
lar ; sometimes this connection is entirely absent and some of
its branches pass backwards in the epidermis to the posterior
margin of the carapace. Whether these branches are prolonga-
tions of the pericardial nerve, or merely branches of the last
cardiac nerve, is difficult to say.

(c) Integumentary Branches.— The lateral expansions of the
carapace are very thin just outside of the bases of the append-
ages, but very thick at the outer margins. The integumentary
branches, therefore, do not show a distinct division into haemal
and neural branches except at their distal ends. Near the
base of each appendage a small nerve is given off posteriorly
to the epidermis. The main integumentary branches (Pl. VI,

No. 1.] STUDIES ON LIMULUS. 163

Fig. 1) proceed diagonally backward and outward to the rim of
the carapace. The fibers show a tendency to separate from
each other as they approach the thicker margin and eventually
break up into several divisions; one branch goes haemally,
another neurally, and small branches go posteriorly and ante-
riorly, but the principal branch continues outward to one of the
six large marginal spines (a.s.?”4), which are attached to the sides
of the abdominal carapace. The branch from the first (4.7.9)
branchial neuromere enters the first of these spines, where it
breaks up into fine fibers. The first five spines are innervated
by nerves from the corresponding five branchial neuromeres.

f. Nerves from the Post-Branchial Neuromeres. — As there
are no appendages in the post-branchial metameres, and the
neuromeres are indistinguishably fused, it is difficult to follow
the metamerism in this region. In the typical neuromeres the
neural nerves supply the appendages exclusively, and the haemal
nerves the remainder of the metamere. In the post-branchial
metameres there are no appendages. If we consider the neural
nerves as absent, the post-branchial nerves fall into three similar
pairs, which partake very strongly of the nature of the typical
haemal nerves.

(1) Merves from the First Post-Branchial Neuromere. — The
first post-branchial nerve (Pls. VI, VIII, and IX, Figs. 1, 3-5,
h.n7#) is very similar to the last haemal branchial nerve. It
passes out from the fused, terminal, ganglionic mass posterior
to the muscles of the last gill and anterior to the last haemo-
neural muscle (4.%.m.74), and goes diagonally backward toward
the margin of the carapace. Like the typical haemal nerve,
it is divisible into an intestinal branch, a post-cardiac branch,
and an integumentary branch.

(a) Intestinal Branches.— In this neuromere the intestinal
branches (2.7.4) arise sometimes at some distance from the gan-
glionic mass, and the muscular and visceral branches may arise
quite independently of each other (PI. VIII, Fig. 4). The
branch supplying the last haemo-neural muscle, which belongs
to this metamere, has not been found.

(b) Post-Cardiac Branch. — The post-cardiac branch (s.c.2."4)
arises at some distance from the proximal end of the nerve and

164 PATTEN AND REDENBAUGA. [VoL. XVI.

passes haemally just anterior to the last entapophysis (evfa.’4).
A branch is given to the lateral sympathetic before the post-
cardiac nerve separates from the main nerve. This is the
posterior limit of the lateral sympathetic (Text-fig. 18). Near
the entapophysis the post-cardiac nerve gives off branches to a
slip of the extensor muscle (¢.¢.1.*) of the caudal spine, which
is attached to the last three entapophyses. In this muscle the
branches anastomose anteriorly with the corresponding branches
from the last branchial neuromere, and posteriorly with a simi-
lar branch from the second post-branchial neuromere. The post-
cardiac nerve terminates in the epidermis in the haemal median
line posterior to the heart.

(c) Integumentary Branches. —The integumentary branch
passes diagonally backward to the thickened rim of the cara-
pace, where it gives off haemal and neural branches and some
small nerves posteriorly and anteriorly, but the main branch
supplies the last large spine (a.s.4) upon the edge of the
carapace.

(2) Nerves from Second Post-Branchial Neuromere. — This
pair of nerves (%.z.), because of their larger area of distribu-
tion, are somewhat larger than the preceding haemal nerves.
They arise from the terminal ganglionic mass just back of the
first post-branchial nerve, and pass posteriorly and outward
posterior to the last haemo-neural muscle (4.2.m.4). This
nerve is also divisible into intestinal, post-cardiac, and integu-
mentary branches.

(a) L[nutestinal Branches.— From the base of the nerve (z.7.’5)
a small fiber passes backward along the intestine to which it
gives off several branches. It then continues backward nearly
to the rectum, where it unites with a nerve (2.7.7) from the
third post-branchial neuromere. No muscular branches have
been found.

(b) Post-Cardiac Branch.— Posterior to the last haemo-neural
muscle a large branch goes haemally between the slips of the
flexors of the caudal spine. Near the haemal side of the body
this branch divides into two; one going outward to the external
slips of the extensors (¢.e.m.*) of the caudal spine, and to the
epidermis in the neighboring region; the other going toward

No. I.] STUDIES ON LIMULUS. 165

the median line to the internal slips of the extensors (¢.e.1.*),
and to the epidermis in the median line. Some of the branches
go to the posterior margin of the carapace over the base of
the caudal spine. Inside the extensor muscles the branches
anastomose with those of the first post-cardiac nerve.

(c) Integumentary Branches. — Besides the integumentary
portion of the post-cardiac, which innervates the epidermis
upon the haemal side of the carapace, there is a large integu-
mentary branch of the main nerve, which innervates the neural
side of the carapace. After giving off the post-cardiac branch,
the main nerve enters the flexor muscles of the caudal spine
and gives off to these muscles branches which anastomose
with similar branches from the third post-branchial nerve (/.7.”°).
The integumentary portion turns posteriorly and supplies the
epidermis in the posterior angles of the abdominal carapace.

(3) Nerves of the Third Post-branchial Neuromere.— The
terminal ganglionic mass sends out a large pair of nerves
(4.n.7°), which pass posteriorly upon each side of the rectum
to the telson. Each of these nerves divides, sometimes almost
at the very base, into two branches. The first goes between
the flexor (¢,f.) and extensor (¢.e.7z.) muscles of the telson and
gives to the flexor (¢.f:.) branches which anastomose with the
branches of the second post-branchial nerve. The distal or
integumentary portion divides, sending one branch to the pos-
terior angle of the carapace, and the other to the telson. This
nerve also sends some fine branches to the epidermis in the
posterior margin of the carapace neural to the base of the
telson.

The second branch, which is much the larger of the two,
breaks up in the telson into numerous branches. At the side
of the rectum it gives off a branch (z.x.7°) which supplies the
anal muscles and the rectum and communicates with the intes-
tinal nerve (2.7.%) of the second post-branchial neuromere. A
branch of this nerve also goes to the epidermis at the base of
the telson, posterior to the anus.

Milne-Edwards describes a small ganglion upon each side of
the anus at the root of the nerve going to the rectum, but we
have failed to find it. This ganglion is supposed to lie inside

166 PATTEN AND REDENBAUGH. [VoL. XVI.

the large blood vessel which accompanies the last haemal nerve
into the telson. In alcoholic specimens white blood clots are
often found in the forks of the arteries, and these might easily
be mistaken for ganglia in a gross dissection. Such a clot is
often found at the spot designated by Milne-Edwards, but
microscopic examination reveals no ganglion cells.

There are no post-cardiac branches in the terminal neuro-
mere.

3. SYMPATHETIC SYSTEMS.

a. Lateral Sympathetic.

Milne-Edwards in 1873 described the lateral sympathetic as
a lateral longitudinal nerve parallel to the ventral cord. Refer-
ring to the abdominal haemal nerves he says: ‘“ Chacun d’eux
envoie un filet qui se dirige en avant, et va se réunir, ou plutot
concourt a former un nerf latéral longitudinal. Celui-ci’’ (the
lateral sympathetic nerve) “s’etend paralleélement a la chaine
ganglionnaire, un peu en dehors de la veine collectrice et entre
les muscles abdominal-oblique et branchio-thoracique; il se pro-
longe en avant jusqu’au thorax, et en arriére il présente un
petit renflement ganglionnaire; dans son parcour il fournit des
filets aux muscles voisins. Ce nerf latéro-abdominal, dont l’ex-
istence n’a jusqu’a present été signalée que chez les Limulus,
rappelle par sa position le grand sympathique des animaux
supérieurs ; mais son réle physiologique est tout a fait different,
puisque au lieu de se distribuer aux organes de la vie de nutri-
tion, il se rend aux organes de la vie de relation.”’

I have found that the lateral sympathetic nerve (Text-figs. 9,
15, 16, and 18; Pls) Vi, VIL, and 1X, Pigs..1).35,and:6, 7.5.7)
which has already been partially described, lies nearly parallel
to the ventral cord, and receives a branch from each of the
haemal nerves, from the eighth to the fourteenth, inclusive. It
is in close connection with the branchio-thoracic muscles (0.2.7.),
and its anterior portion is formed of anastomosing branches
within, and forming the nerve supply of, this bundle of mus-
cles. It extends as far forward into the cephalothorax as do
the branchio-thoracic muscles. The posterior portion consists

No. 1.] STUDIES ON LIMULUS. 167

hnlé
hnlS Np S

NS
sconl# \

in!S

Fic, 18. — Diagram showing the lateral sympathetic nerve and its relations to the haemal and cardiac
nerves and the ventral cord (seen from the neural side).
a.g.9%6, abdominal ganglia ; @.7.z., anterior branch of intestinal nerve of ninth neuromere ;
h.n.7-%6, haemal nerves of the seventh to sixteenth neuromeres; z.7.7-15, intestinal nerves of
the seventh to fifteenth neuromeres ; 2.s.2., lateral sympathetic nerve; 2.n.9%3, neural nerves
of the ninth to thirteenth neuromeres ; #.z.7., posterior branch of intestinal nerve of ninth
neuromere ; s.c.7.7-13, segmental cardiac nerves of the seventh to the fourteenth neuromeres ;
v.¢c., ventral cord.

168 PATTEN AND REDENBAUGH. [Vo.. XVI.

of a single nerve trunk lying, for the most part, neural to, or
outside of, the tendinous stigmata (¢.s5.°”).

The branchio-thoracic muscles do not extend back as far as
the lateral sympathetic nerve goes, but their posterior portions
are replaced by the tendinous stigmata, which are segmen-
tal invaginations of the chitin furnishing attachment for the
branchio-thoracic muscles. The tendinous stigmata, six in
number, occur just posterior to each of the abdominal append-
ages; z.¢., from the eighth to the thirteenth metameres, inclu-
sive. The branchio-thoracic muscles, which are attached to
these stigmata, belong, then, in the eighth to thirteenth meta-
meres, and should receive their nerve supply from the eighth
to thirteenth neuromeres. This is actually the case; the lat-
eral sympathetic nerve, or more properly the branchio-thoracic
nerve, receives a recurrent branch from each of the haemal
nerves from the eighth to thirteenth (47.4%); but it also
receives one, a very fine one, from the fourteenth haemal nerve.
This would indicate that there is a branchio-thoracic muscle
belonging to the fourteenth metamere, which is very probable,
inasmuch as we have a pair of entapophyses, a pair of haemo-
neural muscles, and a pair of typical haemal nerves in this
metamere, but no appendage or neural nerves.

The recurrent branches of the lateral sympathetic arise from
the haemal nerves near the segmental cardiac nerves (s.¢.2.5),
or from the roots of the cardiac nerves themselves. The
exact mode of origin varies considerably in different individuals ;
Fig. 18 (text) shows the relation of the lateral sympathetic to
the haemal nerves in one specimen.

The cardiac nerves (s.c.z.7°°4°) from the séventh and eighth
haemal nerves fuse together, and the root of the eighth cardiac
nerve gives off a branch to the sympathetic.

The root of the ninth cardiac nerve (s.c.z.9) also sends a
branch to the sympathetic.

In the tenth neuromere the roots of the cardiac (s.c.z.7°) and
recurrent branches are flattened out, and some small nerves
are given off to the inter-entapophysial muscles. The origin of
the recurrent branch is, however, essentially the same as in the
preceding neuromere, except that the cardiac nerve does not

No. 1.] STUDIES ON LIMULUS. 169

separate entirely from the haemal nerve before the recurrent
branch is given off.

In the eleventh and twelfth neuromeres the recurrent branches
are given off from the haemal nerves (4.2.7 "472) before the
cardiac branches (s.c.z./7 247) separate from them.

A portion of the cardiac branch (s.c.z.’4) of the fourteenth
neuromere separates from the haemal nerve (/.z.4) near the
ventral cord and, opposite the lateral sympathetic, receives
another fiber which separates from the haemal nerve close to
the origin of the recurrent sympathetic branch.

The last recurrent sympathetic branch arises from the four-
teenth haemal, or first post-branchial nerve (4.2.74). The post-
cardiac (s.c.z./4) separates from the haemal nerve some distance
beyond the origin of the sympathetic branch.

Under favorable conditions small recurrent fibers may be seen
in the angles between the roots of the sympathetic branches
and the outer, or integumentary portions of the haemal nerves.

The ganglionic enlargements referred to by Milne-Edwards
could not be found. He probably referred to the flattened por-
tions which sometimes occur at the junctions of the lateral
sympathetic with the haemal nerves. These flattenings are
likely to occur wherever a nerve passes between two muscles
or other parts in close contact with each other; they do not
contain any ganglion cells.

b. Merves of the Heart.

The only mention we have of the cardiac nerves of Limulus
is found in “ Anatomie des Limulus,” by Milne-Edwards. «Sur
les cétés de l’estomac se trouvent aussi deux filets délicats et
ténus qui se rendent a un nerf volumineux situé sur la ligne
mediane du cceur et dans toute la longueur de cet organ. Ce
nerf cardiaque, qui s’amincit beaucoup vers les extrémités du
vaisseau dorsal, est au contraire trés-large vers la partie moyenne
de celui-ci; effectivement, il présente un certain nombre de ren-
flements situés au niveau de chaque paire d’ouvertures vascu-
laires, et de ces points partent des filets qui se dirigent a droite
et a gauche sur les parois adjacentes.”

170 PATTEN AND REDENBAUGH. [VoL. XVI.

The plexus upon the heart, and its connections with the
central nervous system through the segmental cardiac nerves
have never been described. The ‘deux filets délicats et ténus”’
which Milne-Edwards describes, but does not represent in his
figures, we have not been able to find.

(1) The Cardiac Plexus.—In a cross-section of the heart
(Text-figs. 12 and 14; Pl. 1X, Figs. 6 and 7) three large nerves
(/.c.z. and m.c.m.) are seen in the three angles. They lie upon
the outside of the heart between the longitudinal strands of
connective tissue. In Figs. 5, 8,9, and 10 of the plates these
nerves are seen to better advantage.

A median ganglionated nerve (m.c.v.) traverses the heart
longitudinally upon the haemal side. Along the middle of its
course it is quite large, but dwindles down at the ends. Under
a low magnifying power (Pls. IX and X, Figs. 8 and Q) it ap-
pears as a large bundle of intertwining fibers intermingled with
masses of ganglion cells. Still higher magnification shows that
many of these ganglion cells are bipolar (g.c., Pl. X, Fig. 10).

The lateral nerves (/.c.z.) of the heart are not ganglionated.
They traverse the sides of the heart just above the lateral angles
and communicate with the median nerve by an elaborate plexus,
which is richest upon the haemal sides of the heart. The
neural side of the heart seems to have very few nerves.

The main branches of the cardiac plexus (Pl. IX, Fig. 8)
arise from the median nerve uniformly in pairs opposite the ostia,
but the connections with the lateral nerves seem to be entirely
irregular. Some of the branches of the plexus approach very
closely the origins of the lateral arteries, but no nerves have
been observed running out onto them.

(2) Segmental Cardiac Nerves. — The segmental cardiac
nerves (Text-figs. 8, 9, 12, 14, 15, 16, and 18, Pls. VI-IX, 1-3, 5,
and 6, s.c.z.°5) have been partially described in the foregoing
pages. Those of the five branchial neuromeres (s.c.7.%“3) are
most typical, and they will be considered first. They arise from
the haemal nerves (4.7.9), in close connection with the recur-
rent branches of the lateral sympathetic, opposite the branchio-
thoracic muscles, and pass outside of these muscles to the
haemal side of the body, just anterior to the entapophyses

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No. I.] STUDIES ON LIMULUS. gal

(enta.~3), They then turn toward the median line in the epi-
dermis haemal to the heart, where they dip downward and com-
municate with the median nerve (m.c.z.) of the heart (Pl. IX,
Figs. 5 and 6, and Text-fig. 8) opposite the last five pairs of
ostia (os.7). The connections with the median nerve of the
heart have been actually found only for the cardiac nerves
(s.c.z.773) of the five branchial neuromeres, but similar connec-
tions probably exist in other neuromeres.

Besides the branch communicating with the median cardiac
nerve, the segmental cardiac gives off numerous branches to
the epidermis haemal to the heart, and also an important branch
which goes anteriorly and unites with the pericardial nerve (7.7.),
a longitudinal nerve trunk running parallel to the heart inside
the pericardium.

The cardiac branches (s.c.z.7*"4°) of the seventh and eighth
neuromeres fuse together and form a large nerve, which passes
haemally outside of the branchio-thoracic muscles and anterior
to the large entapophysis (ex¢a.7°44), The root from the eighth
neuromere gives a branch to the lateral sympathetic. Upon
the haemal side of the body this cardiac nerve divides into a
number of branches. A large one enters the inter-tergal mus-
cle and breaks up into anastomosing branches which supply
that muscle. Some small branches pass into the epidermis
haemal to this muscle, and approach the median line. Although
these branches could be traced in the epidermis to points just
above the three anterior pairs of ostia (os.°*), no connections
with the median nerve of the heart could be made out. In this
region the connective-tissue strands which support the heart
upon the haemal side are very numerous, and it is difficult to
trace nerve fibers among them.

The most important branch of this cardiac nerve, the peri-
cardial nerve (7.z.), turns posteriorly in the areolar tissue which
lies above the pericardial sinus. It first gives off a small
branch to the lateral inter-tergal muscle, and then, continuing
posteriorly, gives a branch to each of the cardiac nerves
(s.c.z.73) of the branchial neuromeres. These branches pass
from the outer side of the pericardial nerve toward the proxi-
mal ends of the cardiac nerves. The posterior extremity of the

L72 PATTEN AND REDENBAUGH. [VoL. XVI.

pericardial nerve is lost in a plexus of nerves, some of the
branches of which extend in the epidermis to the posterior
margin of the abdomen.

The cardiac nerve (s.c..43) of the last branchial neuromere
differs from the others in that it gives a branch to a slip of the
extensor muscle of the telson, which is inserted upon the last
three entapophyses. Similar branches from the first two post-
cardiac nerves go to the same muscle, anastomose with this nerve
and with each other, and send a branch in the epidermis to the
posterior end of the abdomen.

The two post-cardiac nerves (s.c.7./4"475) also send branches
to the epidermis near the median line. As the heart does not
extend back of the thirteenth metamere, the post-cardiac nerves
have no connection with this organ.

In the sixth thoracic neuromere a large cardiac branch (s.c..°)
is given off from the sixth haemal nerve (%.z.°). This does not
communicate with the lateral sympathetic, but passes haemally
and anteriorly outside of the branchio-thoracic muscles, and
enters the large inter-tergal muscles haemal to the heart. Here
its branches anastomose with those of the fused seventh and
eighth cardiac nerves (s.c.2.7°°4%), Some of the finer branches
innervate the epidermis also, and possibly communicate with
the median nerve of the heart, although this connection has
not been observed.

A small nerve, which could not be traced out, was found
arising from the fifth haemal nerve (4.%.5) at a point cor-
responding to the origins of the cardiac nerves of other
neuromeres.

It is a curious fact that, leaving out the post-cardiac nerves,
there are as many segmental cardiac nerves as there are ostia
in the heart. The cardiac nerves (s.c.z.73) from the five bran-
chial neuromeres enter the heart opposite the five posterior
pairs of ostia (05.7). This leaves three segmental cardiac
nerves (s.c.z.°*) corresponding to the three anterior pairs of
ostia (os.°%), If these have any connections with the median
nerve of the heart, the connection will probably be found oppo-
site the three anterior pairs of ostia, inasmuch as the connec-
tions of the other five cardiac nerves with the median cardiac

No. 1.] STUDIES ON LIMULUS. me

nerve have been found opposite the five posterior pairs of ostia.
The stump of the cardiac nerve found in the fifth neuromere
would then correspond to the rudimentary ostia (7.0s.).

c. Nerves of the Alimentary Tract.

(1) The Rostral Nerves. — Three rostral nerves, a median
and two lateral ones (Pls. VI-VIII, and X, Figs. 1-3, 11, and
12, /.a.n.), arise from the anterior commissure and innervate the
rostrum, or upper lip.

(2) Stomodaeal Nerves. — A pair of stomodaeal nerves (Pls.
VIII and X, Figs. 3, 11, and 12, s¢.x.) arise from large ganglia
on the inner side of the oesophageal collar and innervate the
oesophagus, proventriculus, and pyloric valve. These have
already been fully described under the Nerves from the Mid-
Brain.

A small ganglion upon the sides of the proventriculus, the
existence of which is doubtful, has been described by Milne-
Edwards. ‘Au point ot cet organe se replie brusquement pour
se porter en arriére, se trouve un trés-petit ganglion aplati,
logé, comme le nerf dans l’artére, prés de l’anastomose de cette
derniere avec la branche gastrique émanée de la convexité de la
crosse aortique.” The point mentioned is a place within the
aortic arch from which several nerves diverge to supply the
proventriculus, and where white blood clots are extremely liable
to lodge. In alcoholic specimens these clots take on the
appearance of ganglia.

“De ce ganglion partent en avant des filets qui se distri-
buent aux parois trés-musculeuses de |l’estomac, et en arriére
deux rameaux dont l'un se rend a la portion pylorique de ce
viscére, et l'autre gagne l’intestin. Ces parties son trés difficiles
a distinguer, car elles sont extrémement gréles, et pour les
degager des artéres ot alles sont logées, il faut procéder avec
un trés grand soin.”” I have found branches running onto the
anterior end of the intestine, but could not trace them beyond the
pyloric valve even with a methylen blue stain. Although
good stains of the nerves of the proventriculus and the anterior
end of the intestine were easily obtained by this method, every

R74 PATTEN AND REDENBAUGH. [VoL. XVI.

attempt to demonstrate a nerve plexus on the intestine has
failed.

According to Milne-Edwards “ Sur les cétes de l’estomac se
trouvent aussi deux filets délicats et ténus qui se rendent a un
nerf volumineux situé sur la ligne médiane du coeur et dans
toute la longueur de cet organe.” All attempts to find these
cardiac branches of the stomodaeal nerves have been unsuc-
cessful.

(3) Jutestinal Nerves.— Milne-Edwards has described, in addi-
tion to the stomodaeal nerves, a pair innervating the posterior
portion of the alimentary canal. <‘L’autre, destiné a l’épine
caudale, passe au-dessous du muscle abaisseur de l’anus et a
point ou l’artére qui le contient s’anastomose avec l’artére
anastomotique, fournit trois ou quatre filets gréles qui remon-
tent sur les parois de l’intestin et se rendent a un petit ganglion
rectal situé un peu en avant du sphincter de l’anus, au-dessus du
faisceau musculaire abaisseur de celui-ci. Ce ganglion, un peu
allongé d’avant en arriére, est logé dans la dilatation arterielle
qui existe sur ce point, et envoie des filets nombreux en avant,
en dessus et en arriére. Ces filets s’enfoncent dans les parois
intestinales. L’existence de ce petit centre ganglionnaire est
trés-curieuse et indique un systeme sympathique rectal qui
n’existe pas, ou du moins qui n’a pas été observé chez les
autres Arthropodes; il es d’ailleurs trés-difficile a isoler des
parois arterielles qui l’engaiment.”

As in the case of the ganglion upon the side of the pro-
ventriculus, the existence of the rectal ganglion is doubtful.
Clots of blood have been observed at this point in dissect-
ing, and careful examination of serial sections through this
region has revealed no ganglion cells. A mass of matter with
many small nuclei was found, but this had exactly the appear-
ance of undoubted blood clots in other portions of the same
artery.

Hitherto the segmental intestinal nerves have not been de-
scribed. These occur in all the neuromeres from the sixth to
the sixteenth, and possibly in the second neuromere. Their

1 More recent trials indicate the presence of an elaborate nerve plexus provided
with minute ganglion cells surrounding the circular muscles of the intestine.

~ No. 1.] STUDIES ON LIMULUS. 175

arrangement is most typical in the abdominal region, and it will
be well to take up one of these neuromeres first.

In the first gill neuromere (Text-fig. 8; Pls. VIII and IX, Figs.
3, 4, and 6) two small nerves arise very close together from the
haemal side of the haemal nerve (Z.7.9) close to the abdominal
ganglion. The anterior one (a.z.z.) enters the adjacent mass of
longitudinal abdominal muscles and communicates with the
plexus supplying these muscles. The posterior one (f.2.7.)
divides into two branches, one going to the haemo-neural muscle
(Z.n.m.9) and the other to the intestine. The latter has been
observed to communicate with the corresponding nerves of other
neuromeres by a plexus lying in the tissue immediately surround-
ing the intestine. Only glimpses of this plexus have been
obtained here and there. The nerves are very fine and not
easily made out without a microscope. In one or two instances,
while working with methylen blue, a fine plexus was seen in the
tissues haemal to the intestine, near the posterior end.

In the tenth, eleventh, and twelfth neuromeres the roots of the
two intestinal nerves arise a little farther apart. In each case
the anterior nerve joins the plexus in the longitudinal muscles.
The posterior nerve gives a branch to the haemo-neural muscles,
and one to the intestine, and also communicates sometimes
with the plexi in the longitudinal abdominal muscles, and in the
tissues surrounding the intestine.

In the neuromeres posterior to the twelfth the intestinal
nerves are very irregular in their origin, sometimes arising
from the haemal nerve at some distance from the ganglion, and
entering the intestine by several branches. In the specimen
represented in Fig. 4 of the plates the posterior branch of the
intestinal nerve (2.7.7*) of the twelfth neuromere traverses the
twelfth haemo-neural muscle (4.7.m.’*) before entering the in-
testine. In this figure the branches going to the intestine are
represented as cut off.

The next intestinal nerve (z.z.%) has no muscular branch.

The fourteenth (2.2.4) upon the right side arises near
the abdominal ganglion and proceeds posteriorly a long dis-
tance, and finally enters the intestine by three branches.
Upon the left side the fourteenth intestinal nerve (7.7.’4) is

I 76 PATTEN AND REDENBAUGH. [VoL. XVI.

represented by three branches arising from the haemal nerve
(Z.n.4) at some distance from the ganglion. Oneof these enters
the longitudinal abdominal muscles, the second anastomoses
with the twelfth intestinal nerve (z.z./?), and the third enters the
intestine.

The fifteenth intestinal nerve (z.z.%) arises near the origin of
the fifteenth haemal nerve (4.z.) and passes posteriorly along the
surface of the intestine, giving off several branches to the intes-
tine, and then anastomoses with the sixteenth intestinal nerve
(CE

The sixteenth intestinal nerve (2.7.7) arises from the caudal
branch of the sixteenth haemal nerve (4.2.7) about midway
between the terminal abdominal ganglion and the anus, and
passes posteriorly a short distance to the side of the rectum, or
proctodaeum, where it divides into several branches. It was
at this point that Milne-Edwards found the “ ganglion rectal.”
One branch goes anteriorly to anastomose with the fifteenth
intestinal nerve (2.7.75), a second one goes posteriorly to the
levator ani (l.a.) and to the rectum, and the third goes to the
epidermis upon the haemal side of the base of the telson.

In one methylen blue preparation the sixteenth pair of
intestinal nerves were joined together by a cross-branch upon
the neural side of the rectum. In some of the anterior neuro-
meres also a similar cross-branch has been found uniting the
intestinal nerves of the right and left sides of the body. This
seems to suggest that there is an elaborate network of nerves
in the tissues surrounding the intestine. As these nerves are
very delicate, it is difficult to trace them by dissection alone,
and many of them with their connections are necessarily
missed.

The intestinal nerves (z.7.°*) of the sixth, seventh, and
eighth neuromeres are so complicated in their relations that it
is impossible to unravel them. The sixth (z.z.°) and seventh
(2.2.7) pass through foramina (f.° and 7-7) in the endocranium
and lose themselves in the anterior portion of the mass of longi-
tudinal abdominal muscles. The eighth (z.7.°) goes directly
into these muscles near the posterior edge of the endocranium.
Sometimes each one of these nerves arises from the haemal nerve

No. 1.] SLODIES ON WINTOLES. N77

by two separate roots, and the seventh has been observed to
pass through the endocranium by two foramina.

They commingle in a rich plexus supplying the longitudi-
nal abdominal muscles. From this plexus numerous branches
emerge and, after ramifying and anastomosing through the tis-
sues outside of the intestine, enter its walls, some of them
extending far forward toward the anterior end.

The intestinal nerve (z.7.”) arising from the second haemal
nerve (Pext-fie!3;" Pls. VIII and Xr tees 3,7 01,and ¥2)
passes median to the anterior cornua of the endocranium and
supplies the tergo-proplastral muscles (¢.p.m.744), but no
branch has been observed going to the intestine. As it has an
origin similar to that of the other intestinal nerves, and supplies
similar muscles, it has been included in the same category.

SUMMARY.

1. The nervous system of Limulus is made up of sixteen
neuromeres exclusive of the fore-brain.

2. Each neuromere consists of a pair of ganglia united by
several cross-commissures, a pair of neural and a pair of haemal
nerves. In the first or cheliceral neuromere we have in addi-
tion a pair of stomodaeal and three rostral nerves. In the
three post-branchial neuromeres the appendages and, conse-
quently, the neural nerves are absent.

3. Each neuromere, as a rule, innervates one metamere; the
neural nerves and their branches supply the appendages and the
haemal nerves supply the remainder or body portion of the meta-
mere, including the epidermis and internal organs.

But there are many cases in which nerves extend through
several metameres either as single nerves or united with others
to form longitudinal connectives. For example, the pericardial
nerve springs from the fused seventh and eighth neuromeres
and communicates with the corresponding nerves of the next
five posterior neuromeres. The nerves which supply longitu-
dinal muscles extending through several metameres and having
muscular slips attached in each of the metameres are united
into longitudinal anastomosing plexuses. The lateral sympa-

178 PATTEN AND REDENBAUGH. [Vou. XVI.

thetic, the plexus in the flexors and in the extensors of the
telson, and the anterior branches of the segmental cardiac nerves
supplying the median inter-tergal muscles, are similar examples.
In fact, wherever muscles of different metameres fuse together,
we find the nerves supplying them united into longitudinal
connectives. The first haemal nerve or lateral nerve is an
exceptional case in that it extends through nearly all metameres
of the body, but does not communicate with any of the other
haemal nerves except the second.

4. The brain may be divided into four regions: (1) the fore-
brain, or cerebral lobes, which is probably formed of three
neuromeres, an olfactory, a median eye, and a lateral eye neuro-
mere; (2) the mid-brain, formed of the cheliceral neuromere ;
(3) the hind-brain, formed of five thoracic neuromeres, those from
the second to the sixth; and (4) the accessory brain, formed
of two neuromeres, the chilarial, or seventh neuromere, and the
opercular, or eighth neuromere, both of which were originally
abdominal neuromeres. |

5. The neuromeres of the accessory brain region, the
chilarial and opercular neuromeres, are more completely united
than those in front of or behind them, and some of their nerves
wander into other metameres than their own. This fact led
Dr. Patten to call the accessory brain the “ vagus region.”

The cardiac nerves of these two neuromeres are completely
fused and on the haemal side of the body form the pericardial
nerve, which extends into the five branchial neuromeres.

6. In the typical cranial neuromere the neural nerve divides
into three groups of branches: (1) the mandibular branches ;
(2) the ento-coxal branches ; and (3) the pedal branches. The
haemal nerve also divides into three branches : (1) the intestinal
branch; (2) the cardiac branch; and (3) the integumentary
branch.

The ventral cord consists of five branchial neuromeres (those
from the ninth to the thirteenth) and three post-branchial neuro-
meres (the fourteenth, fifteenth, and sixteenth).

In the typical abdominal neuromere the neural nerve arises
from the posterior side of the ganglion and divides into there
branches, one to the anterior and one to the posterior side

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Journal of Morphology Vol. Xv. PI. Vi.

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THE MATURATION AND FERTILIZATION. OF
THE EGG OF LIMAX AGRESTIS (LINNE).

ESTHER FUSSELL BYRNES.

TABLE OF CONTENTS.

PAGE

MTDNA RR OTD UT CIE TO Nyse cscs oases dooce eae coco oars cede jam cdg d ana 2 eae RE ee we 201
VAT RAT IONUOR TEE iG Gili... 2222. cascs secs ae eee ee 202

rs Phe ArchiamphiaSter :..2c.cc05. sc. 0 ee 202

Zen @ Variatiell 0S eee Rese tists, See. cet eos “ensbassn Suiigusazehascereeseee tata ea renee eres. 204
3. First Maturation Spindle. Extrusion of the First Polar Body .... 206
4. Formation of the Second Maturation Spindle. Extrusion of the

SeEcOndvROlaty WOW ss 22. wc. tc See ncacnaccce oy eeeee catee eae eae eo aan 207

II. STRUCTURE AND MATURATION OF THE SPERMATOZOON ......002000-2------ 211
I. Structure of the Spermatozoon ...................- higpmiee Mime eee A es 211

Bog) AMEN BUNL AL ANOS Oey a Sse eats ee Recs AEs Veale Se 212

III. OBSERVATIONS ON ABNORMAL OVA .....00.22. cesse20----- dict tite ae eter. 218
14g al KOU SAS) OVS) 10017 ers h ee ee are i OM pees ose peeoreere 218

2 Abnonmalitiess@ther than POlysperny esecscerseeeeeseeesee ere eeneeee enn 219

INVA hOR MARTON TOE) GEE SPTNIDIGE seep. eee Se A leet 220

V. STRUCTURE OF THE CYTOPLASM. ARCHOPLASM ........0.cccsccecessesccess-se--- 222

Wile Dri (CENTROSOME! S252 .2cc6 e005 t,he cro seeeace eee tee meee ee cemrer 226
NYC eo SGI 00 7 NT a ee ee ere ere 227
AVADIEESVAIPPENIDIX esate tact lees ela cP el Nin eA BA ES Apa STL 228

Material. Collecting and Keeping................. ded sbepe Mesrag ues ate sold sown, 228
PreparationtoL Material ss.1:cc02. 2c. ! 2 o2r ese nee ee eee MRE Posters 229
INTRODUCTION.

THE study of the maturation and fertilization of the egg of
Limax agrestis was carried on in the Biological Laboratory of
Bryn Mawr College, under the direction of Prof. T. H. Mor-
gan. I gladly take this opportunity of thanking Professor
Morgan for his kind interest and helpful guidance during the
course of the work.

The development of the egg of Limax agrestis was first
studied by Warneck in 1850; and in 1881 Mark published his

201

202 BYRNES. [Vou eva,

epoch-making classic on the maturation and fertilization of the
egg of Limax campestris. The studies of Warneck on the liv-
ing eggs of Limax agrestis, and of Mark on Limax campestris,
leave little to be added to the descriptions that have already
been given of the phenomena exhibited by the living eggs dur-
ing the early stages of development. When, however, the
eggs are preserved and sectioned, and then stained in haema-
toxylin after iron-alum, they show details of structure that
cannot be seen either in the living egg or in preserved eggs
studied in optical sections.

It will be convenient in describing the maturation stages to
distinguish between the aster as a whole and the center of the
aster, which undergoes a series of changes that are apparently
independent of modifications of the astral rays. I shall, there-
fore, follow the terminology that Wilson has adopted in his
studies on the sea-urchin’s egg, and use the term ‘“astro-
sphaere”’ to designate the astral rays as well as the center
of the aster. For the center of the aster alone I shall reserve
the term “centrosphere.” This terminology is employed only
for convenience in description, and has no significance based on
the recognition of a fundamental distinction between the two
parts of the astrosphaere.

I. MATURATION OF THE EGG.
1. The Archiamphiaster.

The youngest eggs of Limax campestris that Mark studied
were those that had just been deposited. In these eggs a large
amphiaster, the ‘‘archiamphiaster”’ of Whitman and of Mark,
occupied the center of the egg. The eggs of Limax agrestis
agree very closely with those of Limax campestris in forming
the archiamphiaster before the eggs are laid. Sections of eggs
that have just been laid show that in the archiamphiaster stage
the centers of the asters no longer appear structureless, as in the
living egg, but are composed of distinct concentric rings or
zones, which react toward haematoxylin and other staining
reagents very differently from the rest of the cell. During

No. 1.] STUDIES ON LIMULUS. 179

of the proximal portion of the appendage, and one to the distal
portion ; the haemal nerve arises from the anterior side of the
abdominal ganglion and divides into three branches: (1) the
intestinal branch; (2) the cardiac branch; and (3) the integu-
mentary branch.

7. The rostrum, oesophagus, and proventriculus are inner-
vated by the rostral and stomodaeal nerves, which arise respec-
tively from the pre-oral commissure and from the ganglia on the
median sides of the first thoracic neuromere.

The intestine and rectum are innervated by the intestinal
nerves, which arise from all the neuromeres from the sixth to the
sixteenth (possibly from the second also).

The intestinal branches of the sixth to the sixteenth neuro-
meres anastomose with each other and form plexi or longitudinal
connectives parallel to the ventral cord.

8. The heart extends through all the metameres from the sixth
to the thirteenth and is probably innervated by cardiac nerves
from each of the neuromeres from the sixth to the thirteenth,
although actual connections have been made out only in the five
branchial neuromeres, vzz., the ninth to the thirteenth.

The cardiac branches of the vagus and abdominal neuromeres
are joined to each other by longitudinal connectives or sympa-
thetic branches, vzz., the lateral sympathetic and the pericardial
nerves.

9g. The muscles of each metamere are innervated from the
corresponding neuromere. If the muscles of several metameres
become fused with one another, the nerves of the different
neuromeres anastomose with each other to form plexi.

10. The chilaria were regarded by Owen and Lankester as
detached inner portions of the sixth pair of mandibles. This
is without doubt an error, since in the embryo there is a separate
neuromere for the chilaria, and in the adult, though the ganglia
are fused with those of the sixth and eighth neuromeres, there
is still a pair of distinct neural (z.z.7) and haemal nerves
_(i.n.7) belonging to the chilarial neuromere. The appendages
also have a complicated set of muscles of their own, both plastro-
coxals and tergo-coxals.

Moreover, the chilaria have certain characters which place

180 PATTEN AND REDENBAUGA. [VoL. XVI.

them together with the operculum, among the abdominal append-
ages. For example: (1) a bar of capsuliginous cartilage (0.c.7)
acts as an internal support for the appendage, and this bar is
identical in structure with the branchial bars found in the
operculum and gills. The fact that this capsuliginous cartilage
is found only in the branchial bars of the chilaria, operculum,
and gills renders the resemblance the more striking. (2) The
roof of the occipital ring (0c.v.) is very similar to the abdominal
endochondrites (a.e.473), (3) The chilarial muscles (7°) arising
from the neural side of the occipital ring and inserted upon
the insides of the chilaria may be compared to the internal bran-
chial muscles (2.6.1.9) ; and. the long tergo-coxal muscle (7”) of
the chilaria resembles the external branchial muscles (¢.6.m.3).
(4) Its neural and haemal nerves are more like those in the
abdomen than those of the thorax.

If we regard the chilarial and opercular neuromeres as ab-
dominal rather than cranial, and consider that the anterior parts
of the intestine and probably of the heart are innervated from
the sixth, seventh, and eighth neuromeres, then the intestine
and the heart must be mainly abdominal organs, indicating a
greater forward movement of the organs in the haemal than on
the neural side of the body.

11. The innervation of the liver, nephridia, and generative

organs has not been made out.
Wn. A. REDENBAUGH.

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BIBLIOGRAPHY.

1605 C.usius, C. (Charles de l’Ecluse). Exoticorum. Libri x.

1633 DE LAET, J. Novus orbis seu descriptionis Indiae occidentalis.
Libri xviii. Lugd. Batav.

1671 BERNIz, MART. V. Cancer moluccanus. Mit Taf. LZphemerid.
acad. natur. curios. Ann. ii, p. 176. December 1.

1776 SPENGLER, L. Einige neue Bemerkungen tiber die Molukkische
Krabbe. Beschaftig. d. Berliner Gesellsch. naturf. Freunde. Bd. ii,
pp. 446 ff.

1782 ANDRE, W. A Microscopic Description of the Eyes of the Mono-
culus polyphemus. Phzlo. Trans. Roy. Soc. of London. Vol. xxii,
Parti2-;pp- 440 i1., table I, -e:

1818 RANZANI, C. Osservazioni sul Limulo polifemo. Ofusc. scientif.
Vol. ii, pp. 275 ff.

’38a VAN DER HOEVEN, J. Recherches sur l’histoire naturelle et l’anato-
mie des limules. Leyde.

’°38b VAN DER HOEVEN, J. Einige Worte tiber die Gattung Limulus.
Archiv f. Naturgesellsch. Bd. iv, 1, pp. 334 ff.

’°38-'41 DuveERNOoy, G. Sur quelques points de l’organisation des limules
et description plus particuliére de leurs branchies. Compt. rend.
de [1 Acad. de Science. ‘Tome vii, pp. 605 ff.

’°38 MILNE-Epwarps, H. Recherches relatives au développement des
limules. Soc. Philomatique Extr. Proc. l'Institut. Tome vi,
Pp. 397:

‘58 GEGENBAUR, C. Anatomische Untersuchung eines Limules, mit be-
sonderer Berticksichtigung der Gewebe. Miteiner Taf. Adhandl.
ad. naturf. Gesellsch. in Halle. Bd.iv. Halle. 49.

’°66-"79 GERSTAEKER, A. Arthropoden. Broun’s Klassen und Ord-
nungen des Thierreichs. Bd. v, 1. Abtheil. Schwertschwanze:
Poecilopoda, p. 1080.

’67 WoopwarbD, H. On Some Points in the Structure of the Ziphosura,
having Reference to their Relation to the Euripteridae. Qxart.
Journ. Geol. Soc. of London. February.

‘70 Lockwoop, S._ The Horse-Foot Crab. Amer. Vat. Vol. iv, pp. 257—
274.

"71 Dourn, A. Zur Embryologie und Morphologie des Limulus poly-
phemus. /enaische Zettschr. f. Mediz. und Naturw. Bad. vi,
pp. 580-639.

"71 VAN BENEDEN, E. De la place qui les Limulues doivent occuper dans
la classification des arthropodes d’aprés leur développement embry-
onnaire. Communiqué a la Soc. Ent. de Belgique, 14 oct., 1871.
Gervais, Journ. Zoologie. Tome i, pp. 41-44. 1872. Annals and
Mag. Nat. Hist. 1872.

182 PATTEN AND REDENBAUGA. [VoL. XVI.

'72 PACKARD,A.S. The Development of Limulus polyphemus. M/emozrs
Bost. Soc. Nat. Hist. Vol. ii, pp. 155-202. March, 1872.
PRELIMINARY :

The Embryology of Limulus polyphemus. Amer. Vat. Vol.
iv, pp. 498-502. October,1870. Proc. Bost. Soc. Nat. Hist.
Vol. xiv, p. 60. June, 1871.

On the Embryology of Limulus polyphemus. Pvoc. Amer.
Assoc. Adv. Sci., 19th meeting, Troy, N. Y. July, 1871.
p- 8. 8° Quart. Journ. of Micr. Sct. Vol. xi, p. 263.

Morphology and Ancestry of the King Crab. Amer. (Vat.
Vol. iv, pp. 754-756. February, 1871.

'72 OwEN, R. On the Anatomy of the American King Crab. TZvyans.
Linn. Soc. of London. Vol. xxviii.

73. MILNE-EDWARDS, ALPH. Recherches sur l’anatomie des limules.
Ann. des Sct. Nat. Tome xvii, Sér. 5. (Commission Scientifique
du Mexique.)

'78 LANKESTER, E. Ray. Mobility of the Spermatozoids of Limulus.
Quart. Journ. of Micr. Sct. pp. 453, 454.

'80 PacKkaArRD, A. S. On the Anatomy, Histology, and Embryology of
Limulus polyphemus. Memoirs Bost. Soc. Nat. Hist. 1880.

PRELIMINARY :

Further Observations on the Embryology of Limulus, with
Notes on its Affinities. Amer. at. Vol. vii, pp. 675-678.
November, 1873. Proc. Amer. Assoc. Adv. Science, Port-
land meeting, 1874.

On the Development of the Nervous System in Limulus. Amer.
Nat. Vol. ix, pp. 422-424. July, 1875.

On an Undescribed Organ in Limulus, supposed to be Renal in
its Nature. Amer. Vat. Vol. ix, pp. 511-514. September,
1875.

Structure of the Eye of Limulus. Amer. Wat. Vol. xiv,
pp. 212, 213. March, 1880.

Internal Structure of the Brain of Limulus. Amer. Vat. Vol.
xiv, pp- 445-448. June, 1880.

’81 LANKESTER, E. Ray. Limulus an Arachnid. Quart. Journ. of Micr.
562.0) \ViOl. 2cxi-

'82 LANKESTER, E. RAy. On the Coxal Glands of Scorpio, hitherto
undescribed and corresponding to the brick-red glands of Limulus.
Proc. Roy. Soc. of London. No, 221.

’82 PacKARD, A. S. Is Limulus an Arachnid? Amer. Mat. Vol. xvi,
pp. 287-292.

’83 BENHAM, W. B.S. On the Testis of Limulus. Trans. Linn. Soc. of
London. Vol. ii, Part 9.

’84 KINGSLEY, J.S. The Developmentof Limulus. Scz. Record. Vol. ii,

Pp. 249.

No. 1.] STUDIES ON LIMULUS. 183

'84 LANKESTER, E. Ray. On the Skeleto-Trophic Tissues and Coxal
Glands of Limulus, Scorpio, and Mygale. Quart. Journ. of Micr.
Scz. Vol. xxiv.

’'85 LANKESTER, E. RAy. On the Muscular and Endoskeletal Systems of
Limulus and Scorpio, with Some Notes on the Anatomy and
Generic Characters of Scorpions. By E. Ray Lankester, assisted
by W. B. S. Benham and Miss E. J. Beck. TZvans. Zool. Soc. of
London. Vol. xi, Part to.

’'85 LANKESTER, E. Ray. A New Hypothesis as to the Relationship of
the Lung-Book of Scorpio to the Gill-Book of Limulus. Qwart.
Journ. of Micr. Sct. Vol. xxv.

’85 KINGSLEY, J. S. Notes on the Embryology of Limulus. Qdzxart.
Journ. of Micr. Sct. Vol. xxv.

’85 GULLAND, G. L. Evidence in Favor of the View that the Coxal
Gland of Limulus and Other Arachnids is a Modified Nephridium.
Quart. Journ. of Micr. Sct. Vol. xxv.

’85 OszorNn, H. L. The Metamorphosis of Limulus polyphemus. ous
Hopkins Univ. Cire. Vol. v.

’85 Brooks, W. Kk. Abstract of Researches on the Embryology of Limu-
lus polyphemus. /ohns Hopkins Univ. Circ. Vol. v.

'85a PackaRD, A. S. On the Xiphosurous Fauna of North America.
Nat. Acad. of Sct. Vol. iii, 16th memoir.

’'85b PacKARD, A. S. On the Embryology of Limulus polyphemus, III.
Proc. Am. Phil. Soc. Vol. xxit. “1885. “Amer. Vat.) Vol. xix,
p. 722.

’87 CxLaus, C. Prof. E. Ray Lankester’s Artikel “‘ Limulus an ‘Arachnid ”’
und die auf denselben gegriindeten Pratensionen und Anschuldi-
gungen. Ard. Zool. Inst. Wien. Bad. vii.

’89 PATTEN, Wm. On the Origin of the Vertebrates from Arachnids.
Quart. Journ. of Micr. Sci. New Series. Vol. xxxi, Part 3.

’'89 WartTasE, S. On the Structure and Development of the Eyes of
Limulus. Johns Hopkins Univ. Circ. Vol. viii, No. 70, pp. 34-37.
Abstract in Journ. Roy. Micr. Soc. of London. Part 6, pp. 747, 748.
Abstract in Amer. Nat. Vol. xxiv, p. 81. January, 1889.

90 VIALLANES, H. Sur la structure des centres nerveux du limule
(Limulus polyphemus). Compt. rend. de Acad. des Sciences de
Paris. Tome cxi, pp. 831-833. 1890. Abstract in Journ. Roy.
Micr. Soc. of London. Part 1, pp. 36, 37. 1891.

'90 KINGSLEY, J. S. The Ontogeny of Limulus. Amer. Wat. Vol. xxiv,
pp. 678-681. Zool. Anzeiger. No. 345, pp. 536-539. Abstract in
Journ. Roy. Micr. Soc. of London. Part 6, pp. 718, 719.

"91 PacKARD, A. S. Further Studies upon the Brain of Limulus poly-
phemus. Zool. Anzeiger. Vol. xiv, No. 361, pp. 129-133.

"91 SmitH, H. M. Notes on the King-Crab Fishery of Delaware Bay.
Extract Bulletin U. S. Fish Commission. Vol. ix, pp. 363-370.

184 PATTEN AND REDENBAUGH. [VoL. XVI.

’91

92

92

93

93

93

93

93

94

194

"95

96

96

BovuvirErR, E. L. Observations sur l’anatomie du systéme nerveux de
la Limule Polyphemus. Compt. rend. Soc. Philomatique de Paris.
No. 20, p. 1. 8 aoit. Bull. Soc. Philomatique de Paris. (8)
Tome iii, No. 4, pp. 187-198. ‘

KINGSLEY, J. S. The Embryology of Limulus. Journ. of Morph.
Vol. vii, No. 1. 1892.

KISHINOUYE, K. On the Development of Limulus longispinus. Journ.
Coll. of Sci. Imp. Univ. Japan. Vol. v, pp. 53-100.

PRELIMINARY :
Zool. Anzeiger. Jahrg. 14, No. 369, pp. 264-266. Abstract
in Journ. Roy. Micr. Soc. Part 6, pp. 732, 733.

Hyper, Ipa H. The Nervous Mechanism of the Respiratory Move-
ments in Limulus polyphemus. /ourn. of Morph. Vol. ix, No. 3.

PACKARD, A. S. Further Studies on the Brain of Limulus polyphe-
mus, with Notes on its Embryology. Memoirs National Acad. of
Scz. Vol. vi, pp. 287-331. Washington, D.C.

KINGSLEY, J.S. Embryology of Limulus. Part2. /ourn. of Morph.
Vol. viii, No. 2.

PATTEN, Wm. On the Morphology and Physiology of the Brain and
Sense Organs of Limulus. Quart. Journ. of Micr. Sct. Vol. xxxv,
No. 137. July.

VIALLANES, H. Etudes histologiques et organologiques sur les centres
nerveux et les organes des sens des animaux articules. Sixi¢me
Mémoire: I. Le cerveau de la Limule (Limulus polyphemus).
Ann. des Sci. Nat. Tome xiv, Nos. 4, 5,6 (10 mars, 1892). Paris.
1893.

PATTEN, Wm. On Structures resembling Dermal Bones in Limulus.
Anat. Anzeiger. Bd. xvii, No. 14, pp. 429-438.

PATTEN, Wo. Artificial Segmentation of Limulus. Zool. Anzezger.
Bd. xvii, pp. 72-78. 1894.

Tower, R. W._ Brick-Red Glands in Limulus. Zool. Anzeiger. Bd.
xviii, pp. 471, 472. Abstract in Journ. Roy. Micr. Soc. P. 186.
April, 1896.

PATTEN, WM. Variations in the Development of Limulus polyphe-
mus. Journ. of Morph. Vol. xii, No. I.

GASKELL, W. H. British Association. Vaturve. Vol. liv, No. 1406.
October 8.

No. 1.]

STUDIES ON LIMULUS.

185

EXPLANATION OF PLATES.

REFERENCE LETTERS.

In the colored plates nerves are represented in yellow, blood vessels in carmine,
cartilage in blue, and muscles in brick red.
All exponential figures indicate the metamere to which the organ referred to

belongs.
a.
ac.

b.¢.7-8
b.c.c.2-33

b.mem.

b.t.m.a and b

¢.ar.

al. pe.
al pt.
e.b.m.233

endo.
ent.?-6
enta.?-%4
fe 6 and 7
far.
fbr.
bace

h.

anus.

anterior commissure of
brain.

anterior cornu of endo-
cranium.

abdominal
drites.

anterior ento-coxal nerve.

anterior intestinal branch.

alary muscles of heart.

anastomosing muscle
fibers of heart.

aortic arch.

appendages.

abdominal spines upon
edge of carapace.

anterior valve of heart.

base of muscle fiber of
heart.

branchial cartilages.

branchio-cardiac canals.

basement membrane of
heart.

branchio-thoracicmuscles.

collateral artery.

dorso-lateral plastro-enta-
pophysial muscle.

dorso-lateral plastro-
tergal muscle.

external branchial mus-
cles.

endocranium.

endochon-

entocoxites.

entapophyses.

foramina of endocranium.

frontal artery.

fore-brain.

ganglion cells in median
nerve of heart.

haemal branch of integu-
mentary nerve.

h.d.a and b
hn. 6
A.nm>™4

h.pr.

ht.
1.6.m.

2.€.M.

1m.

2.92.26

nt.
la.
la.
lam.

lan.
lar 29
hele

igi
ae

Z.é.
len.
Un.
l.ol.n.

lp.pr.

1.5.H.
mM.
MCN.
mM.e.
m.e.N.
mes.

M.ey 2.

mn 6

m.ol.n.

hepatic ducts.

haemal nerves.

haemo-neural muscles.

haemal processes of endo-
cranium.

heart.

internal branchial mus-
cle.

inter-entapophysial
cles.

inter-tergal muscle (enta-
pophysial slip).

intestinal nerves.

intestine.

labrum or rostrum.

levator ani (muscle).

longitudinal abdominal
muscles.

mus-

labral, or rostral, nerves.

lateral arteries.

lateral cornua of endocra-
nium.

lateral cardiac nerves.

longitudinal connective-
tissue strands of heart.

lateral eye.

lateral eye nerve.

lateral nerve.

left olfactory nerve.

latero-posterior process of
endocranium.

lateral sympathetic nerve.

mouth.

median cardiac nerve.

median eye.

median ento-coxal nerve.

meso-metasoma or abdo-
men.

median eye nerve.

mandibular nerves.

median olfactory nerve.

PATTEN AND

meso - plastral -entapophy-
sial muscle.

nephridia.

neural nerves.

nephridial opening.

occludor ani (muscle).

occipital ring.

oesophagus.

olfactory organ.

ostia of heart.

pericardium.

posterior ento-coxal nerve.

posterior intestinal
branch.

pericardial nerve.

post-oral commissures.

posterior process of endo-
cranium.

proctodaeum.

prosoma or cephalotho-
rax.

proventriculus.

pericardial sinus.

REDENBA UGH.
v.0l.n. right olfactory nerve.
r.0S. rudimentary ostia.
S.a. sphincter ani (muscle).
S.a.ar. superior abdominal artery.
S.C. segmental cardiac nerve.
stn. stomodaeal nerve.
isiz)iad semilunar valves.
tel. telson.

t.em.aandb extensor muscles of tel-

fm.
tp.m.a-c
Tisecae®
v.ar.

UC.

D.C.
Up.m.e*3

x.

7a and 7¢

son.

flexor muscles of telson.

tergo-proplastral muscles.

tendinous stigmata.

ventral artery.

ventral cord.

venous collecting sinus.

veno-pericardiac muscles.

root of supposed cardiac
nerve of fifth neuro-
mere.

four tergo-coxal muscles
of chelicerae.

muscles of chilaria.

STUDIES ON LIMULUS. 187

EXPLANATION OF PLATE VI.

“~~

Fic. 1. A view of the entire nervous system of Limulus from the neural side.
(About natural size.)

The carapace is represented as transparent, and all tissues which would obscure
the nerves and internal organs are left out of the drawing. The appendages have
been removed, but the outlines of the entocoxites (ev/.2*) have been sketched in
upon the left side to serve as landmarks and to show the relations of the nerves
to the thoracic appendages. The positions of the three sensory knobs are indi-
cated by the enclosed areas at their outer extremities. The positions of the
abdominal appendages are indicated by the external branchial muscles (ALES EN
the branchial cartilages (4.c.*"), the tendinous stigmata (¢.s."3), and the abdomi-
nal endochondrites (a.e.*%3). In the cephalothorax (/vos.) all the tergo-coxal
and plastro-coxal muscles have been dissected away, leaving the endocranium
(endo.) with the occipital ring (0c.7.) exposed. Upon the left side of the animal
one of the tergo-proplastral muscles (¢.4.7.a) is represented, and the branchio-
thoracic muscles (6...) are seen extending into the cephalothorax. The longi-
tudinal abdominal muscles (/.a.m.) are seen in the abdomen. Upon the right
side of the animal all the muscles have been omitted except the haemo-neural
muscles (.2.m.*%4). The last two haemo-neural muscles (4.7.2.3 and ™4) are
represented upon the left side also. The large entapophysis (eméa.7 "4 8) of the
cephalothorax and the smaller ones (e/a.>"4) of the abdomen are shown upon
the right side. At the base of the telson (¢e/.) the flexors (¢.m.) and extensors
(¢.e.m.b) of the caudal spine are represented as cut off near their insertions. The
anal muscles, sphincter ani (s.a.), levator ani (/.2.), and occludor ani (0.2.), and
their relations to the anus (a.) are shown in the same region.

The oesophagus (oe.) runs forward upon the neural side of the endocranium to
the proventriculus (fvov.). From this the intestine (éw¢.) passes posteriorly on
the haemal side of the endocranium, and emerges upon the posterior side of this
structure, whence it may be traced to the anus.

The brain lies upon the neural side of the endocranium, and the ventral cord
(v.c.) passes back through the occipital ring (0c.7.), haemal to the abdominal
endochondrites (a.e.4%). All of the neural nerves (7.7.11) are cut off, but the
haemal nerves (4.7.""®) upon the left side are represented entire, as are also the
nerves from the fore-brain (/d7.).

The first pair of neural nerves (z.72.') go to the chelicerae. The second to
sixth pairs go to the next five thoracic appendages, which are represented by the
entocoxites (evz.?). The seventh pair of neural nerves (7.7.7) go to the chilaria,
and the eighth pair (v.7.°) to the operculum. The two latter pairs pass through
the occipital ring. The neural nerves from the ninth to the thirteenth (v.7.9%)
arise from the abdominal ganglia and innervate the five pairs of gills.

From the fore-brain a median olfactory nerve (m.0/.x.) and two lateral ones
(7.0l.2. and r.ol.z.) pass forward to the olfactory organ; a median eye nerve
(m.ey.n.) passes anteriorly and haemally upon the right of the proventriculus
(frov.) to the median eyes; and a pair of lateral eye nerves (/.¢..) sweep around
the outer extremities of the entocoxites (evz.?4) to the lateral eye (/.¢.) upon the
haemal side of the lateral expansion of the carapace.

188 PATTEN AND REDENBAUGH.

The first haemal nerve, or lateral nerve (/..), follows the general course of the
lateral eye nerve, but continues posteriorly far back onto the neural side of the
abdomen.

The haemal nerves of the hind-brain (4.7.?°) radiate from the brain to the
margins of the prosomatic carapace, and each one passes anterior to the append-
age of its own metamere. The integumentary portions divide into haemal and
neural branches of which the haemal branches (4.) are cut off. Each haemal
branch gives off a small nerve, which turns back toward the median line upon the
haemal side of the body.

The haemal nerves (4.7.7 2°48) of the accessory brain pass through the occipi-
tal ring and out to the sides of the body between the operculum and the sixth
thoracic appendage. The seventh innervates the posterior angles of the cephalo-
thorax, the eighth the opercular portion of the abdomen.

The next five haemal nerves (4.7.97) arise from the five branchial neuromeres
and pass out anterior to the five pairs of gills to the sides of the abdominal cara-
pace and innervate the first five spines (a.s.7") upon the sides of the abdomen.

The first post-branchial nerve (4.7.'4) innervates the last abdominal spine
(a.s.™4); the second post-branchial nerve (4.2."°) and one branch of the third post-
branchial (4.7.%°) innervate the muscles of the telson and the posterior angles of
the abdomen; and the caudal branch of the third post-branchial nerve innervates
the telson.

Intestinal branches (z.7.°"°) arise from all the haemal nerves from the sixth to
the sixteenth and pass to the longitudinal abdominal muscles and to the intestine.
Those from the sixth and seventh neuromeres (z.7.° 2847) pass through foramina
in the endocranium.

Cardiac nerves (s.c.z.°-"3) arise from all the haemal nerves from the sixth to the
thirteenth. Those which arise from the seventh and eighth neuromeres (7.7.7 2848)
fuse together. Six of the cardiac nerves (s.c.z.8"3) communicate with a lateral
nerve, which has been called the lateral sympathetic nerve (/.s.7.). A branch (x.)
from the fifth haemal nerve (4.7.5) may also be a cardiac branch.

The lateral sympathetic nerve (/.5..) innervates the branchio-thoracic muscles
(4.2.m.). It receives in each metamere, from the eighth to the fourteenth, a branch
from either the haemal or the cardiac nerve.

Two post-cardiac nerves (s.c.#. 294 15) arise from the first two post-branchial
nerves and pass to the haemal side of the body, where they anastomose with a
branch from the last cardiac nerve (s.c.#."3) and innervate the extensors (¢.¢.7.4) of
the telson, and the epidermis posterior to the heart.

- oy ;
‘a -
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Rw he ' < :
ai 4 i cas. ne
= : : : ok x =
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i - x == _ (eal Bee Tye <4.) eee ae a “oo Vale
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a; =. i. 4 uf ? oe te oa ia te
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Journal of Morphology Vol. PU ME.
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STUDIES ON LIMULUS. 159

EXPLANATION OF PLATE VII.

Fic. 2. A portion of the cephalothorax, showing the brain and the relations
of the nerves to the tergo- and plastro-coxal muscles, sensory knobs, etc., in the
region of the entocoxites (seen from the neural side and magnified about one and
one-half times).

The plastro-coxal muscles (g*.>» © andg, 6a,b,e,f,andg, etc.) hide the whole of the
endocranium except the occipital ring (oc.v.) and the capsuliginous bars (4.c.’).

The outer portions of the entocoxites (ev¢.*®) are indicated with attached tergo-
coxal muscles (4° 4% 5» 1, andi, 6° 4d, i, andj, etc,),

The three areas enclosed by the outer portions of the entocoxites represent the
three sensory knobs. The base of one of the chelicerae, with the attached tergo-
coxal muscles (z) of that appendage, and the bases of the chilaria, with some of
the chilarial muscles (74 and 7¢), are also shown.

The four lobes of the nephridia (z.75), with connecting ducts and nephridial
opening (7.0), are represented lying between the plastro-coxal muscles of the
anterior and posterior sides of the second to fifth thoracic appendages.

' The brain lies neural to the plastro-coxal muscles; and the ventral cord (v.c.),
with the nerves from the accessory brain, passes out through the occipital
ring (oc.7.). The anterior commissure (a.c.), with the three rostral nerves (/a.z.), is
situated in front of the aperture through which the oesophagus passes. Four of
the posterior commissures are also seen.

Upon the left side of the animal the neural nerves (7.7.7) of the hind-brain
are cut off close to the brain to show the ento-coxal branches underneath. The
cheliceral nerves (z.z.') turn forward over the fore-brain (/47.). The chilarial
(7.2.7) and opercular (z.7.°) nerves proceed from the posterior side of the brain
through the occipital ring (0c.r.).

From the fore-brain the three olfactory nerves (o/.x.) pass forward to the olfac-
tory organ (0/.0r.); a portion of the median eye nerve (m.ey.v.) is also represented;
and the lateral eye nerve (/.e.7.) sweeps anteriorly around the entocoxites (ezz.”) to
the lateral eye (/.e.).

The delicate lateral nerve (/..), or first haemal nerve, runs parallel to the
lateral eye nerve and at one point fuses with the second haemal nerve (4.z.?). It
is continued posteriorly onto the abdomen.

The other haemal nerves (4.7.7*) radiate from the haemal side of the brain
and pass to the sides of the carapace, each one anterior to the appendage of its
own metamere. Each divides into a haemal (4.) and a neural branch, and in the
four anterior nerves the haemal branch passes haemal to the lateral eye nerve. A
small branch turns backward toward the median line and innervates a portion of
the epidermis of the haemal side of the carapace. The haemal nerves (4.7.7 "4 ®)
of the accessory brain pass posteriorly through the occipital ring and out toward
the sides of the body.

Intestinal branches (é.7.°*) arise from the sixth, seventh, and eighth haemal
nerves, but the one from the eighth (¢.7.°) is concealed by the overlying chilarial
muscles. :

Cardiac branches (s.c.7.°*) also arise from the sixth, seventh, and eighth haemal
nerves; those from the seventh and eighth (¢.7.74"48) fuse together, and the

190 *“PATTEN AND REDENBAUGH.

cardiac root (s.c.7.°) of the eighth gives a branch to the lateral sympathetic nerve
(Z.s.2.). A branch («.), which may be either a cardiac or an intestinal nerve,
arises from the fifth haemal nerve (4.7.°).

In the third, fourth, and fifth appendages there are three ento-coxal branches
of the neural nerve ; an anterior one (a.e.z.), innervating the muscles of the ante-
rior side of the entocoxite and the anterior sensory knob; a posterior one (#.e.7.),
innervating the muscles of the posterior side of the entocoxite and the posterior
sensory knob; and a median one, going to the median (m.¢.2.) sensory knob.
The median one arises from the neural nerve at some distance from the brain, and
hence is represented with its proximal end cut off.

In the second appendage only the anterior and posterior ento-coxal nerves
(a.e.n.2 and f.e.2.7) have been found.

In the sixth appendage the median sensory knob is replaced by the flabellum,
and the median ento-coxal nerve (m#.e.2.°) becomes the flabellar nerve. This is
also cut off at its proximal end.

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STUDIES ON LIMULUS. IgI

EXPLANATION OF PLATE VIII.

Fic. 3. A Sagittal section of Limulus, showing the nerves and principal
organs in relief (seen from the right side, somewhat larger than natural size).

All the prosomatic appendages (a/.?°), except the chelicera (¢/.') and chilarium
(ap.”) of the right side, are omitted. The operculum (a#.°) and the five gills (af.9*)
are represented in the abdominal region.

All the muscles are also omitted except the fibers running from the occipital
ring (oc.r.) to the posterior side of the oesophagus (ee.), the chilarial muscles (7°),
the sphincter ani (s.a.), and the levator ani (/.a.).

The endocranium (ezdo.), with the occipital ring (0c.r.) and the capsuliginous
bar (4.c.”), is seen from the side, and the positions of the abdominal endochon-
drites (a.e.°"4) are indicated.

The mouth (#.) leads into the oesophagus (0e.), which passes through the
brain and forward to the proventriculus (/vov.). A constriction, which marks the
position of the pyloric valve, separates the proventriculus from the intestine (z77.)
which passes posteriorly to the anus (a.). A pair of hepatic ducts (4.d.4 4nd b)
enter the intestine opposite the endocranium.

The heart (4z.), surrounded by the pericardial sinus (/.s.), lies haemal to the
intestine. The pericardium (/.) is shown between the heart and the intestine.
The ostia (05.673) of the heart and the origins of the four lateral arteries (/.ar.°9)
are indicated upon the sides of the heart; the frontal artery (fa7.) and the aortic
arches (ao.a.), curving down to the brain, arise from the anterior end of the heart;
the superior abdominal artery (s.a.av.), and the opening of the collateral artery
into it, are seen at the posterior extremity of the heart.

The brain surrounding the oesophagus is seen in side view upon the neural
side of the endocranium. The ventral cord (v.c.) passes through the occipital
ring (0c.r.) into the abdominal region. The anterior commissure (a.c.), with the
three rostral nerves (/a..) innervating the rostrum, or labrum (/a.), and four of
the post-oral commissures are represented.

The cheliceral nerve (7.7.!) with the small external pedal branch is shown
entire, but the next five neural nerves (7.7.7) are cut off. The chilarial nerve
(2.2.7), the opercular nerve (z.7.°), and the five branchial nerves (7.7.97) enter
their respective appendages, the two former (.7.7 274°) passing through the
occipital ring.

From the fore-brain the three olfactory nerves (0/.2.) pass anteriorly to the
olfactory organ (o/.or.); the median eye nerve (m.ey..) passes to the right of the
proventriculus (frov.) to the median eyes (m.e.); the lateral eye nerve (/.c.7.)
passes forward and is represented as cut off opposite the proventriculus. The
lateral nerve (/.7.), or first haemal nerve, is also cut off just beyond the point
where it fuses with the second haemal nerve (4.7.”). The stomodaeal nerve
(st.2.) ramifies over the oesophagus and proventriculus.

The second haemal nerve (4.7.”) passes to the anterior extremity of the cara-
pace; its haemal branch is cut off opposite the proventriculus. An intestinal branch
(é.2.?) arises from near its base and disappears behind the anterior cornu of the
endocranium.

The next three haemal nerves (4.7.35) are cut off close to the brain, and the

192 PATTEN AND REDENBAUGH.

following nine haemal nerves (4.7.°"4) are cut off beyond the cardiac branches.
The second post-branchial nerve (/.7.') is cut off beyond its branch to the telson
muscles. Both branches of the haemal nerve (4.7."°) are represented extending
into the telson (¢e/.).

The intestinal nerves (7.7.°7°) are shown arising from the haemal nerves and
entering the intestine. Those from the sixth and seventh neuromeres (2.7.° 27d 7)
pass through foramina in the endocranium and communicate with a plexus in the
longitudinal abdominal muscles before entering the intestine. The eighth passes
just posterior to the endocranium and joins the same plexus. Those from the
first four branchial neuromeres (7.7.97) arise very near the abdominal ganglia and
are double in their origins, the anterior branches joining the above-mentioned
plexus, and the posterior branches entering the intestine. The thirteenth, four-
teenth, and fifteenth are somewhat complicated in their relations and will be
described to better advantage under Fig. 4. The fifteenth extends far back
towards the rectum and anastomoses with the sixteenth (¢..'), which arises from
the caudal branch of the sixteenth haemal nerve (4.7.%) and innervates the
rectum and anal muscles.

The segmental cardiac nerves (s.c.7.°"3) arise from the haemal nerves of the
sixth to the thirteenth neuromeres respectively. The most anterior one (s.c.z.°)
passes haemally to the inter-tergal muscles and epidermis in median line, haemal
to the heart, but the connections with the cardiac plexus have not been made out.
The next two (s.c.z.7 248) fuse to form a large nerve, which likewise passes
haemal to the heart, to the inter-tergal muscles, and epidermis, but has not been
observed to connect directly with the cardiac plexus. It, however, sends poste-
riorly a branch, the pericardial nerve (/.7.), which in turn gives a branch to each
of the cardiac nerves of the branchial neuromeres (s.c.7z.9*5), and then continues
onward to the posterior margin of the abdomen. ‘This nerve lies in the epidermis
haemal to the heart. The median and lateral cardiac nerves (m.c.z. and /.c..) are
seen upon the walls of the heart. The five cardiac nerves (5.c.7.%73) from the
branchial neuromeres pass haemal to the heart, in the epidermis, to the median line,
and dip down to the median nerve (7.c.7.) of the heart opposite the last five pairs
of ostia (0.5.73). They communicate with the pericardial nerve (f..) and also
with the lateral sympathetic nerve (/.s.7.).

Two post-cardiac nerves (s.c.7.!4 "4 15) pass from the first and second post-
branchial nerves (4.7.4 494 15) to the epidermis posterior to the heart.

The last cardiac nerve (s.c.7."3) and the two post-cardiac nerves (s.c.7.14 "4 15)
give off branches which anastomose with each other and innervate the extensors
of the telson.

The lateral sympathetic nerve (/.5.7.) receives branches from all the neuromeres
from the eighth to the fourteenth, either through the cardiac nerves or the haemal
nerves, and innervates the branchio-thoracic muscles, extending with these far
into the cephalothorax.

Fic. 4. This drawing is intended to show the intestinal nerves from the
haemal side, and is made to the same scale as Fig. 3. Most of the heart and a
large portion of the intestine have been removed, leaving exposed the haemal
sides of the endocranium, ventral cord, muscles inserted on the endocranium, and
the plexus of intestinal nerves.

The anterior end of the heart (4¢.) with the aortic arches (a.a.) and frontal

STUDIES ON LIMULUS. 193

artery (fav.), the anterior portion of the intestine (¢¢.) with proventriculus ( fvov.)
and oesophagus (0e.), and the rectum, or proctodaeum (/voc.), with the sphincter
ani (s.a.) and levator ani (/.a.), are left in position.

The muscles are dissected away from the left side of the endocranium (edo.),
so that the various parts are exposed to view, vzz., anterior cornu (a.cor.), lateral
cornu (/.c.), haemal processes (4.f7.), latero-posterior processes (/.f.47.), posterior
process (/.f7.), capsuliginous bars (é.c.”), and the foramina (/-° and /’).

Some of the abdominal endochondrites (a.e.*%) with the attached haemo-
neural muscles (/.7.m.*™4) are also seen.

To the anterior cornu (a.cor.) of the endocranium are attached three tergo-pro-
plastral muscles (¢.4.7.a-c); to the haemal process (4.f7.), the dorso-lateral plastro-
tergal muscles (d./.f.¢.) and the dorso-lateral plastro-entapophysial muscle (d./.f.e.) ;
from the side of the posterior process a large meso-plastro-entapophysial (.f.¢.)
muscle goes to the large entapophysis (e¢a.7 848); longitudinal abdominal
muscles (/.a.m.) go from the endocranium to each of the abdominal entapophyses
(enta.9*4) ; and inter-entapophysial muscles (7.e.7.) go from the first entapophysis
(enta.7 294 8) to the next four (evza.9?).

Two veno-pericardiac muscles (v.f.7.° *"17) are attached to the sides of the
endocranium and the bases of the remaining six (v.f.m.*"3) are seen amongst the
longitudinal abdominal muscles.

The ventral cord (v.c.) emerges from behind the endocranium and presents to
view the abdominal ganglia with the roots of the neural and haemal nerves poste-
rior to the eighth neuromere. The neural nerves (7.7.9') are cut off. The
haemal nerves upon the left are cut off, and those upon the right are all concealed
from view except the last four (4.7.'>"°), but their bases with the origins of the
intestinal nerves are exposed.

The plexus of intestinal nerves upon the abdominal muscles is in life largely
hidden from view within these muscles. All the branches which go to the intes-
tine are represented as cut off at the points where they enter the organ.

The two anterior intestinal nerves (¢.7.° and z.z.”) come through the foramina
(f° and f”), and these with the next one (z.7.*) join the plexus within the muscles
upon the haemal side of the endocranium. From this plexus numerous branches
run forward to the anterior part of the intestine. =

In the abdominal region some regularity about the branching of the intestinal
nerves has been observed. In the ninth, tenth, and eleventh neuromeres two
intestinal branches (q.7.z. and 7.7.7.) arise; the anterior one (a.7.7.) joins the plexus
in the abdominal muscles; the posterior one ( 7.2.7.) gives a branch to the haemo-
neural muscle (4.7.7.) of its own neuromere and a branch to the intestine.

In the twelfth neuromere the posterior branch anastomoses with the plexus in
the abdominal muscles, sends one branch to the haemo-neural muscle (4.7.7.1),
whence one proceeds to the intestine. It also gives off another posteriorly
which anastomoses with a branch from the fourteenth haemal nerve, and then
proceeds to the intestine.

From the thirteenth haemal nerve (4.7.') only one intestinal branch (z.7.")
arises, and this enters the intestine by two branches. «

From the fourteenth haemal nerve (4.7.'4) upon the left side an intestinal
nerve (7.7.4) arises and, passing a long distance posteriorly, enters the intestine
by several branches. From the fourteenth haemal nerve upon the right side three

194 PATTEN AND REDENBAUGH.

the first one anastomoses with the twelfth intestinal nerve; the

nerves arise ;
bdominal muscles; and the third enters the

second enters the longitudinal a
intestine.
From the fifteenth haemal nerve (/.7."5) an intestinal branch (z.7.'5) arises,

which goes posteriorly close to the intestine, gives several branches to this organ,

and then anastomoses with the intestinal branch (z.2.%°) of the last neuromere.
The last intestinal branch (¢.7."®) innervates the rectum and anal muscles (/.a.

and s.d.).

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196 PATTEN AND REDENBAUGH.

EXPLANATION OF PLATE IX.

Fic. 5. The heart of Limulus, with adjoining organs and nerves, seen from
the haemal side (drawn to same scale as Figs. 3 and 4).

The haemal side of the carapace has been stripped off and the epidermis and
median inter-tergal muscles removed in order to show more clearly the heart
and arteries. The nerves, however, lying within these omitted portions are
represented.

The heart (#2) lies in the pericardial sinus, which is indicated by the shaded
area upon each side of the organ. Eight pairs of ostia (os.°7) are seen upon the
haemal side of the heart, and the median (#.c.z.) and two lateral cardiac nerves
(Z.c.z.) are indicated. The striated appearance of the walls of the heart is due to
the longitudinal strands of connective tissue.

Upon the left side of the heart the alary muscles (a/.m.*') are represented,
but upon the right they are omitted in order to show the four lateral arteries
(/.ar.°°) and the positions of the collateral arteries (c.a7.), which unite posterior
to the heart to form the superior abdominal artery (s.a.av.). Another lateral
artery (/.av.5) sometimes arises from the anterior end of the heart in front of the
aortic valve. Upon the outer sides the collateral arteries give off numerous
branches to the muscles and haemal side of the body, and upon the median sides
they give branches to the intestine, the superior intestinal branches (s.7.av.%). The
aortic arches (ao.a.) and frontal artery (far.), running over the proventriculus
(grov.), proceed from the anterior extremity of the heart.

Five pairs of branchio-cardiac canals bring blood to the pericardial sinus; the
first of these is formed of two canals, one (4.c.c.*) from the operculum and the
other (6.c.c.9) from the first gill. The remaining four pairs of branchio-cardiac
canals (4.c.c.1~™) bring the blood from the last four pairs of gills.

The seven pairs of entapophyses (ez¢a.7"™4) are represented, and the prin-
cipal muscles attached to the haemal side of the carapace; three tergo-proplastral
muscles (¢.f.7.4-c), two slips of the branchio-thoracic muscles (4.4.2.2 "4 >), the
dorso-lateral-plastro-tergal muscle (d./.4.¢.), and a slip of the inter-tergal muscle (z.77.)
attached to the first entapophysis (ezfa.7 "4 8) are represented in the cephalo-
thoracic region; in the abdominal region are seen the haemal ends of the seven
pairs of haemo-neural muscles (4.7.m.*"4) and the six external branchial muscles
(e.6.m.°3) of the right side, the levator ani (/.a.) and the extensors (é¢.m.4 2nd b)
of the telson. One slip of the extensor (¢.¢.m.a2) is attached to the last three
pairs of entapophyses (ezza.%*"4).

The end of the median eye nerve (m.ey.z.) appears in front of the proven-
triculus.

Seven pairs of segmental cardiac nerves (s.c.2.°') and two post-cardiac nerves
(s.c.2.4 221 15) come from the neural side of the body and pass up into the epider-
mis haemal to the heart. The most anterior one (s.c.7.°) supplies the omitted
inter-tergal muscle, and also a portion of the epidermis. The next one (5.c.7.7 2°48)
is formed of the fused branches of the seventh and eighth neuromeres; this
also innervates the above-mentioned inter-tergal muscles and sends branches to
the epidermis in the median line; one branch, the pericardial nerve (/.%.), goes
posteriorly in the epidermis, and gives a branch to each of the branchial cardiac

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200 PATTEN AND REDENBAUGH.

EXPLANATION OF PLATE X.

Fic. 9. A portion of the cardiac plexus of Fig. 8 enlarged 4o diameters.

The masses of ganglion cells (g.c.) in the median nerve (7.c.7.) are more dis-
tinct, and the courses of the individual fibers in the plexus and lateral nerve (/.c.x.)
are more apparent. Two of the ostia (os.7 24 8) are represented.

Fic. 10. A small portion of the median cardiac nerve (7.c.7.), enlarged 500 times.

The individual nerve fibers and the ganglion cells (g.c.), with their processes,
are easily made out.

FIGs. 11 AND 12. The brain of Limulus from the neural and haemal sides,
respectively (enlarged about 3 diameters).

The enveloping arterial sheath has not been removed. The anterior commis-
sure (a.c.) with the three rostral nerves (/.a.z.) is seen in Fig. 11, behind the fore-
brain (f.474, and four of the post-oral commissures (/.0.c.15) can be made out
posterior to the central canal through which the oesophagus passes. The first
post-oral commissure (/.0.c.") is separated from the others, and is much longer as
it passes haemal to the oesophagus.

Three olfactory nerves (o/.7.) arise from the anterior side of the fore-brain, and
a median eye nerve (7.ey.7.) comes through the arterial sheath a little to one side
of the median line and near the haemal surface. From the haemal side of the
fore-brain arise the two large lateral eye nerves (/.e.7.) with ganglionated bases
(Fig. 12).

Just back of the fore-brain upon the neural side, the cheliceral nerves (z.7.',
Fig. 11) arise and give off, near their bases, from one to three small nerves to the
tergo-coxal muscles of the chelicerae.

Five more pairs of large neural nerves (7.7.?°) radiate from the circum-
oesophageal collar to the next five pairs of thoracic appendages. Each of these
gives off on the neural side a mandibular branch (m.z.2°) and upon the haemal
side several ento-coxal nerves. The typical number of ento-coxal nerves is three;
an anterior (a.e.7.3), a posterior (/.e.7.3), and a median (m.e.7.3) ento-coxal nerve.
From the second haemal nerve (%.7.?) four ento-coxal branches are given off, in
some cases; but two of these may be regarded as branches of the anterior and
posterior ento-coxal nerves; the median ento-coxal nerve has not been found. In
the case of the sixth neural nerve (v.7.°) the median ento-coxal nerve (m.¢.1.°) is
much enlarged and becomes the flabellar nerve.

The chilarial (7.7.7) and opercular nerves (7.7.*) are much smaller than the
other neural nerves, and arise from the posterior side of the brain (Fig. 11)
neural to the ventral cord (z.c.).

From the haemal side of the brain (Fig. 12) the delicate lateral nerves (/.z.), or
first pair of haemal nerves, arise just back of the lateral eye nerves (/.¢.7.).

The next seven pairs of haemal nerves (4.7.7) radiate from the circum-
oesophageal collar. Of these the anterior pair (4.2.7) are somewhat larger than
the others and give off close to the brain a small nerve (¢.7.?) which innervates
the tergo-proplastral muscles.

The posterior three pairs (4.7.°°) give off intestinal nerves (7.7.°*), two of
which (2.7. 294 7) go through foramina in the endocranium.

A pair of stomodaeal nerves (s¢.z.) arise from small ganglia upon the inner
side of the circum-oesophageal collar. Sometimes small nerves are given off to
the oesophagus from the stomodaeal ganglia.

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STUDIES ON LIMULUS. 197

nerves (s.c.7."3). The pericardial nerve also gives a branch to the inter-tergal
muscle (#.7.) which is attached to the first entapophysis. These two anterior
nerves (s.c.7.° and s.c.m.7"48) have not been observed to connect with the cardiac
plexus.

The cardiac nerves (s.c.7.2%3) from the five branchial neuromeres send numer-
ous branches to the epidermis, and also branches to the median cardiac nerve (7.c.7.)
opposite each of the last five pairs of ostia (os.7"). There is considerable varia-
tion in the arrangement of the nerves in the posterior portion of the haemal
region. Moreover, the branches are very numerous and very fine, so that many of
them are necessarily missed in dissection. Some of the branches are continued
posteriorly to the margin of the carapace.

The two post-cardiac nerves (s.c.7.142"415) send branches in the epidermis to
the haemal line. Branches from the last cardiac (s.c.z.'3) and from the two post-
cardiac nerves (s.c.7.142"4 15) anastomose, and innervate one of the slips of the
extensor (4.2.7.4) of the telson. Other branches of the last post-cardiac nerve
innervate the other extensor (¢.e..b) and the epidermis of the posterior margin of
the abdomen.

Fic. 6. A vertical section through the abdominal region in the ninth and tenth
neuromeres showing the course of one of the cardiac nerves (from the anterior
side, about twice natural size). :

The left half of the drawing represents the posterior portion of the ninth neu-
romere, and the right half the anterior portion of the tenth neuromere.

The heart (4z.) is seen in cross-section showing a central lumen surrounded by
a layer of anastomosing muscle fibers; the median and lateral cardiac nerves
(m.c.m. and /.c.z.) are shown in the three angles of the heart.

Upon the right, one of the alary muscles (a/.7.'°) runs from the lateral angle
of the heart to the pericardium.

The pericardial sinus (/.s.) surrounds the heart and the branchio-cardiac canal
(6.c.c.7°) is seen coming from the upper portion of the second gill (ap.").

A pericardial membrane ( 7.) bounds the pericardial sinus upon the neural side.
It is attached to the entapophysis (e77¢a.9) upon the left and to the branchial carti-
lage (4.c.7°) upon the right. The veno-pericardiac muscles (v.f.m.'°) are attached
to it, and the collateral arteries (c.av.) are found in close connection with it.

The intestine (zzz.) lies haemal to the ventral cord which is enclosed in the
ventral artery (v.ar.).

Neural to the ventral cord is the abdominal endochondrite (a.e.9) which fur-
nishes attachment for the haemo-neural muscles (4.7.m.°) and the internal branchial
muscles (2.4.7.9). The external branchial muscles (e.4.7.9 284 1°) are also seen,
and the longitudinal abdominal muscles (/.a.m.) and branchio-thoracic muscles
(4.¢.m.) are cut across. The veno-pericardiac muscle (v.f.77.'°) is seen attached to
the pericardium, and spanning the venous-collecting sinus (v.c.s.) at its neural end.

An abdominal ganglion with its neural (7.7.9) and haemal nerves (4.7.°) is
represented. The neural nerve and the integumentary portion (zz.7.°) of the
haemal nerve are cut off.

The intestinal nerves (7.7.9) arise from the haemal nerve close to the ganglion;
the anterior branch (a.z.z.) goes to the longitudinal abdominal muscles (/.a.7.),
and the posterior one (/.7.z.) goes to the haemo-neural muscle (4.7.7.9) and to
the intestine.

The cardiac branch (s.c.7.9) arises opposite the branchio-thoracic muscle, and

nd

I 98 PATTEN AND REDENBAUGH.

gives a branch to the lateral sympathetic nerve (/.s.7.), and then continues through
the pericardium to the areolar tissue and epidermis haemal to the heart. After
giving off numerous branches to the epidermis, it dips down in the median line to
the median cardiac nerve (#.c..).

Fic. 7. A cross-section of one corner of the heart of a young Limulus (mag-
nified 100 times).

It shows the three layers of the heart; the layer of longitudinal connective-
tissue fibers (/.c.s.) is seen in cross-section upon the outside of the hyaline base-
ment membrane (é.mem.), and the layer of muscle fibers (a.m.f.) is seen upon the
inside. These are cross-striated and anastomose freely. Their bases are expanded
where they are attached to the basement membrane.

Fic. 8. Heart of a young Limulus (5 in. long). It has been slit open along
the neural side, spread out and viewed from the interior (magnified 10 times).

The specimen, from which the drawing was made, was treated by Lowits’s gold
chloride method, which macerated off the muscles, leaving the nerve plexus intact.

The eight pairs of ostia (0s.°") are indicated, and also the rudimentary ostia
(v.os.) anterior to the aortic valve (a.v.). The two aortic arches (ao.a.) are slit
open. The frontal artery, with its opening in front of the aortic valve, is seen
between the aortic arches.

The lateral arteries (/.av.°°) emptying into the collateral artery (c.av.) and the
superior intestinal branches and superior abdominal artery (s.a.ar.) are also repre-
sented. The anterior lateral artery (/.a7.°) is cut open to show the two semi-lunar
valves (s.v.°).

The median cardiac nerve (#.c.2.), with its blackened masses of ganglion cells,
gives off branches opposite each pair of ostia to the plexus of nerves which com-
municates with the lateral cardiac nerves (/.c.7.).

No. I.] THE EGG OF LIMAX AGRESTIS. 203

the archiamphiaster stage the astrosphaeres of the egg of
Limax agrestis conform very closely to the type of astro-
sphaere described by Van Beneden and by Boveri for
Ascaris.

The centrosphere has a distinct peripheral zone, the “ cor-
tical’”’ zone of Van Beneden, or ‘‘archoplasm”’ of Boveri,
which stains intensely black with iron-haematoxylin. When
stained with Lyons blue and borax-carmine it becomes in-
tensely blue. This outer zone is often very sharply marked
off from the rest of the cytoplasm, although the inner ends of
the astral rays are in direct contact with its periphery (Pl. XI,
Figs: 2,3, and 6). On its mner circumference the outer
‘cortical’ zone of the centrosphere is marked off with equal
distinctness from a perfectly clear structureless zone, the
“medullary” zone of Van Beneden. Sometimes, however,
the concentric layers of the centrosphere are less sharply
outlined than in Fig. 2, appearing as in Figs. 4 and 5. Figs.
4 and § represent sections through an egg that was killed
immediately after it was laid. The egg is of approximately
the same age as that represented in Fig.6. The centro-
sphere in Figs. 4 and 5 is composed of four concentric rings,
which are alternately dark and light, as in the centrospheres
formed by only two rings. As the number of rings increases,
the boundaries between them become less sharply defined.
Here, as in Pl. XI, Figs. 2 and 3, the astral rays diverge from
the periphery of the outermost dark ring or zone.

Within the clear inner zone of the centrosphere there is
a deeply staining central mass, consisting of several granules
that form the centrosome or centrosomes. These granules are
often grouped irregularly, as in Fig. 6, but sometimes they are
arranged so as to appear like two short thick rods in the form
of dumb-bells (Pl. XI, Figs. 2 and 5). In haematoxylin the cen-
trosomes stain almost black; in Lyons blue and borax-carmine
they stain blue. The centers of the astrosphaeres in the archi-
amphiaster sometimes fail to show deeply staining central gran-
ules, but this is probably due either to the method of preser-
vation or to some accident in staining, since the centrosomes
are usually present even at a much earlier period. The inner

204 BYRNES. [VoL. XVI.

clear zone of the centrosphere, with its central granule or
granules, corresponds to the centrosome of Boveri (Pl. XI,
Figs. 2, 3, and 6).

2. Ovarian Eggs.

I have not attempted the study of the ovogenesis of Limax.
My attention was directed to the ovarian eggs in the hope of
finding in them the origin of the granular centrosomes of the
archiamphiaster ; for it was evident that the centrosomes ap-
peared before the eggs were laid. I dissected a number of
individuals and found several of them with eggs arranged in a
row along the oviduct. The capsules had already been secreted
about these eggs, which contained a well-formed amphiaster.
Other individuals were dissected and were found to contain
eggs in the lower part of the duct of the albuminous gland;
these eggs had no capsules, but each egg contained an amphi-
aster. It was evident then that the origin of the centrosome
must be looked for at a still earlier period.

I then sectioned the ovo-testis of Limax and found several
eggs in the archiamphiaster stage. These eggs were in the
lumen of a follicle that was filled with spermatozoa. They
showed nothing of the origin of the centrosome, however, for
the spindle was already fully formed (Pl. XI, Fig. 2). In Fig. 2
no trace of the germinal vesicle remains, the chromatin being
already in the equatorial plate stage. Even at this early period
the centrospheres are precisely like those of the newly laid
ova; they have a clear central (“medullary’’) zone, in which
two oblong deeply staining centrosomes are distinctly shown,
and a periphcral (‘cortical’’) zone which stains almost black
with iron-haematoxylin.

The peripheral zone (‘‘archoplasm”’ of Boveri) shows a very
strong affinity for Lyons blue when stained with this dye and
borax-carmine. Pl. XI, Fig. 1, shows a section of an ovarian
egg in which the center of the aster is still undifferentiated.
There is no distinct centrosphere or centrosome present, and
the rays are short and relatively few in number. Unfortu-
nately the series of sections through this egg is incomplete, so
that I cannot make any statement in regard to the condition

Nowa: THE LEGG OF EIMAX AGRESTIS. 205

of the nucleus, or in regard to the relation of the aster to the
germinal vesicle.

Sections through the hermaphrodite gland show many large
ovarian eggs which are still in contact with the walls of the
follicles, and in which the germinal vesicles are evidently in
various stages of breaking down. Even in the largest of these
ova the germinal vesicle still retains a distinct membrane
throughout the greater part of its circumference, and the large
nucleolus, which is characteristic of all of the earlier stages of
ovarian eggs, still persists.

In none of these ova is there any radiate structure present,
or any evidence of the presence of a centrosome. The germi-
nal vesicle must be lost and the archiamphiaster formed after
the ovum becomes detached from the wall of the follicle ; but
as relatively few ovarian eggs reach maturity simultaneously
in Limax, the different steps in the process of ripening are
extremely difficult to find. Many of the younger ovarian eggs
are oval in outline, with a large surface of attachment to the
follicular wall. The structure of these ova is much denser
immediately around the germinal vesicle than at the periphery
of the cell. This dense region stains more deeply with haema-
toxylin than does the rest of the cytoplasm, and with Lyons
blue and borax-carmine it takes a deep blue stain. The
chromatin of the ovarian eggs also stains blue, as does likewise
the chromatin in the head of the spermatozoa that are still in the
follicles. These facts agree with Calkin’s observations on the
color reactions of chromatin and yolk-nuclei in the ovarian egg
of Lumbricus. Beyond the fact that the chromatin and the
cell substance surrounding the nucleus stain alike, however, I
have no observations that indicate that the deeply dyed mass
around the nucleus has been derived from the chromatin.

While the younger eggs are still oval in outline, the proto-
plasm is peculiarly stringy and irregular in appearance. Numer-
ous bodies which appear circular in section, and which stain
more deeply than the surrounding cytoplasm, are present at
opposite poles of the long axis of the egg; there are also
numerous clear spaces in the cytoplasm which resemble vacu-
oles. What the significance of these structures is, I have not

206 BYRNES. [VoL. XVI.

been able to determine ; they vary in size, number, and position,
as do also the deeply staining bodies which probably correspond
to the yolk-nuclei of other forms. As the ovarian eggs enlarge
and leave the walls of the follicles, these structures disappear
and the cytoplasm becomes more uniform in appearance.

3. First Maturation Spindle. Extrusion of the first
Polar Body.

As long as the archiamphiaster remains at the equator of
the egg, the two polar asters are of the same size and retain the
appearance already described. Within half an hour after the
eggs have been deposited the archiamphiaster, or the first
maturation spindle, as it will be called henceforth, assumes a
radial position at the upper pole of the egg. The arrangement
of the centrosphere in concentric rings often persists until after
the extrusion of the first polar body, as shown in Pl. XI, Figs.
g and 10. Sometimes, however, before the first polar body is
extruded, the centrosphere assumes a very different appearance.
The two distinct zones that are so characteristic of the stage
of the archiamphiaster gradually fade away, and the center of
the aster now appears as a deeply stained, homogeneous body,
surrounded by a less deeply staining zone from which the
astral rays diverge (Pl. XI, Figs. 7 and 8).

The astral rays in the eggs of Limax are formed by filaments
which can be traced far out into the cytoplasm in almost straight
lines (Pl. XI, Figs. 3, 7, and 8). During the stage of the first
maturation spindle, Mark has found that the rays of the periph-
eral aster are slightly bent so as to form a spiral. I have
never seen spiral asters in connection with the first maturation
spindle in Limax agrestis.

The different forms under which the centrosphere of the
first maturation spindle appears (Pl. XI, Figs. 7-9) may be
due to different lengths of time elapsing between the forma-
tion of the archiamphiaster and the extrusion of the first polar
globule. The formation of the first polar body is preceded
by a slight flattening of the upper pole of the egg. As the
distal aster approaches the periphery of the egg, the rays are

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No. 1.] THE EGG OF SIVMAX AGRESTIS. 207

at first pressed sideways against the upper pole (Pl. XI, Fig. 7),
and as the polar globule is protruded, the rays are finally
massed together to form a ‘“‘ Zwischenkorper”’ (Pl. XI, Fig. 8),
which for a long time marks the place of extrusion of the first
polar globule. The process of formation and extrusion of the
polar body requires nearly two minutes for its completion.
Soon after its separation from the egg the first polar globule
collapses so that its position is marked only by a shriveled
membrane at the outer periphery of the egg. Mark has de-
scribed a similar behavior of the first polar globule in Limax
campestris.

4. Formation of the Second Maturation Spindle. Extrusion of
the Second Polar Body.

After the extrusion of the first polar globule the astro-
sphaere that remains in the egg undergoes a series of changes
that are shown in Pl. XI, Figs. 1o-14. The “cortical’’ and
‘‘medullary’’ zones of the centrosphere become less sharply
defined as the sphaere continues to enlarge (Pl. XI, Figs. 12
and 14). The astral rays, which often persist for some time
after the extrusion of the first polar globule, soon begin to
shorten as if by contraction, so that the centrosphere is seen
at one time surrounded by rays of uniform length, as in Pl. X1,
Fig. 12, and again by still shorter rays that terminate at their
inner ends at the periphery of the centrosphere, in a circle
of granular thickenings or ‘“microsomes”’ (Pl. XI, Fig. 13).
Within the circle of microsomes the rays are continued cen-
tripetally as extremely delicate fibers that extend through the
“‘cortical”’ zone to the “medullary ” zone, which now appears
slightly granular.

When the centrosphere reaches its maximum size it becomes
uniformly very finely reticular, so that it appears almost gran-
ular and at the same time loses its strong affinity for staining
reagents (Pl. XI, Fig. 14, a, 4,c). In the center of the sphaere
the centrosomes are often distinguishable as two tiny, deeply
staining granules, as shown in Pl. XI, Figs. 14 @ and 14 ¢.
These centrosomes are evidently the same as those seen in the

208 BYRNES. (Vou. XVI.

archiamphiaster, but their form has changed. Between the
centrosomes a light region (the “centrodesmose”’ of Heiden-
hain) sometimes marks the position of the central spindle, but
at the time of the first appearance of the central spindle there
are no distinct fibers visible (Pl. XI, Fig. 15).

While still within the sphaere the centrosomes may become
surrounded by a distinct peripheral zone that stains less deeply
than the surrounding matrix. Sometimes no centrosomes are
visible within the centrosphere at this stage, as in Pl. XJ;
Fig. 12; they may, however, be present. Soon after their
separation the centrosomes in the centrosphere become the
focal points from which new astral rays diverge (Pl. XI,
Fig. 16). At the same time the rapidly fading rays of the old
aster can be seen terminating in the peripheral ring of micro-
somes that surround the newly formed spindle. The rays of
the new asters elongate rapidly and reach far out into the cells,
so that at the equator of the spindle the rays from opposite
poles meet and cross one another.!

At first the chromatin lies entirely outside the newly formed
spindle, on the periphery of the centrosphere, where it was
left after the extrusion of the first polar body. In the interval
between the extrusion of the first and second polar globules no
nuclear membrane is formed. These relations are shown in
Ply. XI, Figs. 11, 14, and 15. As the’ second maturation
spindle enlarges and the sphaere in which it is first formed
fades away, the chromatin comes to lie on the upper surface of
the central spindle, which at first forms at right angles to the
polar axis of the egg. Finally the chromatin is drawn into the
equator of the spindle, where it forms the equatorial plate. As
the poles still continue to separate, the spindle sinks into the
egg and rotates through an angle of 90 degrees. After it has
reached its maximum growth (Pl. XI, Fig. 19), the second
maturation spindle comes to rest at the upper pole of the egg.
It is worthy of note, in the egg of Limax, that during the forma-

1 This account of the formation of the rays of the second maturation spindle
differs from Mead’s account of the formation of the aster in the second maturation
spindle in Chaetopterus, where the rays of the old aster are merely focussed on
new centrosomes,

No. 1.] DHE GG OPS ELMAX AGRE STIS. 209

tion and rotation of the second polar spindle the astral rays
are always straight. There is not the slightest indication of a
spiral arrangement of the aster, so that mere migration of the
spindle cannot explain the spiral twisting of the astral rays
that follows later. The centrospheres of the second polar
spindle are very similar to the second stage of those already
described for the first polar spindle; compare Pl. XI, Figs. 19
and 8, and Figs. 20 and 7. The centrosphere contains a
deeply staining center, surrounded by a less deeply staining
peripheral zone from which the astral rays diverge; no definite
central granule is distinguishable. The centrosphere may en-
large, but it always retains this general character throughout
the stage of the second maturation spindle, and never develops
the sharply marked zones that are characteristic of the archi-
amphiaster stage.

Notwithstanding the similarity that often exists between the
first and second maturation spindles, they can always be identi-
fied by the presence of peculiar deeply staining bodies that
group themselves around the equator of the second spindle.
Only one of these bodies is represented in Pl. XI, Fig. 19, but
there are many similar structures in the egg. They are more
or less regular in outline, being round or slightly oval, but they
are apparently without any definite structure. In the earlier
stages they are sometimes present as vague bodies lying around
the periphery of the egg, but just before the extrusion of the
second polar globule they tend to aggregate around the spindle
in the upper hemisphere. These bodies probably owe their
origin to the circular rings in the ovarian eggs, and are no doubt
to be regarded as the yolk-nuclei of various authors.

There is no apparent difference in the size of the two
astrosphaeres of the second polar spindle. The distal aster
presses close against the periphery of the egg, which again
becomes slightly flattened just before the extrusion of the
second polar globule. The time required for the formation and
separation of the second polar globule is about two minutes —
the same as that required for the formation and extrusion of the
first. After the second polar globule has been extruded,
the egg-nucleus immediately forms a membrane and becomes

210 BYRNES. [VoL. XVI.

vesicular. Although the nucleus increases in size, it retains its
position with reference to the place of formation of the polar
globules, being frequently held fast to the egg-membrane by
a “ Zwischenkorper”’ (Pl. XII, Fig. 27), which often persists
until both nuclei have reached their maximum growth. The
persistence of the Zwischenkorper is of some importance in
the later stages, being often the only means of distinguishing
the egg-nucleus from the nucleus of the spermatozo6n.

During the formation and growth of the egg-nucleus the
astrosphaere undergoes a series of very remarkable changes,
which may be referred to three distinct periods. These periods
are characterized by the formation, the growth, and the dis-
appearance of the spiral astrosphaere. From the time of the
extrusion of the second polar globule until the astrosphaere
begins to enlarge, the centrosphere retains unchanged the char-
acter of the centrospheres of the second maturation spindle
(Pl. XII, Fig. 22). The astral rays are at first straight, but
while the aster is still small they begin to bend spirally. When
seen from the upper pole of the egg, the rays of the spiral
aster are bent toward the right in the direction of the move-
ment of the hands of a clock. Kostanecki and Wierzejski
have figured an aster in the egg of Physa, which shows the rays
at the inner pole of the second maturation spindle arranged
spirally; the time of the appearance of the spiral aster in
Physa corresponds exactly, therefore, to the time of the forma-
tion of the spiral aster in Limax.

During the second period, the period of growth of the spiral
aster, the centrosphere again becomes arranged in distinct zones
(Pl. XII, Fig. 25). During this period the center of the aster
shows a deeply staining central body, surrounded by a clear
zone (“heller Hof’’), which is traversed by a loose reticulum.
This reticulum connects the central body with the periphery of
the sphaere from which the astral rays diverge, and seems to be
formed by prolongations of the inner ends of the astral rays.
Sometimes an extremely minute granule (the “centriole”’ of
Boveri?) can be seen at the center of the homogeneous central
body (Pl. XII, Fig. 25). This deeply staining central body is
probably homologous to the deeply staining mass of granules

No. 1.] THE EGG OF LIMAX AGRESTYIS. ZAETE

in the middle of the centrosphere of the archiamphiaster (cf
Pie Piss 25, with) Pl. Xi ig.3)) 2 Phe rays) of ‘the spiral
aster continue to lengthen, so that the entire cytoplasm is now
involved in the formation of a spiral, which extends from the
egg-astrosphaere as a center to the circumference of the egg
(el ee Bigs 22). Pl, XI, Figeieeeishows.a,\section taken
through the spiral aster near the equator of the egg. It also
shows that the rays are bent so as to form a right-handed
spiral.

The third period is the period of the disappearance of the
egg-astrosphaere. The central body, or centrosome (Pl. XII,
Fig. 25), gradually fades away (Pl. XII, Figs. 26 and 27) and
gives place to a reticulum which traverses the entire sphaere
(Pl. XII, Fig. 28). In this stage the centrosphere corresponds
exactly to the reticulated centrosphere figured by Wilson for
Toxopneustes,! and by Brauer for Artemia salina. During the
third period the spiral aster is still plainly visible, but it is less
distinct than during the second period. As the nucleus enlarges
it gradually encroaches on the reticulated centrosphere, and
finally comes to occupy the entire site of the sphaere, so that
the astral rays now seem to diverge directly from the periphery
of the nucleus as a center (Pl. XII, Fig. 31). The rays become
more and more vague, until they disappear and nothing remains
of the egg-astrosphaere. The maturation of the ovum is now
complete.

II. STRUCTURE AND MATURATION OF THE SPERMATOZOON.
1. Structure of the Spermatozoon.

The spermatozoa were mounted whole on the slide, and killed
in a % per cent solution of acetic acid, and also in a saturated
solution of corrosive sublimate to which 5 per cent acetic acid
was added. They were then stained with Heidenhain’s iron-
haematoxylin and with various double stains, such as Lyons
blue and borax-carmine, iron-haematoxylin and orange G., acid
green and eosin, eosin and haematoxylin. After all of these

1 Diagram /, Fig. 108, in ‘ The Cell,” Wilson.

212 BYRNES. [VoL. XVI.

methods of staining, the spermatozoén shows always a precisely
similar structure.

In the last stage of the formation of the spermatozoon of
Limax agrestis the sperm-head appears heart-shaped in optical
section. The tailof the spermatozoon is very long and slender,
as is usual among the mollusca. The tail becomes somewhat
thickened near the head and passes into the head as an axial
rod. In cross-section the axial rod is seen to be surrounded by
a mass of chromatin in the shape of a trefoil. The maturation
of the spermatozo6n seems to be completed by the spiral twist-
ing of the sperm-head; the proximal part of the tail is also
involved in the formation of the spiral, as shown in Pl. XII,
Fig. 40. In the mature spermatozooén the chromatin appears
as a delicate though deeply staining cord, twisted spirally round
a refractive, colorless axis. I have not been able to differentiate
a middle-piece in the spermatozodn. When the mature sper-
matozoa are taken from the ducts of the ovo-testis and mounted
on a slide, the slender distal part of the tail often wraps itself
around the proximal part as a whip around the handle.

2. Fertilization.

The youngest fertilized ova that could be found in Limax
were those stored in the lower part of the oviduct. I have
succeeded in finding but very few ova at this stage. It is
impossible to determine how long the sperm-head has been in
the eggs that are taken from the oviduct, for the eggs of Limax
are not laid immediately after copulation. I have dissected
individuals immediately after copulation and have found no ova
in the oviduct. Moreover, a careful watch has been kept over
slugs that have been seen copulating, but they laid no eggs
within the following twenty-four hours. The spermatozoa are,
therefore, probably stored for some time after copulation before
they are used, and the ova are presumably fertilized soon after
leaving the ovo-testis.

Whenever the sperm-head is present in eggs from the oviduct,
it appears merely as a deeply stained oval body at the periphery
of the egg. There is no evidence of an attraction-cone having

No. 1.] THE EGG OF LIMAX AGRESTIS. 213

been formed by the penetration of the spermatozoén. In the
fertilization of the eggs of some of the mollusks, notably in
Arion? and in Physa,? the tail follows the head of the spermato-
zoon into the ovum. When the tail is present in eggs, it is
generally easily recognized by its strong affinity for certain
stains, a characteristic which may be acquired after its entrance
into the egg, according to Van Beneden and Platner. I have
never seen any evidence that the tail of the spermatozo6n
enters the egg of Limax, but the difficulty of obtaining eggs
at the time of the earliest contact of the spermatozo6n with
the ovum makes it impossible to determine whether the tail-
piece has really penetrated the egg and there broken down, or
whether it has been left outside.

I have never been able to detect a middle-piece in the sper-
matozoon after it has entered the egg, or to recognize a rotation
of the sperm-head. Very little change occurs in the sperm-head
from the time it first enters the egg until the eggs are deposited,
although when the eggs are laid the sperm has probably been
in the egg for some hours. All eggs taken from the oviduct
and from the albuminous gland were found in individuals that
were dissected in the late hours of the evening —from 8 to
10 o'clock p.m. As eggs are rarely deposited during the night,
before the early morning hours, it seems probable that at least
several hours must elapse between the time of fertilization and
the depositing of the eggs.

While the archiamphiaster occupies the middle of the egg,
the sperm-head remains quiescent at the periphery, very near
its place of entrance at the lower pole. Sometimes the sperm-
head is oblong, and sometimes it is slightly constricted in the
middle in the form of a dumb-bell, as in Pl. XI, Fig. 4. It is
homogeneous, however, and always stains uniformly. As the
spindle moves toward the upper pole of the egg, preparatory to
the formation of the first polar body, the sperm-head loses its

1 Platner, ‘“‘ Ueber die Befruchtung von Arion empiricorum.” Archiv f. mikr.
Anat., Bd. xxvii, 1887.

2 Kostanecki und Wierzejski, ‘“‘ Ueber das Verhalten der sogenannten achroma-
tischen Substanz im befruchteten Ei: Nach Beobachtungen an Physa fontinalis.”
Archiv f. mikr, Anat., Bd, xlvii, 1896,

214 BYRNES. [Vor. XVI.

homogeneous appearance and becomes slightly granular ; at the
same time it also becomes more or less vesicular, as in Pl. XI,
Figs. 7 and 8. The sperm-head sometimes appears as a deeply
staining mass of chromatin surrounding a clear central core. A
similar appearance of the sperm is often seen when the sperm-
heads are cut transversely in the follicles of the ovo-testis.
From these appearances, and from the absence of any definite
middle-piece to the spermatozoon, and also, as we shall see
later, from the usual absence of sperm-asters during the early
stages, the suggestion arises that the middle-piece may pos--
sibly be surrounded or overgrown, as it were, by the sperm-
head, so that the centrosome or centrosomes, if there be any,
come to lie within the nucleus. There is, however, no direct
proof of this.

During the stage of the first maturation spindle small round
bodies are often found accompanying the sperm-nucleus, as in
Pl; XI, Fig..7, and Pl. XM, Fig. 43. > They are similar imysize
to the large yolk-granules, but they stain very deeply like the
chromatin. They vary in number; generally there are from
two to five of these bodies, but they are always found in the
immediate vicinity of the sperm-head. Small deeply staining
bodies similar to those in Limax are shown occasionally at the
periphery of the egg in Physa, but no function has been ascribed
to them.

Foot has described granular bodies (“ sperm-granules’’) which
accompany the sperm-head in Allolobophora foetida. In this
form the author ascribes their origin to the breaking down of
the original sperm-asters. In the fertilized ovum of Limax the
similar behavior of chromatin and of these tiny bodies toward
certain stains suggests that they may owe their origin to par-
ticles of chromatin that are constricted off from the sperm-
nucleus before it becomes vesicular. I have seen a few cases
in which a portion of the chromatin seemed to be in process of
constricting from the sperm-head, but such cases are not of
very frequent occurrence. After the extrusion of the first polar
globule these deeply staining bodies are rarely found in con-
nection with the sperm-nucleus. Still later they disappear, or
become scattered through the cytoplasm of the egg, as in Pl. XII,

No. 1.] TIRE EGG OF LIMAX AGRESTIS. 215

Fig. 39. What the function of these bodies is I have not been
able to determine. ;

During the stage of the second maturation spindle I have
been fortunate enough to find an apparently normal egg in
which the sperm is accompanied by two tiny asters. The
sperm-head, the sperm-asters, and a part of the second matu-
ration spindle are shown in Pl. XI, Fig. 20, a, 0, 0, and c.
The asters lie some distance away from the sperm, and between
the sperm-head and the maturation spindle. These relations of
the sperm-head, the asters, and the maturation spindle are pre-
cisely similar to those figured by Kostanecki and Wierzejski
for Physa,! and also to those described by Wilson, Boveri, and
Hill for the echinoderm, and by Wilson and by Mead for the
annelid. During the formation of the second polar spindle the
sperm-head becomes more and more vesicular and moves away
from the periphery of the egg, in toward the egg-astrosphaere.
I have found a single egg in which the centrosome and aster
are present, in connection with the sperm-nucleus, immediately
after the extrusion of the second polar globule, but the egg
is evidently abnormal. Sections through the sperm-nucleus of
timis)ece/areshown in Pl: XII, Fig. 21374, 0) and/21)) Dhe
nucleus is enormously distended and completely enclosed by
the rays of a spiral aster, which is formed about a deeply
staining granule—the centrosome. There is so little chro-
matin in the nucleus that it is barely perceptible in the
sections. I have repeatedly studied the early maturation
stages of Limax agrestis in the hope of finding other asters;
but, except in the two cases just described, I have found only
two or three extremely doubtful cases. These cases, however,
are of especial interest, for they serve to throw light on a
problem that in Limax agrestis is extremely difficult of solu-
tion. They show that in all probability, even in these very
early stages, structures are present in the cell which do not
usually manifest themselves until a much later period, although
in most of the forms that have been carefully studied —echino-
derms, mollusks, and worms—the sperm-asters appear very
soon after the spermatozoén has entered the egg. When the

1 Archiv f. mikr, Anat., Bd. xvii, 1896, Pl. XVIII, Fig. 11.

2106 BYRNES. [VoL. XVI.

centrosomes first appear in the eggs of Limax, they are so
extremely minute that it would not be possible to recognize
them were it not for the presence of the centrosphere or the
. astral rays by which they are surrounded. Free from these
structures, they would appear only as minute granules (micro-
somes), from which they are quite indistinguishable.

After the extrusion of the second polar globule the sperm-
nucleus begins to grow rapidly. It becomes vesicular, devel-
ops a distinct membrane, and rises rapidly through the spirally
arranged cytoplasm toward the egg-nucleus, which lies at the
upper pole (Pl. XI, Fig. 28). As the egg-nucleus and sperm-
nucleus enlarge, they keep pace with each other in develop-
ment, and by the time the sperm-nucleus has reached the upper
pole of the egg both nuclei have attained considerable size.
They are often temporarily prevented from coming directly in
contact with each other by the enormous reticulated centro-
sphere, which still persists in connection with the egg-nucleus.

After the centrosphere has disappeared and the two nuclei
have attained their maximum growth, as shown in Pl. XII,
Fig. 32, they lie side by side at the upper pole of the egg.
No difference in size or structure can be detected; they very
frequently, though not always, contain even the same number
of large nucleoli. There is, however, a constancy in the rela-
tive position of the two nuclei, by which they can often be rec-
ognized with perfect certainty. The egg-nucleus retains its
position at the periphery of the egg, where the Zwischen-
korper, that was formed between the second polar globule and
the egg, still persists. Moreover, the sperm-nucleus, though
touching the egg-nucleus, generally lies a little deeper in the
egg.

The appearance of the egg of Limax agrestis at this stage
is precisely the same as that figured by Mark for the egg of
Limax campestris, with the exception that the nuclear mem-
brane of Limax agrestis is always perfectly spherical in outline,
never amoeboid, as Mark had shown for Limax campestris. It
can easily be seen, by a comparison with the living egg, that the
even outline of Limax agrestis is not the result of swelling
caused by the killing fluids, for the nucleus in the living egg

No. I.] THE EGG OF LIMAX AGRESTIS. 217

is also perfectly spherical. Up to the time of the apposition
of the nuclei no asters are usually present in the eggs of Limax,
and the centrosomes, even if they be present, cannot be recog-
nized. When the asters first appear, they are almost always.
in closer connection with the sperm-nucleus than with the
egg-nucleus (Pl. XII, Fig. 36), though sometimes they appear
directly between the two (Pl. XII, Fig. 33). There is no evi-
dence that a central spindle has been formed between the two
centrosomes when both asters are present. Sometimes one
aster appears before the other, as Mark has shown in Limax
campestris. The centrosomes look like homogeneous refractive
bodies, which stain very deeply with Heidenhain’s haematoxy-
lin. When the asters first appear, there is no differentiation of
the center of the aster into a centrosphere. The astral rays
are at first few in number and relatively short, but very distinct.
As the asters enlarge, the rays increase in number and length,
reaching far out into the cytoplasm and sometimes coming into
contact with the periphery of the egg. After the formation of
the asters, the nuclear membrane is often slightly flattened on
the side toward the aster, but the rays do not seem to pene-
trate the nuclear membrane (Pl. XII, Figs. 34 and 38).

When the asters first appear, the chromatin in the nuclei is
in the form of small granules which show no regular arrange-
‘ment, but are scattered irregularly throughout the nuclei (PI.
XII, Figs. 33 and 34). Later, while the nuclear membrane
still persists, the chromatin granules become arranged in rows
along the nuclear filaments within the nucleus (Pl. XII, Figs.
35 and 37); outside of the nuclear membrane, between the
nucleus and the centrosome, the astral rays appear to be directly
continuous with the nuclear filaments, and show distinct vari-
cosities.

As the sperm-asters gradually give rise to the segmentation
spindle, and the nuclei come to lie side by side at the equator
of the spindle, as shown in Pl. XII, Fig. 38, the sperm-nucleus
and the egg-nucleus can no longer be distinguished from each
other. The nuclei soon lose their membranes without forming
a segmentation nucleus, leaving the chromatin lying at the
equator of the spindle in two distinct masses. Later, when

218 BYRNES. [Vou. XVI.

the chromatin is brought into the equatorial plate, the separate
origin of the maternal and paternal chromatin is no longer
recognizable.

When the astrosphaeres of the segmentation spindle first
form, the centrospheres show no differentiation into zones such
as has been described for the centrospheres of the maturation
spindles. After the spindle has fully formed, however, the
‘cortical’? and “medullary’”’ zones begin to develop around
the centrosome, so that the astrosphaeres that appear in con-
nection with the sperm-nucleus are generally structures similar
to those of the maturation spindle.

When the segmentation spindle is completely formed and
the egg is ready to divide, the spindle lies transverse to the
polar axis in the upper hemisphere in a mass of protoplasm
that stains more deeply than the rest of the egg. After the
maturation spindle has divided, and the two resulting daughter-
nuclei have come to rest, all trace of the centrosomes and asters
is lost. When the daughter-cells are again ready to divide, the
centrosomes and asters reappear on the periphery of the nucleus,
and gradually separate to form the segmentation spindle of the
next division, and so on.

III. OBSERVATIONS ON ABNORMAL Ova.
1. Polyspermy.

I hoped to find in polyspermic eggs a clue to the origin of
the sperm-asters. Polyspermy rarely occurs in Limax, how-
ever, and when cases of it are found they throw no light on
the origin of the centrosomes. I have found single capsules
containing as many as twenty-five eggs, some of which were
unsegmented and apparently normal; others had divided into
two cells; still others into four cells. The unsegmented eggs
when killed in Vom Rath’s fluid and mounted whole, unstained,
showed as many as three small vesicular sperm-nuclei in addi-
tion to the usual large egg-nucleus. One set of abnormal eggs
suspected of being polyspermic, when sectioned, showed that
in some cases as many as twelve spermatozoa had entered a

No. 1.] THECE GG OF AHMAX AGRESTIS. 219

single egg. Most of the sperm-nuclei had become vesicular
and had migrated toward the egg-nucleus at the upper pole.
These eggs were very irregular in outline, and the cytoplasm
showed that degeneration had already begun. None of these
polyspermic eggs showed any evidence of division, and in none
of them was there any evidence of the presence of centrosomes or
asters. I have occasionally found isolated cases of polyspermy
among normal sets of eggs, but except for the presence of
more than two nuclei, they gave no evidence of being abnormal.
Polyspermic eggs are evidently incapable of development in
Limax agrestis ; normal development ceasing before the sperm-
nuclei attain their full growth and, therefore, before the asters
appear.

2. Abnormalities Other than Polyspermy.

A few abnormal eggs showed very peculiar bodies in connec-
tion with the sperm-nucleus. I have seen a number of eggs at
the stage when the asters usually appear, in which the sperm-
nucleus is accompanied by two relatively large spherical bodies,
which lie out in the cytoplasm on each side of the sperm-nucleus.
These bodies are on the side of the sperm-nucleus away from
the egg-nucleus, but from their number and evident connection
with the sperm-nucleus one can scarcely avoid the conjecture
that they bear some relation to the centrosomes. I have been
much puzzled by these bodies and thought that I might be
dealing with two species of Limax, in one of which the centro-
somes appeared as naked sphaeres. I am indebted to Prof.
H. A. Pilsbry, of the Philadelphia Academy of Natural Sci-
ences, for having identified all the individuals as belonging to
the single species, Limax agrestis (Linné).

In two instances at least, the eggs in which these structures
occur are known to have been abnormal, for the apposition of
the egg and sperm-nuclei did not occur until five hours had
elapsed after the eggs were laid. Normally this stage was
reached within two hours.

Before the extrusion of the second polar globule the upper
poles of the eggs became very irregular in outline and formed
pseudopod-like projections from the upper surface. After the

220 BYRNES. [Vou. XVI.

extrusion of the second polar globule the eggs regained their
normal outline. In some of these abnormal eggs the egg-
nucleus and sperm-nucleus remained permanently separated,
although both had attained their maximum size.

The sperm-nucleus is sometimes held fast to the egg-mem-
brane at the lower pole, or it may have penetrated only half-
way into the egg. While the sperm-nucleus is thus retarded
in its advance toward the egg-nucleus, the two spherical bodies
appear. Pl. XII, Figs. 41 and 42, show two sections through
different regions of the same egg. The section in Pl. XII,
Fig. 41, which is the last of a series, falls through the sperm-
nucleus on the side away from the egg-nucleus, and shows the
sperm accompanied by the two distinct bodies. These bodies
behave differently toward different staining reagents. This
egg was killed in corrosive sublimate acetic acid (§ per cent)
and hardened in Flemming’s solution. When stained in
Heidenhain’s haematoxylin, the bodies appear as deeply stained
homogeneous structures with well-defined outlines ; but in borax-
carmine and Lyons blue they are highly refractive and almost
colorless. Around one of these bodies is a slightly denser
layer of protoplasm, which bears out the suggestion that they
may be centers of attraction. There is, however, no radi-
ate structure around either of them. Nevertheless, their late
appearance, their connection with the sperm-nucleus, their be-
havior towards staining reagents, their number and general
structure make the suggestion irresistible that they may be due
to the presence of the centrosomes which have taken this pecu-
liar form under abnormal conditions. If these conclusions be
well founded, these abnormal ova furnish important evidence in
regard to the nature of the centrosome in Limax, for they show
that centrosomes may exist as definite structures in the egg apart
from the rays which usually mark their presence.

IV. FORMATION OF THE SPINDLE.

Soon after the apposition of the egg and sperm-nuclei the
chromatin becomes arranged along the nuclear filaments that
lie in the long axis of the forming spindle (Pl. XII, Figs. 36

No. 1.] THE EGGNOR, LELMAX VAGRESTTS:- 221

and 37). This process takes place before the breaking down
of the nuclear membranes. The intimate connection between
the chromatin and the nuclear filaments before the loss of the
nuclear membrane seems to support the view held by Flemming,
Reinke, and Wilson, that the chromatin is in contact with the
mantle fibers from the beginning, and not secondarily brought
into connection with them by the ingrowth into the nucleus of
the polar rays. If, however, the spindle is formed entirely by
a morphological rearrangement of the nuclear filaments that
are focussed at the centrosome, it is not wholly clear why the
nuclear membrane on the side toward the aster should be flat-
tened as if by pressure exerted on the membrane by the astral
rays. Moreover, the explanation of the formation of the spin-
dle by a rearrangement of nuclear substance does not account
for the formation of the second maturation spindle of Limax
agrestis. The second maturation spindle is formed wethzn the
centrosphere (Pl. XI, Fig. 16), while the old astral rays still
persist. The new asters are formed by rays that reach through
the centrosphere out into the cytoplasm, so that immediately
surrounding the centrosphere two sets of radiating fibers can
be detected — those that belong to the central astrosphaere of
the first maturation spindle, and those that belong to the asters
of the second maturation spindle in process of formation.

It is not possible to explain the formation of the second
maturation spindle on any theory of a mere rearrangement of
a preéxisting structure in Wilson’s sense. The process seems
to be exactly the reverse of focussing; for the rays are pro-
jected from the centrosome out into the centrosphere, which
appears almost structureless. Meanwhile the centrosphere in
which the spindle lies is constantly expanding and acquiring an
ever increasing diameter until it finally fades out. The astral
rays seem to be formed de xovo about the centrosome; hence
Wilson’s explanation of the organic growth of the astral rays
may apply not only to their continued growth, but also to their
origin. While the spindle is forming, the chromatin still lies
outside the sphaere, on the upper surface, where it was left
after the extrusion of the first polar body (Pl. XI, Fig. 15).
Later the chromatin is drawn into the equator of the spindle,

229 BYRNES. [Vo. XVI.

but not until after the spindle has already formed. Hence, in
the formation of the second maturation spindle, only the mantle
fibers of the spindle cow/d be formed from nuclear substance, as
Flemming and Reinke maintain.

I am unable to determine the precise method by which the
chromatin is brought into the equator of the second polar
spindle. It seems, however, as if it must be effected by some
of the polar rays that come secondarily into contact with the
chromatin, and thus become transformed into mantle fibers.

V. STRUCTURE OF THE CYTOPLASM: ARCHOPLASM.

The very coarse cytoplasmic network that is often seen in
the egg of Limax is probably not characteristic of the living
egg. A coarse reticulated appearance is nearly always found
in eggs that have been preserved in corrosive sublimate acetic
solution, and is in all probability due, in part, to precipitations
that are formed when the eggs are killed. This structure of
the cytoplasm occurs in eggs that are apparently perfectly
preserved and that show the finest details of structure in the
astrosphaeres (Pl. XI, Figs. 2, 5, and 10). Probably a truer
representation of the structure of the cytoplasm is seen in those
eggs that were hardened in Flemming’s solution after they
had been killed in corrosive sublimate acetic solution. These
eggs show no distinct reticular structure, but minute varicosi-
ties are distinguishable on threads of the utmost fineness. The
general appearance of such a preparation is granular rather
than reticular, although the filamentous nature of the cyto-
plasm in the preserved material is still faintly discernible. In
these eggs the astral rays are extremely delicate, and the details
of the centrosphere are clearly shown.

The peculiar structure of the centrosphere, particularly at
the stage of the archiamphiaster, cannot be due to the method
of preservation of the eggs, for it is constantly present, no
matter with what reagent the eggs have been killed. Prepara-
tions have been made of eggs that were killed in Flemming’s
solutions, in corrosive sublimate acetic (5 per cent), in chromic
acid (1 per cent), and in Flemming’s solution after corrosive

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No. I.] LAE EGG IOF LIMAX AGRESTIS. 222

sublimate acetic (5 per cent), and also in picro-acetic; all these
agree in showing similar structures at corresponding periods.

The archiamphiaster in the egg of Limax is of particular
interest, as the centrosphere corresponds very closely to the
type of centrosphere in which Boveri first described the “archo-
plasm ”’ in the eggs of Ascaris. In Limax the outer zone of the
centrosphere, which corresponds to the “‘archoplasmic”’ zone of
Boveri, appears as a deeply staining homogeneous (‘ cortical’’)
zone surrounding a clear (‘medullary’) zone which is not
stained by any of the methods used in coloring the prepara-
tions. The contrast between the “cortical” zone of the centro-
sphere, or the ‘“‘archoplasm,” and the cytoplasm is sharpest in
those eggs that have been killed and hardened in their cap-
sules in Flemming’s solution. This shows that the appear-
ance of the ‘‘archoplasm”’ is sometimes modified by the killing
reagent. It is not, however, produced by it.

When the sections are treated with the double stain, Lyons
blue and borax-carmine, the centrosome and ‘cortical’’ zone
(“archoplasm’’) of the centrosphere both stain blue, while the
spindle, the astral rays, and the cytoplasmic network take the
red stain of the carmine. After the first polar globule has
been extruded and the egg-centrosphere has become uniformly
granular in appearance, as shown by PI. XI, Fig. 14, the whole
centrosphere takes a faint bluish stain after Lyons blue and
borax-carmine. The second maturation spindle is formed within
the blue staining centrosphere, and if Boveri’s theory of archo-
plasm holds, the second maturation spindle should stain due.
This is not the case, however. The astral rays of the second
maturation spindle are red like the cytoplasm, even while the
spindle lies within the centrosphere. The centrosomes of the
archiamphiaster appear faintly bluish, but even the tendency of
the centrosomes to take the blue stain constantly diminishes.
At this stage the Zwischenkorper of the first polar globule also
stains blue, like the archoplasmic zone in the archiamphiaster
stage.

After the extrusion of the second polar globule the distinc-
tion between those structures in the astrosphaere that stained
blue and those that stained red in the earlier stage is still

224 BYRNES. [Vou. XVI.

more indistinct, until finally the color distinction between
different structures is wholly lost. During the stage of the
archiamphiaster the chromatin of both the egg-nucleus and
the sperm-nucleus stains 4/ue after corrosive sublimate acetic
(5 per cent). When the nuclei have attained their maximum
growth the chromatin stains ved after corrosive sublimate acetic
(5 per cent).

The only generalization that seems justifiable from the color
reactions in the egg of Limax is that the different structures
in the cell periodically undergo chemical changes in their
constitution. Indeed, there is some evidence from the color
reactions that these changes in color may correspond to phases
of a cycle through which the protoplasm of the cell passes
during development. There is, however, no evidence for the
existence of an ‘“archoplasm”’ which is distinct from the gen-
eral cytoplasmic reticulum.

It has been assumed in the case of Ascaris that the cleavage
stages follow one another in such rapid succession “that the
character of the centrosphere has not time to change.”’ Accord-
ing to Boveri’s account of Ascaris, ‘“ Schon wahrend der Bildung
des Richtungskorpers finden wir das Archoplasma wenn auch
weniger verdichtet und nach aussen allmahlich sich verlierend
um das Spermatozoon angehauft; noch friiher dagegen lasst
sich seine Existenz nicht nachweisen.”

In Limax, on the contrary, the peculiar type of centrosphere
that is characteristic of Ascaris is found in ova only during a
long period of quiescence, lasting from the time their devel-
opment in the ovary is completed until the formation of the
first polar globule. After that the centrosphere never again
acquires such a high degree of differentiation, although, as we
have seen, it still undergoes a series of changes that are repeated
during the maturation and fertilization stages of different ova
with the greatest precision. Kostanecki has attempted to
explain the different phases under which the ‘“archoplasm”’
of Physa appears as due to various changes that occur in the
structure of the cell in the course of development. These
changes consist in varying relations between the yolk, the
vacuoles, and the protoplasm, and they show that the formation

No.1.) THE EGG OF LIMAX AGRESTIS. 225

’

of the “‘archoplasm”’ in Physa depends merely on the way in
which the protoplasm is collected around the centrosome.

The cytoplasmic nature of the “archoplasm”’ in Limax is
revealed by various changes that go on in the centrosphere
itself, rather than by changes in the surrounding cytoplasm.
PU bees) tO.and 13; and PlaXxtl Migs 22.125) 27, 28 and
30, show successive stages in the disappearance of the “archo-
plasm”’ and of the “centrosome” of the Ascaris type. Pl. XI,
Fig. 13, shows the centrosphere approaching the type of the
‘“‘attraction sphaere”’ of Van Beneden, or the ‘“ Mikrosphaere ”
of Heidenhain. The outer ‘cortical’ zone of the centrosphere
is bounded by a circle of granules (microsomes) in which the
radially arranged cytoplasmic fibers at first seem to terminate.
Within. the centrosphere the astral rays are continued as
extremely delicate fibers which traverse the “cortical’’ zone
and terminate on the periphery of the “medullary” zone. The
central zone is uniformly finely granular in appearance. It is
without a distinct center and is not penetrated by the radial
fibers. In the second stage of growth of the centrosphere,
after the extrusion of the second polar globule, there is no
trace of a distinct homogeneous (‘cortical’) zone. The astral
rays come closer together as they approach the periphery of
the medullary zone or ‘“‘heller Hof’’ (centrosome of Boveri),
but they always remain distinctly separate. They can be
traced into the reticulum which traverses the “heller Hof”
and which is attached to the centrosome at the center of the
sphaere. The “cortical” zone of the archiamphiaster stage
now corresponds to that part of the cytoplasmic reticulum
which immediately surrounds the “heller Hof,” and which, by
the radial arrangement of its fibers, forms the central rays of
the aster.

Moreover, the transformation of the aster into a spiral that
involves the cytoplasm of the entire cell after the extrusion of
the second polar globule leaves little doubt as to the cytoplas-
mic nature of the aster. As the spiral rays diverge from the
centrosphere, they gradually break up into the cytoplasmic
reticulum. During the period of the disappearance of the
egg-centrosphere the sphaere is still further resolved into the

226 BYRNES. [VoL. XVI.

structure of the cytoplasm, and there is a complete disap-
pearance of any specialized central body. At this stage the
centrosphere corresponds to the type of the “reticulated”
centrosphere that Wilson has described for Toxopneustes, and
that Brauer has figured for Artemia.

The series of successive stages through which the centro-
sphere of Limax passes seems to show that a highly special-
ized astrosphaere can be resolved into the reticulum of the
cytoplasm by a gradual relaxation, as it were, of a tension
exerted on the surrounding contents of the cell from a focal
point, the centrosome. These observations on the eggs of
Limax are in accordance with the view held by Van Beneden,
Heidenhain, Reinke, Wilson, Kostanecki, and others, that the
‘“archoplasm”’ has no existence as a specific substance, but is
only a part of the general cytoplasm.

VI. THE CENTROSOME.

The study of the ova and the spermatozoa of Limax agrestis
throws little light on the origin and nature of the centrosome.
In the ovum the centrosome appears under widely different forms
in different stages. First, it is seen as a small deeply staining
point in the middle of the aster, as in Pl. XI, Fig. 1. In the
archiamphiaster stage, after a long period of growth, the central
body or centrosome is composed of a mass of granules (Pl. XI,
Figs. 2 and 3). After the centrosome has divided in this stage
the two resulting centrosomes appear as dumb-bell-shaped rods
(Pl) XI, Figs. 2 and 4). After the extrusion of )the inset
polar globule the centrosome again appears as a single tiny
granule that stains very deeply with Heidenhain’s haematox-
ylin (PI. XI, Fig. 14). After the extrusion’ of the second
polar globule the central body apparently corresponds to the
granular centrosome of the archiamphiaster stage; it reaches
an enormous size (Pl. XII, Fig. 25), after which it becomes
resolved into a reticulum and finally disappears (Pl. XII, Figs.
27 and 30).

The centrosomes of the segmenting egg appear in connec-
tion with the sperm-nucleus, though I have never been able to

No. I.] THE EGG OF LIMAX AGRESTIS. 227

trace their origin to a middle-piece in the spermatozoén. The
time of their appearance is variable, but they usually are not
visible, even if present, until the egg and sperm-nucleus have
reached their maximum size. There is no reason to believe
that asters that appear during the early fertilization stages in
the egg of Limax are temporarily lost only to appear later,
just before the formation of the segmentation spindle, as in
Allolobophora foetida. The asters once formed in connection
with the sperm-nucleus persist, but only in very exceptional
cases are they formed at all before the apposition of the egg
and sperm-nucleus. Crampton has recently described a some-
what similar case in the egg of an Opisthobranch, Bulla, in
which the centrosomes do not appear in the fertilized egg until
the time of apposition of the pronuclei.

VII. Summary.

The centrosome in the egg of Limax agrestis appears under
different forms during the maturation of the egg. Sometimes
the centrosome appears as a group of granules, as in the archi-
amphiaster stage. The granular centrosome at each pole of
the archiamphiaster is often divided into two distinct groups
or centrosomes, each of which is in turn composed of granules
that seem to be connected with each other, and appear dumb-
bell-shaped. Sometimes the centrosomes appear as single
granules, as in the centrosomes of the second maturation spin-
dle. The centrosome is seen asa large spherical body after
the extrusion of the second polar globule. The centrosome then
becomes granular and finally is resolved into a reticulum without
dividing, after which no definite centrosome can be detected.

The character of the entire centrosphere changes during
the maturation stages. First, the centrosphere appears as a
series of concentric zones; after this as a deeply staining cen-
ter with a single limiting zone; then as a reticulated sphaere
with a large homogeneous body in the middle; and finally as a
reticulated sphaere. The egg-centrosphere then disappears.
The various zones of the centrosphere are formed from the
cytoplasmic reticulum of the egg.

228 BYRNES. [Vou. XVI.

’

There is no evidence of an ‘‘archoplasm’”’ which is distinct
from the cytoplasm of the egg in Limax.

There is no middle-piece to the spermatozoon. The asters of
the segmentation spindle very rarely appear before the apposi-
tion of the egg-nucleus and the sperm-nucleus. Sometimes,
though very exceptionally, they appear before the maturation
stages of the egg are completed. The sperm-asters cannot
be traced directly to the spermatozoon, although they are more
closely connected with the sperm than with the egg-nucleus.
The nuclei do not unite to form a segmentation nucleus.

The spindle in Limax is not formed wholly by a rearrange-
ment of nuclear substance.

While the old astral rays still persist around the centro-
sphere of the first maturation spindle, the second maturation
spindle is formed wzthzm the centrosphere. The polar rays
of the second maturation spindle project through the centro-
sphere out into the cytoplasm; they are not formed by the
focussing about new centers of rays that are already formed.

The asters of the second maturation spindle are newly formed
structures. The mantle fibers of the second maturation spindle
seem to be derived secondarily from polar fibers. The forma-
tion of the spiral aster in Limax agrestis occurs soon after the
extrusion of the second polar globule, and only then. When
seen from the upper pole of the egg the rays of the spiral aster
are bent in the direction of the movement of the hands of a
clock.

The spiral aster is derived secondarily from an aster whose
rays are at first straight. Normally, the spiral arrangement of
the astral rays occurs in connection with the egg-aster. It
may, however, occur in connection with the sperm-aster under
special conditions.

VIII. Appenpix.
Material: Collecting and Keeping.

Limax agrestis, Linné, is a species of Limax common in
Europe. It has been introduced into this country and has
become very abundant in the neighborhood of Philadelphia.

No. 1] PAENEGG OF FIMAX AGRESTIS. 229

During the early fall months I collected large numbers of
Limax in old vegetable gardens in Bryn Mawr and under stones
that lay along the banks of an open drain. These slugs can be
collected out of doors as late as the middle of December. Much
of my material was collected late in the evening, with the aid
of a lantern, and in the early morning hours when the slugs
were on their way to their hiding places. During the winter I
was able to collect Limax in small numbers in the carnation
beds of a neighboring hothouse.

The slugs were kept in the laboratory in a large Wardian
case filled with living plants, and were fed on cabbage leaves,
plantain, dock, and various vegetable roots. Under these con-
ditions the slugs lived for some time in an apparently healthy
condition and yielded eggs in abundance. Sooner or later,
however, slugs that are kept in confinement become infested
with parasites, even when the greatest precautions are taken as
to cleanliness and an abundant food supply. The ovo-testis
becomes infested with parasitic protozoa, which are sometimes
found in swarms in the capsules after the eggs have been laid.
A parasitic thread worm is also found in the reproductive
organs of Limax.

Eggs enclosed in capsules that contain even a great many
parasites are by no means necessarily abnormal in their devel-
opment; indeed, they often give rise to normal embryos. In
order to avoid any complication that might result from the
study of abnormal eggs, however, fresh relays of slugs were
constantly supplied from time to time in place of the old ones.

Preparation of Materral.

The egg of Limax is imbedded in an almost liquid jelly
contained in a large, tough capsule. The egg is immediately
surrounded by a somewhat denser layer of jelly, which adheres
very closely to its surface.

At first I attempted to preserve eggs in the capsules, and for
this purpose Flemming’s solution and chromic acid (1 per cent)
were chiefly used. The killing fluids quickly penetrated the
capsules so that the eggs were perfectly preserved. The cap-

230 BYRNES. [VoL. XVI.

sules containing the eggs were then washed in water and were
afterwards passed through the various alcohols. Preparatory
to imbedding the egg in paraffine, most of the capsule was cut
away, only that part being left which immediately surrounds
the egg. After using this method of preparation it was found
to be almost impossible to section the eggs, owing to the ex-
treme brittleness of the hardened jelly around them, so that
although the eggs themselves seemed well preserved, the
method had to be abandoned as impracticable. It was found
necessary to resort to the tedious method of removing the eggs
one at a time from the capsules. This was done as follows:
Each egg was watched under the microscope until it had
reached the desired stage of development; the capsule was
then quickly placed in a saturated solution of corrosive subli-
mate, to which 5 per cent glacial acetic acid had been added.
As soon as the egg had become white and opaque, the capsule
was removed to a bath of distilled water. The capsules were
then opened under water, and the eggs were returned, free
from the jelly, to the corrosive sublimate acetic solution, where
they were allowed to remain for a few minutes. They were
then passed through the alcohols (35, 50, and 70 per cent suc-
cessively), and finally into 90 per cent alcohol, where they were
kept until used.

By far the best results, however, were obtained from eggs
that were fixed for from 15 to 20 minutes in Flemming’s
weak solution, after they had been killed in corrosive sublimate
acetic solution. This method is always [uniformly] successful,
and the preparations made by it show the minutest details of
structure.

A few good preparations were also obtained by the use
of picro-acetic acid, 2 per cent, as a fixative after corrosive
sublimate acetic. This method was, however, generally unsatis-
factory, as the picric acid often completely destroyed the struc-
ture of the centrosphere. The great advantage of corrosive
sublimate acetic over other killing reagents is that it pene-
trates the capsule very quickly without hardening it. If the
corrosive sublimate acetic solution be allowed to act too long
on the capsules, so that the inner layers become toughened,

No. 1.] THE EGG OF LIMAX AGRESTIS. 231

the difficulty of removing the egg from the jelly is greatly in-
creased. The jelly can still be removed, however, by allowing
the egg to stand in water, but this method should be avoided,
as it is apt to impair the structure of the egg. Picric acid,
chromic acid, and Flemming’s solution toughen the liquid con-
tents of the capsule so rapidly, that after using them it is often
extremely difficult to free the eggs from the capsules without
injuring them.!' For this reason these solutions were avoided
as killing reagents.

On account of the small size of the eggs it was found expe-
dient to stain them before imbedding in paraffine. The eggs
were imbedded in hard paraffine (56 per cent) and cut 3 and
4 thick. Each egg gave from 20 to 25 sections. These
were mounted serially with Mayer’s albumen and water, care
being taken to wash off as much of the albumen as possible.
The sections were afterwards stained on the slide in Heiden-
hain’s and Delafield’s haematoxylin after iron-alum.

Various combination stains were also used, chiefly Kor-
scheldt’s double stain, Lyons blue and borax-carmine, and iron-
haematoxylin with orange G.

Bryn MAwr COLLEGE, June, 1897.
1 In place of needles, fine porcupine quills were used in opening the capsules.

They proved most efficient implements for the purpose, and have the added advan-
tage that they are not acted upon by corrosive sublimate and acids.

232 BYRNES. [VoL. XVI.

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BIBLIOGRAPHY.

’'88 BoveERI, T. Zellen-Studien.

BRAUER, A. Zur Kenntniss der Spermatogenese von Ascaris megalo-
cephala. Arch. f. mikr. Anat. Bd. xlii.

BURGER, O. Ueber Attractionsspharen in den Zellkérpern einer Leibes-
fliissigkeit. Anat. Anzeiger. Jahrg. 6.

CONKLIN, E.G. The Fertilization of the Ovum. &zo/. Lect. Woods
Holl, Mass.

EIsMOND, J. Einige Beitrage zur Kenntniss der Attractionsspharen
und der Centrosomen. Anat. Anzeiger. Bd. x. November 7.
ERLANGER. Zur Befruchtung des Ascaris-Eies nebst Bemerkungen
iiber die Structur des Protoplasmas und des Centrosomas. Zool.

Anzeiger. Bd. xix. March.

Foot, KATHARINE. Yolk-Nucleus and Polar Rings. /ourn. of
Morph. Vol. xii, No. 1.

Foot, KATHARINE. The Origin of the Cleavage Centrosomes. /ourz.
of Morph. Vol. xii, No. 3.

HEIDENHAIN, M. Neue Untersuchungen iiber die Centralk6rper, etc.
Arch. f. mikr. Anat. Bad. xliii.

Hitt, M. D. On Fecundation, Maturation, and Fertilization. Quart.
Journ. of Micr. Sct. Vol. xxxviii, No. 2.

KOSTANECKI and WIERZEJSKI. Ueber das Verhalten der sogenannten
achromatischen Substanz im befruchteten Ei: Nach Beobachtungen
an Physa fontinalis. Arch. f. mikr. Anat. Bad. xlvii.

KOSTANECKI and SIEDLECKI. Ueber das Verhaltniss der Centro-
somen zum Protoplasma. Arch. f. mikr. Anat. Bad. xlviii.

Mark, E. L. The Maturation, Fecundation, and Segmentation of
Limax campestris (Binney). Bull. Mus. Comp. Zool. Vol. vi,
No: 2.

MEAD, A. The Centrosome in the Annelid Egg. Sczence. New
Series. Vol. v, No. IIo.

MEAD, A. The Origin of the Egg-Centrosomes. /ourn. of Morph.
Vol. xii, No. 2.

MeEvEs, F. Ueber die Entwicklung der mannlichen Geschlechtszellen
von Salamandra maculosa. Arch. f. mtkr. Anat. Bad. xlviii.

Moore, J. E. S. Reproductive Elements in Apus and Branchipus.
Quart. Journ. of Micr. Sci. September.

NIESSING. Die Betheiligung von Centralkérper und Sphare am Auf-
bau des Samenfadens bei Sadugethieren. Arch. f. mikr. Anat.
Bd. xlviii.

PLATNER, GusTAv. Ueber die Spermatogenese bei den Pulmonaten.
Arch. f. mikr. Anat. Bd. xxv.

195

50

93

193)

95

95

Eiay THEVEGG OF ETMAX AGRESTYS. 233

PLATNER, GUSTAV. Zur Bildung der Geschlechtsproducte bei den
Pulmonaten. Arch. f. mikr. Anat. Bd. xxvi.

PLATNER, GusTAV. Ueber die Befruchtung von Arion empiricorum.
Arch. f. mikr. Anat. Bd. xxvii.

RUckeErRT, J. Ueber das Selbststandigbleiben der vaterl. u. miitterl.
Kernsubst., etc. Arch. f. mikr. Anat. Bd. xlv, No. 3.

SopoTTa, J. Die Befruchtung und Furchung des Eies der Maus.
Arch. f. mikr. Anat. Bd. xlv, No. 1.

SogoTTa, J. Die Befruchtung des Eies von Amphioxus lanceolatus.
Anat. Anzeiger. Bd. xi, No. 5. September.

VAN DER STRICHT. La maturation et la fécondation de l’ceuf d’Am-
phioxus lanceolatus. Bll. de Acad. Roy. de Belgique. Série iii,
Tome Ixxx, No. II.

WARNECK, N. A. Ueber die Befruchtung und Entwickelung des
Embryos bei Gasteropoden. Sud. Soc. Impér. des Naturalistes de
Moscou. Tome xxiii, No. 1.

WatTASsF, S. Homology of the Centrosome. /ourn. of Morph.
Vol. iii.

WHEELER, W.M. The Behavior of Centrosomes in the Fertilized Egg
of Myzostoma glabrum Leuckart. Journ. of Morph. Vol. x, No.1.

WILson and MATTHEWS. Maturation, Fecundation, and Polarity in
the Echinoderm Egg. New Light on the Quadrille of the Centers.
Journ. of Morph. Vol. x, No. 2.

WILson, E. B. Archoplasm, Centrosome, and Chromatin in the Sea-
Urchin Egg. Journ. of Morph. Vol. xi, No. 2.

234 BYRNES.

EXPLANATION OF PLATE XI.

All figures drawn with camera and lenses of Zeiss. Homogeneous immersion,
1.5 mm. apochromatic compensation, ocular 4.

Fic. 1. Section of ovarian egg containing small astrosphaere.

Fic. 2. Section of ovarian egg containing a well-formed archiamphiaster.
The centrosphere of the aster consists of two zones, a dark “cortical” zone and
a light “medullary”? zone. The “medullary” zone contains two oblong centro-
somes composed of granules.

Fic. 3. Section of egg just deposited. The egg contains an archiamphiaster
similar to that in the ovarian egg. In the “cortical”? zone to the left of the
spindle the central granules are arranged irregularly. In the “cortical” zone to
the right the central granules are arranged in two groups.

Fic. 4. Section through one pole of the archiamphiaster. The centrosphere
consists of four concentric rings. The central granules are grouped so as to form
two dumb-bell-shaped centrosomes. The sperm-head is seen at the lower pole.

Fic. 5. Section through pole of archiamphiaster. No centrosomes are
present.

Fic. 6. Centrosphere and centrosomes of archiamphiaster.

Fic. 7. First maturation spindle. Sperm with deeply staining body at lower
pole of egg.

Fic. 8. Formation of first polar body. Thecentral centrosphere is enlarging
before the separation of the polar body. Sperm-head vesicular.

Fic. 9. Extrusion of first polar body. Centrospheres in the archiamphiaster
stage.

Fic. to. Section through egg-astrosphaere after extrusion of first polar body.
The centrosphere is beginning to enlarge. The centrosomes are single granules
which are beginning to separate.

Fic. 11. Section through egg-astrosphaere after extrusion of first polar body.
Beginning of the disappearance of the “cortical ” and ‘‘ medullary” zones.

Fics. 12 and 13. Later stages than Fig. 11.

Fic. 14 a, 4,c. Three successive stages through the centrosphere just before
the second maturation spindle begins to form.

Fic. 15. Early stage in the formation of the second maturation spindle within
the centrosphere. The astral rays still persist. The chromatin lies on the
periphery of the sphaere where it was left after the extrusion of the first polar
body.

Fic. 16. A later stage in the formation of the second maturation spindle.
The spindle still lies within the centrosphere, which is outlined by granular thick-
enings in which the rays of the old aster terminate.

Fic. 17. The second maturation spindle after the disappearance of the cen-
trosphere. Chromatin on the upper surface of the spindle.

Fic. 18. Later stage than Fig. 17.

Fic. 19. Second maturation spindle.

Fic. 20 a. Section through pole of second maturation spindle.

Fic. 20 6 and 4’. Sperm-asters in the same egg.

Fic. 20¢c. Sperm accompanying sperm-asters. (Fig. 20 4 and 0.)

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236 BYRNES.

EXPLANATION OF PLATE XII.

All figures drawn with camera and lenses of Zeiss. Homogeneous immersion,
1.5mm. apochromatic compensation, ocular 4. X 667.

Fic. 21a. Egg-astrosphaere after extrusion of second polar body. The astral
rays are straight.

Fic. 21 6and c. Two successive sections through the sperm-nucleus, which
is surrounded by rays of a spiral aster.

Fic. 22. Egg-nucleus and spiral-aster after extrusion of second polar-globule.
The centrosphere consists of a deeply staining center surrounded by a light
peripheral zone.

Fic. 23. Section through equator of egg containing spiral-aster. Section seen
from upper pole. Astral rays bent to right.

Fic. 24 a. Section through egg-nucleus and part of astrosphaere.

Fic. 24 6. Second polar globule and “ Zwischenkorper ” of Fig. 24 a.

Fic. 25. Centrosphere and astral rays of Fig. 24 a2. Homogeneous body in
center of clear zone which is traversed by a reticulum. The reticulum is formed
from inner ends of astral rays.

Fics. 26 and 27. Sections through egg-centrosphere showing successive
stages in the breaking down of the central body.

Fic. 28. Section through the reticulated egg-centrosphere after the disappear-
ance of the central body. The sperm-nucleus and the egg-nucleus are the same
size. Egg-nucleus at the upper pole.

Fics. 29 and 30. Successive stages in the disappearance of the egg-astro-
sphaere.

Fics. 31 and 32. Sections through the egg- and sperm-nucleus. The egg-
nucleus lies nearer the upper pole of the egg. Fig. 31 still shows indications of
the astral rays.

Fics. 33 and 34. First appearance of the sperm-asters after the nuclei have
reached their maximum size and have come into contact with each other.

Fic. 35. Radial arrangement of chromatin within the nuclear membrane.

Fics. 36 and 37. Section showing closer contact of asters with sperm-nucleus
than with egg-nucleus. Formation of segmentation spindle.

Fic. 38. Formation of segmentation spindle between egg- and sperm-nuclei.

Fic. 39. Deep-staining granule (chromatin?) in the cytoplasm near the
nucleus.

Fic. 40. Mature spermatozoon.

Fics. 41 and 42. Sections through an abnormal egg in which the apposition
of the egg- and sperm-nuclei occurred five hours after the eggs were laid.

Fic. 41. Section through periphery of sperm-nucleus on side away from egg-
nucleus. The sperm-nucleus is accompanied by two refractive bodies (centro-
somes ?).

Fic. 42. Section through egg- and sperm-nuclei. Egg-nucleus at upper pole
of egg near the second poiar globule.

Fic. 43. Sperm with deeply staining bodies at periphery of egg.

EARVAL) STAGES VOM SCRLOENBACHIA.

JAMES PERRIN SMITH.

STANFORD UNIVERSITY, CALIFORNIA.

CONTENTS.

PAGE

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Method of Phylogenic Research ........... Laden Oe See SO eee 243
INOMENCLALUR ENON S RAGES fOK |G ROW/D Ee ores ete ee ee . 244
Genus Sch loes0achia NOUMIAY Tet. o1. in oo eee 245
BCL CL UL LCL OT CL OTLETES TS es es eee atte ve . 246
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Paraneplonic Gastrzoceras Stabe hoo wee ee ee el Oe 251
Pardtegoreras, Stage. i vias ue eee ee eee eT

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INTRODUCTION.

Law of acceleration of development.— A few years ago natural-
ists were very much given to speculating about the theory of
evolution, reasoning abstractly for or against it, and construct-

237,

238 SMITH. [VoL. XVI.

ing imaginary family trees. To-day a serious naturalist would
just as soon think of taking up cudgels in favor of the theory
of gravitation as of evolution. Speculation is no longer popu-
lar, and now we wish to know, not whether certain organisms
developed out of others, but ow. This change has largely
been brought about by the application of the law of accelera-
tion of development to the study of biology. From the studies
of Louis Agassiz and his followers we know that, theoretically,
each organism in its ontogeny ought to go through stages of
growth corresponding to all its ancestors, and that these stages
ought to appear in the order of its ancestral forms. A part of
this may be verified in biological laboratories, by studying
embryonic and larval stages in animals. Even this is difficult,
because the habits of larvae are so different at successive periods
of growth that, in confinement, it is often impossible to trace
them with certainty through the various stages, and we usually
have to take different individuals to show the successive devel-
opment. And when we attempt to correlate growth stages with
ancestral genera the task becomes still more difficult, for then
we must leave the living organisms, and are thrown back on
paleontology. We often find in a growth stage an association
of characters that never occurred in any ancestral form, thus
obscuring the parallelism. Then, too, the geologic record is
notoriously incomplete, and from the nature of things must
always remain so; thus the paleontologic record is often lack-
ing just where we most need it.

PALEONTOGENY.

In order to make a satisfactory comparison of ontogeny and
phylogeny, the naturalist must select some group in which
living and fossil forms are classified on the same basis; such
groups are the brachiopods and the molluscs. But brachiopods
are scarce, hard to obtain by dredging, hard to rear in marine
laboratories, and most of the families are long since extinct, so
that ontogenic studies of living species have not thrown much
light on the history of the race. Beecher, Schuchert, and J. M.
Clarke have, however, succeeded in working out the ontogeny

a

Novi) LARVAL STAGES OF SCHLOENBACHIA. 239

of a number of fossil species, and have compared the growth
stages of these with the history of the group. Here, again,
comes in the difficulty that ontogenic series can be obtained
only by putting together in a row a number of distinct individ-
uals of various sizes, with the chance of making mistakes in
identification increasing as the specimens grow smaller and
have fewer characteristic marks. Very naturally, closely re-
lated species become more alike as we go down to the younger
stages, until it is impossible to tell which species the very
young larvae belong to. Of course some species have specific
characters thrown back by acceleration until even the earliest
larval stages are recognizable, but usually specific characters do
not appear until the adolescent period is well advanced. This is
true of all marine invertebrates that go through a larval period.

Of living molluscs the gastropods and the pelecypods offer
the same difficulties as the brachiopods, and have been much
less studied; even of the common oyster not all the larval
stages are known, and no other mollusc has been so closely
studied as that has.

Life history of cephalopods.—The chambered cephalopods
offer the best means of comparing ontogeny with phylogeny,
although the one available living form, Vaztz/us, belongs to the
old unspecialized group of nautiloids that has changed little
since its origin. Here, again, we are thrown back on paleon-
tology, but this time the difficulties are not so great, for there
is a great group of cephalopods, the ammonoids, that has left
in the stratified rocks abundant materials for study.

The ammonoids branched off from the nautiloids in the
Upper Silurian or the Lower Devonian, at first small, simple,
and rare, but they developed rapidly, until by the end of the
Devonian all the groups of goniatites were already present.
These increased steadily in numbers, size, and complexity, and
during the Carboniferous gave rise to the first simple ammo-
nites; these latter are a distinctly, although not exclusively,
Mesozoic race, which developed with wonderful rapidity from
the first rare members into numerous families, hundreds of
genera, and thousands of species, reaching their acme in the
Jurassic. In the Cretaceous they gradually declined, dropping

240 SMITH. [Vou. XVI.

off one at a time, until all were gone before the end of that
age. Only the simple radicles or stocks persisted, but from
time to time certain genera branched off from the main line,
became highly specialized, and often gave rise to so-called
abnormal forms, such as Hamites, Baculites, Crioceras, Scaphites,
phylogerontic or degenerate genera (retrogressive), which did
not perpetuate their race, but soon died out without descendants.

Of course there were many other phylogerontic genera that
were not abnormal in form; thus Clymenza branched off in the
Upper Devonian into a variety of species, and disappeared as
suddenly ; Medlicottta reached its culmination in the Permian,
barely managed to live on into the Trias, and disappeared with-
out posterity ; while the main stock of unspecialized Prolecant-
tzdae endured as long as the race.

In the beginning the number of phylogerontic forms was
small, for most of the goniatites left descendants among the
ammonites ; but their number increased during the Mesozoic,
showing a constantly growing tendency to become abnormal,
until before the end of the Cretaceous the entire stock of am-
monites had become phylogerontic, and died out finally from
sheer lack of plasticity to modify itself further with changing
conditions.

The life history of the ammonites is a finished chapter in
biology, and we have in museums and monographs a nearly
complete record of their development. It only remains to
study genetic series of ammonites. One way (that usually
adopted) is to compare a series of adults from successive
geologic periods, and by tracing resemblances to construct
theoretical family trees. Results of this work may be seen
in text-books of paleontology, and its unreliability may be real-
ized if one ever tries to use these tentative genealogies. This
would undoubtedly be the safer way if we had a complete geo-
logic record and if the faunas of the various geographic
provinces had been preserved. But since this is not the case,
we have to meet conditions as they are, not as they might be.

Palingenests.— The other way is to study the ontogeny of
representative species under each genus, and by comparing
each stage of growth with antecedent forms to find out the

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Nosi “LARVAL STAGES OF SCHLOENBACHTA. 241

probable relationship of these genera and the meaning of the
growth stages. From the researches of Hyatt, Branco, and
Karpinsky we have learned that the ammonoids preserve in
each individual a complete record of their larval and adolescent
history, the protoconch and early chambers being enveloped
and protected by the later coils of the shell. Thus by break-
ing off the outer chambers successively the naturalist can, in
effect, cause the shell to repeat its life history in inverse order,
for the ontogeny of the individual is an epitome of the history
of the race, and each stage of growth represents, if not always
an ancestral genus, at least some of the salient characters of
that genus, although unequal acceleration often crowds together
in an ontogenic stage characters that occurred in genera widely
separated in time. But where the parallelism is at all exact
these genera appeared (although not necessarily disappeared) in
the order of their minute imitations in the larval history of
their descendants; thus by comparing larval stages with ante-
cedent adult forms the naturalist finds the key to relationships
and is enabled to arrange genera in genetic series.

The ammonoids were all marine, never parasitic, never fixed
in station, and with them no resorption of the shell has ever
been noted; thus with them, while there often is some slight
obscuring of the record, due to unequal acceleration of certain
characters, there is no “ falsification of the record.’’ Ancestral
characters may not be repeated in the same association in the
history of the descendants, but they occur in the same order in
which they occurred in the history of the race. So far as the
writer’s experience goes, these characters shown in the larval
stages of ammonites are mainly palingenetic; it is a mistake to
give the name of coenogenesis to crowding together by unequal
acceleration in the descendant of characters that occurred in
separate generations of ancestors.

Omission of stages.—The only cases known to the writer
where stages of growth are actually omitted entirely are: (1) by
pushing back remote ancestral stages or characters beyond the
protoconch, where they are either lost entirely out of the ontog-
eny, or at least leave no record in the shell; (2) between the
protoconch and the first larval stage. The protoconch is remark-

242 SMITH. [Vou. XVI.

ably constant in all ammonites, but even in nearly related spe-
cies of the same genus the first larval stages may be quite
different, although the later larval stages may be very similar.
It is a well-known fact that free larvae have a much better
chance of repeating their ancestral history in unabbreviated
form than embryos that go through their development in the
egg.t It is quite probable that different species of ammonites,
just as is the case with living molluscs, were hatched at differ-
ent stages of growth, and that the omitted stages may corre-
spond to a longer period spent in the egg by one species than
that spent by a species that did not omit these stages. Thus
certain species of Schloenbachia reach the glyphioceran stage
immediately after the protoconch, while others go through sev-
eral generic stages between the protoconch and the glyphioceran
stage; this happens in species in the same geologic horizon, so
it cannot be due to difference in removal from the parent
radicle. Such cases as these make it hard to interpret ontog-
eny, but they are not “ falsifications of the record.’ Holzapfel?
has described the young stages of Axarcestes karpinskyi Holz-
apfel, showing that it goes through a typical mimoceran stage,
in which the nepionic shell does not touch the protoconch for
half a revolution. But in the ontogeny of a nearly related spe-
cies, Anarcestes plebeiformis Hall, as described by J. M. Clarke,’
this mimoceran stage is omitted, for the shell is close coiled
from the very beginning. We might say, however, that the
mimoceran character of the open coil is pushed back by unequal
acceleration and lost, while other mimoceran characters are
retained, though so merged with those of Avxarcestes that it
is impossible to recognize them.

Each ammonite went through a larval history that is long
and varied in direct proportion to the length of time from its
period back to the Lower Devonian, when the first of the race
are known. Thus in the Wautzlinidae, the first group of ammo-
noids, the ontogeny is comparatively simple, there being few

1 Balfour, Treatise on Comparative Embryology, vol. ii, p. 362.

2«“Die Fauna mit Maeneceras terebratum Sandberger,” Adhandl. k. Preus-
sischen Geol. Landesanstalt, N.¥F., Heft 16, p. 77, Pl. III, Figs. 15-20, 1895.

3 “Notes on the Early Stages of Certain Goniatites,” 26th Ann. Rep. State
Geologist of New York, pp. 165-168, 1898.

Noni) (LARVAL) STAGES OF SCHLOENBACHIA. 243

changes from the larval period up to maturity. But the higher
Devonian and Carboniferous forms go through several generic
changes before they reach maturity, while Mesozoic genera
have still longer larval and adolescent periods, — that is, longer
in the sense of going through more stages. In Paleozoic spe-
cies, however, one rarely finds ammonoids preserved so that the
inner coils may be separated. In Mesozoic species, while the
preservation is often good, the acceleration is usually so great
that any certain interpretation of the meaning of larval stages
is difficult, not to say impossible.

Method of phylogenic research.— Since ammonites preserve
in each individual a complete record of their ontogeny, one
might work out the life history of each species from a single
specimen by making drawings of each stage before pulling off
the coils representing this stage. In some few cases the writer
has succeeded in taking off the outer coils so as to show almost
the complete ontogeny in a single specimen without destroying
the specimen. But this method usually necessitates the destruc-
tion of those parts that are taken off, and so the original is
lost, and the naturalist has to show for his work only his notes
and drawings, which may or may not be sufficiently accurate ;
his results cannot be verified.

The more satisfactory way is to select a number of well-pre-
served adults, so as to be sure of the identification of the species
and to break off the outer coils until the desired stages are
reached. To do this, finger nails and steel dental chisels are
all the tools needed. After the specimen is reduced to a small
size the coils are pulled off under water to prevent loss. The
material used must be selected with great care, preferably lime-
stone, not so soft as to crumble nor so hard as to shatter. The
young ammonite may be studied under the microscope in three
different mountings: dry on white cardboard to see the surface
markings; on white cardboard in a drop of water to see the
septa and shape; under water in a watch-glass over a strong

1 The results given in this paper are based on the study of about 150 specimens,
illustrating all the life history of Schloenbachia oregonensis, but of course these
could not all be figured, nor even included in the tables. Only the distinct stages
and not the transitions were selected for illustration.

244 SMITH. [Vov. XVI.

condensing lens to see the siphon and other internal characters
when the specimen is translucent. This latter mounting is well
suited to work in direct sunlight with a polarizing microscope,
for the whole field is dark except where the doubly refracting
calcite of the young ammonite allows the light to pass through.

NOMENCLATURE OF STAGES OF GROWTH.

In order to correlate ontogenic stages with generic changes
seen in the development of the race it is necessary to have an
exact scientific nomenclature. The most satisfactory, and one
now being generally adopted, is that given by Professor Hyatt
in ‘ Phylogeny of an Acquired Characteristic.” ?

TABLE OF ONTOGENIC STAGES.

Stages. Stages. Substages. Comparison with Phylogeny.
Embryonic (1) Embryonic ( Protembryo Phylembryonic
Mesembryo
Metembryo
Neoembryo
Typembryo
Phylembryo
Larval (2) Nepionic Ananepionic
{ Metanepionic } Phylonepionic
Paranepionic

Adolescent (3) Neanic ( Ananeanic
Phyloneanic

Epacme

Metaneanic
Paraneanic

Adult (4) Ephebic Anephebic
Metephebic Phylephebic Acme

Parephebic

Senile (5) Gerontic Anagerontic
{ Metagerontic } Phylogerontic } Paracme

Paragerontic

With the embryonic stage the paleontologist can do nothing,
except the very last substage, or phylembryo, when the J/o//usca,
Brachiopoda, and other groups begin to secrete their shells;
but all the later stages are easily accessible in well-preserved
material.

1 Proc. Amer. Phil. Soc., vol. xxxii, No. 143, pp. 391 and 397.

No.1.] LARVAL STAGES OF SCHLOENBACHIA. 245

The best example of correlation of ontogenetic stages with
phylogeny is the genealogy of Medvecottia, worked out by Kar-
pinsky, who has shown that the Carboniferous genus Pronorites
goes through the following stages: latisellate protoconch, phyl-
embryonic; with the second suture it reaches the Anarcestes
stage, nepionic; about the end of the first revolution the /der-
giceras stage begins, paranepionic; second revolution shows the
Paraprolecanites stage, neanic; on the third whorl begins the
Pronorites stage,adult. Thus with regard to Pronorites the genus
Anarcestes is phylonepionic, /dergiceras is phyloparanepionic,
Paraprolecanites is phyloneanic. In the same work Karpinsky
has shown that Med/icottia is a direct descendant of Pronorites
and in its development goes through all the stages of the ances-
tral genus and adds several more. The first revolution of
Medlicottta could not be studied, but on the second revolution
was seen the /bergiceras stage, metanepionic; on the third
whorl the Paraprolecanites stage, paranepionic; at end of the
third whorl the Pronorites stage, beginning of the neanic; on
the fourth whorl the Szcanztes stage, end of the neanic; on the
fifth whorl the Promedlicottia stage, anephebic; and lastly, at
end of the fifth whorl, J/ed/icottza, adult in characteristics,
though not yet in size.

Genus SCHLOENBACHIA Neumayr, Sztsungsberichte k. Akad.
Wiss. Wren (Math. Nat. K1.), Bd. lxxi, 1. Abth., p. 658, 1875.

As originally defined by Neumayr, Schloenbachia was to include
forms with narrow, compressed whorl, strong curved lateral ribs,
a sharp, often notched, keel; septa comparatively little branched,
two lateral, and one distinct auxiliary lobe. The genus was
supposed to be descended from Amaltheus, although Neumayr!
says that we can only assign Schloenbachia with probability to
this group, since it appears suddenly in the Cretaceous as an
immigrant, without local ancestors; it has later been broken up
into a number of genera and subgenera of questionable value,
some of which cannot be sharply differentiated from each
other.”

1 Loc. cit., pp. 654 and 658.
2 F. B. Meek, “ Report on Invert. Cretac. Foss. Upper Missouri,” 1876. Gros
souvre, “ Les Ammonites de la craie supér. de la France,” 1893.

246 SMITH. [Vor. XVI.

K. A. von Zittel! has separated Schloenbachia from the
Amaltheidae, and placed it in a family of its own, the Przono-
tropidae ; which change is quite proper, for Schloenbachia does
not go through in its adolescent period any stages correspond-
ing either to Amaltheus or Oxynoticeras. Zittel regards the
Prionotropidae as an offshoot of the Amaltheidae, and these in
turn from the Prodecanitzdae,; but neither Schloenbachia nor the
Amaltheidae go through larval stages corresponding to this
Paleozoic group, but rather to the Glyphioceratidae.

SCHLOENBACHIA OREGONENSIS Anderson ms., Pls. A-E.

Schloenbachia sp. indet., aff. S. chicoensis Trask ; J. P. Smith, Journ. Geol.,
Pl. A, Figs. 1-7, vol. v, No. 5, p. 521, 1897.

Schloenbachia sp. indet., J. P. Smith, Chapter IX in Jordan’s “ Footnotes
to Evolution,” Pl. C, Figs. 1-11.

The adult is narrow, discoidal, high-whorled, with wide, shal-
low umbilicus, and almost parallel sides. The whorls embrace
about two-fifths of the preceding. The surface is ornamented
with strong ribs that branch in groups of two from strong knots
on the umbilical shoulders, bend forward and form smaller
knots on the angular abdominal shoulders, and then turn
forward in a sharp angle to the keel. These ribs are exceed-
ingly variable, sometimes fine, and sometimes coarse, with
transitions from one to the other. Between these bundles of
ribs there are from one to two single ribs that do not reach the
umbilical shoulders. The keel is rather low, sharp, and slightly
notched by the ribs; the sloping space between the keel and
the abdominal shoulders has no furrow, although the row of
abdominal knots may give that impression. A cross-section of
the adult is shown on Pl. C, Fig. 7, diameter 22.25 mm., six
whorls, on which the increasing relative height and flattening
sides of the whorls may be seen. The septa are comparatively
simple, and not very digitate; they show a wide external lobe
divided by a short and broad siphonal saddle ; a deep, broad, first
lateral lobe; second lateral lobe about one-half as deep as the
first; and a shallow auxiliary lobe. The first lateral saddle is
notched rather deeply near the middle, a character that begins

1 Grundziige d. Palaeontologie, p. 430, 1895.

No.1.] ZARVAL STAGES OF SCHLOENBACHIA. 247

in the early youth of the shell and continues to increase until
the adult stage is reached. Thesepta of a specimen at diameter
18.50 mm. are shown on Pl. E, Fig. 5.

S. oregonensis grows to a diameter of at least 30 mm., although
no perfect specimens of that size were obtained. The measure-
ments of an adult at end of the sixth whorl are as follows :

MM.

Diameter . : : : : 222 bE OO
Height of the last aes ; : : 9.00 = 0.40
Height of last whorl from the top of the preceding 7.28 0.32
Width of last whorl 4 ‘ : 4 : OO 0,22
Involution . , i : : : 1.72 = 0.07
Width of Geabilious 5 5 ; 2 ; 7.64 = 0.34

This species is nearest to Schloenbachia chicoensis Trask,
Eres Calef.. Acad, \Scz., Vol.) 1, p. 92,) Pi Pe ice i r.e50);) ane
Palaeontol. Calzf., vol. i, p. 68, Pl. XIII, Fig. 17, and Pl. XIV,
Fig. 17, to which it was doubtfully referred by Mr. F. M.
Anderson.}

S. chicoensis, as figured by Gabb, has narrower and more
involute whorls than S. ovegonensts, flatter sides, and stronger
nodes on the shoulder keels, and also has the shoulder keels
nearly as high as that on the abdomen. Through the kindness
of Dr. J.C. Merriam, of the University of California, the writer
was able to examine a series of Schloenbachia chicoensis, as
figured and described by Gabb; the following are the dimen-

sions of a typical specimen:
MM.

Diameter . E : : ‘ 4 : 24.0
Height of last ator, : ; : : : : 12.0
Width of umbilicus . ; : : : ‘ : 5.0

The dimensions of a specimen nearing the end of the adoles-
cent stage are as follows:
S. chicoensis (as identified by Gabb),

MM.

Diameter . c - : : : : 13.00 = 1.00
Height of the last Hoel ‘ A : : : 5-3 =0.40
Height of last whorl from the preceding . : 4.3 = 0.33
Width of last whorl . : : 5 ; : 3.50.20
Involution : E 5 ‘ : : : 1.0 = 0.07
Width of umbilicus . ‘ : 3 “ : 4.0 =0.30

1 Journ. Geol., vol. iii, No. 4, p. 467.

248 SMITH. (Vor. XVI.

The adolescent S. chicoensts resembles in appearance and in
relative measurements the adults of S. ovegonensts, and very
probably is a descendant of the latter species.

Occurrence and locality.— The material on which this paper
is based was collected by Mr. Frank M. Anderson, at the Forty-
Nine mine, one and one-half miles southwest of Phoenix, Oregon,
in beds supposed to belong to the Upper Horsetown formation,
top of the Lower Cretaceous, and described by him in “Some
Cretaceous Beds of the Rogue River Valley, Oregon.”’! The
writer’s thanks are especially due Mr. Anderson for the gener-
osity with which he furnished the specimens to illustrate this
work. Ina forthcoming paper, in the Proceedings of the Calt-
fornia Academy of Sctence, Series 3, Mr. Anderson will figure
and describe Schloenbachia oregonensis and the rest of this
interesting fauna.

ONTOGENIC STAGES.
Neptonic or Larval.

Phylembryonic.— The early embryonic stages are shell-less,
and necessarily cannot be represented in fossils, so the paleon-
tologist begins his investigations with the phylembryonic, when
the shell gland becomes functional, and the class or phylum
can be made out. This is represented in the ammonites by
the protoconch, which in this species is a smooth, oval, bobbin-
shaped body, a little wider than high, to which the chambered
coil is attached. With this stage begins the siphon, as a pear-
shaped sac, or caecum, taking up a large part of the entrance
from the protoconch to the chambered shell. The dimensions
of the protoconch are remarkably constant in a large number of
specimens; those of the protoconch figured on Pl. A, Figs.
1-3, are as follows:

MM. MM.

Diameter ‘ : é 0.42 Width ; 4 0.48

This stage is analogous to the protegulum of the brachiopods,
protaspis of the trilobites, and prodzssoconch of the pelecypods,
and corresponds to the primitive cephalopod. The embryonic

1 Journ. of Geol., vol. iii, No. 4.

No.1.] LARVAL STAGES OF SCHLOENBACHIA. 249

shell probably included part of the spiral chamber, but for want
of a natural indication of the end of the stage, the phylembry-
onic is arbitrarily limited to the protoconch.

Ananepionic. — With the formation of the first septum the
animal is considered to cease to be an embryo and to begin its
larval history. This, of course, is purely arbitrary, since the
ammonites are all extinct and we have no way of knowing at
what stage they left the egg. At this period the siphon, which
is in the center, takes up nearly half of the height of the whorl.
The first septum consists of a broad, long, abdominal saddle, a
pair of rather narrow lateral lobes, and a pair of short, narrow
saddles on the umbilical shoulders. This is shown on Pl. A,
Fig. 3, and Pl. C, Fig. 1. It is distinctly nautilian and corre-
sponds to some Silurian nautiloid genus, although it is not
possible to say which one, because the characters are not dis-
tinctive enough. The internal part of the septum is nearly
straight, showing no lobes nor saddles.

Metanepionic. — The second larval substage begins at the
second septum, when the whorl is low, broad, and deeply
embracing. Pl. C, Fig. 1, shows that at the second septum the
broad, abdominal saddle is divided by a deep and broad ventral
lobe; at this stage the shell resembles the Lower Devonian
Anarcestes, one of the first of the typical ammonoids. At the
third and fourth septa little change takes place, but these prob-
ably correspond to Parodoceras and Prionoceras of the Devonian.
At the fifth septum the ventral lobe broadens, showing a tran-
sition from Prionoceras to Glyphioceras (or Gontatites s. str.).
The ananepionic and metanepionic substages take up the first
quarter of a coil. The siphon, during this substage, is still
median, and remains so up to three-quarters of a whorl, when
the paranepionic stage is well along, but always decreasing
in relative diameter as compared with the height of the
successive chambers. The form of the shell at the metane-
pionic stage is shown in the first quarter of a coil from the
protoconch, on Pl. A, Figs. 4 and 5; the septa are shown
on Pl. C, Fig. 1, at the second, third, fourth, and fifth. On
some specimens the metanepionic substage ended with the
fifth septum.

250 SMITH. [VoL. XVI.

Paranepionic. — Hyatt! says that the paranepionic substage
in the later ammonoids begins with the division of the ventral
lobe, and continues as long as only goniatite characters are
shown. In Schloenbachia oregonensis the sixth septum has a
divided ventral lobe and two lateral lobes, like Glyphioceras (or
Gontatites s. str.). This is shown on Pl. C, Fig. 1. If we fol-
low Hyatt’s definition the paranepionic stage will have to be
subdivided, for there are three well-marked goniatite stages in
it, the Glyphioceras, Gastrioceras,and Paralegoceras stages. In
a recent paper? the writer has shown that Glyphzoceras in its
ontogeny goes through as a larva the stages Anarcestes and
Tornoceras (Parodoceras) ; as a youth it is a Prionoceras, and
takes on its own characters at a diameter of about 6 mm.
J. M. Clarke? says that Zornoceras and Parodoceras are dis-
tinct genera, but that they appear along with Axarcestes early
in the Devonian, and, therefore, are probably not descend-
ants from that genus, but have a common origin with it. If
this is the case the genealogy of the Glyphioceratidae will have
to be revised, as will also the nomenclature of the chief genus
of the family, for E. Haug* has recently shown that Gonzatites
de Haan must be retained for the group of G. sphaericus Martin,
while Glyphioceras Hyatt may be retained for the group of
G. diadema.

Glyphioceras stage. — Now Schloenbachia oregonensis goes
through these same preliminary stages, but is so greatly accel-
erated that it reaches the glyphioceran stage at the end of the
first quarter of a coil from the protoconch, and at the sixth
septum, as shown on Pl. C, Fig. 1. The early part of this
stage is shown on Pl. A, Figs. 4 and 5, one-half coil, first eight
septa, and diameter 0.58 mm.; Fig. 6 shows a little more
advanced glyphioceran stage, development of the septa from
the third to the tenth, diameter 0.64 mm.; Figs. 7 and 8 show

1“ Phylogeny of an Acquired Characteristic,” p. 416.

2 Proc. Calif. Acad. Sci., Series 3, vol. i; Geol., No. 3, 1897, “ Development of
Glyphioceras,” etc.

3 «Naples Fauna (Fauna with G. /ztumescens) in Western New York,” z6¢h
Ann. Rep. State Geologist of New York, p. 109, 1898.

4 «Etudes sur les Goniatites,” AZém. 78, Paléontologie, Soc. Géol., France, 1898,
p. 27.

NOI.) LARVAL STAGES OF SCHLOENBACHTIA. 25 I

it with nine septa, diameter 0.68 mm., and three-quarters of a
coil; Fig 9 shows this same stage at a little over three-quarters
of a coil, diameter 0.74 mm., and its septa are shown on PI. D,
Fig. 1. These figures show a gradually increasing height of the
whorl as compared with the width. The glyphioceran stage lasts
up toa diameter of 1 mm., and about one and one-quarter coils,
near the end of which stage, at diameter of 0.80 mm., a deep
sulcation or constriction makes its appearance; this distinctively
glyphioceran character was observed on a large number of speci-
mens near the end of the first whorl, and never after that.

Gastrioceras stage. — Near the beginning of the second
whorl, at diameter of a little over 1 mm., and after the appear-
ance of the constriction, the umbilicus begins to widen, until
at diameter of 1.20 mm. it is proportionally wider than in any
species of Glyphioceras; this is shown on PI. A, Figs. 10 and
II, one and three-eighths coils, and is transitional to Gastrt-
oceras, a genus especially characteristic of the Upper Carbonif-
erous. A somewhat larger specimen, diameter 1.33 mm., one
and five-eighths whorls, is shown on Pl. A, Figs. 12 and 13.
As the size increases the shape becomes more decidedly gas-
trioceran, as shown on Pl. A, Figs. 14 and 15, one and seven-
eighths coils, diameter 1.65 mm.; the septa of this are seen on
Pl. D, Fig. 2. This stage corresponds to that group of Gas¢ri-
oceras that lacks the umbilical ribs and has the second lateral
lobe on the sides of the shell, as in Gastrioceras tllinotsense
Miller and Gurley,! of the Coal Measures.

Paralegoceras stage. — The gastrioceran stage lasts from
near the beginning of the second whorl, diameter a little over
I mm., up to two and one-eighth whorls, diameter 2.15 mm.,
when a third lateral lobe appears on the umbilical border;
then the whorl becomes higher and narrower, and the whole
aspect of the shell is like Pavalegoceras Hyatt, a genus espe-
cially diagnostic of the Upper Carboniferous, and supposed to
be a direct descendant of Gastrioceras This stage is shown

1 Bulletin XT, Illinois State Mus., N.H., p. 42, Pl. V, Figs. 6-8, 1896.

2 For the relations of Glyphioceras, Gastrioceras, and Paralegoceras, see paper
by the writer, “Marine Fossils from the Coal Measures of Arkansas,” Proc. Amer.
Phil. Soc., vol. xxxv, No. 152, 1896.

252 SMITH. [VoL. XVI.

on Pl Biviies. ot) vand) 2,randthe;'septa von gr alow wie se
although the third lateral lobe is considerably exaggerated, on
account of a mistake in drawing. But even if the third lateral
lobe were entirely lacking the stage might still be referred to
Paralegoceras, according to the usage of Hyatt. Throughout
this, as in all preceding stages, each coil embraces about two-
fifths of the preceding. ~By reference to the table of stages of
growth, the widening umbilicus and flattening whorl may be
traced just as in the drawings of the successive stages. This
substage is short, lasting only half a coil, from diameter 2.15
mm. up to two and five-eighths whorls, diameter 2.70 mm.

The decrease in relative size of the siphon in the larval stages
may be seen from the following figures:

At the first septum the siphon is 48 per cent of height of the whorl.

At one-quarter of a coil CON Se AR ‘“ ‘cs
“ one-half be oe OG ei) 6c “ rT
“ three-quarters Gi MER > ‘6 ‘ ‘
«one and one-quarter coils |“ (“24 “ “
‘© one and one-half “ sae ia aoe “ “ “
«¢ one and three-quarters coils ““ “22 « ‘“ “«
“ two coils “ “20 “ rT “
“ two and one-half coils A Ea i Sa “ “

NEANIC OR ADOLESCENT.

Ananeanic.— When an ammonite in its development has
taken on characters that the goniatites never had, it may be
said to have completed the larval stage and to have begun
the adolescent. At the end of the Paralegoceras stage, diame-
ter 2.70 mm., about the middle of the third whorl, the abdomen
becomes sharpened and somewhat higher, and a keel appears.
The smooth sides, simple goniatitic septa, and ventral keel all
remind one of the Triassic genus Styrites1 Mojsisovics. This
stage is shown on PI. B, Figs. 3 and 4, diameter 3.10 mm., three
whorls, with the beginning of the keel at diameter 2.70 mm. ;
the septa are seen on Pl. D, Fig. 4.

1“Das Gebirge um Hallstadt,” Adhandl. k. k. Geol. Reichsanstalt, Wien, Bd.
vi, p. 264, 1893. The ontogeny of this genus is not described here, and we do
not know that it really goes through the preliminary development of the
Glyphioceratidae.

a

No. 1.] LARVAL STAGES OF SCHLOENBACHIA. 253

Schloenbachia oregonensis remains in this stage about a
quarter of a revolution, up to the diameter 3.15 mm., two and
seven-eighths whorls; then without any other change in char-
acters the first lateral saddle suddenly becomes indented, as
shown on PI. B, Figs. 5 and 6, diameter 3.71 mm., and the pro-
jection of the septa on Pl. D, Fig. 5. This stage does not
correspond to any known genus, but the characters have the
nature of Lower Triassic genera, and so it may be referred to
some unknown form of that age; it may be provisionally called
the Parastyrites stage. At diameter of 4.00 mm. the rounded
abdominal shoulders become angular, forming keels. The
Parastyrites stage lasts about half a revolution, to near the
middle of the fourth whorl.

Metaneanic.— At diameter 4.5 mm., three and three-eighths
whorls, ribs appear suddenly on the sides, faint at first, but
rapidly becoming distinct; this is figured on Pl. B, Fig. 7,
diameter 5.60 mm., three and seven-eighths whorls. At first
the ribs, which branch out from nodes on the umbilical shoul-
ders, reach only to the abdominal angles. This stage usually
ends with the fourth whorl, at diameter a little over 7 mm.,
thus lasting about five-eighths of a coil. Near the end of the
fourth whorl the septa, which up to this time have persisted in
their simple goniatitic character, become slightly digitate, or
ammonitic; this is shown on Pl. E, Fig. 1, diameter 6.00 mm.,
and Fig. 2, diameter 6.40, a little over four coils.

Paraneanic.— Near the beginning of the fifth whorl, at
diameter between 7 and 8 mm., the ribs begin to form knots
on the abdominal keels and the nodes on the umbilical shoul-
ders grow stronger. The height of the whorl, in proportion
to its width, grows more pronounced, and, instead of a sharp-
ened abdomen with a keel, the abdominal shoulders become
higher and more angular, and the ventral keel rises little above
them. At the same time the septa become more ammonitic, as
shown on Pl. E, Fig. 3, diameter 8 mm., four and one-half
whorls, and Fig. 4, diameter 9.20 mm., four and three-quarters
whorls. This stage lasts up to a diameter of about 12 mm.,
five whorls. The ribs and the nodes on the abdominal shoul-
der keels become gradually stronger, and the whorl grows

[Vou. XVI.

SMITH.

254

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LARVAL STAGES OF SCHLOENBACHIA. 255

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No.1.] LARVAL STAGES OF SCHLOENBACHIA. 2

steadily higher and narrower, changing gradually to the adult
characters, but with no sudden change to mark the stage.

EpPHEBIC OR ADULT.

It would be purely artificial to divide the adult stage into the
three subdivisions ana-, meta-, and paraphebic, for the change
is too gradual. Near the beginning of the sixth whorl, ata
diameter of 12 mm., the nodes on the shoulder keels grow
stronger and form continuations of the ribs, bending forwards
over the intervening space to the ventral keel, and finally notch-
ing it. Since most species of Schloenbachia have this charac-
ter this may be considered as the beginning of the adult period.
These characters are seen sometimes as early as four and three-
quarters whorls, diameter of 10 to 11 mm. A general descrip-
tion of the adult stage has already been given under the
diagnosis of the species. The adult septa are figured on PI.
E, Fig. 5, at diameter 18.20 mm., and a cross-section of an
adult specimen on Pl. C, Fig. 7, diameter 22.25 mm.

SyNoPsIS OF RESULTS.

Schloenbachia oregonensts is a remarkable species, in showing
its descent so well through its ontogeny ; the only other species
of which larval stages have been figured, S. varicosa Sowerby,
figured by Branco in Palaeontographica, vol. xxvi, Pl. E, Fig. 4,
shows the glyphioceran character at the third septum, the
Anarcestes, Tornoceras, and Prionoceras stages being omitted
by acceleration of development. The omission of stages occurs
just at this point, between the protoconch, which is always con-
stant in any one group, and the larval stages. A kindred form,
Oxynoticeras oxynotum, reaches the glyphioceran stage at the
second septum, having skipped the preceding stages, but going
through the Gastrioceras, Paralegoceras, and Styrites stages
just as does Schloenbachia oregonensis. This seems to the
writer to have been due to the hatching of different genera or
species at different stages of growth, the omitted stages corre-
sponding to a period when the animal remained in the egg after
formation of the protoconch.

258 SMITH. [VoL. XVI.

Schloenbachia oregonensis in its development repeats the
history of Avxarcestes, Parodoceras, and Prionoceras in the first
five septa and one-quarter of a coil from the nautiloid proto-
conch; then for about one whorl it is a Glyphioceras ; for about
one and one-quarter whorls it is a Gastrioceras ; then for a little
more than one-quarter of arevolution it isa Pavalogoceras, and at
two and five-eighths coils ends its goniatite history, takes on a
keel, and becomes an ammonite, but one like the simpler ammo-
nites of the Permian and Lower Trias. The ananeanic stage
lasts up to three and three-eighths whorls, that is, about three-
quarters of a revolution; the metaneanic stage lasts up to the
end of the fourth whorl, and the paraneanic to near the end of
the fifth whorl. With the beginning of the sixth whorl, at
diameter of about 12 mm., the shell begins to take on its own
proper characters, and is then in the ephebic stage, although
adults grow to at least 30 mm. in diameter, and probably larger.

The larval stages may be compared with considerable cer-
tainty to ancestral Paleozoic genera, but the Mesozoic genera
to which the adolescent stages might be compared are probably
mostly unknown as yet, although they will be found among
trachyostracan descendants of the Glyphioceratidae, and not
among the Prolecanitidae.

No more striking demonstration of the law of acceleration of
development, or tachygenesis, is possible than where a shell in
its larval history hastens through, in two and five-eighths whorls,
and in growth up to 2.70 mm., generic changes from Anarcestes,
Parodoceras, Prionoceras, Glyphioceras, Gastrioceras, and Para-
legoceras, an amount of development that its ancestors required
the time from the Lower Devonian to the end of the Car-
boniferous to accomplish. In the succeeding adolescent stages
the changes are not nearly so rapid.

Another fact brought out by the investigation of many
specimens is that individual variation increases greatly with
the advance of the stage. Thus all protoconchs and most
chambered stages are alike up to the end of the larval period.
After that the uniformity ends, for in the adolescent period the
ribs begin at various sizes, as does also the digitation of the
septa. And in the acquirement of adult characters still greater

No.1.] LARVAL STAGES OF SCHLOENBACHIA. 259

variation was observed, not only in time, but also in the char-
acters themselves, so that one would be inclined to make
several species out of one, were it not for the transitions
between the varieties. A parallel study of the ontogeny of two
nearly related species has shown just these same facts, only in
greater degree, for specific variation is only individual variation
carried to extremes.

260 SMITH.

EXPLANATION OF PLATE A.
Schloenbachia oregonensis Anderson.

Fics. 1-3. Protoconch, phylembryonic to ananepionic. 4°.

Fics. 4 and 5. Phylembryonic to paranepionic; diameter 0.58 mm.; one-half
whorl, first eight septa, glyphioceran stage at the sixth. 4°.

Fic. 6. Paranepionic, glyphioceran substage ; diameter 0.64 mm.; third to tenth
septa, five-eighths of a whorl. +42.

Fics. 7 and8. Phylembryonic to paranepionic, glyphioceran substage; diameter
0.68 mm.; three-quarters of a whorl, nine septa. 42.

Fic. 9. Paranepionic, glyphioceran substage ; diameter 0.74 mm.; seven-eighths
of awhorl. +2.

Fics. io and 11. Paranepionic, transition from glyphioceran to gastrioceran
substages ; diameter 1.20 mm.; one and three-eighths whorls. 7°.

Fics. 12 and 13. Paranepionic, transition from glyphioceran to gastrioceran
substage ; diameter 1.33 mm.; one and five-eighths whorls. 2°.

Fics. 14 and 15. Paranepionic, gastrioceran substage ; diameter 1.65 mm.; one
and seven-eighths whorls. 4°.

Journal of Morphology, Vol. XVI. Lie A:

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262 SMITH.

EXPLANATION OF PLATE B.
Schloenbachia oregonensis Anderson.

Fics. 1 and 2. Paranepionic, paralegoceran substage; diameter 2.25 mm.; two
and three-eighths whorls. 2°.

Fics. 3 and 4. Ananeanic, Styvztes stage; diameter 3.10 mm.; two and seven-
eighths whorls. 22.

Fics. 5and 6. Ananeanic, Parastyrites stage ; diameter 3.70 mm.; three and
one-fourth whorls. 1).

Fic. 7. Metaneanic, advanced adolescent stage ; diameter 5.60 mm.; three and

three-quarters whorls, showing beginning of ribs at diameter 4.70 mm. 1).

Y ial fe a0

Journal of Morphology, Vol. XVI.

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204 SMITA.

EXPLANATION OF PLATE C.
Schloenbachia oregonensis Anderson.

Fic. 1. Protoconch of Schloenbachia, showing the first six sutures of the
attached coil. Enlarged thirty times.

Fic. 2. Larval stage of Schloendachia, diameter 0.68 mm.; thirty times enlarged;
three-fourths of first whorl. 2a, side view; 24, front view.

Fic. 3. Larval stage of Schloendachia, diameter 0.64 mm. ; thirty times enlarged.
Showing sutures from the third to the tenth. From above.

Fic. 4. Larval stage of Schloenbachia, diameter 1.20 mm.; fifteen times en-
larged ; one and one-half whorls. 4a, front view; 4é, side view.

Fic. 5. End of larval stage of Sch/oenbachia, diameter 2.25 mm.; fifteen times
enlarged. Paralegoceras stage. 5a, side view; 54, front view.

Fic. 6. Cross-section of Schloendachia, diameter 6.25 mm.; fifteen times en-
larged ; four whorls. Adolescent stage. The protoconch is seen in the center ?.

Fic. 7. Cross-section of Schloenbachia, 22.25 mm.; three and one-half times
enlarged ; six whorls. Adult stage.

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266 SMITH.

EXPLANATION OF PLATE D.

Schloenbachia oregonensis Anderson.

Development of the septa.

Fic. 1. Septum at seven-eighths whorl; diameter 0.75 mm.; glyphioceran
stage; paranepionic. 4°.

Fic. 2. Diameter 1.70mm.; gastrioceran stage; two whorls; paranepionic. 4°.

Fic. 3. Diameter 2.50 mm.; paralegoceran stage; two and one-half whorls;
paranepionic. 4°.

Fic. 4. Diameter 3.00 mm.; Styrvites stage; two and seven-eighths whorls;
ananeanic. 4°.

Fic. 5. Diameter 3.80 mm.; three and one-eighth whorls ; Parastyrites stage. 2°.

Fic. 6. Diameter 4.86 mm.; three and one-half whorls; neanic. 2°.

Journal of Morphology, Vol. XVI.

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268 SMITH.

EXPLANATION OF PLATE E.

Schloenbachia oregonensis Anderson.

Development of the septa.

Fic. 1. Diameter 6.00 mm.; about four whorls; metaneanic. 42.

Fic. 2. Diameter 6.40 mm.; metaneanic. 4°.

Fic. 3. Diameter 8.00 mm.; paraneanic; four and one-half whorls. 135.
Fic. 4. Diameter 9.20 mm.; paraneanic; four and five-eighths whorls. 143.

Fic. 5. Diameter 18.50 mm.; metephebic, early adult; about five and three-
quarters whorls. 7.

Fic. 6. Diameter 5.60 mm.; three and three-quarters whorls; front view of
Fig. 7,0n Pl. IV. 4.

Fic. 7. Glyphioceras (Muensteroceras) oweni Hall. Pal. V.Y.,vol.v. Part II.
Pl. 73, Fig. 6, for comparison with the young stage of Schloenbachia orego
nensis. 4.

Fic. 8. Glyphioceras (Muensteroceras) oweni Hall. Loc. cit., Fig. 3, adult, 3,
for comparison.

Journal of Morphology, Vol. XVI. PU ore:

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Volume XVI, February, 1900. Number 2.

JOURNAL

OF

MOR PP OEnOGY,

STOLONIZATION IN AUTOLYTUS VARIANS.
P. CALVIN MENSCH.

TuE development of the stolons in the chain-forming species
of Syllidians has been referred to as early as 1788 by O. Franz
Miiller (1), and later by de Quatrefages (2), Milne-Edwards (3),
Claparéde (4), Von Marenzeller (5), and Langerhans (6) ; but it
is only in the recent works of de St.-Joseph (7), Pruvot (8),
and more recently in the excellent monograph of Malaquin (9),
that detailed descriptions of this process are presented. In this
paper I shall give results of my observations in a species which
presents some characters different from those described in the
previous works.

I desire to express my gratitude to Dr. M. M. Metcalf, at
whose suggestion I undertook this work, also to Professor
Whitman, director of the Marine Biological Laboratory at
Woods Holl, Mass., for valuable assistance and advice, and
finally to Dr. E. A. Andrews for suggestions as to methods
of collecting the species.

Matertal,— The material for the work was collected at Woods
Holl during the summer of 1895. Individuals of Autolytus
varians in the process of budding may be found at any time

of the year among the hydroids growing on the piles or in the
269

270 MENSCH. [Vou. XVI.

dredgings in and about Vineyard Sound. They occur most
abundantly among the stems of Parypha, and during certain
seasons of the year may be obtained in all stages of develop-
ment by placing the hydroids in vessels of water and allowing
them to remain until the water begins to become stagnant,
when the specimens will collect at the surface or sides of the
vessel. The mature sexual individuals are best collected in the
tow-net at night in the region of piles, though they may also
be found among the hydroid stems.

The abundance of the stolon-bearing forms is very much
dependent upon the condition of Parypha, being most plentiful
when the hydroids are fully developed, and disappearing almost
altogether at the time when the hydroids die down. Occasional
specimens in different stages of stolonization may also be found
among the stems of Bugula and certain algae, but never in any
number, even in localities otherwise favorable to their exist-
ence. In dredgings of loose sand, taken from a depth of from
five to ten fathoms, quite a number of specimens were also
obtained.

Associated with this species may be found two other species
of the tribe Autolytus: the one, A. cornutus, a small form
first described by A. Agassiz (10), which occurs in abundance
on the stems of Eudendrion and Penaria in the early part of
the summer, before the appearance of A. varians; the other,
Procerea ornatus (Verrill), very much larger, and appearing in
numbers among the stems of Eudendrion and Parypha later
in the summer, after A. varians has become less abundant. In
both of these species the phenomena of stolonization are essen-
tially different from that of A. varians. Individual specimens
of these three species may be found at any season of the year
in process of budding.

Methods. — ¥or surface study living specimens were used
almost exclusively, the distortions produced by the killing fluids
frequently being so marked as to make some of the most impor-
tant details uncertain. By allowing the animals to remain in
a small quantity of sea water until it becomes somewhat stag-
nant, they become sufficiently inactive to permit the employment
of pressure and considerable manipulation without producing

No. 2.] STOLONMIZATION IN AUTOLYTUS VARIANS. 2 7

unfavorable contractions. Very dilute solutions of methylene
blue have been of service in studying some of the chains of
living individuals, the stain soon becoming sufficiently deep to
be of service without producing any noticeable irritation.

For sectioning, on account of the small size of the animal,
great care is necessary in killing the specimens, so as to avoid
contortions and separation of the stolons. Several methods
were employed. One was to place the worm in very dilute
alcohol (35%), and in the course of several hours to gradually
increase its strength until the animal has become thoroughly
benumbed, after which it was placed into the killing fluid. This
method gave good results in many cases, but frequently the
process of narcotizing required so much time that the sections
were ruined. Another method which gave good results was to
plunge the worm into 60% alcohol, remove at once into fresh
sea water, and subsequently add alcohol until it was thoroughly
stupefied, after which it was placed into the fixing fluid. The
best method for killing the animal extended, whenever other
fixing fluids than 70% alcohol were employed, was found to be
by placing it on a slide and drawing off most of the water, then
applying a very weak solution of the fluid by means of a small
brush and gradually increasing the strength of the fluid. In
this way frequently almost perfectly straight chains were
obtained.

A number of fixing fluids were tried. Those which gave the
most satisfactory results were Perenyi’s fluid, corrosive subli-
mate, picro-sulphuric with corrosive, Flemming’s stronger and
weaker solutions, and 70% alcohol, the latter giving for general
study uniformly the best results. For staining, borax carmine
and haematoxylin were used.

DESCRIPTION OF THE SPECIES.

Two distinct varieties of A. varians, described and named by
Verrill (11), may be found among the stems of Parypha, both
of which occur in about equal numbers. The larger variety is
from 10 to 20 mm. in length, is flesh colored, and, when exam-
ined under a medium magnification, may be seen to contain a

Ze MENSCH. [Vor. XVI.

large number of red spots extending along the walls of the
alimentary canal through its entire length, but particularly
numerous in the region of the oesophagus and in the posterior
and more mature stolons. The free-swimming stolons of this
variety have a distinctly light red color and are considerably
larger than those of the other variety. The other variety has
a greenish hue, with few or no red spots along the alimentary
canal, and is somewhat smaller and more slender. Intermediate
forms are, however, frequently found, and the distinction between
the two varieties is not so well marked in the parent stock as
it is in the mature stolons. The free-swimming stolons of this
variety, besides being smaller in size, are light green in color
and somewhat iridescent. They differ very little in size from
the free-swimming stolons of A. cornutus, but the male stolons
can readily be distinguished from these by the fact that the
Polybostricus of A. varians has swimming setae wanting on the
three anterior parapodia, while in A. cornutus they are wanting
on the six anterior pairs. The Sacconereis of either species can
also be readily distinguished by the characters common to each
mode of stolonization.

The parent stock of Autolytus varians (Pl. XIII, Fig. 1) con-
sists of a series of setigerous segments varying in number from
nineteen to as many as fifty-eight, the larger and older individ-
uals always containing the larger number of segments. Ante-
rior to the first setigerous segment is a segment (Pl. XIII,
Fig. 2, 4.5.) called the buccal or tentacular segment, in which
parapodia are absent; but instead of these are present two pairs
of cirri, called the dorsal (d.¢.) and ventral (v.¢.) tentacular cirri
respectively. The dorsal tentacular cirri are longer and con-
siderably thicker than the ventral pair, which are short and
slender and more like the dorsal cirri of a setigerous segment.
The segment itself is usually considerably narrower than the
succeeding setigerous segment. Anterior to the buccal seg-
ment is the head, which consists in this species of a rounded
lobe with two pairs of eyes, an anterior larger and posterior
smaller pair, bearing a single dorsal median (d.m.) and a pair
of lateral tentacles (7). A pair of rudimentary palps form the
ventral appendages of this lobe.

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No. 2.) STOLONMIZATION IN AUTOLYTUS VARIANS. oar ae

Attached to the last segment of the parent stock in mature
individuals may be found a chain of stolons (Pl. XIII, Figs. 1
and 6) in different stages of development. Of these stolons
the posterior is the oldest and most matured individual; the
one next to it being somewhat younger and less mature, and
the ones anterior to this being progressively younger and less
mature, so that the most anterior presents only very faintly the
outline of a stolon. The number of such stolons in a single
chain may vary from several to as many as eight, and appears
to be dependent upon the size of the parent stock and the sex
of the stolons. In a larger chain, where the number of stolons
may be as many as seven or eight, the different stages of devel-
opment are progressively represented in each successive stolon.
In a chain of fewer stolons, however, this is not so marked,
and more frequently chains composed of but several stolons
are found in which an almost mature posterior stolon may be
attached to a very young and immature anterior stolon. Fre-
quently specimens of this species may also be found which
contain but a single, often quite mature, stolon, and give no
evidence of a chain formation. Such specimens are usually
smaller and younger in appearance and seem to indicate the
very beginning of the process of stolonization.

Anterior to the youngest stolon and forming the connective
between the chain of stolons and the parent stock are a number
of segments (Pl. XIII, Fig. 6, ~e.) which are still younger and
give little evidence of belonging to a distinct stolon. This
region, since it is composed of the youngest and least devel-
oped segments, I shall designate as the embryonic region. The
segments of this region are successively produced as outgrowths
from the last segment of the parent stock, which segment,
since it presents internal structures relative to this outgrowth
that are different from those of the preceding segments, I shall
refer to as the segment of proliferation.

Description of the Free Stolons.

Before proceeding to trace the external development of the
stolon, it will be well to describe the appearance of the mature

274 MENSCH. [Vou. XVI.

separated stolons, or the so-called sexual individuals, Polybostri-
cus ($) and Sacconereis (?). The Polybostricus of the red
variety (Pl. XIII, Fig. 3) is about 5 mm. long; that of the
green variety, 4 mm. and more slender. (This and the color
being the only marks of distinction between the two varieties,
I shall make no reference to either variety in further descrip-
tions.) It consists of from eighteen to twenty-four setigerous
segments, the first three of which have short parapodia, with
short setae similar to those borne by the parapodia of the
parent stock (Pl. XIII, Figs. 1a. and 16.), and contain the sex-
ual products. All the segments posterior to these, with the
exception of the last or anal segment, have large elongated
parapodia (Pl. XIII, Fig. 4), consisting of a ventral less mus-
cular portion ending in a short process (v.7.) and bearing the
short setae, and a more dorsal thickened and very muscular
portion ending in a process (d.rv.) which contains a tuft of long
swimming setae. Dorsal to the thick musculature belonging
to the dorsal ramus is a thin plate-like structure which forms
the dorsal outline of the parapodium and constitutes the basal
portion of the dorsal cirrus (d.c.). When the stolon is at rest
the parapodia are always directed backward and pressed close
against one another, thus giving this part of the body a very
compact appearance and obscuring the outline of the segments.
The size and direction of these parapodia as compared with the
size and direction of the three anterior pairs produce a contrast
sufficient to divide the stolon into two well-marked regions. At
the junction of these regions the body-wall, consisting of the
posterior part of the third and the anterior part of the fourth
setigerous segments, shows quite prominently, and on exami-
nation appears less firm, and the line of demarcation between
the segments is much fainter than it is in the parent stock.
The anal segment is small, considerably narrower than the
preceding segments, and in place of parapodia a pair of long,
slender caudal cirri form the only appendages of the segment.

The buccal segment is well marked and bears a dorsal and a
ventral pair of tentacular cirri. The dorsal tentacular cirri (@.2.)
are very stout at the base and, when fully extended, reach as
far back as the thirteenth or fourteenth setigerous segments,

No. 2.] SZOLONIZATION IN AUTOLYTUS VARIANS. 275

The ventral tentacular cirri (v.¢.) are much more slender and
about one-fourth as long as the dorsal pair.

The head differs in shape from that of the parent stock in
being broad and emarginate in front. Two pairs of eyes are
present ; those corresponding to the anterior eyes of the parent
stock, being the larger, are placed on the ventral side of the
head, so that they are not seen in a dorsal view of the animat.
The ventral eyes are considerably larger than the correspond-
ing ones of the parent stock, and bear conspicuous lenses which
are directed down and outward. The second pair are smaller in
size and are placed dorsally and directly over the ventral pair.
Small lenses are present and directed upward and forward. A
single median tentacle and two pairs of lateral tentacles com-
prise the appendages of the head, distinct palps being absent.
The median tentacle (d.m.) is about equal in length to the
dorsal tentacular cirrus, but is less stout at the base and is
always directed backward. The anterior lateral tentacles (a./.)
are flattened dorso-ventrally, are very broad at the base, gradu-
ally tapering as they curve outward, and end in bifurcated
processes which are not unlike dorsal cirri. The position and
form of this tentacle have led Malaquin and several other inves-
tigators to regard it as being formed by the fusion of the palp
with the lateral tentacle, the inner ramus representing the
palp, the outer, the anterior lateral tentacle. The posterior
lateral tentacles (f./.) are short and straight, inserted anterior
to the dorsal eyes and usually directed forward, reaching a
little beyond the margin of the head. This pair of tentacles
is not represented in the parent stock. The mouth opening of
the Polybostricus, as also of the Sacconereis, lies a little ante-
rior to the base of the ventral tentacular cirri and is directed
downward.

The Sacconereis of this species (Pl. XIII, Fig. 5) is from 3
to 4 mm. in length and contains from sixteen to twenty setiger-
-ous segments. Swimming setae are usually absent from the
first two setigerous segments, sometimes only from the first.

The buccal segment is narrow dorsally ; ventrally, however,
it is quite well marked. The dorsal tentacular cirrus is absent,
but a small papilla (d.2.) occupying the same position may be

276 MENSCH. [VoL. XVI.

found in most of the specimens, and may be regarded as the
rudimentary representative of this cirrus.

The head is less emarginate in front than that of the Poly-
bostricus, has a median (d@.m.), and but a single pair of lateral
tentacles (a./.), and is very similar in appearance to the head of
the parent stock. Palps and the posterior lateral tentacles are
absent, and the anterior lateral tentacles are not thickened and
bifurcated, as in the male stolon.

The fully matured Sacconereis carries on its ventral side an
egg-sac (0.v.) filled with eggs. This sac consists of a thin
membrane attached to and formed from the tissue of the under
surface of segments 4 to 8 org. The anterior part of the body
as far as, and sometimes including, the fourth setigerous seg-
ment is free, as are also the segments posterior to 8 and 9.
When the animal is at rest the free portions of the body are
usually coiled around the egg-sac in a spiral manner, in this
way forming a protection for the delicate sac. When in motion,
however, the free regions of the body are fully extended and
the egg-sac bulges considerably both ventrally and laterally.
The color of the less mature eggs is bright red; but as they
become more fully matured they assume a darker and almost
black appearance.

In comparing the head structure of the mature male and
female stolons with those of the parent stock it is evident that,
with the exception of the fusion of the palps with the anterior
lateral tentacles, the presence of the posterior lateral tentacles
in the male, the absence of palps and dorsal tentacular cirri in
the female, and the shifted position of the anterior eyes, the
head parts of the stolon are an almost exact reproduction of
the head parts of the parent stock.

The movements of these free stolons as compared with those
of the parent stock are very active, and notwithstanding the
size of the egg-sac, the mature Sacconereis is able to move
about with great agility.

Sexual Characteristics of the Chain of Stolons.— The chain
of stolons is always unisexual, all stolons of a chain being
either male or female. The sex of the chain can, even in very
_ young stolons, be distinguished by the bifurcation, in the male,

ee —

No. 2.] SZOLOMIZATION IN AUTOLYTUS VARIANS. 277

of the anterior lateral tentacle, which becomes evident soon
after the tentacle has made its appearance.

The male and female chains of Autolytus varians also show
another difference very similar to that observed by Malaquin
(9), de St.-Joseph (7), and Pruvot (8), in other chain-forming
species, vzz.: the male chain is always longer and contains
the greater number of stolons. Few female chains of this
species are found with more than four or five stolons, while
male chains may have as many as eight stolons.

The relative number of male chains appears also to be greater
than that of the female chains. This fact is particularly evi-
dent among the free individuals, where five or six males may be
found to a single female.

EXTERNAL PHENOMENA OF STOLONIZATION AS REPRESENTED
IN THE CHAIN OF STOLONS. ;

The successive stages in the development of stolons of
a chain are well shown in a male chain, as is represented in
Pl. XIII, Fig. 6. This chain consists of six well-defined stolons,
with a distinct embryonic region (”e.), and is attached to the
thirty-fifth segment of the parent stock. Externally the thirty-
fifth segment, which is in this specimen the segment of prolif-
eration, presents no characters different from those of the
preceding setigerous segments.

The embryonic region of this specimen consists of five seg-
ments, the two anterior of which are of equal size, and, being
the youngest segments of the series, may be regarded as typi-
cal embryonic segments. These two segments are very much
smaller than the segments of the parent stock and show no
signs of parapodia. The third, fourth, and fifth segments are
slightly more advanced and present on each side small papilla
which represent rudimentary parapodia. These segments are
of equal size and differ sufficiently from the two segments pre-
ceding them and the series following to be regarded as having
been formed in close succession and as developing at an equal
rate toward the formation of a new stolon (St. A). Very fre-
quently instead of three equally developed segments, as repre-

279 MENSCH. [Vou. XVI.

sented in this figure, four such segments are present. In all
cases, however, whether three or four segments be present, the
first and the last are a little larger and more developed than
the intervening segments.

The beginning formation of this stolon is more evident in a
slightly older series of this kind, with four segments, as is rep-
resented in Pl. XIII, Fig. 7. Segments 1, 2,and 3 represent
the three appendage-bearing segments of Fig. 6 (St. A). . Seg-
ments I and 3 have increased in size and bear lateral append-
ages of about equal size, while segment 2 has increased less
and has smaller appendages. Between segments 2 and 3 a new
segment has made its appearance and presents the same embry-
onic characters as do segments I and 2 in the embryonic region
of Fig. 6.

In such a series as is represented in Fig. 7 there are pres-
ent all the segments necessary for the formation of the differ-
ent regions of a mature stolon. Of these, segment I becomes
the first setigerous segment from which will be developed the
head and the buccal segment; segment 3 forms the anal seg-
ment; and segment x represents the region of new growth
which contributes to the elongation of the stolon. The origin
of segment +, as we shall see in the study of a section of this
region, appears to be from the anal segment and is not depend-
ent upon any contribution from segment 2 in the process of
its development. In this way segments 1 and 3 become the
important factors in the development of the stolons, while seg-
ment 2 remains as an indifferent zone which is constantly
being increased by the addition of new segments posteriorly.
Malaquin (9) in describing the development of the stolon in
Myrianidae lays great weight upon the importance of the anal
segment (Pygidium) so far as its early appearance is concerned,
but pays less regard to the part it plays in the formation of the
embryonic segment (x) of this stolon, a fact which I regard as
of no little importance.

The origin of this stolon would then be by (1) the successive
outgrowth from the last segment of the parent stock, or seg-
ment of proliferation, of three embryonic segments similar to
the three segments of stolon A in Fig. 6, followed by (2) the

No. 2.] SZTOLONIZATION IN AUTOLYTUS VARIANS. 279

addition of a fourth segment (7), while at the same time lateral
outgrowths are appearing on segments I and 3 and a little
later on segment 2, thus presenting a stage as represented in
Fig. 7.

Two regions of growth may accordingly be distinguished as
having been-active in this early stage of development. The
first region is that posterior to and including the segment of
proliferation and has contributed three, or, in some specimens,
four segments to the formation of the stolon, these segments
forming the most anterior setigerous, the anal, and one or two
indifferent segments. Of these segments the anal is the old-
est, the others being successively younger from this segment
forward, while the first setigerous is, therefore, the youngest.
The second region of growth is anterior to and includes the
anal segment, and supplies new segments for the lengthening
of the individuals. This region in Autolytus varians does not
contribute to stolon A in Fig. 6, but makes its appearance at a
little later stage, thus not becoming active until after the out-
line of the stolon has been clearly defined.

Posterior to this zone of embryonic segments in Fig. 6 is the
most anterior stolon (St.1), which represents in a rudimentary
condition all the regions of a mature stolon. This young
stolon consists of eight well-marked segments, the anterior
ones being the larger, while the posterior ones are considerably
smaller. The first four segments have rudimentary parapodia,
with small setae just appearing, and distinct dorsal cirri. The
fifth segment has still more rudimentary appendages, similar
to those of stolon A. The sixth and seventh segments are the
most embryonic and show no evidence of lateral appendages,
but present an appearance like the embryonic segments in the
anterior part of the chain. The eighth or anal segment has a
pair of caudal cirri (Fig. 8, c.c.) of considerable length, the
development being sufficiently advanced to bear evidence of
the early origin of this segment.

On the anterior dorsal half of the first segment of this stolon
a new region of growth (c.) has made its appearance. This is
the first indication of the head and consists, in a stolon of this
age, of merely a thickening of the tissue in the anterior half of

280 MENSCH. [Vou. XVI.

the first setigerous segment and its forward extension slightly
beyond the anterior region of this segment. The forward exten-
sion of this new tissue is confined in greater part to the dorsal
surface and has the appearance of a thickened plate, with the
lateral portions bulging out so as to give the appearance of the
two lateral lobes. At the sides this plate tapers into a narrow
rim which is lost in the lower lateral margin of the segment on
a plane with the parapodia. This stage of development has
been reached by a gradual elevation and forward extension of
mostly the anterior part of the first setigerous segment. The
very first indication of this growth is a slight thickening of
almost the whole dorsal wall of the segment, which gradually
becomes more marked anteriorly and soon forms a prominent
ridge over the anterior dorsal and lateral portions of the seg-
ment. Subsequently this ridge begins to bulge forward in the
form of a prominent rim, with two faint lobes laterally, beyond
which it gradually becomes less marked until near the ventral
surface, where it disappears.

Stolon 1, therefore, presents two regions of growth. The
one anterior to and including the anal segment which, as I
have already indicated in the description of the preceding
stolon, is rapidly contributing to the increase of the number
of setigerous segments. The other is a new region, which has
appeared prominently for the first time in this stolon. This
new region is developed secondarily in a segment which has
originally been separated from the parent stock, and appears
shortly after the region of growth anterior to the anal segment
has become active. Thus the different structures of this stolon
have originated in three ways: (1) from segments derived di-
rectly from the parent stock; (2) from segments derived from
the anal region of growth; (3) from outgrowth from the ante-
rior segment of the series. Of these, the last two contribute
to the future development of the stolon.

Stolon 2 represents a stage a little more advanced than
stolon 1, and presents the most important changes which next
take place in the process of development. It consists of fifteen
setigerous segments, the anterior ones of which are consider-
ably larger than those of stolon 1, while the more posterior com-

No. 2.] SZOLONIZATION IN AUTOLYTUS VARIANS. 281

pare well in size with those of the preceding stolon, thus making
the outline of the stolon appear somewhat wedge-shaped. The
parapodia and dorsal cirri in the anterior half of the body are
well developed, while those in the posterior half are progress-
ively smaller from before backward as far as the pre-anal seg-
ment, which segment, as in the preceding stolon, represents
the region of youngest growth. In this and the two preceding
segments, in a progressively less degree however, the parapodia
are quite rudimentary, and the segments present an appearance
very similar to the parapodia-bearing segments of stolon A.
The anal segment has also increased in size, and the caudal
cirri have been considerably elongated. In this figure the
segment is almost completely hidden by the head of the
succeeding stolon.

In comparing the setigerous segments of this stolon with
those of the preceding, it will be noticed that parapodia, either
well developed or rudimentary, are present on all the segments
of the stolon except the anal. This would indicate that the
purely embryonic growth, as represented in the embryonic
segments of the preceding stolon, had passed its stage of
greatest activity and is not as prominent as in the preced-
ing stolon, the stolon having already attained a considerable
length and the addition of new segments from this stage on
progressing very much slower.

The head of this stolon has also advanced considerably, both
in size and in the development of new structure. Its antero-
posterior diameter has increased sufficiently to equal that of
one of the most advanced setigerous segments of the stolon.
The lobed structures, appearing in the preceding stolon and
becoming more prominent in intermediate stages, have given
rise to the rudimentary anterior lateral tentacles (a./.). These
tentacles are thick and knob-like in structure and are directed
forward and outward. The dorsal median tentacle (¢@..) has
also appeared in the form of a slender tentacle, extending as
far back as the second setigerous segment. Two eyes (e.a.)
have appeared, and have in this individual already attained a
considerable size. These are located at the base of the lateral
tentacles, and hence correspond to the anterior eyes of the

282 MENSCH. [Vou. XVI.

parent stock. The first indication of these eyes is marked by
the appearance of minute pigment spots in a stage intermediate
between this and stolon 1, at a time when the lateral and dorsal
median tentacles have become about one-half as large as repre-
sented in stolon 2.

A lateral view of the developing head of this stolon is repre-
sented in Pl. XIII, Fig. 8. In this view it will be seen that
the zone of new growth extends forward considerably more
than in the preceding stolon, and also, that instead of extending
down laterally as far only as the parapodia, it now extends to
the ventral median line. The whole structure at this stage
assumes the appearance of a separate segment, being narrower
above and narrowing down laterally until on the ventral surface
it forms a very narrow strip of tissues, the outlines of which are
lost as it approaches the median line. On the lateral surface
of this new growth, a little dorsal to the insertion of the dorsal
cirri, a small papilla (@7¢.) has made its appearance. This bud
represents the rudimentary dorsal tentacular cirrus, and first
appears at about this stage of development, being somewhat
later than the dorsal median tentacles, which may be found in
stolons of a stage of development intermediate between this and
stolon 1. Ina stolon of this age, then, there are present: (1) a
pair of anterior lateral tentacles ; (2) the dorsal median tentacle ;
and (3) the pair of dorsal tentacular cirri, all having appeared
successively in the order enumerated. Since the tentacular
cirri form a part of the buccal segment, it may be assumed
that already in this stage this new tissue is being differentiated
into the buccal and head segments, even though there are no
external indications of this division.

Between the anal segment of the preceding stolon and the
head parts of stolon 2 (Pl. XIII, Fig. 8) there exists a narrow
structure which belongs, properly speaking, to neither of these
segments, and which I shall designate as the region of separa-
tion (r.s.). This region forms the connection between the two
stolons, and in earlier stages seems to belong to and constitute
an undifferentiated part of the anal segment. Ata stage repre-
sented by these two stolons, however, it seems, externally at
least, to be entirely distinct from the anal segment, and presents

No. 2.]| SZOLONMIZATION IN AUTOLYTUS VARIANS. 283

the appearance of a very narrow lighter band of interposed em-
bryonic tissue. It is in this tissue that the separation of the
stolon from the chain will take place. It becomes plainly visible
for the first time between consecutive stolons of this stage of
development, and appears most prominently here, being in later
stages hidden by the overgrowing head of the succeeding stolon,
and also probably because of the structural changes, to which
I shall refer later.

Stolon 3 shows a few more advances in the process of devel-
opment which make their appearance in an individual of this
size. This stolon consists of eighteen setigerous segments,
followed by the anal segment, not shown in this sketch. The
anterior segments are somewhat larger than those of the pre-
ceding stolon, and the parapodia and dorsal cirri are more
prominent and more fully developed, the whole individual pre-
senting a larger and more mature appearance. The breadth
of the head has increased in conformity with the breadth of
the first setigerous segment, and a second pair of eyes (e.f.)
has appeared, being placed posterior to the first pair and in a
line with the insertion of the dorsal median tentacle. These
eyes are not so widely separated from one another as are the
first pair and are considerably smaller. The arrangements of
the eyes on the head of this stolon very closely simulate the
position of the eyes on the head of the parent stock, although
the shape of the head is decidedly different. The appendages
of the head have also increased in size; the dorsal median ten-
tacle has increased by about the width of two segments and
reaches as far back as the third setigerous segment. The two
anterior lateral tentacles, besides having increased in length,
also show indications of branching by the appearance of a small
bud on the median side of each tentacle. The bifid tentacle
being characteristic of a male stolon, it is possible even at this
stage of development to distinguish the sex of the stolon and
hence of the chain. In a dorsal view of this stolon the outline
of the buccal segment is not more visible than in the preceding
stolon, and were it not for the projection of the dorsal tentacu-
lar cirri (d.¢.) evidence of the existence of this region would be
wanting. Laterally the buccal segment has attained a consid-

284 MENSCH. [VoL. XVI.

erable dimension, particularly in the region of the dorsal ten-
tacular cirrus, and its outline is more easily defined than in the
preceding stolon. More dorsally its outlines, as in the preced-
ing stolon, are lost in the external undifferentiated tissue which
constitutes what is here designated as the head.

While the dorsal and lateral surfaces of the chain thus far
examined have undergone numerous changes by way of the
addition of new tissue and the formation of new structures, the
contour of the ventral surface has remained very much less
disturbed, and a ventral view of parts of stolons 2 and 3 (Pl. XIII,
Fig. 9), in which dorsally great changes have taken place, indi-
cates comparatively little activity in the surface of this region,
the changes being confined to the addition of new segments of
similar character and not to the ventralward extension of dor-
sal thickenings. Such a view of this part of the chain would,
therefore, present a comparatively smooth surface ventrally,
with marked lateral irregularities caused by the difference in
size of the various segments. In this figure may be distin-
guished the exact outline of the anal segment (a.s.) of stolon 2,
the true size of its caudal cirri, and the position of this seg-
ment in relation to the head of the successive stolon. The
zone of separation (7.s.) appears as a very narrow band of tis-
sue, the outlines of which are plainly visible in this view.

Stolon 4 presents a still later stage in the process of devel-
opment. It consists of twenty distinct setigerous segments,
the last four or five of which are quite narrow as compared
with the preceding, and thus gives the outline of this stolon an
appearance quite different from that of the preceding ones and
makes it conform more closely to the outline of the free Poly-
bostricus. As compared with the preceding stolon, the seg-
ments of the anterior two-thirds of this stolon have increased
in breadth and a greater number of parapodia in an advanced
stage of development have become visible. The appearance of
one of the more advanced parapodia of a stolon of this age is
represented in Pl. XIII, Fig. 10. At this stage the ventral branch
of the parapodium (v.7.) has attained its full development and is
not unlike the lateral appendages which constitute the entire
parapodium in the parent stock so far as the position and num-

No. 2.] SZTOLONMIZATION I[N AUTOLYTUS VARIANS. 285

ber of setae are concerned, and differs but little in size. The
dorsal cirrus (d.c.) is, however, placed a little more dorsally
than that of the parent stock, and between the dorsal cirrus
and this ventral branch of the parapodium a new growth of
tissue (@.7.) is arising. This new growth is the rudiment of the
dorsal branch of the parapodium and becomes visible for the
first time in a stolon of this stage of development.

In a dorsal view the appearance of the head has remained
unchanged, except that the anterior eyes have approached more
closely the lateral margin of the head. The shifting in posi-
tion of the anterior eyes at this stage is very slight, but still
sufficient to mark the beginning of important changes that are
about to make their appearance in stolons of this size. These
changes consist in the gradual outward shifting of the anterior
eyes, in process of which they are first carried laterally, then ven-
trally, until finally in the free-swimming individual they occupy
a position directly ventral to the posterior eyes. The shifting,
as we will see later in the study of transverse sections of this
region, is due to the large increase in the number of nerve cells
in the middle region of the head, the region occupied by the
eyes being in consequence carried successively lateral and ven-
tralward. In a lateral view of a stolon a little more mature
(Pl. XIII, Fig. 11), the anterior eyes (e.a2.) occupy a lateral posi-
tion, and the changes in the contour of the head have been
such as to bring the anterior and the posterior eyes nearer a
transverse line with one another. The appendages of the head
of stolon 4 have increased considerably in size, the dorsal
median tentacle reaching as far back as the sixth setigerous
segment, while the anterior lateral, besides elongating, have
also increased in thickness and are bifurcated for more than
one-half their length. The outlines of the buccal segment
have not as yet appeared dorsally, although the dorsal tentacu-
lar cirri (d.¢.) have attained a considerable length. In a lateral
view of the slightly older stolon, represented in Fig. 11, this
segment is quite well marked, and besides the presence of the
now elongated dorsal tentacular cirri (@.7.), a rudimentary ven-
tral tentacular cirrus (v.¢.) has also made its appearance. In
this figure may also be noticed the region of separation (~s.),

286 MENSCH. [Vou. XVI.

which is still visible in a lateral view of a stolon of this age.
On comparing it with the same region of younger stolons, it
will be noticed that it has become more constricted and forms
a much narrower bond of union between the two stolons, the
constriction at this stage being noticeable both ventrally and
laterally. It is at this stage of development also that the young
stolon shows the first indication of independent movement.
Stolon 5 represents a stage in which the stolon has attained
its fullest length and breadth, and in which the general contour
of the Polybostricus is becoming more apparent. The most
striking difference between this and the preceding stolon exists
in the large increase in the size of the parapodia, particularly
in the middle region of the body, the development of the para-
podia being so great as to give the segments an entirely differ-
ent appearance in a surface view. If the anterior or posterior
surface of such a parapodium (Pl. XIII, Fig. 12) be examined,
it will be noticed that the dorsal ramus (d@.7.) has increased
very much in length and slightly exceeds the ventral ramus
both in length and breadth. The two rami remain fused in
the line of junction and thus form a broad, flattened appendage
with a tuft of setae protruding from the ventral angle. In the
outward growth of the dorsal ramus the dorsal cirrus has been
carried outward from its position on the side of the body-wall,
and now occupies a position on the dorsal angle of the para-
podium. As compared with the more simple parapodium of
the parent stock (Pl. XIII, Fig. 1a.), this parapodium is not
alone very much larger, but by the outward growth of the
dorsal ramus the position of the ventral ramus has been so
changed _as to cause it to assume a more transverse position
instead of inclining ventralward, as it does in the parent stock.
Normally, when at rest these parapodia are directed slightly
backward, thus already assuming the position of the parapodia
of the free forms. The parapodia of the first three setigerous
segments, however, do not undergo so complete a change.
They remain considerably smaller, and instead of being di-
rected backward, are placed directly at right angles to the
axis of the stolon, the growth of the dorsal ramus being con-
fined to an elongation of that region sufficient to carry the

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No. 2.] SZTOLONIZATION IN AUTOLYTUS VARIANS. 287

insertion of the dorsal cirri to a position almost dorsal to that
of the setae.

The head of this stolon has also assumed a different appear-
ance, and in a dorsal view has attained the size and shape of
the head of the Polybostricus. The anterior eyes have shifted
laterally and now occupy a position very similar to that repre-
sented. im Pl. XIII, Fig. 11.') The posterior eyes have’ been
carried forward to nearer the anterior margin of the head and
have increased considerably in size. The appendages of the
head have undergone no changes save an increase in length.
The buccal segment is now visible dorsally as a narrow seg-
ment considerably less in transverse diameter than the head
and very indistinctly separated from it. The dorsal tentacular
cirrus has increased very much in length, while the basal por-
tion has increased to a thickness greater than the dorsal width
of the buccal segment. The ventral tentacular cirri have
attained a length about equal to that of a dorsal cirrus.

Stolon 6 represents the stage of advancement shortly before
the stolon is separated from the chain. As compared with the
preceding stolon the most striking change is the still greater
increase in the size of the parapodia and the presence of swim-
ming setae in all the parapodia except the three anterior. The
increase of the parapodia in thickness as well as in length has
given the posterior three-fourths of the stolon a more compact
appearance, thus making the distinction between the three
anterior and the posterior segments very much greater. The
larger parapodia are directed backward still farther, and in this
way a considerable space is left between the third and fourth
pair of parapodia. In an anterior view of such a parapodium
it will be seen that the different parts of this appendage have
assumed the appearance of those of a parapodium of a free-
swimming individual; the dorsal ramus extending somewhat
beyond the ventral ramus and containing at its extremity a tuft
of long, slender swimming setae; the dorsal cirrus inserted
some distance in from the insertion of the setae ; the ventral
ramus ending in a small tubercle containing a tuft of setae;
while posterior to this tubercle the two rami unite to form a
broad plate made up by the musculature of this appendage.

288 MENSCH. [VoL. XVI.

In the head the anterior eyes have shifted more ventrally
and now lie almost beneath the posterior pair. A new pair of
tentacles, the posterior lateral (7.7), have made their appear-
ance simultaneous with the appearance of the swimming setae.
These are inserted anterior to the dorsal eyes and consist of
bud-like processes that extend a little beyond the margin of
the head. The buccal segment has become more prominent
and can be more readily distinguished from the region consti-
tuting the head. Ina lateral view of the anterior part of this
and the lateral portions of the preceding stolons, it would be
noticed that the ventral surface of the anal segments of the
preceding stolon no longer lies in the same plane, but that the
buccal segment and even ventral parts of the succeeding seg-
ment are elevated considerably above the plane of the anal
segment, thus causing the attachment of this stolon to the
preceding one to appear to be more on the dorsal surface of
the anal segment than posterior, as in the preceding unions.
This is made very evident in specimens that are in a state of
partial contraction, but is also well marked in the normal state
and is due to the increase in size of the head, which, thicken-
ing only dorsally, pushes ventralward the tissues constituting
the zone of separation.

After this stage of development has been attained the inde-
pendent movements of the stolon become very active and sep-
aration from the chain soon takes place. The separation of
the stolon takes place in the region of separation previously
referred to and which already prior to this stage, as will be
seen in the examination of sections of this region, has under-
gone a ventral degeneration by which this structure is gradu-
ally weakened so that when the stolon becomes more active,
rupture of the tissue is easily effected.

The stolon when liberated from the chain has attained the
size of a mature free-swimming stolon and possesses all the
appendages, most of which have attained their full size and
undergo but few external changes in becoming completely
mature. The most important change concerns the carrying of
the anterior eyes exactly ventral to the posterior pair. This
process seems to be completed immediately after the separation

No. 2.]| SZTOLOMIZATION [IN AUTOLYTUS VARIANS. 289

of the stolon and is effected by the contraction of the ruptured
tissue and a consequent drawing down of the ventral tissues of
the head. The shifting of the head tissues has a slightly rotat-
ing effect upon the anterior lateral tentacles so that the plane
of bifurcation, instead of being horizontal, is more vertical. At
the same time the tentacles are bent outward and become still
more thickened at the base. The posterior lateral tentacles
also increase in length and are extended forward some distance
beyond the margin of the head. As the spermatozoa which
are contained in segments I, 2, and 3 ripen, the walls of these
segments become somewhat distended and the outlines of the
segments remain quite definite. At the same time the walls
of the segments posterior to these more or less lose their out-
line, and it is with difficulty at times that the outlines of the
segments can be distinguished at all save by the location of
the parapodia. This condition marks the complete maturity
of the sexual products and hence also of the stolon, and may
appear shortly or some time after its separation from the chain.

Development of the Female Stolon.— The development of the
stolons of a female chain presents external changes very simi-
lar to the development of those comprising a male chain. Since
the female chains contain fewer stolons, the different stages in
the progress of development are not so well marked in the
same chain, and the chain usually is composed of successive
stolons which exhibit greater differences in age than in a male
chain; the shortness of the chain being due, therefore, not to
an earlier separation of the stolon, but to a greater lapse of
time between the periods of stolon formation at the beginning
of the process. The early stages of the processes are identical
with those in the male chain, except for the bifurcation of the
anterior lateral tentacle and, in the older stolons, the appearance
of the dorsal tentacular cirri. The ventral tentacular cirri ap-
pear for the first time in a stolon which has reached a stage of
development corresponding to that of stolon 4, Pl. XIII, Fig. 6.
In a stolon of this age the parapodia of the female stolon
are not unlike those of the male. At a somewhat later stage,
however, in a stolon corresponding in age to stolon 6, they
present an appearance quite different from those of the male

290 MENSCH. [VoL. XVI.

(Pl. XIII, Fig. 13). In comparing it with a parapodium of
the Polybostricus, it will be noticed that, besides being smaller,
the dorsal ramus (d.7.) has remained quite short, while the ven-
tral has pushed out some distance beyond it, thus carrying the
insertion of the short setae external to that of the swimming
setae and presenting a condition reverse to that of the male
parapodium. The insertion of the parapodium does not extend
as far dorsal as it does in male stolons, and hence the dorsal
contour of the stolon remains more like that of the parent
stock. As no further growth takes place after this stage of
development has been reached, such a parapodium presents all
the characteristics of a parapodium of a fully matured Sacco-
nereis. The sexual products appear very early, and even in
stolons no older than stolon 1, Fig. 6, small ova are plainly
visible through the thin body-wall, and by the time the stolon
reaches a stage corresponding to that of stolon 6, it assumes a
darker color and becomes very much more opaque, due to the
accumulation of a large number of ova in the body cavity, the
outlines of which can be distinctly seen in a surface view.

The separation of the female stolon takes place some time
before the formation of the egg-sac, and at the time when the
stolon breaks from the chain it is well distended with closely
packed ova which extend even into the cavities of the para-
podia, and the swimming setae have been fully developed. Such
specimens at once acquire the habits of the fully matured indi-
vidual and may be found swimming at the surface associated
with the fully matured individuals. Shortly after the libera-
tion of the stolon the ventral cuticle begins to become more
distended, and before long the ova begin to accumulate in the
ventral sac and the stolon presents the appearance of the
mature Sacconereis.

HISTOLOGICAL PHENOMENA OF STOLONIZATION.

Internal Structures of the Mature Stolon.— Prior to taking
up the histological changes in the development of the young
stolons, I shall briefly describe such structures of the ma-
ture stolon as form the regions in which the most important

No. 2.] STOLOMIZATION IN AUTOLYTUS VARIANS. 291

processes of stolonization take place. As the principal phenom-
ena are those which are connected with the development of the
head and buccal segment, while those concerned in the devel-
opment of the remaining portion of the body present no impor-
tant characteristics different from direct development among
Syllidians, a section of the anterior segments of a mature
stolon will present all the important structures concerned in
the process of stolonization.

Pl. XIII, Fig. 14, represents a longitudinal median section
through the head, buccal, and the anterior part of the first setig-
erous segments of a mature male stolon. The first setigerous
(sg.1) and buccal segments (d.5g.) are separated by a distinct
dissepiment (dzs.). Between the buccal segment and the head
the musculature of the dorsal median tentacle (¢.7.) forms the
only partition, and laterally where these fibers do not exist, the
‘coelomic cavity of the buccal segment extends forward into
the tissues of the head for a short distance, so that internally
these two regions are not so distinctly separated. Dorsally, as
well as laterally, the body-wall of the buccal and setigerous
segments consists of an epidermis covered by a thin cuticle
(cw.) and overlying a layer of circular and longitudinal muscle
fibers. The epidermis (ect.) consists of a single layer of cells
distinctly separated from the underlying tissues, and its out-
lines are clearly defined. Ventrally, however, the epidermal
cells are very indistinctly outlined, and, particularly in the
regions of the ganglia of the ventral cord, are so intimately
associated with the ganglion cells (c.z.) of the ventral cord that
the epidermis cannot be distinguished from the underlying
tissues by means of the ordinary methods of preparation.
The endodermal cells, as they approach the mouth-opening (0),
become smaller, and where this structure joins the ectoderm
the outlines of the cells are lost. The body space of the first
setigerous segment is filled with sperm cells. The cavity of
the buccal segment also contains sperm cells (s.f.), but they
are usually fewer in number and less compact.

The head (c) is an almost solid structure, its only cavity
being the space on either side of the muscles of the dorsal
median tentacle, which is in reality a forward extension of the

292 MENSCH. [VoL. XVI.

buccal cavity and not a separate cavity. In a median section
like the one represented in Pl. XIII, Fig. 14, this space is not
seen, and the posterior part of the head in this plane is made
up entirely of the muscular structures of the dorsal median
tentacle (¢.7.), which reach as far ventralward as the junction
of the epidermis with the endoderm. Anterior to the muscu-
lature of this tentacle is the brain structure, which consists of
a central brain mass surrounded by a dense mass of ganglion
or brain cells (¢.z.). The central brain mass or medullary sub-
stance consists of two distinct lobes, the anterior of which (cd.a.)
is the smaller, and gives off a pair of nerves to the palps and
sends fibers into the circumoesophageal nerve ring. The poste-
rior lobe (cé.g.) is the larger, and is partly divided into two
smaller lobes by a fissure that extends more than two-thirds
across the lobe. It supplies the tentacles and eyes with nerves
and gives off fibers which form the greater part of the circum-
oesophageal nerve ring. Examined under a high power, this
central brain tissue is found to consist of very minute fibers
interlacing one another and having no distinct direction except
in regions from which nerves are given off, where they assume
a more parallel course. The tissues surrounding this central
brain substance consist of a mass of cells imbedded in a
network of very fine fibers, not unlike the fibers of the central
mass, but less densely arranged. The brain cells are not scat-
tered uniformly around the medullary substance, but are so
crowded as to form centers—these centers occurring at the
origin of the nerves and at the fissures in the medullary sub-
stance. The outlines of these cells, particularly in the region
of the centers, are very indistinct, and even away from these
centers it is difficult, by means of the ordinary methods em-
ployed, to distinguish the exact shape of the cells. In the
less dense regions of the eyes, however, as has been observed
by Malaquin in other Syllidians, distinct unipolar cells may be
_ distinguished.

The epidermis overlying this cortical substance, just as in
the case in that underlying the nerve cells of the ventral cord,
cannot be distinguished from the cortical substance, the cells
of both being so intimately associated that the outline of the

No. 2.] SZTOLONIZATION IN AUTOLYTUS VARIANS. 293

epidermis is entirely obscured. Posterior to and over the
median tentacle, as well as over the other tentacles, the epi-
dermis is present as a distinct layer of tissue; but where the
base of the tentacle encroaches upon cortical tissues the outline
of this layer becomes abruptly lost, and in regions anterior to
this tentacle the cortical substance seems to be composed of
cells of uniform structure, and to extend from the medullary
substance to the borders of the cuticle without presenting even
a peripheral line of nuclei to indicate the presence of such a
layer. This intimate association of cortical and epidermal cells
was first pointed out by Fraipont (12) in Protodrilus, and it
appears to be a condition common to all Syllidians. In
sections of the head of Autolytus pictus and Eusyllis there
exists, according to Malaquin (9), a peripheral arrangement
of cells, by the orientation of which he is able to recognize
an epidermal layer. Such an arrangement of cells is not
sufficiently apparent to justify the recognition of a distinct
epidermal layer in a section of the head of a mature stolon of
Autolytus varians ; and in a similar section of the head of the
parent stock the orientation, while being more evident, is not
sufficiently marked to indicate the presence of a true epidermis.
What results are to be obtained in the distinguishing of these
two structures by the employment of special methods, hitherto
not tried, I shall endeavor to demonstrate in a later paper.
The internal structures of the head of the parent stock are
so similar in character and arrangement to those of the stolon
that it would be useless to repeat the figure. The medullary
substance of both are alike in size and divisions into lobes,
while the arrangement of ganglion cells into centers is as well
marked in one as in the other, and the only way by which a
section of the adult head might be distinguished from a similar
section of the head of a mature stolon would be by differences
in the alimentary canal, the increase in size of the median ten-
tacle, and the absence posterior to the median tentacle of a
ciliated region known in species of Autolytus where this is
more marked as the epaulets. Thus it is evident that as far
as internal structures are concerned, the head of the stolon is
an exact reproduction of the head of the parent stock, and the

294 MENSCH. [Vou. XVI.

development of the head of the stolon, therefore, means the
appearance by a different method of such structures as are
present in the head of the parent stock and which have been
formed in the ordinary processes of development.

Region of Proliferation.

The changes brought about by the phenomena of stoloniza-
tion are already evident anterior to the chain of stolons in the
internal structures of the last segment of the parent stock
(Fig. 6, 35) in an area which I have designated as the region
of proliferation. In a transverse section of this region (PI.
XIII, Fig. 15) it is at once apparent that structures closely
associated with the processes of stolonization have made their
appearance which are not present in sections of segments ante-
rior to the segment preceding the chain. These structures
consist of masses of mesodermal tissue (7.¢.), which completely
fill the coelome laterally and even extend into the cavities of
the parapodia. Dorsally and ventrally the tissue is scantier,
and well-defined areas of body space (coe.) are visible. In the
anterior parts of the segment this mesodermal tissue is almost
entirely absent, and a section of this region would not be at all
unlike a similar section through any of the preceding segments.
In all preceding segments the body cavity is spacious, and such
mesodermal masses can be observed only in a segment imme-
diately preceding a chain of stolons. Examined under a higher
magnification, this tissue will be seen to consist of cells which
appear embryonic in structure and are in no wise different from
those constituting the mesodermal structures of embryonic seg-
ments. Associated with this tissue are the muscles of the
parapodia (7.ac.), while dorsally and ventrally may be seen the
dorsal (m.d.) and ventral (#.v.) muscle bands, all of which
structures, as well as those of the dorsal (d.v.) and ventral
(v.v.) blood vessels, can readily be distinguished from this
tissue. In regions a little posterior to the section here rep-
resented this tissue becomes more dense, fills the entire
coelome cavity, and is directly continuous with similar tissues
of the succeeding segments.

No. 2.] STOLONIZATION IN AUTOLYTUS VARIANS. 295

In addition to the appearance of this mesodermal tissue the
epidermis, particularly in the dorsal region, is somewhat denser,
and the cells seem more crowded than in sections of preceding
segments. Ventrally the epidermis in this section is not unlike
that of preceding segments, the lateral outline of its cells being
quite distinct, while in the region of the ventral cord the cells
are so closely associated with the ganglion cells (c.z.) of this
region that the two structures cannot be differentiated.

Pl. XIII, Fig. 16, represents a section through the posterior
part of segment 35, Pl. XIII, Fig. 6, in a plane just anterior to
the first segment of the embryonic region. In this section the
mesodermal tissue (.¢.) so nearly fills the coelomic cavity as
to leave but very small dorsal and ventral spaces (coe.). The
epidermis (ec¢.) has also increased considerably in density, both
laterally and dorsally, and the cells are no longer arranged in a
simple layer, but are so compactly placed as to give the appear-
ance in transverse section of several layers. (In this section,
owing to the strong contractions produced by the fixing fluid,
the thickness of the epidermis is somewhat exaggerated, par-
ticularly dorsally and on the one side.) Compared with the
epidermal cells in any other segment of the parent stock, the
cells of this region appear to be smaller, less regular, and
present more fully the characters of newly dividing cells.

The alimentary canal (ew¢.) of this section also presents
characteristics different from that in preceding segments. Its
calibre is very much smaller, the constriction being already
apparent in Pl. XIII, Fig. 15, and the decrease in the size of
the cells is quite well marked. The abundance of nuclei in
the cells would also demonstrate a rapid formation of new
cells in this region.

The general appearance of the tissues of these sections
would, therefore, indicate that the posterior part of this seg-
ment has undergone a complete change from that of an
ordinary segment of the parent stock, and that, while its
external appearance remains unchanged, its internal structure
has become so modified as to convert its posterior regions into
a distinct embryonic center, in which new tissue is being rapidly
formed and pushed back to furnish the embryonic tissue, from

296 MENSCH. [Vov. XVI.

which the new segments formed in the anterior part of the
chain are built up.

The gradual transformation of the tissues of this segment
into embryonic tissue is well shown in a longitudinal median
section of this and succeeding segments of the chain. Pl. XIII,
Fig. 17, represents a longitudinal sagittal section through seg-
ment 35, the embryonic region and the anterior part of stolon 1,
Fig.6. The plane in which the section represented in Fig. 15
was taken is shown at A, while the plane of Fig. 16 is shown
at B. The limit of this segment (35) is well marked anteriorly
by the dissepiment (dzs.); posteriorly, however, the internal
limits of the segment are lost in the masses of embryonic
mesoderm (.¢.), which completely fill the body cavity at the
posterior extremity of this segment, and the external constric-
tions alone define its exact limit. The dorsal epidermis at B,
and posterior to this plane, shows quite a transformation when
compared with that anterior to the plane A, the cells appearing
considerably narrower and possessing more the characters of
embryonic ectoderm. Ventrally the character of this ectoder-
mal tissue, as seen in the longitudinal sections, is even more
distinct, and in the posterior part of this segment (e.¢.), in a
plane with the denser masses of embryonic mesoderm, the out-
lines of these ectodermal cells are quite apparent. The tissues
in the regions belonging properly to segment 35 are composed
of elongated spindle-shaped cells closely crowded together, but
having a clearly defined outline, particularly at the very poste-
rior border of this segment. More anteriorly their outlines
become gradually fainter, until, in a plane a little anterior to B,
they assume the appearance common to the region ventral to
the nerve cord in preceding segments.

From a longitudinal section of segment 35 it is, therefore, quite
evident that the posterior part of this segment has become con-
verted into an embryonic center, in which new tissue is con-
stantly being added to the three embryonic layers, which new
tissue, in the process of formation, is carried back and forms
the most anterior embryonic segments of the chain. In this
way the formation of all the segments preceding stolon A,
Pl. XIII, Fig. 6, can be accounted for.

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No. 2.] STOLONIZATION IN AUTOLYTUS VARIANS. 297

Embryonic Region.

In this region (Pl. XIII, Fig. 6, ~e.) I have included in the
description of the external characters of the chain the very
youngest segments which have been developed from the region
of proliferation, and also such older segments which have already
indicated by their general appearance the positions they will
occupy in the mature stolon, and in which can already be dis-
tinguished the outlines of a stolon (St. A), but all of which I
regard as having been derived as outgrowths from the region
of proliferation. Pl. XIII, Fig. 18, represents a transverse sec-
tion through the segment immediately following segment 35, and
possesses the characters presented by a very young setigerous
segment just after its external outlines, as represented in PI.
XIII, Fig. 6, have become distinct. Its structures differ very
little from the embryonic structures of the preceding segment,
and it presents the very simplest and youngest structures which
can form a distinct segment. The mesodermal tissue is quite
prominent and fills the entire coelomic cavity. Dorsally and
ventrally the tissue appears quite loose, but laterally two dis-
tinct denser regions may be distinguished; the cells of the
dorsal region (m.e¢.') being the less compact and forming a
larger area, while those of the ventral region (m.e.') are more
compactly arranged and are confined to a smaller area near the
ventral cord. Between these two regions the tissue presents
the looser appearance seen in the dorsal and ventral regions.
The dorsal (d@.v.) and ventral (v.v.) blood vessels can readily
be distinguished from the surrounding tissue, while but a few
poorly defined fibers appear to mark the position of the circular
and longitudinal muscles. The ectoderm can readily be distin-
guished from this mass of mesodermal tissue by the size of the
nuclei and the direction and appearance of the cells and muscle
fibers. As in the region of proliferation, this tissue is some-
what thicker and contains many nuclei placed close together,
and indicates by the numerous outlines of cells the rapid forma-
tion of new ectodermal tissue. Ventrally the nerve cord (v.c.)
passes through this tissue in three distinct strands, all of which
are separated from one another and are surrounded by cells

208 MENSCH. [Vou. XVI.

similar to those comprising the remainder of this tissue. The
appearance of the ventral cord in this section is not unlike the
appearance of the cord in sections of the parent stock passing
through the region of the cord between the ganglia, except that
in the parent stock these strands are never so widely separated.
This section is, however, taken through a plane, in which, in a
more mature segment, a ganglion is present, and the examina-
tion of successive sections of this segment will demonstrate the
entire absence of a ganglion in a segment of this stage of
development ; neither are the nuclei surrounding the cord more
densely crowded than they are in any other parts of this tissue.
The entodermal cells show no important differences from those
of the preceding section, which represents a zone equally active
in growth.

Pl. XIV, Fig. 20, represents a section through the segment
immediately following the one just described, or the second
segment of the chain. The most important characteristics of
a section of this segment, as compared with the preceding,
appears in the greater thickening of the ectodermal tissue and
the scanty appearance of the mesoderm. Neither of these
layers presents structures that would indicate any difference
in the age of this and the preceding segments, the tissues of
both being quite alike in general arrangement and appearance.
The ectoderm of this segment has increased considerably in
thickness and would indicate, by the more crowded appearance
of the nuclei, a region in which the cells are smaller, and hence
a segment in which growth seems more active than in the pre-
ceding segment. Besides this, however, no differences can be
distinguished in a transverse section. The narrowing of the
band of mesoderm is confined mostly to a lateral decrease in
the tissue, while dorsally and ventrally no difference can be
observed from that of the preceding segment.

The structural differences of these two segments are made
more apparent in the longitudinal sections of Pl. XIII, Fig. 17.
The plane of Pl. XIII, Fig. 18, is represented at C; that of
Pl. XIV, Fig. 20, at D. If these two segments be compared,
it will be seen that in passing from plane C to plane D the
dorsal ectoderm becomes rapidly thicker, and the cells appear

No. 2.] SZTOLONMIZATION IN AUTOLYTUS VARIANS. 299

smaller and more numerous. Ventrally there is even a more
marked difference between the cells of the two segments.
In the anterior segment (Sg. 1) the outlines of the cells are
quite indistinct, and in this section of the ventral ectoderm
appear very little different from those of a segment of the
parent stock. In the second segment, on the other hand, this
ventral ectoderm presents an appearance entirely similar to the
ventral ectoderm of segment 35 in the plane of B (e.e.); the
outlines of the spindle-shaped cells (e.e.f.) are quite distinct,
and this region appears as distinctly embryonic as does the
ventral ectoderm of the posterior part of segment 35.
These two segments accordingly present a stage in which
the three embryonic layers have undergone very little change
toward the formation of any structure of the mature segment.
In the ectoderm the ventral cord forms the only distinctly
differentiated structure. The cells beneath it have, in the
anterior segment, assumed the appearance of the nerve cells
common in this region; while in the second segment, as seen
in longitudinal section, the cells have not undergone this differ-
entiation, but still possess the characters of the purely embry-
onic cells of the zone of proliferation. The mesoderm of these
segments, with the exception of the dorsal and ventral blood
vessels and the faint bands of the muscle tissue, presents no
differentiated structures, but differs in the two segments in the
amount of tissue present. Both segments being embryonic in
structure and still possessing characters which make them
appear different from one another, if these differences are not
due to the differences in age of the segments, we have presented
in these two segments two distinct phases of embryonic seg-
ment formation. If these differences were due to the variance
in the ages of the segments, the second segment, being the
elder, would present the greater differentiation ; while, as is
quite evident in the longitudinal section of these regions, the
anterior segment in reality presents the more differentiated
structures. There must be present, therefore, already at this
early stage, two distinct types of embryonic segments, each
of which is destined by its internal formation to occupy a dis-
tinct position in the mature stolon. As will be seen later,

300 MENSCH. [VoL. XVI.

the second segment presents characters common to the last
segment of each stolon, and as the segment following already
shows ectodermal thickenings indicative of a head formation,
this segment may be regarded as the anal segment.

Pl. XIV, Fig. 21, represents a transverse section through the
posterior part of the first segment of stolon A, Pl. XIII, Fig. 6,
and indicates a condition less embryonic than that presented
by the two preceding segments. The ectoderm in this section
is very little thickened dorsally, though in the anterior parts
of this segment, as will be seen in another section, the thicken-
ing is considerable. Laterally the ectoderm has pushed out in
the form of small processes which form the rudiments of the
parapodia (/.). Ventrally the ectodermal cells have increased
in number, and in the region of the nerve cord the number of
nuclei (c.z.) has increased sufficiently to suggest a region of
ganglion cells. The cord itself has increased in thickness and
presents an appearance common to the ganglionic region. The
mesodermal tissue has also undergone some differentiation. A
coelomic cavity has made its appearance dorsally, and in part
laterally, while the dorsal and ventral muscle bands (m.d. and
m.v.) appear much more prominent than they do in the preced-
ing segments. In the ventral lateral regions, however, the
embryonic masses of mesoderm (w.e.) still exist and form quite
a dense undifferentiated structure.

Sections through the second segment of stolon A show no
characters different from those presented in the segment just
described, save a slight decrease in the thickness of the dorsal
ectoderm; while sections of the third segment of this embry-
onic stolon repeat almost exactly the structures presented by
Pl, XIN, Fic. 20.

In a longitudinal section (Pl. XIII, Fig. 17) this stolon (St.
A) appears as quite a distinct stolon. Anterior to the plane
of Fig. 21 (£), the dorsal ectoderm of the first segment (¢.)
bas become considerably thicker, and spindle-shaped cells
similar to those which appeared beneath the ventral cord
in the embryonic region of segment 35 are beginning to make
their appearance in this region. Over the anterior part of the
second segment the ectodermal cells are less spindle-shaped

No. 2.] STOLONIZATION IN AUTOLYTUS VARIANS. 301

and are arranged in a single layer, but posteriorly, and over the
third segment, the tissue is again thickened and the cells again
present a spindle-shaped appearance. Ventrally the ectodermal
cells of the first and second segments present very much the
appearance of this tissue in the mature stolon, but in the third
segment the spindle cells (¢.¢.f.) again appear, and this tissue
assumes an appearance similar to that in the zone of prolif-
eration. The mesoderm presents dorsally and, less distinctly,
ventrally a coelomic cavity for segments I and 2, but in seg-
ment 3 the cavity is wanting.

In these three segments, which have in Pl. XIII, Figs. 6 and
17, been designated as stolon A, the external appearances sug-
gestive of a distinct stolon have been confirmed by the inter-
nal structures of these segments; and, these segments being
typical of the very earliest stolon differentiation, there can,
therefore, be recognized in their structures: (1) a thickened
ectoderm from which the head will later on be differentiated ;
(2) an anal segment of purely embryonic tissue which, as will
be seen later on, contributes to the elongation of, (3), the indif-
ferent region in which the ordinary setigerous segment is being
formed. All of these segments are, as may be seen in the
study of longitudinal sections of the different stages in the
development of these segments, derived from the region of pro-
liferation, so that all the tissue of that region which I have
designated as the embryonic region owes its origin to the single
center of growth within the last segment of the parent stock.

Development of the Head and Buccal Segment.

The first differentiation toward the development of the head
makes its appearance in the thickened dorsal ectoderm of stolon
A (Pl. XIII, Fig. 17, ¢.), already referred to in the preceding de-
scription. In stolon I of the same figure this dorsal thickening
(c.) has become more pronounced, and in its structure may be seen,
for the first time, the fully developed spindle cells as they appear
in the early stages of this dorsal thickening. Here the cells
have become quite long, and are compactly arranged to form a
somewhat rounded dorsal elevation of the ectodermal tissue.

302 MENSCH. [Vor. XVI.

Posterior to this region, in the second segment of the stolon,
the ectodermal cells are less spindle-shaped and are arranged in
a single row, while more posteriorly they present the characters
of ordinary epidermal tissue. Anterior to this thickening, how-
ever, the dorsal ectoderm comprising the anal segment of stolon
A, as we have seen in the study of the embryonic region, is
also thickened and presents similarly arranged spindle cells.
Since these cells appear in regions in which either nerve cells
or simple epidermal cells will be differentiated, they cannot be
regarded as being of a purely nervous character, even though
they attain their greatest dimensions in positions which later
on will be occupied by nerve cells. On the other hand, they
may rather be regarded as the typical embryonic cells of this
ectodermal tissue in general, the outlines of which are more
easily distinguished in regions where the ectoderm is thickest.
The appearance of the tissue beneath the ventral cord in the
embryonic region would also support this view.

Pl. XIV, Fig. 22, presents the appearance of the developing
head of stolon I in transverse section, in the plane of F, Pl.
XIII, Fig. 17. Dorsally the ectoderm is considerably thickened
and presents two lateral prominences which mark the lateral head
regions, while between these is a small ectodermal projection
which indicates the origin of the median tentacle (@.m.). Lat-
erally the ectoderm is also somewhat thicker, though this
becomes less marked toward the ventral surface. The out-
lines of the spindle cells, as in all other transverse sections, are
quite indistinct; but in the lateral prominences, particularly
near the surface, the nuclei of these cells (c.z.) are very much
more numerous than in the region of the median tentacle and
the dorsal mesoderm. These cell groups indicate the appear-
ance of the first pair of eyes. Ventrally the ganglion cells (c..)
present a grouping very similar to the groupings in the dorsal
lateral prominences, and the cells present a very similar appear-
ance at this stage. Along the dorsal borders the regular arrange-
ment of nuclei in this section would suggest the presence of a
distinct epidermis, as Malaquin has been able to distinguish in
the mature heads of certain other species of Autolytus; but
as this disappears in later developments, it would appear to be

No. 2.] SZTOLOMIZATION IN AUTOLYTUS VARIANS. 303

of no significance. In a similar section of a stolon, a little
older than stolon I, another group of nuclei has made its
appearance in the cerebral tissues directly over the dorsal
vessel, while the lateral groups have become somewhat denser.

Pl. XIV, Fig. 23, presents a longitudinal median section
through the head of stolon 2. At this stage the cerebral
thickening is very much greater than in the preceding stolon,
the outline of the head has become quite distinct, and its divi-
sion from the tissues of the anal segment is quite well marked.
The median tentacle (d.m.) appears quite prominent, and its
internal structures are not unlike the surrounding structures
of the head proper. The cerebral tissues have, however, at
this stage already begun to be differentiated into a small cen-
tral medullary region (c.d.) and the peripheral cortical area (c.z.)
of the mature head. The medullary region has appeared
directly over the mesodermal tissue in the region occupied by
the group of nuclei, which I have described as appearing in
a line with the median tentacle in a transverse section of a
stolon a little older than stolon I, and owes its origin to changes
which have taken place within this group of nerve cells. Ex-
amined under a high power, this young medullary tissue pre-
sents an appearance similar to the tissue along the border of
the medullary substance in the head of the mature stolon, and
does not as yet possess the dense fibrillated arrangement com-
mon to the medullary substance of this region in more mature
individuals. The cortical substance in this plane is composed
of cells evenly distributed throughout the region of the head
and showing no differences in the median tentacle. The
arrangement of the nuclei for the formation of a distinct epi-
dermis over the anterior part of the head is not as evident as
it was in the transverse section of the preceding stolon; but in
the median tentacle not only have the nuclei an arrangement
suggestive of epidermal tissue, but the outlines of the cells are
also more plainly visible, and form a marked contrast with the
underlying cells. Posterior to the median tentacle the ecto-
dermal cells still retain their embryonic spindle characters, and
this region is already beginning to contribute toward the for-
mation of the buccal segment.

304. MENSCH. - [Vor. XVI.

The mesoderm beneath this thickened tissue has already
undergone some changes. Posterior to the medullary sub-
stance and beneath the area in which the spindle cells blend
with the brain cells, a new growth of mesodermal tissue (c.me.)
_ has made its appearance and is pushing up toward the base of
the median tentacle. This tissue results as an outgrowth from
the mesoderm of this segment and will form the musculature
of the dorsal median and the other tentacles of the head.

A transverse section of the head of a stolon of this age, pass-
ing through the plane G of the figure just described, is rep-
resented in Pl. XIV, Fig. 24. [This section is not exactly
transverse, and ventrally includes parts of the anal segment
and caudal cirrus (c.c.).]__ Dorsally the brain cells (c.z.) form a
thick mass of tissue which projects considerably beyond the
lateral margins of the segment. On either side of the plane
occupied by the eyes, the anterior pair of which are quite well
developed, the brain cells are uniformly distributed, and along
the external border so irregularly placed as to give no indica-
tion of the presence of an epidermal formation. The medul-
lary substance (¢.d.) appears as a small area immediately
overlying the faint dorsal muscle fibers (m.d.) and does not
project beyond the lateral border of the mesoderm.

Pl. XIV, Fig. 25, represents a section through the posterior
part of the head of stolon 2 taken in the plane H, Fig. 23. In
this region the cortical substance is less prominent than in the
preceding section, and the ganglion cells (¢.z.) are less abun-
dant. Along the dorsal margins, as well as laterally, the nuclei
of these cells are again regularly arranged and seem to form a
layer of tissue that is directly continuous with the epidermis
over the parapodia (f.). The medullary substance (c.d.) forms
a narrow plate in this region and is also less prominent than in
more anterior sections. Laterally the cerebral tissue in this
region gradually blends with the less thickened ectodermal
tissue of the first setigerous segment, and as yet shows no
signs of the circumoesophageal nerve ring.

Beneath the cerebral tissue, on either side of the area of
medullary substance, a small cavity (coe.’) has made its appear-
ance. This cavity is the forward extension of the coelomic

No. 2.] SZTOLONIZATION IN AUTOLYTUS VARIANS. 305

cavity (coe.) on the first setigerous segment, and has been
formed by the upward growth of the mesoderm for the forma-
tion of the tentacular muscles, a part of which tissue (c.me.)
may be seen on either side of the dorsal blood vessel in this
section.

The next important stage in the development of the head
is represented in a longitudinal median section of stolon 4
(P]. XIV, Fig. 26). At this stage the structures of the head,
as has been seen in the dorsal view of this stolon (Pl. XIII,
Fig. 6), have become quite well developed, and the rounded
mass of cerebral tissue presses down into the underlying tissue,
so as to appear partly imbedded within it. At the same time
the line of junction of the cerebral tissue and ectoderm of the
anal segment anterior to it, which, as seen in Pl. XIV, Fig. 23,
appeared as a deep groove, has by the increase in the size of
the head been completely covered, and is now placed almost
ventral to the middle region of the head. The head itself in
this section appears quite distinct from the surrounding tissues,
and protrudes considerably beyond the lower limits of the sto-
lon, the tissues directly underlying it being those belonging to
the region of separation. The buccal segment (d.sg.) has also
appeared, and forms a distinct segment separated posteriorly
from the first setigerous segment (Sg. 1) by a dissepiment,
while anteriorly it is indistinctly separated from the structures
of the head and anal segment by the mass of undifferentiated
mesoderm occupying this region. The cortical substance of
the head is clearly defined from the surrounding structures,
and the brain cells are somewhat more densely crowded than
in the preceding sections, while the arrangement of the periph-
eral cells suggestive of the presence of an epidermis has disap-
peared, and a distinct epidermis in the head structures of this
stage of development is visible only in the tentacles. The
medullary substance (c.d.) has become more prominent, and
dorsally has sent up two processes which at the base of the
median tentacle unite and form the nerve (w.7.) of the tentacle.
Similar processes, as may be seen in more lateral sections of
this stolon, are also sent out to innervate the lateral tentacles
and the eyes.

306 MENSCH. [Vou. XVI.

The ectoderm on the dorsal surface of the buccal segment,
as is also the case in the anterior part of segment 1, is still
thickened and somewhat embryonic in character; while in the
anterior ventral region the faint outline of spindle cells may
be seen immediately posterior to the degenerating tissues of
the region of separation (7s.). A short time after the buccal
segment has attained its full length, the dorsal cells give way
to a distinct epidermis and ventrally to the ganglia cells com-
mon to this stage. The mesoderm of the buccal segment has
been differentiated into a somatic and splanchnic layer, and
both ventrally and dorsally a distinct coelomic cavity is present
throughout the greater part of the segment. In the anterior
region, however, the cavity in this section is broken up by the
musculature of the median tentacle (c.me.), but in more lateral
sections it is in direct communication with the cavity of the
head already present in stolon 2 (Pl. XIV, Fig. 25).

The development of the circumoesophageal nerve ring in the
ectodermal tissues of the buccal segment is very similar to the
development of the medullary substance in the head. It first
appears in a stolon of a stage of development a little earlier
than stolon 4, and at this age it has already placed the brain
into direct communication with the ventral cord. In a trans-
verse section of the head of this stolon, passing through the
anterior basal parts of the median tentacle and ventral tentac-
ular cirri (Pl. XIV, Fig. 27), the more dorsal parts of the cir-
cumoesophageal nerve ring (c.oes.) may be seen on each side as
a branch of the medullary substance extending through the
mass of nerve cells directly over the mesodermal tissue; while
external to these branches, just as over the medullary substance
in the brain, are placed a number of nerve cells (¢.z.), some of
which will in the further development of the stolon add to the
thickening of the nerve ring, while the others will remain as
the nerve cells that surround these nerve fibers in the mature
stolon. In the ventral ectoderm of this figure, which represents
a part of the region of separation (7s.), the ectodermal cells
have undergone a partial degeneration, thus obscuring the
ventral cord and the cells beneath it; and it is just posterior
to this region that the nerve ring blends with the ventral cord.

No. 2.] SZTOLONIZATION IN AUTOLYTUS VARIANS. 307

As may be seen in the earlier stages of the development of the
nerve ring, the nerve fibers composing this ring make their
appearance as downward growths from the medullary substance
of the brain, while the fibers of the ventral cord remain passive.

The remaining important stage in the development of the
head consists in the shifting of the anterior eyes from the
dorsal surface of the head to a position almost directly ventral
to the posterior eyes. The change in position of the eyes first
becomes apparent in a stolon a little younger than stolon 4.
At this stage a transverse section in a plane with the anterior
eyes will reveal a mid-dorsal area of rapid growth, extending
from this plane forward to the anterior part of the head. Ina
transverse section of the head of a stolon, corresponding in
development to that of stolon 5 (Pl. XIV, Fig. 28), this growth
has already been sufficiently great to carry the anterior eyes
(e.2.) from their original dorsal position to a lateral ventral
position, and the lens (/), instead of being directed upward, is
directed almost directly downward. In later stages this process
will be continued by the active increase of the brain cells in the
mid-dorsal region (cz.d.) until the anterior eyes occupy a posi-
tion almost ventral to the posterior eyes, the complete location
of the eyes taking place immediately after the separation of the
stolon.

The development of the eyes in the stolon of Autolytus
varians is so similar to the development of the eyes in the
embryo, and so identical with that described by Andrews (13),
that I shall not enter into a description of this process.

Development of the Mesodermal Structures.

The development of most of the mesodermal structures of
the stolon is also identical with the development of similar
organs in the embryo, and hence needs but little mention here.
The mesodermal tissue of the embryonic region, even in the
youngest segment, is not uniformly embryonic throughout,
for at this stage there may already be distinguished in the
dorsal regions indistinct bands of sparsely scattered muscle
fibers, which are a continuation of the dorsal musculature of

308 MENSCH. [VoL. XVI.

the last segment of the parent stock. Later these fibers are
increased in number by additions of tissues from the dorsal
dense area of cells (Pl. XIII, Fig. 18, m.e.'), and at the same
time the tissues of this region divide into the somatic and
splanchnic layers, so that at a slightly older stage (Pl. XIV,
Fig. 21) there is present a dorsal coelomic cavity (coe.) with
distinct overlying muscle tissue dorsally (w.d.), while laterally
it gradually merges into the more differentiated tissue. The
ventral dense area of the embryonic mesodermal cells, seen -in
Pl. XIII, Fig. 18 (m.e.'’), still persists in Pl. XIV, Fig. 21 (m.e.'),
and it is from this region of cells that the reproductive organs
and nephridia will be formed. The nephridia can very frequently
be distinguished in a stage of development represented by stolon
2, but more frequently they can be clearly defined only in later
stages.

The male reproductive products may be detected in small
numbers in a stolon corresponding in external development to
stolon 4. A little later these sperm cells become quite numer-
ous, and in a transverse section of the second setigerous segment
of a stolon of the age of stolon 5 (Pl. XIV, Fig. 20, s.g.) they
occupy a great part of the body cavity. Ata later stage these
cells become still more abundant and completely fill the body
cavities of the first three setigerous segments, and at the time
the stolon is liberated from the chain the spermatozoa have
completely ripened.

The ova may be distinguished in the developing stolon even
at an earlier stage than the male reproductive products, and not
infrequently in a stolon in which the head is just beginning to
appear small ova may already be distinguished in the lateral
body cavity. Near the time when the stolon separates from
the chain, the ova become so numerous as to completely fill the
body and even fill the cavities of the parapodia, after which, by
the relaxation of the ventral tissues, caused by the pressure of
the increasing ova upon this region, the egg-sac is formed,
and the ova from all parts of the body are transferred into
this sac.

No. 2.] STOLONIZATION IN AUTOLYTUS VARIANS. 309

ELONGATION OF THE STOLON.

After the outlines of the young stolon (A, Pl. XIII, Fig. 6),
formed entirely of tissues derived from the zone of proliferation,
have appeared, it has been seen in the external description of
the phenomena of stolonization that a new region of growth
makes its appearance; and that it is this region, which I have
designated as the region of elongation, that contributes to the
increase in the number of setigerous segments of the stolon.
The external appearance of the segments of the embryonic
region in its different stages of development up to a stage
of differentiation equal to that of stolon A indicates, according
to the description I have given, the absence of such a mode of
segment formation. This fact is also supported by the appear-
ance of the tissues of these segments in a longitudinal section
(Pl. XIII, Fig. 17), where in the ventral ectoderm the embry-
onic cells (¢.e.g.) in every section I have examined are confined
to a single segment, which, as has been seen, forms the anal
segment of the forthcoming stolon. In a longitudinal section
through the last two segments of the stolon represented in
Pl. XIII, Fig. 7, in which a new segment formed by this region
first occurs, the ectodermal and mesodermal structures of both
segments are exactly like the structures of the anal segment of
stolon A, Pl. XIII, Fig. 6, the two segments being distinguish-
able from one another only by their external constriction, and
hence composed of embryonic tissue. A little later, however,
the tissue of the newly formed segment (7) undergoes a higher
differentiation, the spindle cells disappear, a coelomic cavity
appears in the dense mass of mesoderm, and the segment pre-
sents in a longitudinal section an appearance very similar to
that of the segment (Sg. 1) in the embryonic region of Pl. XIII,
Fig. 17. The anal segment, in the mean time, retains its embry-
onic characters, begins to elongate, and before long adds another
segment to the series. Different stages of this process are
represented in the longitudinal sections in Pl. XIII, Fig. 17,
Pl. XIV, Figs. 22 and 25, and sufficiently explain the descriptions
of Pl. XIII, Fig. 7,to make additional figures useless. In all these
sections it will be seen that the anal segment is the only seg-

310 MENSCH. [VoL. XVI.

ment of the setigerous series that retains the purely embryonic
characters, the segments preceding it, in very young stolons at
least, as soon as formed undergoing a sufficient differentiation
to make the distinction quite marked. This is particularly evi-
dent in the stolons represented in Pl. XIII, Fig. 17, and even
in Pl. XIV, Fig. 23, where a new segment has just been formed;
and in these stolons it may be safely said that the anal segment
forms exclusively the region of elongation. In stolons 2 and 3,
where elongations of the stolon by the addition of new segments
are very active, the tissues of the young segments anterior to
the anal have, in longitudinal section, an embryonic appearance
similar to that of the anal segment, and in these segments the
different tissues, as well as the external constrictions in the
mid-region of some of these segments, indicate the possibility
of an increase in the number of segments by division of some
of the newly formed setigerous segments. Later on, however,
when the formation of new segments becomes less rapid, the
embryonic tissues are again confined to the anal segment (PI.
XIII, Fig. 26), and in all stages of development the tissues of
the anal segment appear to be active centers from which are
developed the new segments of this region. The center of
growth in the anal segment is similar to that in the region of
proliferation in the last segment of the parent stock, and in ex-
amining the anal segments of stolons in different stages of
development it will be found that the embryonic tissues are
retained as long as new segments are added to the stolon.
Malaquin (9) regards the formation of new stolons in the
posterior part of the stolon as taking place in a region anterior
to the anal segment which he calls the Zoonzte formateur. In
describing the formation of new segments in the stolon of
Myrianida he says : “Tl semble donc trés probable que ce
phénoméne est bien constant et que la production de stolons se
fait chez la Myrianide dans la zone terminale méme d’accroisse-
ment de l’adulte, c’est-a-dire dans la zone précédant immédiate-
ment le pygidium, et que j’ai désignée déja sous le nom de
Zoonite formateur. ... La zone active de prolifération, le
zoonite formateur, est donc bien distinct du pygidium, en tant
que zoonite ; si, en effet, le pygidium avait le pouvoir de former

No. 2.] STOLONMIZATION I[N AUTOLYTUS VARIANS. 311

les segments nouveaux, s’il renfermait les tissus embryonnaires
en voie active d’accroissement, la zone de prolifération des
nouveaux segments, et par conséquent des nouveaux bourgeons,
serait irrémédiablement entrainée avec lui. D’autre part, nous
verrons plus loin qu’il existe des zoonites formateurs trés éloignés
du pygidium et absolument indépendants de ce dernier.” In
describing the stolon of Autolytus edwarsi he makes the same
observation : “Il est inutile d’insister ici sur l’accroissement
du corps, c’est-a-dire sur la formation de nouveaux anneaux,
qui est toujours identique a ce qu'elle est partout.

“C’est le Zoonite formateur qui en se multipliant, produit de
nouveaux anneaux et fait s’accroitre le corps d’avant en arriére.
Les caracteres de l’individu et de l’espéce restent identiques
pendant cette période a’ accrotssement qui s’étend de 1l’individu
jeune a l’individu adulte possédant 50 4 60 segments.”

My observations in sections of Autolytus varians do not
accord with this view. In young stolons corresponding in age
to stolon 1, Pl. XIII, Fig. 6, sections of the posterior part of
the stolon plainly indicate the absence of any embryonic tissue
anterior to the pre-anal segment (Pl. XIV, Fig. 23), and hence
the absence of a region in which the formation of new seg-
ments could take place. The pre-anal segment in this stolon
furthermore presents the appearance of having quite recently
been separated from the anal segment, this accounting for the
embryonic tissue seen in this segment, and also indicating
the manner in which new segments make their appearance.
In stolons with a greater number of segments than the one
just referred to, the pre-anal and several segments anterior to
it do contain embryonic tissues very little more advanced in
the more anterior segments than those of the anal segment
itself; and it is in such segments that we found traces of ex-
ternal segmentation that would indicate the formation of new
segments in a region anterior to the anal segment. In older
stolons, however, where the formation of new segments is no
longer as rapid, this embryonic tissue (Pl. XIV, Fig. 26) is
again confined to the anal segment and new segments must
hence be derived from this region. Since the anal segments
contribute the very first segments toward the elongation of the

le MENSCH. [Von. XVI.

stolon and also take part in the formation of the last seg-
ments that are added to the almost mature stolon, the forma-
tion of new segments in a region anterior to the anal segment
must be regarded as a secondary process and not the primary
factor in the elongation of the stolon; and the Zoonite forma-
teur cannot in this species, at least, have the significance that
Malaquin attributes to it in the Syllidians in which he has
made these observations.

SEPARATION OF THE STOLON.

As the young stolons of the chain become more differentiated,
it has been seen in the lateral and ventral views of the united
stolons that the region of the anal segment adjoining the head
parts of the succeeding stolon becomes somewhat distinct from
the anal segment in external appearance, and forms what I have
designated as the region of separation. In sections this region
forms in young stolons an indistinguishable part of the anal
segment, and even in older adjoining stolons, in which it is
externally visible as a distinct band, sections of this region
merely show a decrease in the amount of mesodermal tissue
ventrally and laterally (Pl. XIV, Fig. 23, ~s.), and a similar
decrease in the entire ectodermal tissue. (The ventral projec-
tion of the ectoderm in this section is due to abnormal contrac-
tions.) The tissues of this zone, however, do not remain as an
intrinsic part of the anal segment, but at a later stage are made
distinct by the appearance of a degenerative process in the ecto-
dermal cells, particularly in the ventral region (Pl. XIV, Figs.
26-28, ~.s.). This degeneration of the ectodermal tissue in the
ventral region also extends to the nerve cord, effects its sever-
ance, and at the same time so weakens the tissues in this place
that, by the independent movements of the stolon, separation
from the chain is readily accomplished.

Claparéde (4), de St.-Joseph (7), and Pruvot (8), in different
species of Syllidians have described the separation of the stolon
as taking place between two segments. In chain-bearing Syl-
lidians, like Autolytus varians, in the separation of its stolons
from the chain, it appears to me incorrect to make this state-
ment; for, even if the zone of separation be regarded as an

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No. 2.] SZTOLONMIZATION IN AUTOLYTUS VARIANS. 313
intrinsic part of the anal segment, this segment, as compared
with the other segments of the stolon, is so embryonic in char-
acter, and its posterior outlines so poorly defined, that at the time
of separation it has hardly attained the value of a distinct seg-
ment. The appearance of this region in longitudinal section at
once seems to suggest that the separation of the stolon does not
take place in the tissues forming a part of a true segment, but
rather in a mass of embryonic tissue, which in the development
of the head and buccal segment has been formed as an embry-
onic union of these and the anal segments, and constitutes a nar-
row undefined band of embryonic tissue anterior and posterior to
which similar embryonic cells are undergoing differentiation,
while at the same time the embryonic cells of this region are be-
ginning to degenerate. The embryonic regions of the buccal
and anal segments are hence continuous, and it is in the midst
of this undifferentiated region that the degeneration of the cells
appears and separation takes place.

POSITION OF THE CHAIN.

The position of the chain of stolons in Autolytus varians, as
I have already indicated, is not constant, but varies consider-
ably in different specimens. Like the chain-forming species,
A. ehbiensis and A. edwarsi, described by de St.-Joseph, the
region of stolonization in this species has a similar range,
although the position of the chain is never as far forward as
in the species described by this author. In a hundred speci-
mens examined, the greater number of chains were found to
be attached to segments between the 32d and 38th setigerous
segments. A small number bore the chain posterior to seg-
ments 45-48, and a still smaller number bore the chain of
stolons as far forward as segments 23-28. A few specimens
were found, in which the chain was placed as far forward as
the 19th and 21st setigerous segments, while a few others were
also found, in which the chain was borne as far back as seg-
ments 56 and 58. The range of stolonization in this species,
therefore, is between setigerous segments 19-58.

In describing the position of the chain of stolons, Malaquin
and de St.-Joseph appear to regard this great difference in the

314 MENSCH. [VoL. XVI.

position of the chain in the same species as being accidental
rather than due to any particular cause. The appearance of
several specimens observed, and the difference in size and
apparent age of the parent stock in individuals in which there
is a great difference in the position of the stolon, together with
the fact that in Autolytus cornutus and in Procerea the position
of the stolon is so constant, has caused me to regard the range
in the position of the chain as being due to more than mere
accident. In this species there is, without a doubt, a distinct
difference between the age of the parent stock with 56 or 59
segments and that of I9 or 20 segments. ‘This is indicated,
both by the size and more mature appearance of the parent
stock, in which the largest number of segments occur, and also
by the distribution of the red color-spots, which equally indicate
the difference in age between the young individuals without
stolons, or those in which stolonization is just appearing, and
the chain-bearing individuals. Furthermore, specimens are- oc-
casionally found in which the line of demarcation between the
posterior segment of the parent stock and the anterior segment
of the chain is not sharply defined by the great difference in
the size of the segments as it is in the specimens usually found.
One specimen of this kind which I have observed showed an
anterior segment of the chain that was very much larger than
the second segment of the chain, and had attained a size nearer
to that of the last segment of the parent stock. No parapodia
were present in this segment, and the only indication of a con-
dition differing from that of the ordinary was the increased size
of the segment. In another specimen, however, I found a simi-
larly located segment bearing parapodia, and presenting all the
characteristics of a segment of the parent stock, but the small
size of the parapodia, and the less mature appearance of the
segment, gave evidence of its having more recently been added
to the parent stock. Such specimens would accordingly indi-
cate that the embryonic region not alone furnishes segments
for the formation of stolons, but also contributes segments for
the elongation of the parent stock.

Evidence that additions to the number of segments of the
parent stock may occur abnormally can occasionally be obtained

No. 2.] STOLONMIZATION IN AUTOLYTUS VARIANS., 315
from previously injured specimens. Pl. XIII, Fig. 19, repre-
sents the posterior part of such a specimen of Autolytus varians
with a chain of stolons attached to the 44th setigerous segment,
presenting the normal phenomena of stolonization. In this indi-
vidual, presumably after injury, a second series of embryonic
segments has made its appearance between segments 4o and
41, and consists of six well-marked segments, in every way
similar in structure to that of the embryonic region of a
normal chain of stolons. All the segments have advanced
sufficiently far in the course of development to indicate their
future condition, and there can without difficulty be marked
out a developing stolon (St. 1), in which the segment from
which the head will be formed has already developed well-
marked parapodia. Segment 41 has also undergone changes,
and gives unmistakable evidence of a head formation, thus con-
verting the four segments of the parent stock (41-45) into part
of another stolon. Anterior to stolon I is a segment (me.)
which is similar to and represents the embryonic segments of
an ordinary chain. Anterior to this is a segment (s.a.) which
is larger than the embryonic segment, and in addition shows
developing parapodia, thus demonstrating without a doubt that
it is being added to the parent stock.

This figure gives additional evidence that the zone of embry-
onic segments contributes segments, not only posteriorly for
the formation of the stolon, but also anteriorly for the length-
ening of the parent stock. In this way I think the great
length of some of the parent stocks (a length which I have
not been able to find in slender specimens) may be explained,
and in consequence the process of stolon formation in this
species may be regarded as being confined to a range between
segments 18 to 38, the range in which I have noticed the for-
mation of the first stolon of the chain in young individuals,
and not limited to accidental occurrence between segments 18
and 51.

ORIGIN OF THE CHAIN.

The first indication of the phenomena of stolonization
appears in a young individual from 8 to 10 mm. in length and

316 MENSCH. [VoL. XVI.

containing from 4o to 48 setigerous segments, all of which,
with the exception of the anal, are similar in structure, and
many of which have reached a stage of development equal to
that of the segments of a chain-bearing parent stock. It con-
sists in a thickening of the ectoderm of one of the segments
posterior to the 18th to 38th, and the subsequent development
of the head in a manner very similar to the development
of the head in the: embryonic segments of the chain. The
appearance of the head at once marks a division of the indi-
vidual into an anterior parent stock, and a posterior stolon
formed of segments originally belonging to the young indi-
vidual and hence formed in a manner different from the forma-
tion of the stolons of the chain. The manner in which this
stolon is formed has been described as that of fission (sczssz-
parite, de St.-Joseph) in forms in which the production of a
single stolon is followed by its separation and a subsequent
regeneration of separated parts. In chain-bearing forms, how-
ever, this so-called fission, besides producing a stolon that is
different from the stolons separated from the chain, does not
consist of the separation of the stolon after it has matured;
but in the course of the formation of the head and buccal seg-
ment, new tissues and later new segments begin to appear
between the segment upon which the head has been formed
and the segment anterior to it. In this way the stolon is car-
ried back just as is the case in the maturing stolons of the
chain, and finally becomes separated as the first stolon of the
newly formed chain. Before the stolon has separated, the new
segments may have become sufficiently numerous and their
development advanced sufficiently far to show the presence of
one or two distinct stolons similar in appearance to the young-
est stolon represented in Pl. XIII, Fig. 6. Subsequently, while
these young stolons are maturing, the chain is elongated by the
addition of new segments and the development of new stolons
in the manner described by de St.-Joseph and Malaquin in similar
chain-bearing species as that of budding (dourgeonnement).
Stolonization accordingly makes its appearance in the young
so-called asexual individuals in the formation of a single stolon,
by a process somewhat similar to that of fission in chainless

No. 2.] STOLONMIZATION IN AUTOLYTUS VARIANS. 317

forms. This is followed by the successive formation by bud-
ding of a number of stolons which owe their origin, as has
been seen, to an outgrowth of tissue from the last segment of
the parent stock. The maturing and separation of the stolon
would complete the cycle of stolonization in this species, but
frequently specimens may be found which give evidence of the
formation of a second stolon by fission anterior to the position
of the chain and at the expense of the segments of the parent
stock. This stolon appears after the stolons of the chain have
all been separated, the remaining embryonic segments of the
preéxisting chain forming the posterior region of this stolon.
The formation of such a stolon in this species is always by true
fission, such as described by de St.-Joseph; and in no instance
have I found an embryonic region indicating the formation of a
chain as occurs in the development of the first stolon.

The cycle of stolonization in Autolytus varians, therefore,
consists in :

1. The development of a first stolon on the young asexual
individual by a process akin to that of fission.

2. The development of a chain of stolons from the last
segment of the parent stock by the process of budding, and
the successive separation of an unknown and possible variable
number of stolons.

3. The development of possibly a single stolon posterior to
the middle region of the parent stock by a true fission.

The subsequent fate of the parent stock from which the last
stolon has been separated by fission and which may consist of
from 19 to 24 setigerous segments, I have not been able to
satisfactorily determine in this species. Some of these indi-
viduals may be found to contain eggs as far forward as the seg-
ment posterior to the gizzard, thus suggesting the conversion
of the parent stock into a sexual individual by a process similar
to that described as Epigamie by Malaquin, to which I have
previously referred in another species (14). None of the speci-
mens found, however, would warrant this assumption.

URSINUS COLLEGE, COLLEGEVILLE, PA.
November 20, 1897.

318 MENSCH. [VoL. XVI.

_

Il.

LITERATURE.

. O. FRANZ MULLER. Zoologia Danica. Hafniae. Vol. ii. 1788.

DE QUATREFAGES. Etudes sur les types inférieurs de l’embranche-
ment des Annelés. Mémoire sur la génération alternate des Syllis.
Ann. d. Sct. Nat., Zool. Série 4, tome ii. 1854.

MILNE-EDWARDS. Observations sur le développement des Annélides.
Ann. a. Sct. Nat., Zool. Série 3, tome ii. 1845.

CLAPAREDE. Les Annélides Chélopodes du golfe de Naples. J/ém.
de la Soc. de Phys. et @ Hist. naturelle de Geneve. Tome xix.
1868.

. Von MARENZELLER. Zur Kenntniss der adriatischen Anneliden.

Sitzungsber. der Akad. zu Wien. 69, 70,72. 1875.
LANGERHANS. Wurmfauna von Madeira. Zedtschr. fiir Wiss. Zool.
Bd. 32. 1879.
DeE St.-JosEPH. Sur les Annélides polychétes des cétes de Dinard.
Compt. Rend. Acad. Sct. Tomeci. 1885.

. PrRuvot. Sur la formation des stolons chez les Syllidiens. Com#t.

Rend. Acad. Sct. Tome cviii. 1890.

. MALAQUIN. Recherches sur les Syllidiens. Lille. 1893.

AGassiz. On Alternate Generation of Annelids and the Embryology
of Autolytus Cornutus. Boston Journ. of Nat. Hist. Vol. vii.
1862.

VERRILL. Notice of Recent Additions to the Marine Invertebrata of
the Northeastern Coast of America, with Description of new Genera
and Species and Critical Remarks on Others. Part I. Proceedings
of United States National Museum. Vol. ii. 1879.

. FRAIPONT. Recherches sur le Systéme nerveux central et périphé-

rique des Archi-annélides. Archives de Biologie de Van Beneden.
Tomev. 1884.

. ANDREWS. Compound Eyes of Annelids. Journal of Morphology.

Vol. v. 1893.
MeEnscH. Fate of the Parent Stock in Autolytus Ornatus. Zool.
Anzeiger. 505. 1896.

No. 2.] SZTOLONMIZATION IN AUTOLYTUS VARIANS. 319

AP. UT.
a.t., Ut.

2.U., UU.

o

REFERENCE LETTERS.

Aciculus.

Anterior lateral antennae.

Anal segment.

Buccal segment.

Head.

Medullary substance.

Caudal cirri.

Ganglion cells.

Coelome.

Nerve ring.

Cuticle.

Dorsal cirri.

Dissepiment.

Dorsal median tentacle.

Dorsal and ventral ramus of
parapodium.

Dorsal and ventral tentacu-
lar cirri.

Dorsal and ventral blood
vessels.

C.Q., C.D.
ect.

Anterior and posterior eyes.

Epidermis.

Ectoderm.

Entoderm.

Lateral tentacles.

Muscles of parapodia.

Dorsal and ventral muscle
bands.

Mesoderm.

Nephridia.

Nerve tentacle.

Mouth.

Parapodia.

Posterior lateral tentacles.

Embryonic region.

Region of separation.

Suboesophageal ganglion.

Sperm cells.

Tentacle muscles.

Ventral cord,

320 MENSCH.

EXPLANATION OF PLATE XIII.

Fic. 1. Autolytus varians. Parent stock with a chain of five distinct
stolons. X 5.

Fic. 1. a. Enlarged parapodium of the parent stock.

Fic. 1. 64. Enlarged setae of the parent stock.

Fic. 2. Enlarged head of the parent stock. x I5.

Fic. 3. Mature male stolon. x 30.

Fic. 4. Parapodium of the mature male stolon.

Fic. 5. Mature female stolon. x 30.
Fic. 6. Chain of six distinct male stolons. x 18.

Fic. 7. Very young isolated stolon.

Fic. 8. Lateral view of the posterior part of stolon 1 and the anterior part
of stolon 2. X 40.

Fic. 9. Ventral view of the adjacent parts of stolons 2 and 3.

Fic. to. Parapodia of stolon 4.

Fic. 11. Lateral view of the posterior part of stolon 3 and anterior part of
stolon 4. X 40.

Fic. 12. Parapodia of stolon 5.

Fic. 13. Parapodium of a mature female stolon.

Fic. 14. Longitudinal median section through the head of a mature male
stolon. X 140.

Fic. 15. Transverse section through the last segment of the parent stock in
the region of a parapodia. x 200. ;

Fic. 16. Transverse section through the posterior part of the last segment of
the parent stock. xX 200.

Fic. 17. Longitudinal median section through segment 35, the embryonic
region, and the anterior part of the stolon 1, Fig. 6. X 175.

Fic. 18. Transverse section through the anterior segment of the embryonic
region of the chain. x 200.

Fic. 19. Posterior part of a specimen showing the formation of new segments
between segments of the parent stock. x 18.

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EXPLANATION OF PLATE XIV.

Fic. 20. Transverse section through the second segment of the chain. x 200.

Fic. 21. Transverse section through the posterior part of the first segment
of stolon A. X 175.

Fic. 22. Transverse section through the head of stolon 1. x 200.

Fic. 23. Longitudinal median section through the posterior part of stolon 1
and the anterior part of stolon 2. X 175.

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X17 5.

Fic. 25. Transverse section through the posterior part of the head of stolon 2.
x 175.

Fic. 26. Longitudinal median section through the anal segment of stolon 3
and the anterior part of stolon 4. X 175.

Fic. 27. Transverse section through the head and buccal segment of stolon 4.
X 200.

Fic. 28. Transverse section through the anterior part of the head of stolon 5.
X 200.

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ON) THE HEART OF) LUNGLESS) SAL AMANDERS?
HENRY L. BRUNER, Pu.D.

In the Asnerican Naturalist for 1896 Hopkins (14) announced
the discovery of a septum atriorum in the heart of certain lung-
less salamanders. Beginning with the heart of salamanders
with lungs, Hopkins calls attention to various parts of that
organ, but omits entirely the valve at the sinus-atrium opening.
Later, however, in his study of the heart of the lungless sala-
manders, Hopkins observed this valve, but considered it to be
a rudimentary septum atriorum. The conclusion of Hopkins,
that such a septum is present in lungless salamanders, has
been accepted by Bethge (1), but apparently without personal
investigation. A similar view occurs also in the recently pub-
lished “‘ Vertebrate Zodlogy”’ of Kingsley (17). On account
of these facts I wish to report some observations which I had
already made on the heart of lungless salamanders before the
paper of Hopkins came to my notice. The investigation was
undertaken at the suggestion of Professor Wiedersheim, in
whose Institute in Freiburg the work was begun, and to whom
I am indebted for the necessary material.

Fitstorical.

The general structure of the heart of amphibians was de-
scribed by Rusconi (29), Briicke (7), and Stannius (28), about
the middle of the present century.

An excellent account, published somewhat later, is that of
Fritsch (11), whose work is made, to a considerable extent, the
basis of the description in Bronn’s ‘““Amphibien,” where also
the figures of Fritsch are reproduced. In his work on the frog,
Fritsch studied the structure of the ventricle and the arrange-

1 A short abstract with the above title was published in the Proceedings of the

Indiana Academy of Science (3) for 1897. Later a similar notice appeared in the
Anatomischer Anzeiger (4).

325

324 BRUNER. [VoL. XVI.

ment of valves in the heart, and pointed out for the first time
the function of the spiral valve in regulating the direction of the
flow of blood.

Langerhans (21) described the perforations in the septum
atriorum of Urodela. His results were later confirmed by
Huxley (15) and Boas (6).

Boas (5) investigated the spiral fold of the salamanders, and
showed it to be formed by the fusion of a row of valves. Indi-
cations of this origin are still to be found in some forms (Triton
cristatus), in which the valve is represented by a row of knots
on the conus wall.

Gompertz (12) published an excellent account of the physi-
ology of the anuran heart in 1884.

Langer (19) worked out the development of the proximal
and distal valves of the conus arteriosus. Thickenings repre-
senting these valves appear in a very early stage of the larval
development. Later these thickenings are hollowed out next
to the conus wall.

The literature of the lungless salamanders begins in 1894,
when Wilder (35) announced the discovery of a number of
species. The same year Camerano (9) discovered two lungless
Italian forms. In 1896 Wilder (36) and Lonnberg (22) each
added a number of species to the list, which, according to
Wilder, includes at least half of the known species of Sala-
mandrinae.

Camerano (9) attempted to determine the mode of respira-
tion of Salamandrina perspicillata and Spelerpes fuscus, follow-
ing the method of Marcacci (37) with the frog, by hindering the
bucco-pharyngeal respiration. As the animals experimented
with quickly died, in spite of the fact that the cutaneous res-
piration was undisturbed, Camerano concluded that the latter
is relatively unimportant in sustaining life, while the bucco-
pharyngeal respiration is really essential thereto.

Bruner (2) found special muscles for opening and closing
the external nares in the lungless salamanders. In salaman-
ders with lungs these muscles constitute an important part of
the mechanism of inspiration. In lungless forms, however,
their function seems to be restricted to the protection of the

No.2.) HEART OF LUNGLESS SALAMANDERS. 325

nasal cavities. The use of the muscles for respiratory pur-
poses, such as the inflation of the pharynx or oesophagus, has,
at any rate, not been observed. Study of Salamandrina in
water also failed to show such an inflation for hydrostatic
purposes.

Maurer (23) reported the occurrence of blood capillaries in
the buccal epithelium of both Anura and Salamandrinae (Rana,
Bufo, Hyla, Salamandra, Triton). Maurer is of the opinion
that this vascularization is primarily a response to the demand
of a many-layered epithelium for proper nourishment. The
closer proximity of the capillaries to the surrounding medium
would, however, also favor the more rapid aération of the blood,
and thus assist in respiration. This fact might then account
for the further development of epithelial vessels for respiratory
purposes (e.g., in lungless salamanders).

Such a special development of the capillaries of mouth and
oesophagus Bethge (1) found in the lungless Spelerpes fuscus.
He says : “Schon mit guter Lupe erkennt man, dass sie (the
capillaries) nicht glatte Gefasse sind, sondern in ihrer ganzen
Ausdehnung ein fast traubenformiges Aussehen zeigen, an
manchen Stellen so deutlich, dass man einen gemeinsamen
Stiel und daran sitzende Beeren unterscheiden kann. .
Auf Schnitten durch den ganzen Kopf lasst sich die Lage der
Kapillaren erkennen. Wir finden ein mehrschichtiges Epithel ;
die Zellen der unteren und mittleren Lage zeigen unregel-
massig kubische Form, die Zellen der aussersten Schicht sind
von cylinderformiger Gestalt. Zwischen den Epithelzellen der
mittleren und oberen Lage erstrecken sich Becherzellen. Die
Kapillaren breiten sich nun zwischen den Zellen der basalen
Lage aus und treiben Ausstiilpungen zwischen die mittleren
Zelllagen hinein, die haufig bis an die oberste Schicht heran-
reichen.” This peculiar development of the capillaries must
greatly facilitate respiration ; nevertheless Bethge considers
the respiratory function of the skin of still greater importance,
and opposes the view advocated by Camerano.

Bethge compared also the larger blood vessels of Spelerpes
with those of salamanders with lungs, and found the chief dif-
ference in the development of the pulmonary vessels. In the

326 BRUNER. [VoL. XVI.

lungless form the pulmonary vein is wholly wanting; the pul-
monary artery, on the other hand, has been preserved because
it supplies other parts than the lungs. In regard to the heart,
Bethge accepts the conclusions of Hopkins.

Miss Woldt (33) found a pulmonary artery in Plethodon
erythronotus and P. cinereus, in which it supplies both oesoph-
agus and skin. Beyond the point of origin of the last
oesophageal branch no trace of the true pulmonary trunk could
be found.

W.E. Ritter and Loye Miller (26) find in the toes of Autodax
lugubris great blood sinuses, which they consider an important
seat of respiration. ‘The toes have, in fact, assumed the func-
tion of external gills.”

The Heart of Salamanders with Lungs.

The heart from which Fig. 1, Pl. XV, was drawn had been
injected immediately after death with 70% alcohol, until all
blood was washed out. The large vessels were then ligatured
and the entire animal, with the heart, placed in 70% alcohol.
After hardening, the heart was in excellent condition for study.
Figs. 3 and 4, Pl. XV, represent sections of a heart injected
with blood only. The amount of distention is less here than
in Fig. 1.

The heart of Salamandra maculosa, which will be used as a
type in the following description, is composed of two different
sections —one for the reception and one for the expulsion of the
blood. The former includes the sinus venosus and two in-
completely separated auricles; the latter includes the single
ventricle and the truncus arteriosus. The right auricle receives
blood from the general circulation; the blood from the lungs
pours into the left auricle. Into the latter the pulmonary vein
enters directly, while the blood from the body passes first
into the sinus venosus, which lies on the dorsal side of the
heart and toward the left side. The sinus is formed by the
fusion of three large veins — the two venae cavae superiores,
which open into the sinus by separate mouths, and the vena
cava inferior, which enters the sinus at its posterior end. The

No. 2.] MEART OF LUNGLESS SALAMANDERS. Bey

sinus venosus is provided with muscular walls, whose contraction
marks the first stage of the heart-beat. The return of the
blood from the auricle into the sinus is prevented by a valvular
contrivance, which, according to Fritsch (11), corresponds to
the valvula Eustachii of higher vertebrates.

The two auricles of Salamandra show externally no evidence
of separation. A furrow on the ventral auricular surface indi-
cates the position of the truncus arteriosus and does not corre-
spond to that of the septum, which lies farther to the left. An
examination of the interior of the heart shows that the septum
is somewhat oblique to a median vertical plane, the inclination
being from left above to right below. Dorsally it attaches close
to the edge of the sinus-atrium opening; caudally it hangs with
a free margin over the atrio-ventricular opening. According
to Langerhans, the septum atriorum of Salamandra maculosa is
always perforated, particularly in its dorsal third. It is supplied
with an abundance of muscular tissue, which contracts with the
walls of the auricles.

Of the three openings in the auricular walls, two are provided
with valves, the third is valveless. Of the former, the atrio-
ventricular opening is guarded by two fibrous pouches, whose
margins are connected by means of cords to the wall of the
ventricle. The anterior surface of each valve is attached at its
middle to the margin of the septum atriorum. Between the
two points of attachment the septum hangs free.

The sinus-atrium opening lies in the dorsal wall of the right
auricle. The plane of the opening is almost transverse to the
axis of the body, but its left margin lies usually somewhat
anterior to the right. Immediately in front of the opening we
find the pulmonary vein, which is formed on the dorsal side of
the heart by the union of two vessels, one from each lung. At
the dorsal margin of the sinus opening the vein penetrates the
atrial wall, on whose inner surface the now flattened vessel ex-
tends forward and toward the left, until, at the anterior margin
of the sinus-atrium valve, it reaches the septum, through which
it discharges into the left auricle. From the sinus opening to
the septum, the vein is closely united to the atrial wall; the
latter, however, is dorsal to the vein, as is shown by the struc-

328 BRUNER. [Vou. XVI.

ture of the entire region. This fact explains the manner in
which the vein projects into the atrial cavity (see Pl. XV, Fig. 3).

The sinus-atrium opening of amphibians is furnished, in typi-
cal cases (Perennibranchiata), with two transversely placed valves,
which are attached on the right to the atrial wall, on the left to
the septum atriorum. In Salamandrinae, however, one of these
valves, the ventral one, becomes rudimentary or wholly disap-
pears, while the other is greatly enlarged. The form and rela-
tions of the latter valve in Salamandra are shown in Pl. XV,
Figs. 1, 3, and 4. In this species a second valve is wholly
wanting. The enlarged valve is a sail-shaped membrane, which
extends from the sinus-atrium opening to the atrio-ventricular
opening, where it is attached, along with the septum atriorum,
to the middle of the inferior valve. The convex margin of the
valve is fixed by means of muscular trabeculae to the septum ;
the concave margin is wholly free. The attachment of the
dorsal end of the valve follows the ventral wall of the pulmo-
nary vein from the septum to the sinus opening, where the valve
bends toward the right, covers the opening, and attaches to its
dorsal half.

The sinus valve, like the septum and atrial walls, is contrac-
tile, its muscular bundles being in part continuous with those
of the supporting structures. In the neighborhood of the sinus
opening these bundles are strongly developed in a dorso-ventral
direction, which is here the direction of greatest tension.

The truncus arteriosus projects forward from the ventricle
with a slightly sigmoid curve. Its proximal portion (conus
arteriosus) contains two circles of valves—a posterior one,
which lies at the ventricular opening and consists of three
pouches, and an anterior set, which includes normally four
valves. One of the valves of the distal circle is produced cau-
dalward as a low ridge of connective tissue, which terminates
in front of the proximal set of valves. This ridge is the
so-called spiral fold of the salamander.

Beyond the conus we find the bulbus arteriosus, from which
arise the great arterial trunks.

With the foregoing description as a basis for comparison, we
may now turn to the study of salamanders without lungs.

No.2.] MEZART OF LUNGLESS SALAMANDERS. 329

The Heart of Lungless Salamanders.

In the following description I shall use Salamandrina perspi-
cillata and Plethodon erythronotus as types; other forms, how-
ever (Plethodon cinereus, Desmognathus fusca, Spelerpes
fuscus), have essentially the same structure. The prepara-
tion followed in Fig. 2, Pl. XV, was treated as that used for
Fig. 1. The sections represented in Figs. 6 and 7, Pl. XV, are
from a heart naturally injected with blood.

In all of the lungless species mentioned above, the external
arrangement of the parts of the heart is very similar to that of
the heart of Salamandra maculosa; but the pulmonary vein is
not to be seen, and a careful microscopic examination has failed
to reveal a trace of it. Similar results have been obtained by
Hopkins (14) and Bethge (1) with certain lungless forms.

In the interior of the heart of Salamandrina still other modi-
fications of structure may be readily seen, the most important
of which is due to the disappearance of the septum atriorum,
which, like the pulmonary vein, seems to have left no trace of
its former existence. We find a well-developed sinus-atrium
valve, which extends from the sinus opening to the ventral
aspect of the ostium atrio-ventriculare ; its convex antero-
ventral margin, however, is fixed to the left atrial wall. The
posterior end of the valve is attached to the middle of the
lower atrio-ventricular valve, while its dorsal end extends from
the left atrial wall obliquely caudalward toward the sinus open-
ing, to whose dorsal margin it is fixed. This relation of the
valve to the sinus opening is essentially the same that we saw
in Salamandra, and has occasioned the remark of Hopkins,
that the sinus opening of the lungless salamander leads into
the left auricle.

The conus of Salamandrina shows the same general structure
that we found in the conus of Salamandra. A spiral valve is
distinctly recognizable in the lungless form.

Let us now consider the significance of the facts presented
above. We have found in the heart of lungless salamanders
certain changes of structure which must be attributed directly
or indirectly to the loss of lungs. One of these changes —

330 BRUNER. [VoL. XVI.

the shifting of the attachment of the sinus-atrium valve — is to
be accounted for by the disappearance of the septum atriorum.
The valve itself shows no sign of degeneration, which indeed
could not be expected to occur here, for the condition which
requires the presence of a valve at the sinus-atrium opening is
certainly not affected by the loss of lungs.

It is worthy of note that the loss of lungs has affected the
pulmonary artery and the pulmonary vein in an altogether
different way. As already stated, the survival of the artery
in lungless salamanders has been reported by Hopkins (14),
Bethge (1), and Miss Woldt (33). I have observed the artery
also in Salamandrina, where, however, it receives its blood only
through the ductus Botalli, the proximal portion of the vessel
having disappeared. In all of the forms studied, the pulmo-
nary artery supplies certain parts (oesophagus, skin), to which it
is distributed also in salamanders with lungs. Such an acces-
sory saving function the pulmonary vein of lungless salaman-
ders did not possess.

The fact that the septum atriorum disappears with the lungs
indicates clearly that in salamanders with lungs the septum
performs a function which becomes superfluous or impossible
after the loss of those organs. This function is the separation
of the venous blood of the right auricle from the aérated blood
of the left auricle. But what is the significance of this separa-
tion if the two sorts of blood are afterward mixed during their
passage through the ventricle and conus? Or is there, after
all, in salamanders with lungs, a partial separation of aérated
and venous blood in its entire course through the heart?
Such a separation occurs, as is well known, in the heart of
Rana. Nowas regards the atrium and ventricle we find essen-
tially the same structure in Salamandra as in Rana. It is true
that the septum atriorum of the salamander is perforated, while
that of the frog is not. But during the brief stay of the blood
in the auricles the small perforations which have been described
in Salamandra would permit little mixing of the blood. There
would be a much better opportunity for this to occur in the
ventricle ; but here we have the same spongy condition in Sala-
mandra and Rana. So far, then, Rana does not seem to have

Noo2:|)\ (BEART OF LUNGLESS, SALAMANDERS. 331

a decided advantage over the salamander in respect to the
separation of venous and arterial blood in the heart. We may,
therefore, conclude that in the salamander, as in Rana, the
first blood passing from the ventricle into the conus during
the ventricular systole is chiefly venous. In Rana this blood
is directed into the pulmonary artery. In the salamander,
however, the structure of the conus does not indicate that it
could influence the direction of the blood current. We must
turn, then, to the bulbus arteriosus and the great arterial ves-
sels for further light on our problem. Here, however, peculiar
difficulties present themselves whose solution I shall not attempt
at this time. But it seems not improbable that, in salamanders
with lungs, a difference of blood pressure in the arterial trunks
at the time of the ventricular systole may lead to a distribution
of blood similar to that occurring in Rana.

The spiral valve of the salamanders can have no control
over the direction of the blood which passes through the conus.
Its function seems to be rather to prevent the collapse and
obstruction of the conus, which might, in the absence of the
valve, arise either as a result of the strong contraction of the
conus walls, or from outside pressure.

soe

N

IO.

IZ.

16.

17,
18.

BRUNER. [VoL. XVI.

LITERATURE.

BETHGE. Das Blutgefasssystem von Salamandra maculata, Triton
taeniatus und Spelerpes fuscus; mit Betrachtungen tiber den Ort
der Athmung beim lungenlosen Spelerpes fuscus. Zedtschr. fiir
qwiss. Zool. Bad. lxiii. 1898.

BRUNER. Ein neuer Muskelapparat zum Schliessen und Oeffnen der
Nasenlécher bei den Salamandriden. Arch. fiir Anat. und Phys.
Anatomische Abtheilung. 1896.

BRUNER. On the Heart of Lungless Salamanders. Proc. /udiana
Acad. Sci. Indianapolis. 1897.

BRUNER. On the Heart of Lungless Salamanders. Axat. Anz.
Bd. xv. 1899.

Boas. Ueber den Conus arteriosus und die Arterienbogen der Am-
phibien. Morph. Jahrb. Bd. vii. 1882.

Boas. Beitrage zur Angiologie der Amphibien. J/orph. Jahrb.
Bd. viii. 1883.

BRUCKE. Ueber Mechanik des Kreislaufs des Blutes bei Fréschen.
Wiener Sitzungsber. 1851.

BrUcKE. Beitrage zur vergleichenden Anatomie und Physiologie des
Gefiasssystems der Amphibien. Denkschr. Acad. Wien, 1852.
CAMERANO. Ricerche anatomo-fisiologiche intorno ai Salamandridi
normalmente apneumoni. JLzdbveria della Acad. della Sci. Torino.

1894.

CAMERANO. Nuove ricerche intorno ai Salamandridi normalmente
apneumoni ect. Anat. Anz. 18096.

FritscH. Zur vergleichenden Anatomie des Amphibienherzens.
Arch. fiir Anat. und Phys. 1869.

GOMPERTZ. Ueber Herzund Blutkreislauf der nackten Amphibien.
Arch. fiir Anat. und Phys. Leipzig. 1884.

HOFFMANN. Bronn’s Classen u. Ordnungen des Thierreichs. Bd.
vi. II. Abth. Amphibien.

Hopkins. The Heart of some Lungless Salamanders. Amer. (Vat.
Vol. xxx. 1896.

HuxLey. On the Structure and of the Skull and of the Heart of
Menobranchus lateralis. Proc. Zool. Soc. 1874.

Hyrti. Berichtungen iiber den Bau des Gefasssystems von Hypoch-
thon Laurentii (Proteus anguineus). Med. Jahrb. des Oesterr.
Staates. 1844.

KINGSLEY. Vertebrate Zodlogy. New York. 1899.

LAMBOTTE. Observations anatomiques et physiologiques sur les
appareils sanguins et respiratoires des Batraciens anoures. J7ém.
Cour. de Ll’ Acad. de Bruxelles. 1838.

t bs f
F A
s a
: =
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No.2.] HEART OF LUNGLESS SALAMANDERS. 333

19.

20.

25.

26.

LANGER. Ueber die Entwicklungsgeschichte des Bulbus cordis bei
Amphibien und Reptilien. J/orph. Jahrb. Vol. xxi. 1894.

LANGERHANS. Zur Histologie des Herzens. Virchow’s Archiv.
Bd. lviii.

LANGERHANS. Notiz zur Anatomie des Amphibienherzens. Zeztschr.
fiir wiss. Zool. Bd. xxiii. 1873.

LONNBERG. Notes on Tailed Batrachians without Lungs. Zool.
Anzeiger. Nr. 494. 1896.

MAvRER. Blutgefasse im Epithel. Morph. Jahrb. Bd. xxv. 1897.

OwEN. On the Structure of the Heart in the Perennibranchiata.
Trans. 2001. Soc: Vol. 1.1834"

PREVOST et LEBERT. Mémoire sur la formation des organes de la
circulation et du sang dans les batraciens. Ann. Sct. Vat. Zool.
3¢ série. 1844.

RITTER and MILLER. A Contribution to the Life History of Auto-
dax lugubris Hallow. Ammer. Wat. Vol. xxxiii. 1899.

Rose. Beitrag zur vergleichenden Anatomie des Herzens der Wirbel-
thiere. JMJorph. Jahrb. Bd. xvi. 1890.

STANNIUS. Handbuch der Anatomie der Wirbelthiere. Berlin.
1854.

Rusconi. Histoire naturelle, développement et metamorphose de la
Salamandre terrestre. 1854.

VAILLANT. Mémoire pour servir 4 "histoire anatomique de la Sirene
lacertina. Ann. Sct. Vat. 4¢ série. Tome xix. 1863.

WEISSMANN. Ueber die Musculatur des Herzens beim Menschen
und in der Thierreihe. Arch. fiir Anat. und Phys. 1861.

WIEDERSHEIM. Grundriss der vergleichenden Anatomie der Wirbel-
thiere. Jena. 1893. .

Wo.Lpt. The Pulmonary Arch of Lungless Salamanders. Proc.
Indiana Acad. Sci. 1897.

Wyman. On the Heart and Respiration of Menobranchus and
Batrachians. Pyroc. Boston Soc. Nat. Hist. Vol.v. 1856.

WILDER. Lungenlose Salamandriden. Anat. Anz. Bd. ix. 1894.

WILDER. Lungless Salamanders. Second Paper. <Axat. Anz.
Bd. xii. 18096.

Marcacci. L’asphyxie chez les animaux 4 sang froid. Arch. [ta-
liennes de Biologie. Bd. xxi, Turin. 1894.

334

BRUNER.

DESCRIPTION OF FIGURES.

Undivided atrium of lungless salamander.
Atrio-ventricular aperture.
Right auricle.

Left auricle.

Septum atriorum.

Sinus venosus.

Truncus arteriosus.

Right vena cava.

Vena cava inferior.

Left vena cava.
Pulmonary vein.
Sinus-atrium valve.

Spiral fold.

BRUNER.

Oo
ios)
OV

EXPLANATION OF PLATE XV.

Fic. 1. Heart of Salamandra maculosa, viewed from the right side. The
perforations of the septum atriorum are not indicated.

Fic. 2. Heart of Salamandrina perspicillata, also viewed from the right side.
Septum atriorum and pulmonary vein are wanting.

Fic. 3. Frontal section through the heart of Salamandra maculosa. The
plane of the section passes through the anterior part of the sinus-atrium valve.
The spongy structure of the ventricle is not shown in the drawing.

Fic. 4. Section a short distance behind Fig. 3, and from the same series.
The transverse portion of the sinus-atrium valve and the dorsal margin of the
sinus opening lie in the plane of the section.

Fic. 5. Section through the conus arteriosus and spiral valve of Salamandra
maculosa.

Fic. 6. Frontal section through the heart of Plethodon erythronotus; the
direction and location as in Fig. 3.

Fic. 7. Frontal section through heart of Plethodon erythronotus ; correspond-
ing to Fig. 4.

Fic. 8. Conus and spiral valve of Spelerpes fuscus.

Journal of Morphology. Vol.XVI. PU.XV.

2. Salamandrina

8. Spetlerpes

7. Plethodon

HL. Bruner del

Tith. Werner 2 Winter, Frankfort 7M.

\\

ON THE STRUCTURE OF TWO FISH TAPEWORMS
FROM’ THE GENUS PROTEOCEPHALUS
WEINLAND 1858.)

HARRIS M. BENEDICT.

THIS paper consists of an anatomical and histological study
of a little-known native species of Proteocephalus and an inves-
tigation of P. fi/zcollis, a well-known species first described by
Rudolphi, in which some additional points are elucidated.

The investigations were carried on in the years 1895-97, and
the paper has been accepted as the thesis for the Master’s
degree at the University of Nebraska.

To Dr. Ward my sincerest thanks are due, not only for the
material with which I worked and the use of his private library,
but also for the valuable assistance which in other ways he has
given me. I am also indebted to the kindness of Prof. Dr.
Zschokke, of Basel, in sending to Dr. Ward, specimens of
P. filicolts.

In the study of the genus of fish taeniae, to which Lonnberg
(94) has given the name /chthyotaenia, the paper by Weinland
(58) has evidently been overlooked. In this paper, which was
brought to my notice by Dr. Charles W. Stiles in private cor-
respondence, the following occurs:

“Gen. 2. Proteocephalus, Weinl. (The name is derived
from ‘mp@reus,’ the ever-changing principle in the old Greek
mythology, and ‘xepady,’ head.) The shape of the head of
this genus is extremely changeable. There is no proboscis nor
hooklets. The eggs are provided with two shells, the outer
shell being mucilaginous. These taeniods live in reptiles and
fishes. The type of this genus is Taenta ambigua Dujardin.
Here belong Yaenza filicollis and Taenia dispar.”

. 1 Studies from the Zodlogical Laboratory, The University of Nebraska, under
the direction of Henry B. Ward, No. 33.

337

338 BENEDICT. [VoL. XVI.

I have not been able to obtain specimens of amdbigua, and
the literature which contains a more detailed description than
that of Dujardin is inaccessible. Hence I could not determine
from personal investigation whether amdbzgwa was a true type
of the genus which has been called /chthyotaenia. Riggenbach
('96), however, lists ambzgua as one of the /chthyotaeniae, and the
excellence of his work on this group warrants me in accepting
this designation. The rules of priority in nomenclature, there-
fore, demand the adoption of the generic name Proteocephalus.

The Genus Proteocephalus.

The oldest literature on the Proteocephalus is found in the
works of Rudolphi (10), Dujardin (45), Diesing ('50), van
Beneden (’61), and von Linstow (78). All of these authors
confine their descriptions to a few details concerning the
size and form of parts, particularly of the scolex and certain
proglottids.

Zschokke (’84) was the first to make any study whatever of
anatomical structure, and therefore his work is of much greater
value than that of the older authors. The next author to make
an anatomical and histological study was von Linstow ('91), who
described 7. longicollis. He was the first to note the relation-
ship between the various fish taeniae. In the following year
Kraemer (92) made a very exhaustive study of Cyathocephalus
truncatus, P. torulosa, and P. filicollis, and established the iden-
tity of P. jilicollis and P. ocellata. He also pointed out other
common characteristics of the fish taeniae. Lonnberg ('94) was
the first to establish the group under the name /chthyotaenia,
and made its characteristics still more distinct and accurate.

By far the best work yet done on this group is found in the
paper by Riggenbach ('96), who, in addition to a careful descrip-
tion of two new species, has for the first time made an accurate
and discriminating summation of the present knowledge of this
group. At the present time a great amount of confusion exists
and the accuracy of the specific determinations is doubtful
in over half of the species of Jchthyotaenia so far described.
Riggenbach shows that out of a list of twenty-nine species

NOM2) SPROCTORE OF) TWO FISH TAPEW ORAS, 339

only seven have supposedly been accurately determined, and
this paper makes evident grave mistakes in the description of
one of these forms. As an example of inaccuracy should be
cited the reference by both Kraemer (92) and von Linstow ('78)
to a description of 7. zorwlosa in a work of van Beneden(’61).
As a matter of fact, the form described is not even given by
the author as ¢orulosa, but as porulosa, and from the figures
could not possibly be ¢orulosa. Until all doubtful species are
further investigated, no satisfactory synopsis of the entire genus
can be made.

Proteocephalus ambloplitis (Leidy).

The specimens of this cestode which came into my posses-
sion proved to be different in internal morphology from any
carefully described species of Proteocephalus, and were at first
believed to represent a new species. In reviewing the litera-
ture on fish cestodes, I noticed an account by Leidy (’87) of
the discovery of a cestode in a rock bass, Ambloplitis rupestrts,
which he named J7aenia ambloplitis. His description was lim-
ited to a few external measurements which did not agree with
the dimensions of my specimens. He also stated, however,
that the cestode closely resembled Zaenza ocellata Rud. Dr.
Ward, through the kindness of Dr. Hassell, acting curator of
the Helminthological Collection of the United States National
Museum, obtained for me Leidy’s type specimens. An accurate
study of one of these by the section method showed that in
spite of certain external differences this species was identical
with my specimens. This is simply another illustration of the
worthlessness of a description dealing merely with external fea-
tures and external measurements in the determination of the
species of cestodes. Exclusive of external form and measure-
ments, the only difference shown by a careful anatomical and
histological comparison of Leidy’s specimens with my own was
the presence of a greater number of calcareous bodies in the
parenchyma and the smaller size of the sphincter muscle. This
latter particular was not important because the younger anterior
proglottids in my specimens showed the same condition. This

340 BENEDICT. [VoL. XVI.

difference then, as well as the size, indicates simply that Leidy’s
specimens were younger than the ones I studied. The descrip-
tion of this form is therefore to be emended as follows:

Specific Characteristics. —Total length from 280 to 410 mm.
Greatest breadth from 1.75 to 2.18 mm. Surface of body very
rough, with transverse ridges and furrows. Scolex prominent
.82 to .88 mm. in greatest diameter just behind the posterior
margin of the suckers. A minute depression, with a slight
elevation in the center, is situated on the rounded apex of the
scolex. The suckers are large, directed outward and forward,
and separated by deep longitudinal furrows, which continue
throughout the length of the short neck. Proglottids closely
joined together, with edges very slightly rounded. Immature
proglottids much wider than long; majority of proglottids
nearly square; posterior proglottids slightly longer than wide ;
last proglottid with a concave posterior margin. Genital sinus
irregularly alternate, situated on the margin, one-fourth of the
length of the proglottis from the anterior end. Vagina with
exceedingly large sphincter muscle and forming a mass of coils
immediately anterior to the ovary. Cirrus formed by an in-
vagination of the distal end of the cirrus pouch and lined by
cuticula continuous with that covering the body. Vas deferens
intricately coiled within the pouch and forming a very large
complex mass of coils near the center of the proglottis. Excre-
tory canals form in the last proglottis a network, which is con-
nected with the exterior by a multitude of minute ducts passing
through the cuticula.

General External Morphology.

The specimens studied were collected by Dr. Ward, while
engaged in the biological examination of Lake St. Clair, during
August of 1893. During this investigation (Ward, '94) ninety
fish of twenty species, caught at New Baltimore on Lake St.
Clair, were examined. Half of them proved to be free from ces-
todes, while the remaining contained two hundred and twenty-
seven specimens of several species. All of the six individuals
of Micropterus dolomieu examined were found to be infested by

No. 2.] STRUCTURE OF TWO FISH TAPEWORMS. 341

this species of cestode, which was found in no other fish.
The parasites occurred in the pyloric caeca and in the intes-
tine, and varied in number from one to four in each fish. The
specimens were killed with corrosive-sublimate solution and
preserved in 83% alcohol—a process which gave very satisfac-
tory results.

The specimens thus preserved varied in length from 280 to
410 mm., and the breadth of the same from 1.75 to 2.18 mm.
The shape of the proglottids at the same stage of development
was not the same in different individuals. Thus, in one speci-
men certain proglottids were nearly four times as wide as
long, while in another individual those of the same develop-
ment were nearly square. An individual which best repre-
sented the greater number of my specimens gave the following
measurements :

Total length of body, 280 mm., containing 332 proglottids.
Breadth of scolex, .84 mm.; length, .55 mm.; suckers, .4 mm.
in antero-posterior diameter, .32 mm. in transverse diameter.
Breadth of neck, .45 mm.

BREADTH. LENGTH.

Proglottids 10 mm. from scolex — 75 mm. .o8 mm.

ue 25 ‘s ss 1.2 58

e 50 fe sf 1.25 -75 to .90

“ 75 “sé ee 1.45 xe) to I

“4 100 ie ss 1.5 1.2 tor.4

f 150 Ye a 2 I.5 to2

“ 280 “ 6c 2 2

It will be seen from these figures that all the proglottids in
the posterior half of the body are nearly of the same size;
and yet in a few specimens there was a gradual and very con-
stant increase in breadth and length from anterior to posterior
ends. However, too much importance has been attached to
simple external measurements by older observers, who, in
many cases, limited their descriptions to statements of the size
of body and the shape of scolex and proglottids. The only
external measurements which are of real value are the dimen-
sions of the scolex and the size of the unsegmented neck.

The scolex of P. ambloplitis is very prominent and plainly
visible to the naked eye. It is .84 mm. in lateral diameter,

342 BENEDICT. [VoL. XVI.

.80 mm. in dorso-ventral, and .85 mm. in length (Pl. XVI, Figs.
1and 2), From the anterior face it presents a nearly square
outline, with a deep notch in the middle of each side, dividing
the surface into quarters. Each quarter contains a large sucker
which is directed outward and upward. The apex of the head
is a smooth, rounded prominence with a small depression in the
top (Pl. XVI, Fig. 3). No hooks are present. A fairly good
idea of the shape of the scolex can be obtained by placing two
truncated pyramids base to base. One of the smaller bases will
represent the beginning of the neck, the other the prominence,
while on the slope just beneath this would lie the suckers.
The notches seen in the anterior view of the scolex form fur-
rows down the four sides of the head, which gradually decrease
in depth and vanish on the first few proglottids.

The surface of the proglottids is very rough, being thrown
into longitudinal and transverse folds, as shown in the margin of
Pl. XVI, Fig. 4, where the transverse furrows are so deep as
to obscure the divisions between the proglottids. The pro-
glottids are also closely joined together, so that it is difficult to
distinguish the divisions between them. The color of the body
in the alcoholic specimen is a watery white, with a yellowish
tinge toward the posterior end.

A small genital sinus, having a depth of .o25 mm. and a
diameter of .o5 mm., is situated about one-fourth the length of
the proglottid from the anterior end, on the margin. It is
irregularly alternate in position (Pl. XVI, Fig. 4).

Anatomical and Hitstological Structure.

Cuticula. —The cuticula can be resolved into two layers ;
an outer thinner portion, .oo15 mm. thick, forms the external
surface (Pl. XVI, Fig. 11, c¢’.). It is very rough, with minute
projections extending from the surface, which are coarser,
blunter, and more irregular than cilia, and which stain darkly.
Within this layer is another, .0075 mm. thick, which appears
nearly homogeneous (Pl. XVI, Fig. 11, ¢¢.). This does not
take the stain so vigorously as the first. Within the cuticula
is a layer of transverse muscles, .oor mm. thick, which appears

No.2 “STACCTORE OF TWO FISH TAPEWORMS. 343

as a dark line in transverse sections (Pl. XVI, Fig. 11, m2.c.cz.).
Beneath this layer is a row of dots, which mark the sections
of longitudinal muscle fibers (Pl. XVI, Fig. 11, m.Zcz.). In
frontal sections, cut somewhat obliquely, these subcuticular
muscles can be best observed. Here the transverse fibers are
seen to be thick and in the form of a fibrous mat, while the
longitudinal fibers are separate and cross the others at right
angles.

The subcuticular cells are slightly pyriform, with a large
nucleus (Pl. XVI, Fig. 11, sd.c.). None of them are closer to
the muscle layers than .0075 mm., but each sends one or more .
processes into the subcuticular muscle layers, beyond which
they can no longer be traced.

The parenchyma cells are irregular in shape and rather
closely packed, presenting the appearance of a fine meshwork
with nuclei in the spaces. Scattered throughout the paren-
chyma are numerous calcareous bodies of various oval shapes,
none larger than .o07 mm. in diameter (Pl. XVI, Fig. 16, ca.).

Musculature. — The longitudinal body muscles are very
prominent ; they are arranged in irregular bundles, from fifty
to sixty in all, which vary in thickness from .o5 mm. to a
fourth that size (Pl. XVI, Fig. 16, Z..).

As the scolex is approached, the bundles become smaller
and the fibers are not so closely compacted, many of them
leaving the bundles and extending separately through the
parenchyma. In the scolex the fibers are no longer arranged
in bundles, but penetrate the parenchyma as scattered fibers.

Within these bundles is another muscle layer. In trans-
verse section that portion of the proglottis which is filled with
sexual organs is seen to be bounded by a thin sheet of wavy
fibers (Pl. XVI, Fig. 16, #.c.). From the sheet single fibers
dip down irregularly and pass between the organs. The ends
of the fibers are coiled like a corkscrew. Other fibers go
outward between the large muscle bundles and take a wavy
irregular course toward the subcuticular cells. In the scolex
these fibers show a radiate arrangement (Pl. XVI, Fig. 3, m.c.),
and transverse sections in this region show the diagonal fibers
crossing at the center.

344 BENEDICT. [VoL. XVI.

The musculature of the suckers consists of three layers.
Beneath the cuticular lining of the suckers is a layer of circular
muscles, precisely as in the proglottids (Pl. XVI, Fig. 3, m.s.).
Within these is the principal layer composed of fibers running
perpendicularly to the surface for a distance of .065 mm. at
the center and gradually decreasing to .04 mm. at the edges.
Prominent nuclei are situated in the median zone of this layer
(Pl. XVI, Fig. 3, mp.). The proximal ends of these fibers are
woven in with a second circular layer of considerable thickness,
but of loosely arranged fibers. A thin, dense, basement mem-
brane covers the inner surface of the sucker.

In the body the partitions between the proglottids are sheets
of muscle fibers, running transversely, with a much smaller
number of dorso-ventral strands.

Nervous System.—In the scolex a thick band of nervous
tissue is arranged in a circle, somewhat anterior to the center
of the head. From a point on the ring directly beneath the
lateral furrow project two branches, one to each adjacent
sucker, so that each sucker receives two trunks, which enter
the lining membrane at two distinct points (Pl. XVI, Fig. 8,
m.r.). The band varies from .o2 mm. in breadth, and .or mm.
thick in one specimen, to a thick mass surrounding the pecul-
iar spherical bodies described hereafter, and having a thickness
nearly twice as great as in the first instance.

From the scolex two longitudinal trunks run down the
lateral margins of the proglottids just within the muscle
bundles (Pl. XVI, Fig. 16, z.). In many transverse sections it
appears as if the nerve sent off at certain places many
branches of various sizes; but until live specimens can be
obtained this cannot be proved. In the end of the last pro-
glottis the nerve trunks bend toward each other and break
into fine spreading fibers.

Excretory System.— Just behind the suckers in the scolex is
a complicated system of rather regularly curving tubes of vary-
ing size and connected by curving cross branches. From the
outer parts of the ducts smaller branches lead to the exterior
and open by excretory pores through the cuticula (Pl. XVI,
Fig. 11, g.ex.). From the scolex eight longitudinal excretory

No. 2.) STRUCTURE OF TWO FISH TAPEWORMS. 345

ducts can be traced through those proglottids in which no
sexual organs are yet developed. One pair lies in the median
frontal plane, two pairs are situated below and outwards, — the
last being close to the cuticula,—and one pair in the dorsal
half of the body, about halfway to the cuticula. Many minute
branches are given off from these which open to the outside
of the body by pores .oo5 mm. in diameter, exactly the same as
the excretory pores of the scolex. The pores which connect
the excretory system with the exterior are simply minute canals
through the cuticula, with no special walls. They do not re-
semble the very prominent pores found by Riggenbach (96) in
P. fossata and Corallobothrium lobosum, being much smaller and
without any muscle fibers. They differ also in being much
more numerous and entirely irregular in position.

Throughout the chain of proglottids the minute excretory
tubes are connected by these pores in the cuticula with the
exterior at various places in each proglottis, being especially
numerous near the posterior end of each.

In the region of sexually immature proglottids a regular
arrangement of the longitudinal ducts cannot be so plainly
traced. The two which lie in the median frontal plane are
the smallest and most regular of all. The others are larger,
with many branchings and connections. A transverse section
through sexually mature proglottids and those posterior shows
a varying number of the sections of the ducts. From larger
longitudinal tubes small branches may be traced which curve
around the muscle bundles, and either terminate in branches
too small to be traced or join some other branch.

Around the cirrus pouch and vaginal opening near the dorsal
surface is a network of rather large ducts arising from longi-
tudinal tubes (Pl. XVI, Fig. 15).

There is no connecting vessel at the posterior end of each
proglottis, connection, as already stated, being accomplished
at various places by irregular branches. This condition resem-
bles the description by Kraemer ('92) of the excretory vessels of
Cyathocephalus truncatus.

The cuticula of the last few proglottids is fairly honeycombed
with excretory pores; especially is this true of the posterior

346 BENEDICT. [Vou. XVI.

concave margin of the last proglottis, which resembles a sieve.
The longitudinal vessels in this proglottis terminate in small
sinuses, which branch and anastomose, much like the ducts in
the scolex, but more irregularly. This network of ducts is
in communication with the exterior by means of a multitude
of pores which are found at this place (Pl. XVI, Fig. 13).

Such a posterior outlet differs from any previously described,
since there is no terminal muscular reservoir. The tendency
of the Proteocephali to the formation of excretory complexes
is very well illustrated by this form.

Directly beneath the apex of the scolex is a sac of cuticular
structure enclosing a small number of circular masses, closely
pressed together (Pl. XVI, Fig. 3, ca.). The masses seem to
be of a calcareous nature, and are penetrated by numerous fine
canals. No connection whatever could be traced between this
sac and any outside system, although the excretory ducts form
a thick network about it. I have found no mention elsewhere
of such a structure, which is different in many respects from
the simple calcareous bodies common among cestodes.

Sexual Organs. — The genital sinus, as before stated, is
irregularly alternate in position, and is situated on the margin
about one-fourth of the length of the proglottis from the ante-
rior end. It resembles in position and relative size that of P.
fossata, P. abscissa, and Corallobothrium lobosum (Riggenbach,
96). In P. torulosa and P. filicollis, as described by Kraemer
('92), no common genital sinus is present, and the genital open-
ings are exactly in the middle of the margin. The vagina
opens directly in front of the male opening, a characteristic of
the Proteocephalt.

The Male Genital Organs.— The testes are from seventy-
five to one hundred in number, arranged irregularly through-
out the space within the longitudinal muscles of the proglottis
(Pl. XVI, Fig. 9, z.). Such a position and number is described
as obtaining in P. torulosa, P. filicollis, P. abscissa, P. fossata,
Corallobothrium lobosum, and many others which have been
less accurately described. When the uterus becomes distended
the testes are crowded to the ventral surface, and the uterus
lies in a broad irregular sheet above them. The testes are

No.2.) STRUCTURE OF TWO FISH TAPEWORHS. 347

crowded at all times enough to give each a polygonal shape.
They vary in size from .o5 to .o65 mm. Each is surrounded
by a very delicate structureless membrane, which is continuous
with the wall of the vas deferens and vasa efferentia. The
testes are the first organs to mature and the first to degenerate.
The spermatozoa are collected by very minute ducts, which take
a general direction towards the center of the proglottis, joining
with others in their course. The larger ducts, which finally
meet, are about .o1 mm. in diameter, and these, after making
more or less intricate coils, unite to form the distended vas def-
erens, which is .o5 mm. in diameter (Pl. XVI, Fig. 9, v.d.). This
is also coiled and twisted, forming a mass a little anterior to the
center of the proglottis, extending to the cirrus sac on one side,
and from the dorsal to the ventral body-walls. The vas deferens,
in addition to the tunica propria, possesses a delicate epithelial
layer. As will be seen by comparison, the mass of the coils of
the vas deferens in this species is much larger and more com-
pact than in any form yet described. At all times in ripe
proglottids the vas deferens is filled with mature spermatozoa
in thick bunches. Just before it enters the cirrus pouch the
duct decreases in size to a diameter of .o15 mm., and the wall
becomes thicker and acquires a layer of longitudinal muscles.

The cirrus pouch is roughly wedge-shaped, with the apex at
its distal end. Its wall is .0035 mm. thick, composed of a
longitudinal and a transverse muscle layer, and lined and cov-
ered with a delicate epithelium. It is intimately connected
with surrounding tissue and never shifts its position. The
wall of the sac extends distally to within .o6 mm. of the body
margin, then turns inward, and the longitudinal muscle layer
is reflected back in the wall of the cirrus. It is in the action
of these reflected muscles that the cirrus pouch aids the pro-
trusion of the cirrus. When the cirrus is protruded the con-
tents of the cirrus sac are fairly pulled away from its walls,
which are never moved.

The vas deferens, where it enters the cirrus pouch, is .o1
mm. in diameter, and has a wall .oo3 mm. thick, with faintly
marked muscle fibers. Just within the pouch it is surrounded
by a cluster of glandular cells. The duct slightly enlarges to

348 BENEDICT. [Vou. XVI.

a diameter of .o12 mm., while the walls gradually increase in
thickness. Its course is a most intricate one, coiling and
twisting, and not confined to any particular part of the pouch
(Pl. XVI, Fig. 17). Its course is not identical in different
proglottids, but all have much the same complicated twisting.
At about the middle of the course of the vas deferens in the
pouch a delicate cuticula, lining the cavity, makes its appear-
ance; the longitudinal muscle fibers have become more promi-
nent, forming a well-defined layer, and circular muscles are first
plainly seen. Proceeding distally, the cuticula constantly but
slowly increases until, at the male genital opening, it is con-
tinuous with the body cuticula, and of equal thickness.

The windings of the vas deferens are invested with a delicate
structureless tissue, which is very elastic. It wraps the coils
of the duct and holds them in a constant relation to each other,
but is connected with the walls of: the pouch only by delicate
filaments.

The distinction between vas deferens and cirrus is simply a
matter of size. The structure of vas deferens within the pouch
in its distal half is as follows (Pl. XVI, Figs. 18 and 20): lining
the lumen, which is about .o15 mm. in diameter, is the cuticula,
.002 mm. thick, and quite regularly cast into folds, like the bel-
lows of a camera (Pl. XVI, Fig. 20, cz.). Without the cuticula
is a very minute layer of transverse muscle fibers, which follows
exactly the contour of the transverse folds (Pl. XVI, Fig. 20,
m.s.). The longitudinal muscle layer, which lies next, is of
unequal thickness, dipping down into the folds (Pl. XVI, Fig.
20, mp.). Above the muscle layers comes a loose layer, con-
taining pyriform glandular cells, with narrow processes pro-
jecting down through the muscle layers, beyond which they
could not be traced (Pl. XVI, Fig. 20, c/.g/.). Surrounding the
entire duct is a delicate single layer of flattened epithelial cells
with prominent nuclei (Pl. XVI, Fig. 20, ¢.).

The last coil of the vas deferens brings its distal end back to
the proximal end of the pouch near the ventral side. .A sudden
enlargement to .o7 mm. in long diameter then takes place, and
the duct bends upward and outward (Pl. XVI, Fig. 17). The
cirrus begins at the sudden enlargement, which is slightly bulb-

No. 2:] STRUCTURE OF TWO FISH TAPEWORMS. 349

shaped. The size increases very abruptly to .2 mm., then grad-
ually narrows down to the genital opening, roughly conforming
to the shape of the pouch (Pl. XVI, Fig. 17). At the largest
part the cirrus in transverse section is .2 mm. in long diameter
and .075 mm. in short diameter, being of elliptical shape. The
cuticula is .005 mm. thick, and its folds are large and rounded.
The circular muscle layer is .oo2 mm. thick, the longitudinal
muscle layer is .0075 mm. thick, and where it dips into the
folds increases to .o15 mm. The layer of tissue next to the
muscles is .02 mm. thick, and around this is the delicate epi-
thelium. At the distal end of the cirrus the longitudinal
muscle fibers reach nearly to the body cuticula and are inserted
in the parenchyma. ‘The greater part of these fibers, however,
are reflected back in the longitudinal muscles of the pouch.
That part of the cirrus which extends from the distal end of
the pouch to the genital pore is a simple hollow stalk with
very thick cuticula, and surrounded by the two muscle layers.
The cirrus, when protruded, is cylindrical in section and longer
than a proglottis (Pl. XVI, Fig. 4). In a transverse section of
the extended cirrus, proceeding from without inward, the follow-
ing layers will be found: a thick cuticula, circular and longitu-
dinal muscles, a layer of gland-like cells, then a space, then an
epithelial layer, glandular cells, longitudinal muscles, circular
muscles, and finally the lining cuticula. By comparing this
with a section of the retracted cirrus and with the vas def-
erens, it will be seen that the cirrus is evaginated and that
the end which was proximal is now distal; while from the distal
end runs back within the cirrus the vas deferens, with its layers
in the normal position. By the action of the circular muscles
the flat cirrus has become cylindrical, and the pull of the longi-
tudinal muscles has turned it wrong side out. That portion of
the cirrus which penetrated the body-wall is now seen as a
muscular stalk (Pl. XVI, Fig. 5).

A cirrus somewhat resembling this has been described in
Diplobothrium simile by Lonnberg ('92), being simply an invagi-
nation of the external cuticula, encircled by its muscle coats
and connected with the vas deferens at its proximal end.
Riggenbach ('96) describes in P. fossata a cirrus which closely

350 BENEDICT. [VoL. XVI.

resembles that of P. ambloplitzs, the chief difference being in
the more delicate structure, and the lesser number of coils of
the vas deferens within the pouch.

Female Generative Organs.— The opening of the vagina is
directly anterior to the male opening and is of variable size.
The duct passes directly inwards for about .05 mm., and then
enlarges to twice the size of the opening. The lumen remains
constant in diameter for about .35 mm., during which distance
the tube curves anteriorly, and then posteriorly again, taking a
course which varies from a nearly straight one to a semicircle,
but which always ends at about the same level as the opening.
Throughout the region just described the vagina possesses an
extraordinarily developed sphincter muscle, which is .03 mm.
in greatest thickness. This muscle is very compact and must
possess great strength. It is by far the most powerful sphincter
about the vagina yet described in any cestode. The absence
of any hooks and the shape of the cirrus render this structure
an important one in most of the Proteocephali. This sphinc-
ter makes the terminal region of the vagina nearly as large
and prominent in sections as the cirrus pouch (Pl. XVI, Fig.
18, va.).

On emerging from the sphincter muscle the cavity of the
vagina enlarges suddenly, forming an elongated chamber with
convoluted walls, which is directly downward and backward,
narrowing as it nears the median body line (Pl. XVI, Fig. 9,
va.). From the opening to the end of this enlargement the
vagina is lined by a continuation of the body cuticula, which
becomes very thin in the chamber, and there bears delicate
cilia (PI. XVI, Fig. 18). Enclosing the sphincter is a layer of
loose glandular cells resembling those of the cirrus (Pl. XVI,
Fig. 19, ms. and cl.gl.). Outside of these cells is a delicate
epithelial layer (Pl. XVI, Fig. 19, ¢f.)._ The chamber with con-
voluted walls is undoubtedly for receiving at the time of copu-
lation a mass of spermatozoa, which may be passed on as the
capacity of the remaining vaginal duct will permit. It is impos-
sible to trace the transition from the lining cuticula to the free
epithelial layer which lines that portion of the vagina extending
from the chamber to the oviduct. This portion of the vagina

No. 2.] STRUCTURE OF TWO FISH TAPEWORMS. 351

passes down the median line of the proglottis near the ventral
side, forming a number of turns and coils (Pl. XVI, Fig. 9). The
inner portion is slightly larger in diameter. The mass of the
coils is just anterior to the ovaries, differing in this respect from
other described species, in all of which the coils are further pos-
terior. The end of this portion of the vagina is marked by a
minute receptaculum seminis (Pl. XVI, Fig. 10, ~sem.). From
thisregion the vagina continues as a heavy-walled duct of
decreased diameter, supplied with a layer of gland cells, and
more heavily ciliated than before. This heavy-walled portion
of the vagina by a series of curves reaches nearly to the poste-
rior end of the proglottis, and there unites with the oviduct (PI.
XVI, Fig. 10). Further investigation will probably show that
in Proteocephalus the vagina usually opens into the oviduct and
not into the odtype. Such is undoubtedly the case in P. fii-
collis, although Kraemer ('92) describes the vagina as entering
the odtype. VP. abscissa and P. fossata Riggenbach ('96) both
show the former condition.

The presence of spermatozoa can be plainly seen in most
specimens; in some cases the vaginal chamber near the
sphincter will be packed with them; other specimens show
this portion with walls collapsed and lumen empty, while the
coils near the ovary are distended.

The ovary lies in the posterior end of the proglottis, close to
the ventral surface. It is a bi-lobed organ, each half extending
from the median line, where they are joined together, to the
vitellarian glands on the side. Each half is somewhat retort-
shaped, with the small end inward, whether viewed in frontal
or transverse section (Pl. XVI, Fig. 9, 0.).

The ovary is surrounded by a delicate epithelial layer; the
ova are closely packed together, those at the extreme outer end
being clearly less developed than those toward the center. The
ova are .OI mm. in diameter, with a nucleus measuring .005
mm., and containing a very prominent nucleolus. The ovary
is penetrated by a few delicate sheets of tissue, which divide
the eggs into irregular groups, extending from dorsal to ventral
surface, but all open at the inner end, thus leaving the passage
free into the oviduct. The two lateral wings of the ovary are

age BENEDICT. [VoL. XVI.

joined at their inner extremities by a portion of varying size,
which is antero-dorsal to the rest of the ovary. From this
common portion projects a muscular organ, the odcapt,! com-
monly known as the “ Schluckapparat”’ of the German investi-
gators, which marks the beginning of the oviduct (Pl. XVI,
Fig. 6). This organ is of oval shape, .025 mm. wide, and
.03 mm. long. It is composed of a layer of circular muscles,
.008 mm. thick, surrounding a lumen, .0075 mm. in diameter,
which is lined by cubical epithelial cells. The contractions of
this organ force the eggs forward into the empty oviduct, as the
diameter of the outer end of the lumen is .002 mm. greater than
that next to the ovary. About the circular muscles is a coat
of fibrous tissue, .007 mm. thick, in which, at fairly regular
distances, are long, spindle-shaped glandular cells, with narrow
processes running toward the lumen.

The odcapt has been described in P. filicollis by Kraemer
(92), in P. coryphicephala by Monticelli, in P. longicollis by von
Linstow (91), and in Corallobothrium lobosum by Riggenbach
(96). The oviduct emerging from the odcapt is .015 mm. in
diameter. It is lined by cubical epithelial, ciliated cells, and
surrounded by loose tissue containing gland-like cells.

The course of the oviduct differs from that of species hitherto
described in that it never passes directly backward into the
ootype, but always passes toward the posterior margin of the
proglottis, and then curves forward into the odtype (Pl. XVI,
Fig. 10, od.). Starting from the odcapt it makes a wide curve
laterally, then turns to the ventral side close to the margin of
one of the ovarial lobes, thence follows a wide sweep towards
the opposite lobe of ovary, from whence it curves forward and

1 To my knowledge there exists in English no name for this structure which is
of such importance and general occurrence in the cestodes. Some authors have
recently endeavored to transfer the German name directly into our scientific termi-
nology. The unsatisfactory character of such usage needs no demonstration here.
I am accustomed to use the form given in the text to designate this organ. The
name is derived from wov, egg, and xdmrws, the gulper or swallower, being thus
analogous to the German term, and of similar formation to the designation
ootype, used for an associated organ. Since the name has never appeared in
print, this formal statement of its derivation and meaning seemed advisable.

HENRY B. WARD.

No. 2.] SZTRUCTURE OF TWO FISH TAPEWORMS. 353

upward, passing into the odtype about the center of that space.
Its course is never exactly the same in different proglottids.
Throughout its course the oviduct is supplied with gland-like
cells, enclosed in a single-layered epithelium and lined by a
layer of ciliated epithelial cells (Pl. XVI, Fig. 6). The oviduct
is easily distinguished from other ducts in this region by reason
of its greater size, darker staining, and more regular course
(Pl. XVI, Fig. 16, od.). The diameter remains about the same
throughout. The ova, as they pass down the oviduct, are fer-
tilized by the spermatozoa from the vagina before they reach
the odtype.

The o6type is a direct continuation of the oviduct (Pl. XVI,
Fig. 12, o¢.andod.). It possesses a circular muscle layer, and for
its entire length is surrounded by the shell gland. Its position
is about the center of the interovarial space. It receives the
common vitellarian duct at its beginning, and the secretion of
the surrounding shell gland throughout its course, resembling
precisely the odtype of P. torulosa and P. filicollis (Kraemer, ’92).
It has exactly the same structure, with the addition of a mus-
cular coat, as has the oviduct, even to being provided with the
gland cells, which are embedded in the shell gland (Pl. XVI,
Fig. 12). Its entire length is only .06 mm., and its diameter
.o15 mm. Its walls are no thicker than those of the oviduct,
the cubical epithelial cells not being so large, and this decrease
being equalized by the muscle layer. The odtype opens directly
into the uterus, there being here no intermediate duct, such as
is commonly described in other species. It is possible that one
of the coils of the thick-walled portion of the vagina has been
mistaken in some cases, at least, for the duct leading to the
uterus. There can be no doubt in P. ambloplhizs that the uterus
extends directly forward from the odtype, yet many sections
seem to show such a duct, which, on careful reconstruction,
always proves to be nothing more than one of the coils of the
vagina. The walls of the uterus are very delicate, of a fibrous
texture, and the lumen at its beginning is .o5 mm. in diameter.
It runs directly anteriad to just in front of the ovary, where it
turns back and proceeds in a dorso-posterior direction to near
the posterior end of the proglottis. It then makes a turn toward

354 BENEDICT. [Vor. XVI.

the anterior, and spreads out in all directions, increasing in size
with the development of the proglottis. The uterus spreads
first over the dorsal portion of the space, which contains the
sexual organs, and when fully developed occupies nearly this
entire space, with the exception of the rudiments of other
sexual organs. The branches of the uterus are few, and vari-
able in size and form. The tissue remaining is in the form of
thin partitions, which stand out perpendicularly from the side.

The shell gland is more compact than that of most of the
Proteocephali, and extends equally on all sides of the odtype.
It appears as an irregular granular mass, with scattered nuclei
distinguishable within it, and large glandular cells grouped
irregularly in the outer portion. No trace of collecting tubules
can be distinguished, and the fibrous appearance indicates a
process to the odtype from each cell. This shell gland is much
larger and more compact than that described in any other species
of the genus (Pl. XVI, Fig. 12).

The vitellarian ducts occupy the lateral margins of the pro-
glottids, just within the longitudinal muscles and partly encir-
cling the nerve (Pl. XVI, Fig. 9, vt.g/.). Two large ducts run
from the anterior portion toward the posterior end of the pro-
glottis. Numerous branches of various sizes join them in their
course. About the level of the anterior margin of the ovary
the main duct on each side curves inward and passes along the
dorsal wall until it is over the odtype; here the two unite to
form the first vitellarian receptacle (Pl. XVI, Fig. 10, v4.7). Such
a union of the yolk ducts from the two sides seems to be a
characteristic of the genus Proteocephalus. Riggenbach ('96)
describes such a condition in the forms he has studied, and
says that only in P. filicollis do the ducts enter the odtype
separately. PP. filicollis, however, is mot an exception, as my
own work has proved that the ducts unite exactly as in
P. ambloplitis (see p. 364).

There are many variations in size, and some in the number
of these ducts; there may be two from one side and only one
from the other, or those from one side may be very large, while
the others are narrow. The yolk cells are spherical masses
containing small, densely staining globules.

No: 2, S7ROCLTORE OF TWO\FTSH) TAPEWORMS, 355

From the first vitellarian receptacle a duct runs ventrally
and expands into a prominent second yolk reservoir (Pl. XVI,
Fig. 12, vt.7.), which is situated close to the beginning of the
oétype. From this reservoir, which is larger than the first, a
narrow, heavy-walled duct leads into the odtype.

As the proglottis nears maturity the dermo-muscular sac
begins to get thinner for a short distance in the mid-dorsal
line. As the uterus becomes distended with eggs, it pushes
up into this portion. With the further growth of the uterus
and development of the eggs, the body-wall at this point be-
comes thinner and thinner, until it breaks apart and a cleft
is formed. Then the pressure on the wall of the uterus at this
point causes its rupture and the eggs escape.

Proteocephalus filicollis Rud.

The early investigators, Rudolphi ('10), Dujardin (45), Diesing
('50), and von Linstow ('78), all described Zaenia filicollis and
Taenia ocellata as distinct species, as also did Zschokke ('84),
The last author, however, added something of the anatomical
structure to the details of size and external form given by the
older writers. Kraemer (92) was the first to study by the sec-
tion method these two forms, making an exhaustive anatomical
and histological investigation, which showed 7. filicollis and
T. ocellata to be identical. As 7. filicollis has the precedence
in Rudolphi’s synopsis, this name should always apply. Rig-
genbach ('96) unaccountably states that oce//ata has the prece-
dence, although 7. ji/zcol/is is described in Rudolphi’s work ('10)
on p. 106, and 7. ocellata on p. 108.

In P. filicollis Kraemer established the presence of a nervous
mass in the scolex, and two longitudinal nerve trunks through
the body. The excretory system was shown to consist of a net-
work of ducts in the scolex and neck, and four longitudinal
vessels through the proglottids. He demonstrated the similar-
ity of the cuticula with that of other cestodes. The vagina was
shown to open in front of the male opening, and to possess at
its distal end a sphincter muscle. He established the presence
of a bi-lobed ovary in the posterior end of the proglottis and

350 BENEDICT. [VoL. XVI.

of an oviduct leading from it to an odtype —a duct with a mus-
cular coat. The lateral position of the vitellaria, the elongated
acinous structure, and the ducts which proceeded from them
to the odtype were described. The uterus was shown to be
a thin-walled sac with lateral branches in the median line of the
proglottis. A shell gland was demonstrated surrounding the
odtype. He pointed out the number and scattered position of
the testes, and the ducts which collected the spermatozoa. He
also showed that these ducts unite to form the vas deferens,
which is coiled in the center of the proglottis, and functions as
a seminal vesicle. Finally he described the termination of the
vas deferens in a muscular cirrus, enclosed in a well-developed
cirrus pouch. These characteristics show it to be a typical
species of Proteocephalus.

The specimens which I received from Dr. Ward’s collection
were in four lots, two of these having been taken from Cove-
gonus nigripinnis, one from C. prognathus, and one from C. artedi.
They were obtained by Dr. Ward while in charge of a bio-
logical examination of Lake Michigan, under the auspices of
the Michigan Fish Commission, during the summer of 1894.
All of the fish were caught in the Traverse Bay region, near
Charlevoix, Mich. The cestodes were found in the stomach
of C. artedi, and of one specimen of C. xigripimnis, and in
the intestine of C. prognathus and of the other specimen of
C. nigripinnis.

General External Morphology.

In external form those from C. artedi and C. prognathus were
alike (Pl. XVI, Fig. 21), while those taken from C. xigripinnis
were longer than the first, and the neck was not only much
longer, but also slenderer (Pl. XVI, Fig. 22). In the shorter
specimens the length varied from 12 mm. to 16 mm., while the
breadth at the widest point was .8 mm., and the neck was about
two-fifths the total length of the body. In the other form the
length varied from 15 mm. to 25 mm., the breadth was .8 mm.,
while the neck ranged from three-fifths to four-fifths of the
entire body length. In one of the two lots which came from
C. nigripinnis was a specimen very much larger than the others.

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No. 2.] STRUCTURE OF TWO FISH TAPEWORMS. 357

Its total length was 38 mm., the length of the neck was 7 mm.,
but the body was no broader than the others. This specimen
corresponds more closely to Kraemer’s description than the
others, both as to total length of body and as to the compara-
tive length of the neck. The fact that the above forms are
identical in all other respects shows how little importance can
be attached to size alone as a means of identification.!

The scolex is .12 mm. in diameter, being of a somewhat
globular shape. It resembles, under low magnification, a
rounded, slightly swollen end of the neck. The four suckers
are .o4 mm. in diameter, directed outward and upward, and
situated a little above the equatorial plane of the scolex
(Pl. XVI, Fig. 24).

The fifth sucker is in every respect a true sucker, since it
has the same structure and musculature that the four large
suckers possess. The whole region of the scolex anterior to
the suckers is capable of considerable motion. Some specimens
show the whole region retracted until it is “‘dished”’ below the
anterior margin of the suckers. In another it may be very
prominent, with the opening hardly visible. The scolex is flat-
tened dorso-ventrally, and slight furrows separate the suckers.

The neck is .10 mm, to .12 mm. broad at its anterior extrem-
ity, and varies from 5 mm. to 10 mm. in length. The first
proglottis is very faintly marked off from the neck, and in most
cases is longer than wide ; in a few specimens, however, the first
proglottis is nearly square. This proglottis varies in breadth
from .12 mm. to .20 mm., and in length from .12 mm. to.16 mm.
The total number of proglottids ranges from nine to twenty,
though the single very large specimen spoken of had forty.
The sexually mature proglottids come about the middle of the
chain. These vary in number from four to seven, and are not

1 The many differences which I found between P. fi/icol/is, as described by
Kraemer, and my own specimens led me to doubt the accuracy of their identifica-
tion. Through the kindness of Professor Zschokke, Dr. Ward obtained some
specimens of the true P. //icollis, which he permitted me to examine. Unfortu-
nately, however, during the passage the alcohol had escaped from the vials, and
the specimens arrived in a dry state. The most careful treatment only partially
restored them, but an investigation convinced me that they resembled my own
specimens too closely to justify me in founding a new species.

358 BENEDICT. [Vou. XVI.

identical in shape in different individuals, varying in form from
nearly square to an oblong in which the length is twice the
breadth. The greatest breadth of the body comes in the next
to the last proglottis. The posterior proglottis makes a gradual
taper toward the posterior end, which is smoothly rounded.
The excretory pore can be seen at the tip as a cup-shaped
indentation.

Anatomical and Histological Structure.

Cuticula. — The cuticula is very delicate, .0035 mm. in thick-
ness; the external layer presents a very rough appearance (PI.
XVI, Fig. 29, cz’.). It takes stain more deeply than the other
layer. There is no smooth outline on the exterior, as drawn
by Kraemer. What he pictures asa homogeneous layer, pene-
trated by perpendicular canals, is in reality a ragged, exceedingly
irregular layer, with clefts and breaks reaching down to the
next layer. Its appearance suggests the sloughing off of the
cuticula. The layer of cuticula beneath this is .oo2 mm. thick,
and stains lightly (Pl. XVI, Fig. 29, cz.).

Beneath the cuticula is a layer of circular muscles .oO1 mm.
thick, and within these is a layer of longitudinal muscles, which
in cross-sections shows as a row of dots between the outer ends
of the cell ‘processes: of ‘the next layer (Pl: XVI, Fig-)20;
m.l.ct. and mt.c.ct.).

The subcuticular cell layer is very prominent, having a thick-
ness of 1025 mm.) (Pl. XVI) Fig. \20,' sd:c.): ) The fells vare
narrow, fusiform, with long delicate processes on the distal
ends. The subcuticular cell layer in P. f/zcollts is much more
prominent than in P. ambloplitis. In transverse sections the
cells form a band, over half the thickness of the dermo-mus-
cular sac in P. filicollis, while in P. ambloplitis the band only
constitutes one-fifth of the thickness of the dermo-muscular
sac (Pl. XVI) Figs, 16jand, 23, s0.2.)-

The parenchyma is very open and loose in texture, looking
like a coarse, irregular meshwork. Muscular fibers are scat-
tered throughout it, extending in a transverse direction. In the
area enclosed by the longitudinal body muscles the parenchyma

No. 2.] STRUCTURE OF TWO FISH TAPEWORMS. 359

presents the appearance of strands interwoven, being still more
open in texture than the outer parenchyma.

Calcareous bodies are not abundant and are very small, none
being longer than .oo5 mm.

Nervous System.— The central nervous system consists of a
ring a little anterior to the middle of the scolex; a large short
trunk runs from the median dorsal, the median ventral, and
two lateral points of the ring about half the distance to the
cuticula, where it divides and half goes to each adjacent sucker
(Pl. XVI, Fig. 25, 27. and w.). From the central nerve ring
two longitudinal trunks pass down the body just within the
longitudinal muscle bundles at the sides. The nervous sys-
tems of P. filicollis and P. ambloplitis are thus almost exactly
identical.

Excretory System.— There are four regular longitudinal canals
traversing the body. The walls of these ducts are much thicker
than in P. ambloplitis, and the course of the duct is more regu-
lar, with fewer branches. Branches of various sizes are given
off at irregular intervals and take a more or less tortuous
course among the organs. The branches which extend toward
the cuticula are straighter, give off fewer branchlets, finally be-
coming lost in the subcuticular layer. In some cases the ducts
seem simply to unite with parenchyma spaces and can be
traced no further. In the cuticula, at rare intervals, an open-
ing may be found through which the excretory system connects
with the exterior. These openings are neither so large, so
numerous, nor so well defined as in P. ambloplitis. In the scolex
and anterior portion of the neck the four vessels are much
branched, the branches forming a network much as in the latter
species. Yet the network so formed is not so complex as in
P. torulosa.

Musculature. —The musculature is not so strongly developed
as in P. ambloplitis. The longitudinal muscles are arranged in
numerous small bundles, appearing in transverse sections of
proglottids as irregular rows of dots parallel to the outline of
the section (Pl. XVI, Fig. 23, m.¢.). The sections of the bun-
dles are of irregular shape and vary in breadth from .oo15 mm.
to .004 mm.

360 BENEDICT. [Vo. XVI.

A loose sheet of circular muscle fibers weaves around the
longitudinal bundles. Large fibers pass in a transverse direc-
tion between these muscle sheets. The divisions between the
proglottids are formed by the interlacing of these fibers with
similar ones which cross them at right angles, both sets being
here much more complicated than in other regions.

In the scolex the longitudinal muscles are represented by
scattered fibers, not arranged in compact bundles, passing ante-
riorly between the suckers, a few extending to the anterior part
of the scolex. Other fibers, arranged in a radial direction, ex-
tend between the suckers, and are connected with the muscula-
ture of these organs.

Sexual Organs.— A common genital sinus is present, situated
a little anterior to the middle of the margin of the proglottis.
Its position is irregularly alternate in the chain of proglottids.

Male Organs. — The testes are from thirty-five to fifty in
number, and about .05 mm. in diameter. They are scattered
throughout the space between the longitudinal muscles and the
vitellaria. Each testis is closely invested by the tunica propria.
Minute ducts collect the spermatozoa and join together, making
larger ducts near the center of the proglottis. These larger
ducts unite to form a dilated thin-walled vas deferens .o2 mm.
in diameter, which forms a convoluted mass of coils in the
middle of the proglottis (Pl. XVI, Fig. 27, vd.). The wall of
the vas deferens consists of the tunica propria and an epithe-
lial covering, while the coils are closely bound together by
parenchymal strands. The vas deferens is always distended
with spermatozoa.

The distal end of the duct passes upward to the dorsal muscle
bundles, and there enters the cirrus sac. At a distance of
.10 mm. from its entrance to the cirrus it undergoes a sudden
diminution in size and receives a delicate muscular coat as well
as an epithelial lining. At the point where the vas deferens
enters the cirrus sac it is only .o15 mm. in diameter, with
walls .oo5 mm. thick, the greater part of which is a muscular
coat. The duct bends sharply as it enters the cirrus pouch.

The cirrus sac is an elongated oval in shape, slightly con-
stricted in the middle. It extends from the edges of the geni-

No. 2.] STRUCTURE OF TWO FISH TAPEWORMS. 361

tal pore to the median dorsal line of the inner parenchymal
space, where a number of heavy muscle strands attach the
proximal end of the inner tube of the pouch firmly to the
dermo-muscular sac. The walls are thickest about the middle
of its length, and consist of a circular and transverse muscle
layer, with a delicate epithelium covering the outer and inner
surface (Pl. XVI, Fig. 34).

At the anterior end the wall of the pouch bends back, form-
ing a tube through the middle of the sac (Pl. XVI, Fig. 31).
This tube is lined, in the distal half, by cuticula, which is con-
tinuous with that covering the surface of the body. At the
posterior end no cuticula can be distinguished, and the muscle
layer is very thick. The space between the inner and outer
walls of the cirrus sac is filled with fibrous tissue, with many
nuclei (Fig. 31). The fibers run from wall to wall in transverse
direction toward the proximal end, while near the distal portion
the fibers are curved, with the concave portion directed proxi-
mally. At the posterior end of the inner tube the thick muscle
layer sends out large strands, which pass out of the proximal
end of the cirrus pouch and join the dermo-muscular sac, as
before stated.

About the tube, within the sac, are many gland cells, pro-
jecting perpendicularly from its walls, and most numerous in
its proximal half.

The cirrus is straight, cylindrical, and of approximately equal
diameter throughout, with the exception of the distal termina-
tion (Fig. 31, cz.) This portion is a muscular knob .o2 mm.
in greatest diameter, composed principally of circular muscles.
In its normal position this terminal portion rests in the end of
the inner sac tube, like a ball on a cup. The sides of the dis-
tal fourth of the cirrus are not in direct contact with the cuti-
cula lining the inner tube, and throughout this distance the
wall of the tube is quite thick, .co5 mm., and the cuticula is
covered with fine, stiff, bristle-like projections. The cirrus has
a lumen of .or mm., which is lined by a thin epithelial layer.
The organ here presents a very peculiar appearance, due to the
circular muscles which compose its walls, and which are here
.004 mm. thick. Throughout most of its course the cirrus is

362 BENEDICT. [VoL. XVI.

very closely joined to the walls of the inner tube, and it seems
probable that it does not move through the tube at all.
Kraemer’s description is widely different. His drawing shows
a slender tube with hooks, representing the cirrus, and followed
by a proglottis-like arrangement, and then several coils of the
vas deferens within the pouch. The drawing has an unreal
appearance on the first glance. The cirrus, with its curved
hooks, is imbedded in the tissue, which must be torn through
before it could be protruded. The proglottis-like appearance
of a portion of the cirrus, as he drew it, was undoubtedly due
to the way in which the circular muscles were cut in sectioning
(PLOXOVI, Fig. 31; 92.8.) He \drew the jeavity) of the jciaus:
into which the cut ends of the circular muscles projected, as
the external outline of the cirrus. The coils would be neces-
sary, according to his theory, but are not to be found. He
says that the muscles, which he calls “the roots,” are for
retracting the pouch, which is sometimes thrust outwards for
some distance through the opening. This protrusion is really
due to an evagination of the free distal end of the inner tube.
When the cirrus is entirely protruded it extends a length of
.10 mm. from the male opening. The distal end is the muscu-
lar knob at the end of the free part of the cirrus, which is
.035 mm. long. At the proximal end this free cirrus enters a
muscular protuberance of the shape of a truncated cone, whose
exterior is covered by minute, bristle-like structures. The
cirrus passes through the center of this and back through the
pouch in very intimate connection with the walls of the inner
tube of the sac. The mechanism of the protrusion seems to
be as follows: The longitudinal muscles of the cirrus sac, which
are reflected at the distal end of the pouch into the wall of the
inner tube, by contraction, would evaginate that distal portion
of the tube which is free from the cirrus, and pull the rest of
the inner tube outward through the sac, stretching the “roots”
(Pl. XVI, Fig. 32). Because of the close connection between
the walls of the cirrus and those of the inner tube, the latter
would carry out with it the cirrus (Fig. 32). The evaginated
distal portion of the tube forms the muscular protuberance, the
former rough cuticular lining of which is now on the outside.

No. 2:] STRUCTURE OF TWO FISH TAPEWORMS. 363

Retraction would be accomplished principally by the roots,
which, as has been seen, are attached to the proximal end of
the inner tube. The vas deferens for a short distance without
the cirrus sac is of the same size as the cirrus itself, and could
be drawn into the pouch, thus providing for the extension.

Female Generative Organs.— The vaginal opening is .008 mm.
in diameter. About .oo5 mm. back of the opening, a sphincter
muscle is found, .oo8 mm. in thickness and hemispherical in
cross-section. The vagina then bends backward and inward,
passing under the middle of the cirrus sac. It is of nearly
equal size throughout, and is lined by ciliated epithelial cells.
Outside of this is a thin, circular muscle layer bounded by deli-
cate epithelium. The whole vagina, toward its distal extremity,
is surrounded by a very prominent layer of glandular cells, with
long processes extending toward the duct. The vagina follows
the median line of the proglottis, close to the ventral body mus-
cles. Its course continues straight until it passes under the
ovaries, where it forms a number of coils ; from there passing
to the posterior end of the proglottis, and opening into the ovi-
duct (at od., Pl. XVI, Fig. 28). Kraemer (92) wrongly states
that it opens into the odtype.

The ovary is a bi-lobed organ, composed of two retort-shaped
parts, connected in the median line by a narrow portion, corre-
sponding to the neck of the retort (Fig. 28, 0.). Each half of
the ovary is covered by a delicate membrane, composed of a
single layer of cells. The ovary is closely packed with ova,
which are .o1 mm. in diameter when they are ready to leave
the organ. The germinal vesicle is very large, measuring
.0075 mm. in the mature ova, with a prominent nucleolus
.005 mm. in diameter. The eggs which are found in the ute-
rus have adiameter of .o2 mm. This last measurement agrees
with that of Kraemer, but it will be noticed that the immature
ova in this specimen seem to be nearly twice the size he
describes. This difference might be accounted for by his over-
looking the delicate protoplasmic mass, which forms the outer
portion of an ovum. The ova pass from the ovary into the
odcapt, which is .025 mm. long and .o2 mm. in diameter. It
is lined by cubical epithelium, which is surrounded by a muscu-

364 BENEDICT. [Vor. XVI.

lar band, oval in section (Pl. XVI, Fig. 35). This organ in
P. filicollis resembles closely that of P. ambloplitis.

The oviduct is lined by cubical, thickly ciliated cells. Around
this layer is a thin stratum of what appear to be muscle ele-
ments running transversely ; then comes an epithelial covering,
and outside of this are-scattered gland cells. The course of
the oviduct depends somewhat on the direction of the odcapt,
requiring another curve if this organ is directed anteriad; but
in all cases the oviduct passes in slightly curving lines nearly
to the posterior margin of the proglottis. Here it may make
a coil or two, or bend directly forward again to the center of
the inter-ovarial space. The vagina usually enters the oviduct
near the posterior margin of the proglottis, although the union
is not at exactly the same place in different proglottids.

The odtype seems to be simply a specialized portion of the
oviduct. The muscle layer is a little thicker; the cilia are very
small, or entirely lacking; and the surrounding gland cells are
more numerous. The organ is .0125 mm. long, and for the
entire distance is surrounded by the shell gland, which in this
form is exceedingly irregular in shape. Around the odtype is
a dense portion with a striated appearance, as if composed
mainly of cell processes (Pl. XVI, Fig. 30, sh.gl.). Long
strands extend outward from here, leading to bunches, or
masses, of cells of irregular shape. There is, moreover, no
similarity in the form of the shell gland in different pro-
glottids.

The vitellaria are found on the lateral margins of the space
within the dermo-muscular sac. Each is formed of a loose
mass of vesicles, joined to a central longitudinal duct by fine
transverse branches (Pl. XVI, Fig. 27, vt.gl.). The central
ducts at the base turn directly inward, and meet a little ante-
rior to and above the odtype. At their junction a small vitella-
rian reservoir is formed of variable size. From this reservoir
a narrow duct leads downwards and backwards to a larger
spherical reservoir, lying just above the odtype, into which it
empties by a very narrow duct (Pl. XVI, Fig. 30, vt.7.).

The uterus extends as a delicate walled tube anteriorly from
the end of the odtype. It passes forward under the connecting

No. 2.] SZRUCTURE OF TWO FISH TAPEWORMS. 365

portion of the ovaries, then turns upward and spreads out ina
broad sheet, close to the longitudinal muscles above the testes.
Lateral outpocketings are formed as the proglottis becomes
more mature, and these branches grow larger until the uterus
in the posterior proglottids nearly fills the entire space within
the dermo-muscular sac (Pl. XVI, Fig. 27, wz.).

The ova escape by a cleft in the mid-dorsal wall of the pro-
glottids. The formation of the cleft is very gradual and seems
to result from a gradual thinning of the dermo-muscular sac at
that place.

In the relations of glands and ducts P. filicollis and P. amblo-
plitis are remarkably similar. .

366 BENEDICT. [VoL. XVI.

‘61

"50
45

92

87

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89

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92

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94

58

84

88

WORKS ClrED,

VAN BENEDEN, P. J. Mémoire sur les Vers Intestinaux. Supplement
aux Compt. Rend, Acad. Sci. Tomeii. 4to, 376 pp., 27 pls. Paris.

DiesinG, C. M. SystemaHelminthum. 2vols.,680and 591 pp. Vienna.

DvuJARDIN, F. Histoire Naturelle des Helminths ou Vers Intestinaux.
654 pp., 12 pls. Paris.

KRAEMER, A. Beitrage zur Anatomie und Histologie der Cestoden
der Siisswasserfische. Zeztschr. f. wiss. Zool. Bd. liii, pp. 647-
722, FIs, XXVIII) XXVIII.

LEIpy, J. Notice of some Parasitic Worms. Proc. of the Acad. of
Nat. Sci. of Philadelphia. Pp. 20-24.

von Linstow, O. Compendium der Helminthologie. Ein Verzeich-
niss der bekannten Helminthen. 382 pp. Hannover.

von Linstow, O. Compendium der Helminthologie. Nachtrag.
I51 pp. Hannover.

von Linstow, O. Ueber den Bau und die Entwicklung von Taenia
longicollis Rud. /Jenaische Zeitschr. f. Naturwiss. Bd. xxv, N.F.
xviii. Pp. 565-576, 1 pl. 1891.

LONNBERG, E. Anatomische Studien tiber skandinavischer Cestoden.
Zwei Parasiten aus Walfischen und zweiaus Lamna cornubica. Kgl.
Svens. Vet.-Akad. Handl. Bd. xxiv, pp. 1-28, 1 pl. Stockholm.

LONNBERG, E. Ueber eine neue Tetrabothriumspecies und die Ver-
wandschaftsverhaltnisse der Ichthyotaenien. Centralbl. f. Bakt. u.
Parasttenkunde. Vol. xv, No. 21.

RIGGENBACH, E. Das Genus Ichthyotaenia. Inaugural Dissertation,
Geneva. Revue Suisse de Zool. Bd.iv. Pp. 165-276, 3 pls.

RvuDOLPHI, C. A. Entozoorum sive Vermium Intestinalium Historia
Naturalis. 3 vols. Vol. iii, pp. 106-108. Amsterdam.

Warp, H. B. A Preliminary Report on the Worms (mostly parasitic)
collected in Lake St. Clair in the Summer of 1893. Bull. of Michi-
gan Fish Commission. No. 4, pp. 49.

WEINLAND, D. F. Human Cestoides. An essay on the Tapeworms of
Man, to which is added an appendix, etc. 93 pp., 12 figs. Met-
calf & Co., Cambridge, Mass.

ZSCHOKKE, F. Recherches sur lorganization et la distribution des
vers parasites des poissons d’eau douce. Archives de Biologie.
Vol. v., pp. 153-241, 2 pls.

ZSCHOKKE, F. Recherches sur la structure anatomique et histologique
des Cestodes. 396 pp.,9 pls. Geneva.

No. 2.] STRUCTURE OF TWO FISH TAPEWORMS. 367

EXPLANATION OF PLATE XVI.

The figures are drawn at the scale indicated and, unless otherwise stated, with
the Abbé camera lucida. Sections from which drawings were made were stained
with haematoxylin.

REFERENCE LETTERS.

ca. calcareous bodies. 0. ovary.
ct. cirrus. od. oviduct.
¢.m. circular body muscles. 0c. o6capt.
cp. cirrus pouch. ot. ootype.
cl’. external cuticular layer. pa. parenchyma.
ct. internal cuticular layer. p.ex. excretory pore.
cl.gi. gland cells. sé.c. subcuticular cells.
dex. excretory ducts. sh.gl. shell gland.
ep. epithelial layer. sp.va. sphincter of vagina.
ep.cil. ciliated epithelium. t testes.
lm. longitudinal body muscles. tr.m. transverse muscles.
m.c.ct. circular subcuticular muscles. ut. uterus.
m.i.ct. longitudinal subcuticular mus- | v.d. vas deferens.

cles. va. vagina.
ms. circular muscles. vtd. vitellarian duct.
mp. longitudinal muscles. vt.gl. vitelline gland.
2. nerve. vt.r. vitelline reservoir.
Mr. nerve ring.

Fics. 1-20. Proteocephalus ambloplitis.

Fic. 1. Lateral view of scolex. x 25.

Fic. 2. Anterior view of scolex. X 25.

Fic. 3. Median sagittal section of scolex. X 50.
Fic. 4. Proglottids showing extruded cirrus. x 25.
Fic. 5. Proximal portion of extruded cirrus. X Ioo.
Fic. 6. Odcapt and beginning of oviduct. x 500.

Fic. 7. Dorsal view of scolex, showing portion of nerve ring and the longi-
tudinal nerve trunks.

Fic. 8. Transverse section showing nerve ring with branches to suckers.

Fic. 9. Reconstruction of frontal sections showing the organs in a proglottis.
Camera outlines. xX 50.

Fic. to. The intra-ovarial region with enclosed female organs. x I0o.

Fic. 11. Section through cuticula and subcuticular structure. x 2000. Nota
camera drawing.

Fic. 12. Section through odtype, shell gland, and vitelline reservoir. x 250.

Fic. 13. Termination of longitudinal excretory ducts in the posterior proglottis.
x 25. Not a camera drawing.

Fic. 14. Transverse section of very young proglottis, showing the eight longi-
tudinal excretory ducts. x 25. Not a camera drawing.

368 BENEDICT.

Fic. 15. Longitudinal dorsal excretory vessels. 25.

Fic. 16. Transverse section near posterior end of proglottis. x 50.

Fic. 17. Course of vas deferens and cirrus within the cirrus sac. Reconstruc-
tion from camera outlines. X 250.

Fic. 18. Frontal section through cirrus pouch and vaginal opening. ™% 100.

Fic. 19. Transverse section through vaginal sphincter. x 200. Notacamera
drawing.

Fic. 20. Transverse section of vas deferens at its distal end. x 1000. Nota
camera drawing.

Fics. 21-35. Proteocephalus filicollis.

Fic. 21. Outline of short form of P. filicollis. x 4.

Fic. 22. Outline of long form of P. filicollis. x 4.

Fic. 23. Transverse section of proglottis through ovarial region. Xx I00.

Fic. 24. Lateral view of scolex. x 200.

Fic. 25. Transverse section of scolex showing nerve ring. x 200. Nota
camera drawing.

Fic. 26. Median frontal section of scolex showing nerve trunks. x 200. Not
a camera drawing.

Fic. 27. Reconstruction of frontal section showing anatomy. Camera outlines.
ei his

Fic. 28. Female sexual organs of the intra-ovarial space. x 225.

Fic. 29. Section through cuticula and subcuticular structures. x 2500. Not
a camera drawing.

Fic. 30. Section of odtype, shell gland, and vitelline reservoir. X 500.

Fic. 31. Cirrus pouch and cirrus in retracted condition. x 200.
_ Fic. 32. Cirrus pouch with cirrus protruded. x 200.

Fic. 33. Frontal section through distal end of vagina. X 200.

Fic. 34. Transverse section through the middle of the cirrus pouch. x 600.
Not a camera drawing.

Fic. 35. Section of odcapt and proximal end of oviduct. x 250.

Posterior «25

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THE EARLY DEVELOPMENT OF PLANORBIS.

SAMUEL J. HOLMES.

CONTENTS.
Part I. DESCRIPTIVE PORTION. ihe
Wie HOGS io oss ccs. eeeewcce tls CL SEA Eee seca rang ete eet acaslcttonsccaeucet ce 370
INOMENCIAEUTE | oo. is ese sdsec ies. nes seusutacsesvecccacoed wns eupositescanesseteesb beseseventastenstastocectulazetuncedeesa 373
MhewE ges rand Hep: Masses ois ccccch sense me acee ieee tan ae an ok a es 373
Mhewmbirst’ Cleavage s.r ee oie ares de Paune eras Mesa an DUE Ren NN Sal een 375
Mey Second) | Cleavage :2..:5, scsstececcsessscanceecssassaecestectsseataeue sense eras ant secon mer uate 376
mbhe AE bird! | Cle aware i. fse5. cok eeve ss ceteb ses ots ieucustsechcapoesesometeiuaeteet eaten PeneAE et ere ee 378
Promithey Hight) toythes Mwenty-tour Cell Stage rice scee eet eseete renee nee en eee 379
CleavageyEavity andiViacuol ese ea ecesecneeceeeceh cece ee een ee 385
Hromytheyaiwenty-four tothe) Vorty-nine) Cell) Stage pass eee 386
The Division of the Second Quartette and the Formation of the Cross 386
Mle mM rochO blasts ieee eceetsscesncesceecocssa-dousveesbessasecedobectecuh sce ate anes peste em nuca a a eee 387
Ahem Mivasionsob ether mMibird \@uartette: vos eve we suet ieine seen aneeee se eee aaa ae 390
Mhewhormationwor the verimeaty) MGSO as tececescestetreerere seems ee nce ea 391
Mhewhourth Ouarte tte eo ce races enter eae eee ee 393
The History of the Cross ......... esse fhevececbinties sovesuatbhoweseeartecensceeaess dogiaecstsszoesvenchaueebes 393
mhen second): Quartette ec cccss sere clececce cence eee cac cae t e eee eonae an ee eae 403
sEhemMesoblastic’ Bam sis serch ac caclee Selece aces ocdencadoues soteeee weneeee ae eee ere 407
The Third Quartette and the Secondary Mesoblast............:...-.s--ssceceeeeeereeeee- 408
General Considerations on’ the Secondary Mesoblast............2...:::ssscscescesecseees 411
Mthen BnEOMer es ee ast No call cal tse ac eceach act uae es alee cee ee cetee teaatas ea aaa ath Ei 413
The Rudiments of the Cerebral Ganglia and Eyes......0........sscsssessececeoreeeases- 415
Mhe Apical Plate nists A ta ee aan see eee eee cebea castes pee 416
MhexCelliiitinease: of the lead Vesicle ss te ererceencteeesstee see aeaeeseeenaetene tices aaa 417
mhe | Cell@itineageof theiBroto troche ee Nee eee eee pense aren ee ceeeaee een nae 418
mihenshellsGlandy and they Hootie. ec. ccee ee nee cee emer ne 421
GG Peart vial ACI ln Cyr sen face csis Sede aca ce socecneastraneeScatan eo rete taunesenscttnntn ecu santa pancesete sates 422
Gastrulationy.s Hatemotl the: Blastopore ites ressese sere cere ecetetecee ee eecemara tetera eee 425
ParT II. GENERAL CONSIDERATIONS.

Reversal) of |Cleavage) and) Reversed) Asymmetry. cece. seccecssecsenedsceeeenteecnccenteneene 426

The Relation between Reversed Cleavage and the Direction of the First
CleavasepPlaneee i: ese BURN J cost ny avs be ehc ees Ss meee nate ae 429
General Considerations oni spiralel@leavalge) erect en ccna ase eee eee eee 430
heversaly of \Gleavage and) Cell MEOmOLOsI1eS iat ceeeecnpesctsteseissee necro cnen seers 440
Bibliography ............... ICE Arete eer Meret SN ek A BONS, PEER Beamer Aeolian Sahn ve Sadar tt 445

Resp lanati cm! Of te wip Une ise scsi, Mae eae yo 2 sc Saw IN eaeea eee bene aeaesl (AIGO)

370 FLOLIM ES. [VoL. XVI.

THE investigation, the results of which are recorded in the
present paper, was carried on under the supervision of Prof.
C. O. Whitman, at the University of Chicago and at the Marine
Biological Laboratory at Woods Holl, Mass. It is a source of
gratification to acknowledge the generous treatment I have
received in both these places at Professor Whitman’s hands.
I wish also to express my appreciation of the many suggestions
I have received from Dr. E. G. Conklin, both in person and
through his admirable paper on the ‘‘ Embryology of Crepidula.”’

The species studied is Planorbis trivolvis Say. This species
occurs in great abundance in the ponds of South Park, Chicago,
and it was also found in a pond near Falmouth, a few miles
from Woods Holl.

Part I, DESCRIPTIVE PORTION.
Methods.

The eggs of Planorbis, when brought into contact with water,
swell quite rapidly, so it is best to tease them out of the cap-
sules directly into fixing fluid. The eggs may, however, be
teased first into normal salt solution, to which it is better to
add a small quantity of the fixative, and then treated with the
fixing fluid alone. Some fixing fluids coagulate the albuminous
substance around the egg so quickly, after or during its escape
from the capsule, that it usually becomes surrounded with more
or less coagulated albumen, from which it is difficult to free it.
By teasing directly into normal salt solution, mixed with only
a small quantity of the fixative, the eggs may be obtained free
from any foreign material. Normal salt alone often causes more
or less swelling.

Formalin does not appear to coagulate the albuminous sub-
stances in the capsules. The egg masses may be kept in a 5%
solution of this substance for several days, the jelly remaining
perfectly fluid and transparent. Unfortunately, formalin does
not otherwise prove a satisfactory fixing agent. Kleinenberg’s
stronger picro-sulphuric gave good results, especially when
followed by the method of staining with acidified Delafield’s

Nor 2) | 2AAZLY DEVELOPMENT: OF PLANOREIS. B71

haematoxylin, used by Conklin. Lithium carmine proved a good
nuclear stain when the eggs were first overstained, and the
color extracted by a long treatment with acid alcohol. Haema-
toxylin has the disadvantage, for the later stages of cleavage, of
staining very intensely the small globules of albuminous mate-
rial, which become very numerous and render the nuclei difficult
to observe.

By far the most useful reagent that was employed was silver
nitrate. In fact, were it not for the beautiful and clear prepa-
rations obtained by using this stain, I should probably have been
unable to follow the cell lineage of Planorbis to the stages here
described. The eggs are teased from the capsules directly into
a .75% solution of silver nitrate, and exposed in a watch glass to
the sunlight, the brighter the better. The eggs may be exam-
ined from time to time with the microscope, and when the cell
boundaries stand out clearly, the nitrate is removed and the eggs
washed quickly in water. The water being mostly removed, a
few drops of a }% solution of hyposulphite of soda is added and
allowed to act only three or four seconds. In fact, I begin to
remove the hyposulphite as soon as it is poured on the eggs.
The object of this treatment is to prevent after-blackening of
the eggs by dissolving out the unreduced silver. Otherwise the
eggs are liable to become in time so dark that they are useless
for study. A too prolonged treatment with the hyposulphite,
on the other hand, will destroy the silver stain entirely. I have
found it unsafe to wash the hyposulphite out with water. The
eggs often suddenly swell to twice their normal volume and are
thereby spoiled. Instead of water, a saturated solution of picric
acid may be used. This acts at the same time as a fixative and
does not injure the stain. After afew minutes’ treatment with
this reagent, the eggs may be passed through the grades of
alcohol, cleared in xylol, and mounted in balsam.

I have used to support the cover glass strips of paper of the
proper thickness gummed to the slide. By moving the cover,
the eggs may be rolled so that they can be studied from all
sides. When the balsam becomes hard, it can readily be soft-
ened by applying a drop or two of xylol to the edge of the cover
glass.

Oye HOLMES. [VoL. XVI.

Eggs treated by the foregoing method will keep indefinitely,
neither fading nor becoming opaque. When the egg is mounted,
the stain is usually safe. In successful preparations the cells
of the egg are not strongly darkened, but the cell boundaries
stand out in a remarkably sharp and clear manner. Some cells,
however, stain much darker than others. The trochoblasts and
the cells of the head vesicle remain almost perfectly transparent,
while the cells of the cross are colored brown. Owing to the
transparency of the trochoblasts the cross appears with a won-
derful distinctness, enabling one to orient the egg at a glance.
Nuclei are not stained, but they can usually be seen, and the
spindles: of dividing cells are often visible. The eggs may,
however, be stained so that the nuclei show fairly well, but I
have generally dispensed with nuclear staining when using this
method. The treatment often injures the impregnation and
renders the egg more opaque, so that it is more often a nul-
sance than a benefit. Individual differences in the impregna-
tion of different eggs are quite decided. Even among eggs
from the same capsule subjected to exactly the same treat-
ment, some will be strongly stained, while the staining in
others is faint. If eggs are left too long in the nitrate, they
not only become too dark, but also become brittle, and the cells
break or become separated when the eggs are rolled. Cell
boundaries take the stain at all periods in the development of
the egg, even in the very first cleavage stages. The method
is of special value, however, in following the cell lineage of
organs in the later periods of cleavage. I have obtained won-
derfully clear preparations of gastrulae, which show the bound-
aries of each cell, when there are several hundred cells in the
egg, with diagrammatic distinctness. The cells of the proto-
troch in the gastrula stage form a conspicuous transparent
band, which appears in marked contrast to the adjacent cells
—a circumstance which proved very helpful in tracing out the
cell lineage of this organ.

NO: 2.) ZARLY DEVELOPMENT OF PLANORZSIS. B73

Nomenclature.

In the matter of nomenclature I have followed the system
used by Conklin in his paper on the ‘Embryology of Crepidula,”’
as this method enables one readily to follow the type of cleavage
found in annelids and mollusks. Besides, the comparisons con-
stantly to be made throughout this paper, with the work of
Dr. Conklin, render it highly desirable, aside from other con-
siderations, that the same system of nomenclature be em-
ployed. The word ‘“‘quartette”’ is used to designate the products
of a generation of cells given off from the four cells at the
vegetal pole of the egg. The term ‘‘quartette” has been used,
however, in a different sense by several writers, who employ
it to designate any four cells of radially symmetrical origin.
Thus, according to the latter usage, the four outer cells arising
from the division of the first generation of ectomeres would con-
stitute one quartette, and the four apical cells another, while,
according to the usage here employed, all of the eight cells
resulting from this cleavage would still belong to the same
quartette. As a substitute for the word “quartette” in the
latter sense, the word ‘tier’? has been employed; thus the
products of cleavage of the first generation of ectomeres would
be called the upper and lower tiers of the first quartette.

The different quartettes in Conklin’s scheme are designated
by coefficients, and the genealogy of the cells of a quartette is
indicated by exponents. The upper cell, or the right one when
the cleavage is equatorial, is indicated by the smaller exponent;
2a’, for instance, indicates the upper cell in the a quadrant of
the second quartette, 2a° the lower. The upper product of the
cleavage of 2a’ would be 2a", while 2a** would represent the
lower cell.

The Eggs and Egg Masses.

The egg masses of Planorbis are flattened and rather firm,
and are usually found adhering to stones or aquatic plants. The
eggs proper are found in relatively large capsules, which are
imbedded in a jelly-like mass, outside of which is a somewhat
tough enclosing membrane. The amount of jelly between the

ota HOLMES. [Vo. XVI.

capsules is quite small; in fact, the sides of the capsules are
generally in contact, leaving only the interstices to be filled
by this material. The amount of albumen in the capsules, in
comparison with the size of the egg, is, on the other hand, very
large. The diameter of the egg measures about .13 mm., while
the diameter of the capsule is 66mm. The egg mass is of a
yellowish color in the species studied, and sufficiently transparent
to enable one to successfully study the living embryo zz szzz.
There is less jelly than in the egg masses of Physa and
Lymnaea, and the membrane surrounding the capsule is less
tough. The eggs may be readily teased out of the capsules,
whereas in Physa and Lymnaea the capsules come out of the
jelly entire, and slip around like rubber balls when the attempt
is made to tease them apart by needles.

The time during which P. ¢rzvolvzs lays its eggs extends from
early spring until the fall. The eggs are deposited in the great-
est abundance in the spring. Where the snails are abundant,
stones may frequently be found almost entirely covered by the
egg masses. Often the snails themselves have several clusters
of eggs attached to different parts of the shell, and sometimes
the egg masses may be found adhering to the bodies of aquatic
insects. When kept in an aquarium, the snails, in the early
part of their laying season, readily deposit their eggs on the —
glass ; but later in the year, when the eggs are produced in less
abundance, the animals become apparently more particular as
to where they lay, and seldom deposit their eggs unless upon
stones or aquatic plants, upon which they find minute algae,
which serve for food.

Usually a capsule contains but one egg, but sometimes it
may have two, and rarely three or more. When there are more
than one egg in a capsule, often only one of them develops
normally, yet two embryos in a late stage of development may
sometimes be found in the same capsule. It is common to
find some of the eggs in a cluster developing abnormally, and
the proportion of such eggs is increased when the water
becomes contaminated. If eggs are obtained from snails kept
in an aquarium, care has therefore to be taken that the water
is kept fresh and pure. With proper feeding and attention,

No: 2: LARLY DEVELOPMENT, OF PLEANOREIS. 375

Planorbis can be made to lay eggs even in the winter months.
Some of the snails I kept in glass dishes deposited eggs in
the latter part of January, and the cleavage of these eggs was
perfectly normal.

The unsegmented eggs of Planorbis are almost spherical in
form and of a bright yellow color. The yolk, which gives the
egg its yellow color, is somewhat more dense at the vege-
tal pole, although the difference is not strongly marked. The
fresh egg, when seen through the microscope by transmitted
light, shows a somewhat darker lower pole —the future ento-
derm— shading gradually into a lighter upper hemisphere—the
future ectoderm. The opaque matter of the egg consists of
two elements, small granules and larger globules of deutoplasm.
The spheres of deutoplasm are found scattered through all
portions of the egg with the exception of a very small, clear,
protoplasmic area at the animal pole. The polar bodies are
small and clear. The first one is the larger and of almost
spherical form, and is carried on the top of the second polar
globule. The polar bodies remain in connection with the egg
until quite a late period of cleavage, when they drop off and
disappear. The nucleus is situated in the small protoplasmic
area at the animal pole.

The First Cleavage.

At the beginning of the first cleavage the clear protoplasmic
area at the upper pole of the egg increases in size and elon-
gates as the chromosomes are separated. The asters form
dense radiating masses of fibers which are clearly visible in
the living egg. Their general appearance is very similar to
those of Physa, which are figured by Kostanecki ('96). The
cleavage furrow appears first at the animal pole and gradually
extends downward on either side, finally surrounding the egg.
The constriction is deeper at the animal pole, as is the rule
with yolk-laden eggs in which the spindle usually lies above
the center. After the separation of the parts of the egg is
complete, the daughter-cells become nearly spherical in form
and come in contact at only a small portion of their surface.

376 HOLMES. [VoL. XVI.

The two cells are equal in size, as is the rule in gastero-
pod eggs in which there is not a very large amount of yolk.
The nuclei are large and vesicular, containing, in proportion
to their size, only a small amount of chromatin. The yolk
spheres again encroach upon the protoplasmic area around the
nuclei, from which they had been extruded during mitosis,
and the cells gradually flatten against each other until they
resemble a single undivided sphere. The behavior of the yolk
in relation to the processes of cell division seems to indicate
that the regions around the poles of the spindle are the seat of
a tension which squeezes out the deutoplasm spheres as the
contraction of a sponge would squeeze out the water contained
in its meshes. With the disappearance of the astral radiations
and the decrease of tension in those regions, the yolk spheres
become free to distribute themselves more uniformly through
the egg. It seems probable that the rounding off of the cells
after division is due to the persistence for a time of the same
central tension which excluded the deutoplasm spheres from
the region of nuclear division, and that the subsequent flat-
tening of the cells after the resting period has begun is due to
a relaxation of the tension which at the same time permits the
more even distribution of the yolk, and allows those agencies
tending to draw the cells together to become dominant. This
flattening of the two blastomeres upon each other occurs very
soon after their complete separation, and it continues until
each blastomere becomes almost a hemisphere. A lenticular
cleavage cavity, if we may call it such, makes its appearance
at this stage, reaching its maximum development just before
the next cleavage.

The Second Cleavage.

The two cells usually begin to divide at the same time.
The division of one cell sometimes occurs a short time before
that of its fellow, but the cleavage is never completed before
the process of division in the other cell is well under way.
Both spindles are at first parallel to the plane where the cells
join. When the elongation of the cells occurs, the spindles

No.2.) LARLY DEVELOPMENT OF PLANORBSIS. 27.

turn slightly towards the right, and the cells themselves, as
they lengthen, undergo a twisting around in the same direction.
The first cleavage plane, when viewed from the animal pole, is
bent first to the left and then to the right. The reason of
this is that the cleavage is not perfectly horizontal, but two
of the cells lie a little higher than the others. When the
division is completed, there result four cells nearly equal in
size, two of which, & and PD, come in contact below, in the
ventral cross furrow, while the other two, A and C, meet in a
cross furrow at the upper pole, which is nearly at right angles
to the lower one. The ventral cross furrow makes a negative
angle of about 45° with the first cleavage plane, while the
upper cross furrow makes with this plane an equal positive
angle. The bending of the first cleavage furrow at the lower
pole of the egg is the reverse of what occurs in Crepidula and
other mollusks with dexiotropic cleavage. It is a rule, holding
good for all known cases, that in dexiotropic cleavage the
ventral cross furrow, when viewed from the animal pole, bends
to the right, while in forms with reversed cleavage it bends to
the left. The cross furrow at the animal pole of the egg,
however, does not show such a constant relation to the first
cleavage plane. In many cases it may be absent entirely, the
four blastomeres meeting above ina point. In some cases it
is parallel to the ventral cross furrow (Crepidula), in others
it is nearly at right angles to it (Nereis, Planorbis, Physa,
Lymnaea). These variations obviously depend upon the fact
that in some cases the two cells B and D meet above as well
as below,—in which case the two polar furrows would be
parallel, — while in other eggs the alternate cells A and C meet
at the animal pole and form a cross furrow at right angles to
the lower one. It is not surprising, therefore, that the polar
furrows should sometimes present different relations to each
other in eggs of the same species.

The four cells round off after division like those produced
by the previous cleavage. They become almost spherical, but
they subsequently draw together and assume very nearly the
form of a single undivided sphere. A cleavage cavity occurs
between the two pairs of cells 42 and CD; that is, between

378 HOLMES. [VoL. XVI.

those cells which were not separated by the last cleavage. In
addition, a central cleavage cavity occurs somewhat later which
reaches a considerable size and assumes a quadrilateral form.
All of these cavities disappear during the next cleavage.

The Third Cleavage.

The third cleavage in Planorbis is laeotropic. As in some
other forms, the amount of rotation is more pronounced during
the later stages of cell division, and the cells of the upper quar-
tette finally lie in the angles between the larger lower cells.
After the shifting has taken place the cells flatten, and there
results once more an almost spherical mass of cells. A central
cleavage cavity again makes its appearance, and again disap-
pears during the next cleavage. The four upper cells resulting
from this division, or the first quartette of ectomeres, are formed
of clear granular protoplasm, which gives them an appearance
quite distinct from the lower cells which contain the yolk.
While much smaller than the lower cells, or macromeres, they
are considerably larger than the corresponding cells in the eggs
of most gasteropods. The cells 1a and Ic meet in a polar fur-
row, which is inclined at a considerable angle to the polar
furrow at the vegetal pole.

It seems probable, as suggested by Lillie, that Rab] has
made some errors in the orientation of the eggs figured in his
first plate, which tend to produce confusion regarding the rela-
tion of the two polar furrows. In the first place, Rabl’s figures
indicate that, in the four-cell stage, the upper and lower cross
furrows are parallel, which is the reverse of what occurs in the
species here described. In case 8 and D meet above, as well
as below, we might expect that their derivatives, 14 and Id,
would also meet in a cross furrow, whose angle with the lower
furrow would approximately measure the amount of rotation of
the micromeres. Kofoid’s Fig. D ('95, p. 53) would represent
their relation under such a supposition. Rabl’s Fig. 11A indi-
cates a further rotation of the upper polar furrow until it lies
at right angles to its original position. Fig. 124, on the other
hand, shows this furrow at right angles to its position in 11A.

Nowe] | (LARLY) DEVELOPMENT, OF PLANORBIS. 379

There is doubtless an error in the orientation of this figure, if not
in the figures of the cross furrow in some of the preceding stages.

In P. ¢rivolvis the upper polar furrow would lie at right
angles to the lower one, were it not that the shifting of the
micromeres from right to left lessens this angle to one of about
45°. The eight-cell stage in P. ¢rivolvis differs from that fig-
ured in Kofoid’s Fig. D in that the upper polar furrow lies
between Ia and Ic (or the cells which Kofoid has called d** and
d**) instead of 10 and 1d (a*”’, 6*’, Kofoid), and is placed at right
angles to the one there figured. The upper polar furrow makes
a positive instead of a negative angle of 45° with the lower
polar furrow taken as an axis.

From the Eight to the Twenty-four Cell Stage.

The four macromeres are the next cells to divide. This
cleavage is dexiotropic, z.e., in the reverse direction to the
previous one. The second quartette of ectomeres thus formed
is, like the first, composed of rather large clear cells, which,
however, are markedly smaller than the macromeres. The cells
flatten out after division, as after the preceding cleavages, so
that the furrows between the cells almost disappear, and the
cell outlines are marked only by narrow clear lines.

From the twelve-cell stage the egg passes quickly to that of
twenty-four cells. The first quartette of ectomeres are the first
cells to divide, the division occurring in a right-handed spiral.
Very soon spindles occur almost simultaneously in the cells of
the second quartette and in the macromeres. The cleavage of
the cells of the second quartette is laeotropic, and the resulting
cells are nearly equal in size. The division of the macromeres,
which is likewise laeotropic, gives rise to the third generation
of ectomeres. The cells of the third generation are large, and
are marked off sharply from the macromeres, even before the
division is completed, by their clear protoplasm. From their
superficial outline they appear equal in size to the macromeres,
or even larger. Their actual bulk, however, is less, as may be
seen in optical sections, for they do not extend so far into the
interior of the egg.

380 HOLMES. [Vou. XVI.

The twenty-four-cell stage, which is reached by these divi-
sions, marks a resting stage of considerable length in the devel-
opment of the egg. A cleavage cavity is formed at this period,
which may attain quite a large size. In the arrangement of the
cells the egg presents a perfect radial symmetry. The macro-
meres, or entomeres, as it is better now to call them, are some-
what larger than the cells above them, and their yellowish color
and greater opacity, due to the yolk they contain, render them
easily recognizable. Lying in the angles between the four
entomeres are the cells of the third quartette. These cells
are elongated in a meridional direction, the direction of their
next cleavage. Alternating with these cells, and hence lying
opposite the entomeres, are the lower cells of the second quar-
tette, 2a’, 20°, 2c’, 2d’. The four entomeres are, therefore,
surrounded by a circle of eight cells, of the second and third
quartettes. The four upper cells of the second quartette, when
viewed from the upper pole, lie a little to the left of the lower
ones, and do not come into contact with the cells of the ento-
derm. At the upper pole of the egg there are eight cells, the
products of the cleavage of the first quartette of ectomeres ;
the four lower cells, the trochoblasts, alternate with the cells
of the second quartette, and lie opposite those of the third with
which they are in contact. The relation between these cells
corresponds essentially to the relation between the groups of
cells arising from them, even in a late stage of development.
The descendants of the four upper cells go entirely into the
formation of the cross, which will be described later; below
these cells lie those of the second quartette, and below these
again the entomeres. Alternating with these groups, there
occur, in vertical arrangement, the trochoblasts, the third quar-
tette, and the angles between the entomeres. This arrangement
of the various quartettes, which is typical for molluscan spiral
cleavage, is a great aid in following their further history.

There are a few points in which the cleavage of P. trivolvts
differs from Rabl’s account of the cleavage of the form studied
by him, which it may be well to point out. Rabl’s different
account of the cross furrows has already been discussed. The
passage from the twelve to the twenty-four cell stage takes

NO2) ) LALLY DEVELOPMENT, OF \PLANORBLS. 381

place, according to Rabl, by the simultaneous division of all
of the cells of the egg. In P. ¢rzvolvis the first quartette
divides before the others, forming a sixteen-cell stage. Finally,
although Rabl says nothing concerning the direction of the
cleavage of the cells of the second quartette, his Figs. 12A and
12£ indicate that the division was dexiotropic. In P. ¢rzvolvzs
this cleavage is plainly laeotropic, and the cells lie much more
nearly in a vertical plane than they are represented in Rabl’s
figure. There can be no doubt that the first quartette in the
forms studied by Rabl is given off in a laeotropic direction.
He expressly states this, and it is also indicated by his figures.
The second quartette, according to the law of alternation of
spirals, should be given off in a dexiotropic direction. If, as
Rabl’s figures indicate, the second quartette divides dexiotrop-
ically, there would result two dexiotropic divisions in immediate
succession. It seems more probable that Rabl’s figures are mis-
leading on this point than that there should be an exception to
the law of alternation of spirals at this early period of cleavage.
It is not unnatural that, not having in mind any significance
attached to this point, an observer should be in error regard-
ing it.

The cleavage of the eggs of Physa and Lymnaea as far as the
twenty-four-cell stage is essentially the same in almost every
point as that of Planorbis, and there is a remarkable agreement
between the cleavage of Planorbis and Limax, as described by
Kofoid and Meisenheimer, as far as the cell lineage in the latter
forms was carried. The cleavage of the eggs of the pulmonates,
so far as they have been studied, seems, in fact, to be charac-
terized by several points of marked similarity. The amount of
yolk in the eggs is not great ; the ectomeres are large; there
is a recurrent cleavage cavity ; and vacuoles are often formed
between the blastomeres. In the twenty-four-cell stage in the
above forms the cells have essentially the same relative size and
arrangement, and the entomeres are scarcely larger than the
other cells of the egg. The eggs of pulmonates complete their
development in capsules which contain a very large amount of
fluid albuminous substance, which serves to nourish the embryo.
As the amount of food in the form of albumen is large, it is

382 HOLMES. (Vou. XVI.

natural that the amount of food in the form of yolk should be
small, and the more nearly equal size of the blastomeres in the
early cleavage stages of pulmonates, in comparison with the
eggs of most marine forms, is probably due to the relatively
small amount of yolk in the egg. It is the rule among gastero-
pods that the greater the amount of yolk in the egg the smaller
are the ectomeres in relation to the entomeres. A comparison
of such yolk-laden eggs as those of Purpura and Nassa with the
eggs of Crepidula and Umbrella, or these again with the eggs
of Paludina or the pulmonates, will illustrate this principle very
forcibly. It has been pointed out by Kofoid, as a rule holding
for a great variety of forms, that “the greater the amount of
yolk, the greater seems to be the tendency of the cells of a
yolk-laden quartette to divide before those of the smaller quar-
tette.”’ For instance, in the eggs of Limax, which have little
yolk, both macromeres and ectomeres at the eight-cell stage
divide almost simultaneously, and the egg passes at once to
the sixteen-cell stage. In the eggs of Planorbis, Physa, and
Lymnaea, which contain somewhat more yolk, the macromeres
divide before the ectomeres, giving rise to a stage with twelve
cells. In the eggs of Umbrella and Urosalpinx, which contain
a still larger amount of yolk, even the third quartette is given
off before the first has divided. These facts can scarcely be
said to show, however, that the presence of yolk in cells actually
accelerates their division, as Kofoid seems to imply. It is a gen-
eral rule that the larger a cell is, the sooner it tends to divide.
There are numerous exceptions to this rule, some of which will
be pointed out later, but it expresses a more or less dominant
tendency in the cleavage of the egg. A large amount of yolk
in the egg, moreover, would determine the micromeres to be of
small size, and the small size of these cells would tend to delay
their cleavage. The yolk may, and probably does, delay the
cleavage of the cells containing it, but the small size of the
ectomeres in yolk-laden eggs delays their cleavage even more.
It is probably for this reason that we find a delayed cleavage of
the ectomeres in yolk-laden eggs. And this conclusion is in
harmony with the fact that the cleavage in yolk-laden eggs is
usually slow.

No.2.] EARLY DEVELOPMENT OF PLANORBIS. 383

As, perhaps, in all other mollusks, except the cephalopods,
and in the annelid worms, all of the ectoderm is contained in
the first three quartettes of micromeres. The three macro-
meres, A, 4, and C, are entirely entodermic, while the poste-
rior one, J, contains both entoderm and mesoderm. The cases
in which more than three quartettes of ectomeres are said to be
formed I think we must regard, with Conklin, as open to seri-
ous question. In the case of Fulgur, in which a large number
of quartettes of ectomeres was said to be formed, Conklin has
shown that there is, in reality, only the usual number, three.
In Nassa, Bobretzky held that the macromeres budded off a
large number of ectomeres ; but Conklin finds that in the closely
related genus, Ilyanassa, the usual number of quartettes is pro-
duced. Salensky has asserted that more than three quartettes
of ectomeres are formed in Vermetus, and Erlanger has made
the same statement regarding Bythinia. But in Serpulorbis
squamata, from the coast of California, —a form very closely
allied to Vermetus, in which genus it was, in fact, originally
placed,—I have found that the whole ectoderm arises from
but three quartettes.

It is not so much the fact that the cases reported of the
formation of more than three quartettes of ectomeres are
exceptions to a general rule that makes them so improbable —
it is the very definite and similar fate of these quartettes in all
the forms in which their history has been traced. Speaking of
this rule that the ectoderm in annelids and mollusks arises from
three quartettes, Dr. Conklin says, ‘‘ The cause of this remark-
able phenomenon is to be found in the fact, I believe, that each
of these quartettes of ectomeres is the protoblast of definite
regions of the embryo.” Each quartette forms essentially the
same parts of the embryo in every mollusk that has been studied
in thisregard. An additional quartette would necessitate a con-
siderable modification of the fates of the preceding quartettes.
Were the fates of the different cell generations to a large extent
indeterminate, a variation in their number would not seem so
great animprobability. Since, however, each quartette has essen-
tially the same destiny, not only in widely separated groups of
the Mollusca but also in annelids, we have very strong reasons,

384 HOLMES. [Vou. XVI.

I believe, for holding the accounts of the formation of four or
more generations of ectomeres to be erroneous.

The last author who records more than three generations of
ectomeres is Fujita, who studied the cleavage of the pulmonate
Siphonaria. After describing the formation of the four-cell
stage, he says: “ During the next following stages four succes-
sive generations of micromeres are budded off from each of
the above-mentioned segments, now to be called macromeres.
Hereupon the macromere D is entitled to the name of ento-
mesoderm, and the remaining three macromeres may be called
entodermic macromeres. Synchronously with the formation
of the fourth generation of micromeres, each member of the
third generation divides, thus giving rise to a fifth generation.
At this stage there are twenty micromeres and four macro-
meres, the relations of which may be seen in Fig. 2. Next
comes in order the formation of a sixth generation of micro-
meres again from the third, followed by that of a seventh
from the fifth.” It seems to me quite certain that Fujita over-
looked the division of the first generation of micromeres and
concluded that the four outer cells resulting from this cleav-
age arose from the macromeres. This is a very natural and
easy error to make, as I can testify from experience, having
been deceived, for a time, on just this point, when working on
another form. Fujita describes no division of the first quar-
tette until after the stage in which the egg contains thirty-four
cells, when the cells of the second quartette have divided twice,
and the posterior cells of the third have divided. Moreover,
Fujita’s figure of the twenty-four-cell stage shows that it corre-
sponds exactly, so far as the relations of the cells are concerned,
with the same stage in Planorbis, Physa, and Crepidula. If we
assume that the cells marked 2 in Fujita’s Fig. 3 are the tro-
choblasts, the genealogy of all the cells would exactly corre-
spond to that in the above forms, and the following divisions
up to the forty-third-cell stage, which Fujita describes, would
correspond point for point with those of Planorbis. The cells
2 are smaller than the apical cells, and their position indicates
that they arose by a laeotropic division, asswould be expected
according to the principle of alternation of spirals. All of these

Wo.2.) (AREY DEVELOPMENT OF PLANORSETS. 385

facts, taken in connection with the antecedent improbability of
Fujita’s conclusion, make it appear very probable that the egg
of Siphonaria contains but the usual number of quartettes of
ectomeres.

Cleavage Cavity and Vacuoles.

The existence of a recurrent cleavage cavity seems to char-
acterize, in an especial manner, the cleavage of the pulmonate
gasteropods. It has been observed by several workers on these
forms (Warneck, Fol, Rabl, Brooks, Schmidt, Meisenheimer),
and has been described so fully in the case of Limax by Kofoid,
that it will only be briefly considered here. In Planorbis the
cleavage cavity does not attain nearly such extensive develop-
ment as in Limax. Whether this relation obtains between the
aquatic and terrestrial pulmonates in general is uncertain, though
in the aquatic forms, Physa, Lymnaea, and Planorbis, the cleav-
age cavity is not so large as in the terrestrial genera, Limax and
Succinea. In Planorbis, as in all the above forms, the cleavage
cavity first appears in the two-cell stage. This disappears at
the next division ; and, in the four-cell stage, two cavities appear
between the two pairs of cells AB and CD, and, in addition, there
develops a central cleavage cavity which assumes a quadrate
form and finally merges with the other two. A similar occur-
rence repeats itself at the eight-cell stage. With each cleavage
the cavity disappears, then forms again, and gradually increases
in size until the rounding off of the cells during the next cleav-
age causes a break in the wall and allows the fluid contents to
escape. From the twenty-four-cell stage on, the cleavage cavity
appears to be permanent.

Small intercellular vacuoles occur in a late period of cleavage,
as in Limax, and they are found most abundantly in the ecto-
dermic portions of the egg. All of these cavities, as pointed
out by Kofoid, seem to be the result of excretory activity.
(For a discussion of the function and occurrence of the cleavage
cavity in different forms, see Kofoid ('95), p. 81.)

386 HOLMES. [VoL XV

From the Twenty-four to the Forty-nine Cell Stage.

The Division of the Second Quartette and the Formation of
the Cross. — The transition from the twenty-four to the forty-
nine cell stage occurs very quickly. The upper tier of the sec-
ond quartette divides in a dexiotropic direction. The upper
cell, as in Crepidula, Umbrella, and Unio, is the smaller, and
Meisenheimer’s Fig. 31 shows the same is true also in Limax.
These upper cells, as in the above forms, form the tips of the
arms of the cross presently to be described. Blochmann was
doubtless wrong in his derivation of these tip cells in Neritina,
and I think Conklin’s correction of this mistake is to be fol-
lowed, rather than that given by Kofoid, as it brings the cleav-
age of Neritina into complete harmony with that of other
mollusks (see Conklin (97), p. 64). The tip cells of the lateral
arms of the cross in Neritina were found by Blochmann to have
a peculiar granular appearance, and were held by him to give
rise to the velum. It is probable, however, that only a portion
of the velum is formed from these cells, as in both Crepidula
and Planorbis, and also in Ischnochiton, certain cells of the first
quartette, and other cells from the second also, go into the
formation of this organ. The granular character of these tip
cells in Neritina has not, I believe, been seen in any other
form.

The division of the upper tier of the second quartette is soon
followed by that of the lower. This cleavage is also dexio-
tropic, but the smaller cell is now the lower one. The upper
cell, resulting from this division, lies at the side of the lower
cell, arising from the previous division. There are now four
groups of four cells each, or sixteen cells, in the second quar-
tette. In each group there is a pair of large cells situated side
by side, and a smaller cell above and one below. The lower
cells lie opposite the entomeres.

The apical cells of the first quartette now divide in a laeo-
tropic direction. When this division is completed, the arrange-
ment of certain cells of the upper pole becomes such as to give
the appearance of a cross. The outer cells of the first quar-
tette, 1a"’, 15’, etc., form the bases of the arms of the cross,

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No. 2:.]) ZARLY DEVELOPMENT OF PLANORSIS. 38 7

the inner ones forming the center, and the cells of the second
quartette forming the tips. At its first appearance the cross,
therefore, contains twelve cells, eight belonging to the first,
and four to the second quartette. From this stage until the
period of gastrulation, the cross is a very conspicuous feature of
the egg. Its further history will be traced in a later section.
The Trochoblasts.— The cleavage of the apical cells of the
egg is soon followed by a division of the four outer cells of
the first quartette, the trochoblasts, 1a’, 14°, etc. This division
is nearly radial, the upper cell lying in the angles between the
arms of the cross directly above the lower one. The two cells
are about equal in size and they soon begin to enlarge and
become clear. One peculiarity of this cleavage is that it occurs
at a much earlier stage than in Umbrella and Crepidula. In
the first genus the trochoblasts do not divide until the egg
contains more than seventy cells; in the second the anterior
trochoblasts do not divide until the number of cells in the egg
is over one hundred, while the posterior trochoblasts do not
divide until later, if at all. In Unio, however, they divide at
about the fifty-cell stage, and at a still earlier stage in Chiton
(Metcalf) and Ischnochiton (Heath). If we compare the egg
of Planorbis with that of Limax in this respect, we find a close
agreement. The time at which this division takes place in
Limax is, according to Kofoid, quite variable, but it occurs,
speaking roughly, at about the forty-cell stage. The cells in
Limax are comparatively large, as in Planorbis, but, strangely
enough, they divide in an entirely different direction. ‘The
axis of the spindle,” says Kofoid, “lies parallel to the plane of
the equator. There is every indication that the division is
nearly meridional (horizontal).’’ Meisenheimer’s Fig. 31 shows
horizontal spindles in all four trochoblasts in the egg of Lzmax
maximus. The cells resulting from this division lie nearly side
by side, instead of the one above the other, as in Planorbis,
there being only a slight dexiotropic tendency in the cleavage
(see Kofoid, '95, p. 59, Fig. 41). In Conklin’s Fig. 50 the cells
of the two anterior pairs of trochoblasts lie in nearly the same
horizontal plane, and their symmetrical position in relation to
the anterior arm of the cross indicates that they were produced

388 HOLMES. [VoL. XVI.

by a bilateral division. At a late period the anterior trocho-
blasts in Planorbis become so shifted as to lie in very nearly
the same position as in Crepidula. It is probable that there is
no other group of cells which presents, in different mollusks,
such a remarkable degree of variation, both in the time and in
the direction of their cleavage. Yet they have essentially the
same fate not only in mollusks, but also in annelids. There is,
I believe, no reasonable escape from the conclusion expressed
by Conklin, that the trochoblasts in annelids and mollusks are
truly homologous. Having the same origin, position, and fate
in both these groups, the evidence of their homology is as com-
plete as ontogeny can furnish.

In most gasteropods the trochoblasts are of small size. This
is especially the case in Neritina, Umbrella, and Crepidula, and, ©
according to Conklin, they are small also in Urosalpinx and
Fulgur. In the pulmonates, however, their size is larger. In
the preceding cases the cells of the first quartette are very
small in relation to the macromeres and they divide unequally,
the outer cells being much smaller than the apical ones. In
Limax agrestis (Kofoid) and in Planorbis the cells of the first
quartette are not only much larger than in the above forms, but
they divide into almost equal parts. In the twenty-four-cell
stage of both forms the trochoblasts have about the same size
as the other cells of the egg. Their bulk is, therefore, vastly
greater than that of the corresponding cells in Umbrella or
Crepidula. Has not the relatively large size of the trochoblasts
in Limax and Planorbis some causal connection with their pre-
cocious divisions? Is not their size dependent largely on the
size of the cells of the first quartette, and this, again, upon the
relatively small amount of yolk in the egg? It seems probable
that the amount of yolk in the egg may indirectly influence
the size of the trochoblasts and the time at which they divide.
It is not contended that this is the only factor in the case. It
does not explain, for instance, why the cells of the first quartette
divide almost equally in some cases and very unequally in
others. But when we compare the eggs of Unio, Planorbis,
and Limax with those of Umbrella and Crepidula, it is difficult
to resist the impression that yolk is, in great measure, respon-

Nol 2 (LARLY DEVELOPMENT, OF PLANORESTS. 389

sible for the great difference in the period at which the division
of the trochoblasts occurs.

It might be urged that if the large size of the trochoblasts
were the cause of their early division, in forms with a rela-
tively small amount of yolk, they ought to continue to divide,
since they grow more rapidly than perhaps any other cells
of the egg. In Crepidula these cells are at first “much the
smallest cells of the entire egg”; finally they become, with
the exception of the yolk cells, the largest cells of the egg;
and their greater relative size is due not only to the fact that
the other cells are getting smaller as division proceeds, but
their absolute bulk is greatly increased. Yet neither in Crepi-
dula nor in any other gasteropod has more than one cleavage
of these cells been observed, while in Ischnochiton, accord-
ing to Heath (99), and in Amphitrite, Lepidonotus, and Clyme-
nella among annelids, according to Mead, they stop dividing
entirely after the second cleavage. The cessation of the
cleavage of these cells is doubtless connected, as Conklin
maintains, with their prospective destiny. They soon acquire
cilia and become a part of a specialized organ. This necessi-
tates a special modification of their structure, which, setting in
at an early period, checks the tendency to division. It is well
known that when the differentiation of a cell is carried very far
it generally ceases to divide, or if it does divide, its specialized
structure in great part disappears and it returns to a more
embryonic condition. And we probably have in the cessation
of the division of the trochoblasts simply an exhibition of this
rule. The trochoblasts in Planorbis, after they have divided,
become much flattened or thinned out. In eggs stained with
silver nitrate they are almost transparent, while the cells of the
cross are stained brown, causing this structure to appear in
conspicuous contrast to the cells between the arms. With the
growth of the trochoblasts the arms of the cross become more
narrow, as if they were pressed together at the sides. They
thus take a somewhat darker stain and stand out in a more
conspicuous manner than before. The further history of the
trochoblasts will be described in the section on the cell lineage
of the prototroch.

390 HOLMES. [Vou. XVI.

Although only the anterior pair of trochoblasts in Planorbis
enters into the formation of the prototroch, the term “ trocho-
blasts’? has been applied to the posterior pair as well. The
latter form a portion of the head vesicle. As this structure
may be considered as consisting mainly of the enlarged poste-
rior portion of the prototroch, it would scarcely be incorrect to
call these cells trochoblasts also.

The Division of the Third Quartette.— The cells of the
third quartette are large and elongated in a meridional direc-
tion. Their cleavage is almost exactly radial, as in Limax.
In Crepidula, according to Conklin, “the direction of the
cleavage is nearly radial, though after the cleavage has occurred
it is seen to be plainly laeotropic in 3a, 34, and 3c, and dexio-
tropic in 3d, z.e., the cleavage is nearly bilateral on the posterior
end, of the ovum’ Yetthe \spindle in. (3¢@ s)sometimes
laeotropic, as Conklin adds, and the nuclei may show this
relation to each other, while ‘the cell body may show reversal
of cleavage.’ ‘This,’ to quote the same author again, “is
but another illustration of the fact that bilaterality first appears
on the posterior side of the egg, that it is due to the change
in direction of one out of four cells, and that it is not per-
fect when it first appears, but is merely a deviation from the
spiral type toward the bilateral.” In Planorbis the two cells
on the posterior side of the egg, 3a and 3d, divide before the
anterior ones, 34 and 3c, but I have been unable to find any
constant deviation from the radial direction of their cleavage.
A radial cleavage may, however, be considered an approach
toward the bilateral type, and we may view the earlier division
of the cells 3a and 3d as the first foreshadowing of bilateral
cleavage.

The lower cells of the third quartette are somewhat smaller
than the upper ones and have their long axis horizontal, while
the long axis of the upper cells is still radial. In Neritina and
Umbrella the cleavage of the cells of this quartette is also
nearly radial, and the lower cell is the smaller, as is also the
case in Crepidula, and, according to Kofoid’s and Meisen-
heimer’s figures, in Limax. In Physa, according to Wierzejski,
the cleavage is also meridional and unequal, but in the anterior

Nor2)) ZARLY DEVELOPMENT OF PLANORBIS:, 391

quadrants the smaller cell is nearer the animal pole, while on
the posterior side of the egg the reverse is the case. Which
of the cells are the first to divide is not stated. In another
pulmonate, Siphonaria, the division of the posterior cells of
the third quartette occurs before that of the anterior ones,
each cell giving off a small cell toward the vegetal pole. The
division in both cells is laeotropic, but less so in the cell on the
left side (Fujita, '95, p. 91, Fig. 5, 6x). This cleavage, accord-
ing to Fujita, occurs at the thirty-two-cell stage; the corre-
sponding cells in the anterior quadrant have not divided at the
stage in which the egg contains forty-three cells, which is as
far as the cell lineage of this form is described.

The first division of the cells of the third quartette seems to
mark the point in the cleavage of gasteropods where bilateral
cleavage makes its first uncertain appearance. In no case is
there a typical spiral cleavage of all the four cells. The cleav-
age may be radial (Physa, Limax, Planorbis, Neritina, Umbrella),
slightly bilateral in the posterior quadrants, and spiral in the
anterior ones (Crepidula), or spiral in the posterior quadrants,
but with an approach toward the bilateral type (Siphonaria).
The posterior quadrants generally divide before the anterior
ones. In Limax, however, the order of cleavage in the differ-
ent quadrants seems to be inconstant (Kofoid). In Crepidula,
Umbrella, and Planorbis the posterior cells divide only a short
time before the anterior ones, while there is a long interval
between these divisions in Siphonaria. The cleavage of the
cells of this quartette is, in all the above forms, unequal, and,
except in the anterior quadrants in Physa, the smaller cell lies
nearer the vegetal pole of the egg.

The Formation of the Primary Mesoblasts. — Soon after the
twenty-four-cell stage is reached, the posterior macromere D
divides into unequal parts. The upper moiety is much larger
than the lower one, and, from the first, lies partly pushed into
the cleavage cavity, so that only a small portion of it appears
at the surface of the egg (Pl. XVII, Fig. 11). It is dark and
granular, and contains a large amount of yolk, like the cells of
the entoderm. The division of D is dexiotropic, as Crampton
found it to be in Physa. The primary mesomere lies, therefore,

392 HOLMES. [Vou. XVI.

to the left of D in forms with reversed cleavage, and to the
right of D in forms in which the cleavage is not reversed.
Before the division of J, the two macromeres, 4 and LD, were
equal in size, and the anterior and posterior sides of the egg
could not be distinguished. The position of 4d or JZ enables
one henceforth to easily locate the posterior side of the egg.
The primary mesomere soon divides in a nearly horizontal direc-
tion, though the cleavage is slightly oblique. This division is
completed, as in Siphonaria, before the other cells of the fourth
quartette arise. The two mesomeres gradually sink further into
the egg and lose connection with the surface at about the sixty-
four-cell stage.

One cannot compare the formation of the primary mesoblast
in the different pulmonates, in which its origin has been traced,
without being struck with the very close similarity of the proc-
ess in the several forms. The exact cell origin of the primary
mesoblast has been determined in Limax, Physa, Lymnaea, Pla-
norbis, and Siphonaria. In all these forms the mesomere arises,
shortly after the twenty-four-cell stage is reached, by the oblique
division of D; it lies, from the first, partly pushed into the cleav-
age cavity; it is much larger than the entomere J, and of an
Opaque appearance; and it soon divides in a nearly horizontal
direction and gives rise to the mesoblastic bands. A similar
origin of the primary mesoderm is found in several other gas-
teropods, but, with the exception of Umbrella, the mesomere is
considerably smaller than the entoderm cell D (Bythinia, Crepi-
dula, Neritina, Ilyanassa, Fulgur). There are at present many
divergent accounts of the origin of the mesoblast in the mol-
lusca. These accounts have recently been reviewed by different
writers on the subject (Heymons, ’93, Tonniges, '96, Schmidt,
'95, Meisenheimer, '96), and fully discussed in Korschelt and
Heider’s Embryology, so that it would be superfluous to
devote space to the subject here. In almost every case, how-
ever, in which the cleavage has been followed with sufficient
care, the primary mesoderm has been found to arise, as in the
above forms, from the posterior macromere J. Yet careful
study has failed to discover pole cells in Paludina vivipera,
but the fate of the cell 4d in this form has never been traced.

Nor2.| LARLY DEVELOPMENT OF PLANORSIS. 393

What becomes of this cell in this form would be a matter of
considerable interest.

The Fourth Quartette. —The fourth quartette is produced by
a dexiotropic cleavage of the entomeres. All the cells of this
quartette are large and full of yolk; the four cells at the vege-
tal pole are small and clearer than the others. They are quite
thin and, therefore, less in bulk than their superficial area would
indicate. The general appearance of the entoderm cells at this
stage is very similar to those of Limax, and markedly different
from those of most marine forms, in which there is usually
much yolk.

The number of cells in the egg when the fourth quartette is
formed is forty-nine.

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One of the most conspicuous features of the egg at this stage
is a belt of large cells around the equator. It is composed of
twelve cells, the four middle pairs of cells of the second quar-
tette alternating with the four upper cells of the third. All of
these cells are more or less oblong, with their long axes verti-
cal. A similar belt of cells may be seen, though less plainly, in
Crepidula and Limax.

The History of the Cross.

At the time of its first appearance the cross contains eight
cells of the first and four cells of the second quartette. Its
center is at the apical pole of the egg, and its arms are anterior,
posterior, right, and left. The tips of the cross lie over the
large entomeres of the fourth quartette, the cells of the second
quartette lying between. The median plane of the egg would
cut through the middle of the anterior and posterior arms, and
the entomere 40, and pass between the two mesoblasts. Pre-
vious to the formation of the fourth quartette, this plane would

oF HOLMES. [Vou. XVI.

have cut the ventral cross furrow at almost a right angle. After
the fourth quartette is formed, owing to the shifting of the small
cells at the vegetal pole, the cross furrow would be cut at an
oblique angle. Viewed from the animal pole, the cross furrow
has been turned several degrees in a laeotropic direction. This
rotation is obviously a result of the dexiotropic cleavage of the
macromeres. Of course, if we take the small cells at the vege-
tative pole as fixed, we might consider that the rest of the egg,
the fourth quartette and the ectomeres, had rotated in an oppo-
site, or dexiotropic, direction. In an egg like that of Crepidula,
in which the homologues of these small cells form the greater
part of the bulk of the egg, it is natural to regard the furrows
between these cells as fixed, and to speak of a laeotropic rota-
tion of the ectoblast rather than a dexiotropic rotation of A, B,
C,and D. Whichever pole of the egg we regard as fixed, — and
this is the essential point, —it is certain that the rotation in the
two forms has taken place, in accordance with their reverse
types of cleavage, in opposite directions.

The next cleavage in the cross occurs in the basal cells at
about the sixty-four-cell stage. This division is nearly radial,
though slightly laeotropic. It is an interesting fact that we
have here a violation of the rule of alternation of spirals
exactly where it first occurs in Crepidula and Neritina, but
with this difference, that in Planorbis the two successive cleav-
ages are laeotropic, while in Crepidula and Neritina they are
dexiotropic.

The apical cells 1a", 1d"', etc., next divide. There seems to
be a tendency to a dexiotropic cleavage in these cells, but the
direction of the division appears to be more or less inconstant.
To the extent that this cleavage may be considered dexiotropic,
it is in accordance with the rule of alternation of spirals. This
division marks the close of the period of definite spiral cleavage
in the cells of the cross. The subsequent cleavages, to which
this may be considered a transition, are all of the bilateral type.
With the completion of this division the number of cells in the
egg reaches 104. In Crepidula this cleavage is laeotropic, and
Heymons mentions the fact that in Umbrella these cells divide,
but says nothing concerning the direction of the cleavage. The

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Nov2.) \/ZARLY DEVELOPMENT, OF PLANORETS. 395

next cleavage occurs in the basal cells of the arms of the cross,
and takes place in a radial direction. Each arm of the cross
now contains three cells, besides the tip cells, which belong to
the second quartette. This division is in the same direction as
the preceding division of the basal cells, so there occurs a sec-
ond exception to the rule of alternation of spirals. It is a note-
worthy fact, also, that the direction of this division is at right
angles to the corresponding cleavage in Crepidula. In Crepi-
dula, when there are three cells in each arm of the cross, the
two cells behind the small tip cells are much elongated trans-
versely to the long axis of the arms, and it is natural that their
cleavage should be at right angles to their longest diameter.
The middle cells of the arms in Crepidula divide first; this
division is followed by the cleavage of the cells at the apical
pole of the egg, and soon afterward the division of the basal
cells takes place. In Planorbis it is the apical cells that first
divide; then the basal cells divide radially, so that before the
longitudinal splitting of the arms occurs each arm has one more
cell than in Crepidula; a radial division, lengthening the arms
in Planorbis, takes the place of a transverse division, splitting
the arms in Crepidula. Moreover, the cells lying just behind
the tip cells of the cross, which in Crepidula divide first, in
Planorbis, except in the anterior arm of the cross, zever divide
again.

The inner median cell of the anterior arm of the cross next
divides transversely and marks the beginning of the splitting
of the arms of the cross. After a short interval the splitting
of the lateral arms also begins; the cleavages are slightly
oblique, the lines of division pointing toward the anterior end
of the cross. This nearly transverse division occurs only in
the basal cells and the ones lying next to them, the inner
median cells. Zhe basal cell in the anterior arm undergoes no
further divisions. Its history will be described later. The
transverse splitting in the anterior arm at this stage is, there-
fore, limited to a single cell. The posterior arm of the cross,
as in Crepidula, remains undivided throughout its entire history.
With the longitudinal division of the basal cell above described,
the history of the cleavages in the posterior arm is completed.

396 HOLMES. [Vou XVI.

The composition of the cross at this period is as follows
(Pl. XX, Fig. 37): Four rather small cells in the center,
1a", 16°", ‘etc.,) around the apical pole of the ere’; around
these are four other cells, the intermediate cells, 1a**’, etc.,
in the angles between the arms; the anterior arm, which is
broader than the others, consisting of a single basal cell, 16°*"",
then’ a pair of cells, 10°%"*", 10°°"*", resulting from the trans-
verse division of the inner median cell; then a single outer
median cell, 14°*”, and finally the tip cell, 26"; the two lateral
arms consisting of two pairs of cells at the base, an undivided
outer median cell, and a tip cell; the posterior arm, consisting
of a row of four cells. In all, the number of cells in the cross
is twenty-nine. The anterior arm is shorter than the others,
and the tip cell is more or less clear. The arms of the cross
are slightly oblique; and it is worthy of note that the direction
of their inclination is laeotropic, while in Crepidula and Isch-
nochiton the arms show a slight dexiotropic twist. This differ-
ence is doubtless connected with reverse types of cleavage of
these forms.

The tip cells of the arms have enlarged and become trans-
parent. The tip cell of the right arm is widest behind, while
that of the left is widest in front ; the outer median cells show
the reverse relation. The cells of the posterior arm of the
cross are usually rhomboidal in outline. The posterior tip cell
is large and elongated in the direction of the arm, while the long
axis of the other tip cells is transverse to the arm. The ante-
rior tip cell is the smallest of the four. As the tip cells in eggs
stained with silver nitrate are clear, the portion of the cross
which stains dark is that derived entirely from the first quar-
tette. This portion is very conspicuous, as it is surrounded
on all sides by transparent cells.

It may be well, before tracing the history of the cross further,
to compare it briefly with the cross in other mollusks. Bloch-
mann traces the history of the cross in Neritina to a stage in
which each arm contained three cells, except the posterior arm,
which was composed of four. Blochmann’s probably incorrect
derivation of the tip cells has already been mentioned, and
Kofoid has shown also that it is almost certain that his deri-

Nori) (ZARLY DEVELOPMENT, OF PLANORBIS: 397

vation of the basal cells of the cross was likewise incorrect.
Interpreting Blochmann’s figures in the light of what is known
to be the rule in other forms, it is evident that the cell lineage
of the cross in Neritina, at the stage when there are three cells
in each arm, corresponds exactly to that of Umbrella, Crepidula,
and Planorbis. In the lengthening of the posterior arm of the
cross by the addition of another cell, there is a further point of
agreement with the history of the cross in the last two forms.
In Crepidula both the basal and the tip cells of the posterior
arm of the cross divide radially at about the same time, so that
this arm comes to have one cell more, instead of one less than
the other three. Two of the four cells in this arm, therefore,
belong to the first quartette and two to the second. Whether
three of the four cells of the posterior arm in Neritina belong
to the first quartette, or whether there are two cells each of
the first and second quartette, is uncertain. Blochmann says
nothing about the derivation of this additional cell. Should we
argue from analogy with Crepidula, we should be led to accept
Conklin’s scheme of the probable derivation of this cell, and
derive the two posterior cells from the second quartette. If,
on the other hand, we should draw our conclusion from a com-
parison with Planorbis, we should infer that the three anterior
cells belonged to the first quartette, while only the tip cell
belonged to the second. In Planorbis the four cells in each
arm have the same derivation and are produced at nearly the
same time. In both Neritina and Crepidula, owing to a delay
in the division of the basal cell, the posterior arm contains at
first but two cells, while each of the other arms contains three;
this stage is followed by a stage in which the posterior arm
contains four cells, while the number in the other arms remains
the same. The tip cell in the posterior arm in Crepidula is
larger than those of the other arms and divides first, and in a
different direction from the others; that is, radially instead of
transversely. The divisions of the cross cells in Planorbis are
such that they preserve the radial symmetry of the cross much
longer than in Crepidula and Neritina. There is little differ-
ence, either in the time or direction of the cleavages, in the dif-
ferent arms until after the period in which each arm is composed

398 HOLMES. [VoL. XVI.

of over four cells. The radial symmetry of the cross is not de-
stroyed until the egg contains over one hundred blastomeres. In
Neritina the cross becomes a bilaterally symmetrical structure,
by the lengthening of the posterior arm, when the egg contains
only about fifty cells. In Crepidula the cross may be said to be
bilaterally symmetrical from the very beginning of its formation,
owing to the smaller size of the basal cell and the larger size of
the tip cell in the posterior arm. At about the forty-eight-cell
stage there are only two cells in the posterior arm and three in
the others; the divisions which increase the number of cells in
the posterior arm to four occur when the egg contains sixty-seven
cells, at the same time that the first transverse division in the
other arms is taking place. The appearance of bilateral sym-
metry in the cross in Umbrella, Heymons did not describe.
The striking differences in the history of the cross in Planorbis
and Crepidula are most interesting. It is natural to seek for
some explanations of the problems which these differences pre-
sent to us. Why is it that the cleavage of the cell, which in
Crepidula results in a splitting of the arm of the cross, produces
in Planorbis a lengthening of the arm of the cross? Why does
the posterior tip cell in Crepidula divide before the others, and
in a different direction, while it does not divide at all in Planor-
bis? Why is the radial symmetry of the cross retained longer
in Planorbis than in Crepidula? And what is the cause of the
very different behavior of all of the tip cells in the two forms ?
These are a few questions which suggest themselves when we
compare the history of the cross in these forms. A complete
solution of these problems is at present impossible, but we
may, perhaps, determine some of the proximate causes of these
differences of behavior. ‘The transverse division of the cells,
1a™*", etc., in Crepidula is doubtless the result of the fact that
the long axes of these cells are transverse to the arm of the
cross. In Planorbis the basal cells in the arm, at the time they
begin to divide, have their longitudinal axes in the contrary
direction. Hence it is natural that their division should be
radial and not transverse. Now the different shape of these
cells in Planorbis is apparently due to the growth of the trocho-
blasts, which, as they increase in size, crowd the adjacent cells

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Nor2i LARLY DEVELOPMENT OF (FLAVORS. 399°

together. Thus the arms of the cross would be subjected to a
lateral pressure, which would tend to give the cells an elonga-
tion in a radial direction. In fact, as the trochoblasts increase
in size, the arms of the cross actually do become narrower, as
may readily be seen by comparing the figures of the earlier and
later stages in the history of this structure. In Crepidula the
trochoblasts are relatively much smaller than in Planorbis, and,
at the time that the divisions above described occur, have not
attained a sufficient size to exert much influence upon the arms
of the cross. Thus the proximate cause of the difference in the
direction of the division of the basal cells of the cross in the two
forms seems very probably to lie in the different character of
the trochoblasts. The cause of these differences in the trocho-
blasts may, as has been suggested above, lie partly, at least, in
the relative size of the first quartette of ectomeres in the two
forms, which, in turn, is largely dependent on the amount of
yolk in the egg.

Are there any facts which throw any light on the different
behavior of the cells in the posterior arm of the cross in the
two forms? It will be remembered that in both Crepidula and
Planorbis the posterior arm always remains undivided ; ze.,
it consists of but a single row of cells. In Crepidula the
development of this arm of the cross lags behind the others,
the first basal cell not dividing until a considerable time after
the others. Why isthis? The explanation appears to lie in the
fact that the cell 1@*’ is smaller than the other three basals.
Conklin, I believe, does not mention the fact, but in all of his
figures this cell is uniformly represented as smaller than the
others (Figs. 23, 25, 26, 29-31), in one case (Fig. 31) the dif-
ference being very marked. Further back than this it is to be
noted that the division of the cell from which the basal cell 1d**
arose seems to be delayed, a fact which would indicate the
smaller size of this cell, although this is not otherwise notice-
able. It is certain, however this may be, that this cell 1a’
divides more unequally than the others, and the principal cause
of the delayed cleavage of the posterior basal must, therefore,
be sought in whatever agency gives rise to the unequal division
of 1d’. In Planorbis the division of this cell is no more unequal

AOO HOLMES. [Vou. XVI.

than that of the others in the same tier, and the basal cell of
the cross which arises from it, being, therefore, of the same size
as the other basals, divides approximately at the same time.
We have, therefore, in the different character of the division of
1@* in Crepidula and Planorbis, the beginning of the difference
in the course of development of the posterior arm of the cross in
the two forms. We may be unable to explain the differential
character of this cleavage of 1d'. Why one out of four proto-
plasmic cells, identical, so far as we can determine, in size and
structure, should divide much more unequally than the others
is certainly not apparent. Nevertheless it is desirable to find
the precise point where the histories of structures in different
forms begin to diverge, although we are unable to discover the
antecedent phenomena which give the direction to the different
lines of divergence. The earlier cleavage of the posterior tip
cell in Crepidula is probably connected with its larger size.
Its cleavage is radial, and each of the daughter-cells divides
again in a radial direction, giving rise in all to six cells in the
posterior arm. The cause of these successive longitudinal
divisions may possibly be the growth of the posterior trocho-
blasts and the cells lying below them, which at this period
have reached a considerable size. The enlargement of these
cells would naturally subject the arm to pressure at the sides
and give the cells a longitudinal elongation. A comparison of
Conklin’s Fig. 49 with Fig. 53 shows that the trochoblasts in
the latter figure are larger, and the basal and tip cells longer
and narrower. The shape of the tip cells just before their
division is not figured, but it is probable that they have become
more or less compressed like the basals, after the stage shown
in Fig. 51. However, after their division they have been
narrowed to about one-half their former diameter (Fig. 53).
The posterior tip cell in Planorbis has quite a different history.
In the first place it is no larger than the tip cells of the other
arms, but the marked difference it presents from the cor-
responding cells in Crepidula is that it never divides. It
increases enormously in size and becomes transparent. Its
shape changes entirely; at first it is elongated transversely to
the arm of the cross; gradually, as it enlarges, it becomes

Nor2.| ZARLY DEVELOPMENT OF PLANOREIS, 401

elongated in the opposite direction and takes part in the form-
ation of the head vesicle. The elongation of this cell goes
hand in hand with the growth of the posterior trochoblasts,
whose increase in size would naturally subject it to a lateral
pressure. The peculiar history of this cell is evidently corre-
lated with the large size which the head vesicle attains in this
form. It affords another case of precocious specialization of
function such as occurs in the trochoblasts. In fact, the fate
of this cell and the posterior trochoblasts is identical, as they
all go to form the same organ. The head vesicle in Planorbis
develops early, and reaches a much larger size than it attains
in Crepidula. The posterior tip cell becomes differentiated at
an earlier period and stops dividing. In Crepidula it divides
twice, perhaps many times, and the products of these divisions
remain for a long time apparently little modified. Their pre-
cise fate is uncertain; probably some of them, at least, enlarge
and enter into the formation of the head vesicle, as in Planorbis.

The other tip cells divide twice in Crepidula, forming a row
of four small cells lying across the tips of the arms of the cross.
All of these cells, accordingly, go into the upper row of cells,
forming the prototroch. Except in the anterior arm of the
cross, the tip cells in Planorbis do not divide at all. The
lateral tip cells, like the posterior one, enlarge, become trans-
parent, and go to form a part of the head vesicle. There is
probably no other group of cells in these two forms which
present such marked differences of behavior as the tip cells of
the cross. In Crepidula they are at first very small and of
unequal size; they grow very little and divide several times.
In Planorbis they are at first quite large and of equal size;
they grow quite rapidly, and, with the exception of the anterior
one, never divide at all. In Crepidula those of the anterior and
lateral arms go into the prototroch; in Planorbis only the ante-
rior one goes into the formation of this organ, and this cell, as
far as could be determined, undergoes only one division.

The next cleavage, after the stage to which the history of the
cross has been traced, occurs in the four cells lying in the angles
between the arms. These divisions are bilateral; in the ante-
rior pair of cells the left cell divides dexiotropically, the right

402 HOLMES. [VoL. XVI.

cell laeotropically ; in the posterior pair the cleavage of the right
cell is dexiotropic, that of the left one laeotropic. The two cells
in the anterior arm)/1G/777) 4 0417 74) ext divide, the meht Gell
in a dexiotropic, the left in a laeotropic direction. The poste-
rior cells on either side resulting from this division come to lie
to the outside of the small, outer, intermediate cells, so that they
no longer lie in contact with the trochoblasts (Pl. XX, Fig. 42).
(Compare the cleavage of 16°** and 16°** in Crepidula.) Next
the anterior median cell, 14"*’, divides transversely, and each of
the daughter-cells divides bilaterally, in the same direction as
that of the pair of cells just described.

At this period the general form of the cross has undergone
marked changes. The cells of the posterior arm have increased
greatly in size and become distorted in shape. The posterior
trochoblasts enlarge unequally, and the posterior arm may be
pushed either to the right or the left. The posterior tip cell
first increases in size; afterward, all the cells lying in front of
it also enlarge. The enlargement of cells does not stop with
the cells of the posterior arm, but the four apical cells increase
in size also (Pl. XX, Fig. 42). The central and anterior portions
of the cross are pushed forward more rapidly than the lateral
arms, which thus appear to be bent backwards. The darkly
staining portion of the cross has now the shape of a V, with the
apex pointing anteriorly. The two anterior tip cells, as the
cross is pushed forward, come to lie more and more nearly side
by side, and, finally, they become arranged transversely across
the tip of the cross. As they were originally somewhat oblique,
this arrangement would very naturally result from a pressure
due to the forward rotation of the apical cap of cells.

The process of enlargement of cells extends forward to the
cells lying in front of the apicals, and eventually forms a tract
of large clear cells, which separates the two halves of the cross
and reaches the prototroch in front. The basal cell of the ante-
rior arm of the cross, since it lies in the median line, takes part
in this enlargement of cells; as it increases in size, it be-
comes pushed forward, and the cells in front of it, which lie
symmetrically on either side of the median axis of the arm, are
forced aside. The fate of this cell has been carefully traced, as

No 2) "ZARLY DEVELOPMENT OF PLANORBIS: | 403

it has a peculiar and most interesting history. At first it lay
at the base of the anterior arm of the cross. When the cells
10****" and 16°***” divide, this cell comes to lie between the
cells produced by these divisions, and soon it is seen further
forward, between the posterior pair of cells arising from the
third cell of the arm, 10%’. Pl. XX, Fig. 42, shows this cell
where the anterior cells are in contact, and Pl. XX, Fig. 48,
shows it pushed still further forward, until it has forced the
cellsno™"" and 10""*** to. either side’ andcome’ into contact
with the tip cells. Its journey does not end here, but it appar-
ently pushes aside the tip cells as well, and comes in contact
with the cells of the second quartette, which lie below them
(Pl. XX, Fig. 46). Thts cell has, therefore, pushed its way
through the split anterior arm from the base to the tip. In
later stages it becomes much elongated transversely, and forms
a part of the prototroch. The origin of this upper median cell
of the prototroch was for a long time a puzzling problem. I
have fortunately found all stages of the process by which it
travels through the middle of the anterior arm of the cross to
its definitive position.

The further history of the cells of the cross is very difficult
to follow. The cells in the center enlarge unequally in differ-
ent cases, and the position of the cells is altered considerably
by this process, which makes it very difficult to follow their
lineage. I have observed a bilateral division of the cells
Pea aniel td , the lines of division converging anteriorly.
Both products of this division then divide at right angles to the
preceding cleavage.

1.2,1.1.1

TG

The Second OQuartette.

The cleavage of the cells of the second quartette has already
been traced to a stage in which there are four cells in each
quadrant. The two middle cells in each quadrant, which are
larger than the upper and lower cells, are the first to divide;
the division of both cells in each pair is laeotropic, the cleav-
age of the right cell occurring a little before the left. These
divisions occur between the fifty-two and the sixty-four cell

404 HOLMES. [VoL. XVI.

stages; the lower cells resulting from these divisions are some-
what larger than the upper ones. When the egg contains about
seventy-five cells, the lower cell in each quadrant divides, gen-
erally in a laeotropic direction, but the direction of the cleavage
does not appear to be constant. Pl. XIX, Fig. 32, shows that
the division of 2a** was probably dexiotropic, but shifting of the
position of cells has of course to be allowed for. The cells
2a”, 20°", and 2c’, previous to their division, have become
more and more flattened in a direction transverse to the ver-
tical axis of the egg. After their division their daughter-cells
become flattened still more in the same direction; one of these
usually lies above the other, which alone comes in contact with
the entomeres; sometimes they lie obliquely, especially in the
a and ¢ quadrants, the outer cell touching the entomeres by a
small portion of its boundary. These cells form the anterior
and lateral boundaries of the blastopore, the cells of the third
quartette lying at the angles. It will be convenient to apply
the term “stomatoblasts”’ to these cells, 2a**, 26°’, and 2c*’, and
their derivatives, as they form a part of the margin of the blas-
topore, and later take part in the formation of the stomodaeum.
The term “stomatoblasts,’ however, was used by Wilson to
designate the cells 2a’, 20°, and 2c’, and their derivatives, which
in Nereis form a ring of cells around the blastopore. In Pla-
norbis the cells 2a’, 26°", and 2c** never come in contact with
the blastopore, and probably do not take part in the formation
of the stomodaeum, so that the term ‘‘stomatoblasts’”’ would
hardly seem applicable to them. The term “stomatoblasts”
is used, therefore, to designate only the lower product of the
division of the cells which correspond to the stomatoblasts of
Wilson. In Nereis the division of the cells 2a’, 20°, and 2c
is transverse to the polar axis and all of the products of the
cleavage border on the blastopore. In Planorbis these cells
divide in nearly the opposite direction, so that 2a*’, 26°", and
2c" lie above the ventral cells and never come into relation
with the entomeres. The same cells divide into an upper and
a lower moiety in Unio, Neritina, Umbrella, Crepidula, Limax,
and Ischnochiton. In none of these forms, except Ischnochiton,
has the cleavage of 2a", 26°", 2c** been described. The cell

Nor2.|| \ZARLY DEVELOPMENT OF PLANORSIS. 405

2d@°° differs in shape and in the direction of its cleavage from
the other cells of the same tier. Before it divides, it becomes
elongated in a vertical instead of a horizontal direction. It
divides laeotropically, its daughter-cells lying obliquely side by
side, with their long axes nearly radial. Later, owing to the
approach of the large cells 3° and 3a’, the cell 2d*** becomes
pushed upwards, losing its connection with the entoderm when
these cells meet in the middle line.

The next divisions in this quadrant occur in the cells 2a**’,
2a°**, etc. The direction of the cleavage is nearly radial, but
slightly dexiotropic. The corresponding cleavages in the 6
quadrant are delayed until a later period. The upper pair
of cells in the quadrants a, c, and d@ next divide, the right
cell laeotropically, the left dexiotropically. The cells from the
adjacent arms of the cross push under the trochoblasts from
either side and often meet each other, thus separating the
trochoblasts entirely from the cells of the third quartette
(Pl. XIX, Fig. 32). Owing to the forward rotation of the
apical cap of cells, the cells 24"*' and 26**" become pushed
apart, so that they come to lie on either side, instead of above
the’cells 20°-7 and. 20°"* (see Pl. XX, Fig, 30).) @hevcleavace
of the tip cell 26™ has already been mentioned ; its two daughter-
cells, owing to the rotation of the apical pole, come to lie side
by side and in contact with the cells 26"** and 26*"*. The cells
BUPULZO. 420,20, 20, and 26°." all) become aroe; and
clear and enter into the formation of the prototroch. In the
anterior quadrant the three upper cells of the four, therefore,
enter into the prototroch as in the annelids, and their products
undergo, I believe, no further divisions beyond the stage just
described. The cell 20*** divides horizontally ; this is the last
cleavage in this quadrant that could be observed. The cleavage
of the second quartette has been followed to a stage in which
there are eleven cells in each of the quadrants a, c, and d, and
ten in the anterior, or 4, quadrant. After this stage the divi-
sions of this quartette become very difficult to follow. I have
seen a nearly horizontal cleavage of 2c***", and have been able
to recognize the cells of this quartette when there are fifteen
cells in each quadrant, but only hypothetical derivations of these

406 HOLMES. [Vou. XVI.

cells can be given. The group of cells in the 4 quadrant of the
second quartette is shorter and broader than the other groups.
This is doubtless due to the forward rotation of the apical pole
of the egg, which would exert a vertical pressure on this group
of cells. The slower cleavage in this group may be also due,
in a certain degree, to the same cause, though it is probably
correlated with the fate of these cells. The portion of this
quadrant which does not go into the prototroch forms only an
exceedingly small portion of the body of the embryo, being
used, as far as could be determined, to form a part of the
stomodaeum.

The early cleavages of the second quartette in Planorbis are
very similar to those of other gasteropods. Kofoid has traced
the cleavage of this quartette in Limax to a stage in which
there are four cells in each quadrant, and these cells agree
almost exactly in relative size and arrangement with those in
Planorbis at the same stage. Blochmann ('82) figures a stage
in which there are seven cells of this quartette in each quad-
rant, and their arrangement is very similar to that found in
Planorbis. Heymons has traced the lineage of this quartette
to a stage in which there are eleven cells in each quadrant, but
he does not describe the direction of the cleavage of the tip
cells. Conklin has followed the cell lineage of the second quar-
tette to a stage in which each quadrant contains eleven cells,
and has traced the cleavage of the tip cells somewhat beyond
this point. The agreement of the direction of the divisions of
this quartette in Planorbis with those in the above forms is quite
close, but there are some minor differences. In general, we
may say that the cleavages are more nearly radial than in the
two last forms. For instance, the cleavages of 2a**’ in Crepi-
dula and 2a*** in Umbrella are nearly horizontal, while in Pla-
norbis they are nearly radial. All the cells lying between the
tip cell and the lowest cell of the group have, at a stage when
there are eleven cells in each quadrant, exactly the same lineage
in Umbrella, Crepidula, and Planorbis. In the first two forms
the cleavage of the tip cell has occurred, while the lowest cell,
2a°’, 20°", etc., remains undivided ; in Planorbis the lowest cell
has divided, while the tip cell, except in the anterior quadrant,

No.2.] LARLY DEVELOPMENT OF PLANORBIS. 407

remains entire. In both Umbrella and Crepidula the divisions
of the second quartette, as far as they have been traced, are,
with the exception of the cleavage of the tip cell in the latter
form (and, possibly, also in the former), perfectly similar in
every quadrant. This may be due to the fact that the forward
rotation of the apical pole occurs later in these forms. The fact
that when this rotation is delayed the similar character of the
division of the quadrants of the second quartette is maintained
for a longer period, lends additional support to the view that in
Planorbis the delayed cleavage in the anterior quadrant is caus-
ally connected with this rotation. The early beginning of this
rotation in Planorbis, caused as it is by the more rapid growth
of the posterior trochoblasts and the posterior arm of the cross,
may be viewed as a result of the early formation of the head
vesicle. This structure develops early in Planorbis, and reaches
a larger size than in Crepidula or Umbrella. The different be-
havior of the cells of the anterior quadrant of the second quar-
tette in these different gasteropods may thus be considered a
sort of indirect effect of the different degrees of development
which the head vesicle attains in these forms.

The Mesoblastic Bands,

The mesoblastic bands in Planorbis have been fully described
by Rabl, who was the first to derive the mesoderm in the gas-
teropods from a single cell. A division occurs, however, after
the first cleavage of the primary mesomere, which it seems that
Rabl overlooked. After the two mesomeres have come to lie
entirely in the cleavage cavity, each buds off at the anterior end
a minute clear cell, which is often quite difficult to observe.
The spindles are inclined slightly towards each other at their
anterior ends, and the small cells that arise lie almost in con-
tact with each other. The next cleavage of the mesomeres is
horizontal and equal, and at right angles to the preceding divi-
sion. There thus result an inner and an outer pair of large
mesodermic cells, which are figured by Rabl in Figs. 17a and
176. The inner pair of cells are the mesoblastic teloblasts.
The next cleavage of the teloblasts is in the same direction as

408 HOLMES. [Vou. XVI.

before, but this time the division is unequal and gives rise to
a small cell lying between the middle and outer cells. The next
cleavage occurs in the outer pair of cells and in the same direc-
tion as the preceding division. In fact, all the divisions in the
mesoblastic bands are henceforth in the same direction, until a
considerably later period of development. At the time when
there are three cells in each band the mesomeres form a con-
cave row of cells in the posterior half of the egg. As the
bands lengthen by teloblastic budding, they assume the shape
of a horseshoe. Later they become resolved into scattered
cells.

The Third Quartette and the Secondary Mesoblast.

At the stage in which the egg contains forty-nine cells the
cells of the third quartette are eight in number, arranged in
four vertical pairs, lying over the angles between the cells of
the fourth quartette. The first cleavage of this quartette forms
a transition from the spiral to the bilateral type, and the subse-
quent cleavages show a bilateral character in a more marked
degree. At nearly the same time the /ower pair of cells, 30°,
3c’, in the two anterior quartettes, and the wffer pair of cells,
3a", 3a’, in the posterior quadrants, divide in a nearly horizontal
direction into equal moieties. Later, at about the sixty-four-cell
stage, the upper pair of cells in the anterior quadrants, 34’, 3c’,
divide in the same direction as the lower pair. The lower pair
of cells in the two posterior quadrants, 3a’, 3¢@*, remain undi-
vided until a much later stage. In each of the two anterior
quadrants there are now two pairs of cells, the one pair lying
directly above the other. In each of the posterior quadrants
there is a pair of cells lying over a large undivided cell. It
is easy to orient the egg from the lower pole at this stage by
the bilateral arrangement of these cells. Definite spiral cleav-
age, which appears in such a marked way in the early divisions
of the first and second quartettes, seems entirely absent in the
third. The first cleavage is radial, and all the succeeding divi-
sions appear to be bilaterally symmetrical with reference to the
median plane of the future animal.

NerZz "LARLY DEVELOPMENT OF PEANORBIS. 409

The next divisions in this quartette occur in the cells 3a",
32,132, 32, om the posterior ‘side of the eseand ini307,
30°", 3c°", 3c°*, on the anterior side. These divisions are radial
and similar in character, each cell budding off a small cell toward
the vegetal pole. There thus arise four pairs of small cells, the
two anterior pairs lying in the angles between the entomeres
on either side of the median plane, the two posterior pairs lying
above the large cells, 32° and 3d°. The upper pair of cells in
each of the four quartettes next divide in the same direction
as before, forming a vertical series of four pairs of cells in the
anterior quadrants, and a similar series of three pairs of cells
above the large cells, 3a° and 3@’, in the posterior quadrants.
The third quartette now contains thirty cells, eight in each
anterior quadrant, and seven in each posterior one.

dhe) twoxpaits of cells,30*"",,. 30°", 3c°"’, andi ges.) became
pushed in towards the cleavage cavity and become partly cov-
ered by the surrounding cells. They divide in a nearly hori-
zontal direction, and their daughter-cells become pushed into
the cleavage cavity still further. They form an irregular row
of four small cells lying above the pairs of small cells. in the
angles between the entomeres. The period of their division is
quite variable. In one case (Pl. XIX, Fig. 27) a division has
evidently occurred in 3c**" before the upper cells have divided,
but this does not usually occur. In Pl. XIX, Fig. 33, 3c**" has
divided, while the cell lying beside it is entire, as are also the
corresponding cells in the 4 quadrant. The lower products of
the division of the uppermost cells in the anterior quadrants,
30, 30°») 36, 3é°, divide ina nearly) radial ‘direction.
Pl. XIX, Fig. 33, shows 3c""’ dividing, while the cleavage in
3c’** is completed. The number of cells in each anterior quad-
rant is now twelve; the small cells in the angles between the
entomeres have remained undivided since their origin; above
this pair are the four cells, which are partly sunk into the cleav-
age cavity, arranged in a transverse row, and above these again
are three pairs of cells in a vertical series. The number of cells
in each posterior quadrant is still seven, and the whole number
of cells in the third quartette is thirty-eight. The entire egg
contains at this period about 150 cells. The four cells in each

410 HOLMES. [VoL. XVI.

of the anterior quadrants, which lie partly pushed into the cleav-
age cavity, finally lose connection with the ectodermic wall and
come to lie in the blastocoel. The cells from the two sides
nearly meet, forming a curved row of cells, the posterior ends
of which nearly meet the anterior ends of the mesoblastic bands,
which curve forward from the posterior side of the egg. The
anterior row of cells forms what Wierzejski calls the secondary
mesoderm. As far as can be judged from the very brief descrip-
tion of this process in Wierzejski’s preliminary paper, the forma-
tion of the secondary mesoderm in Physa is very similar to, if
not identical with, its formation in Planorbis. The first cleav-
age of the cells of the third quartette in Physa is radial, as in
Planorbis, and the lower cell in the two anterior quadrants divides
horizontally into a right and left cell. The cleavage of the upper
cell is not described. The next division of the lower cells is
the same as in Planorbis, each giving off a small cell toward
the vegetal pole. The second pair of cells, 30°", 30°", 3¢°7,
3c°°", divide again in a radial direction, the upper cells going
to form mesoblast; the fate of the lower cells was not deter-
mined. In Planorbis this cleavage is nearly transverse, the
outer cells being somewhat higher than the inner ones, and
probably corresponding to the upper cells (Mutterzellen) in
Physa.';, lf the lowericells)/ 300777 and 307), 3¢1 4.36) 17, agse
fate Wierzejski did not determine, also form secondary meso-
blast, the cell origin of the secondary mesoblast in the two
forms would be identical.

The last cleavages observed in the cells of the third quartette
were those of the large cells, 3a* and 3¢@°. These divide bilat-
erally and in a nearly horizontal direction, a small cell being
given off from each at the outer end. Later these cells give
off another small cell in the same direction as before. After
these two divisions these cells are considerably reduced in size,
but at the time gastrulation begins they form a rather conspicu-
ous pair of cells, lying behind the nearly circular group of ento-
meres. The third quartette at this stage is composed of forty-two
cells.

No.2.) 2ZARLY DEVELOPMENT OF PLANORBIS. 4II

General Considerations on the Secondary Mesoblast.

The origin of the secondary mesoblast in the Mollusca is a
subject to which, for many reasons, considerable interest is
attached. We possess, however, at present very few accounts
of the process by which the secondary mesoblast arises. The
first case in which a double origin of the mesoderm has been
carefully and accurately traced is that of Unio, studied by Dr.
Lillie. In this form both the primary and secondary mesoblast
are segregated at an early period. The primary mesoblast arises
from 4d, as in other mollusks; the secondary or “larval meso-
blast,” as it is called by Lillie, arises asymmetrically from a
cell of the second quartette, 2a’, on the left side of the egg.
This cell is gradually overgrown by the surrounding cells, and,
after budding off two or three small cells to the surface, comes
to lie entirely in the blastocoel. Although the secondary meso-
blast arises asymmetrically, it afterwards becomes disposed in
a symmetrical manner by the migration, apparently, of some of
the cells to the opposite side of the egg. The larval mesoblast
forms a kind of mesenchyme, which gives rise to certain larval
structures, which disappear in later development, and its early
segregation appears to be correlated with the early development
and importance of these organs in larval life. It is a significant
fact that the cell from which the larval mesoblast arises is larger
than the corresponding cell on the other side of the egg.

Secondary mesoblast was discovered later in Crepidula by
Conklin. It was found to arise near the edge of the blastopore
in the three quadrants, a, J, and c, in which no other mesoblast
was produced. The exact cell origin of this mesoblast Conklin
was unable to trace, owing to the large number of cells in the
egg at that stage, but, from the position of the mesoblast cells,
it was shown to have arisen from the cells of the second quar-
tette. There is an anterior mesoblast cell in the 4 quadrant,
and a right and left cell bilaterally placed in the a and c quad-
rants. The d quadrant produces no secondary mesoblast unless
at a*very much later period. The mesoblast in Crepidula arises,
therefore, in each of the four quadrants.

The secondary mesoblast in Physa, according to Wierzejski,

412 HOLMES. [VoL. XVI.

and in Planorbis, according to my own observations, has yet a
different origin, arising from the cells of the third quartette in
the two anterior quadrants. It arises, as in Crepidula, at a late
period of cleavage, and its origin is likewise bilateral. As the
cells of the third quartette are arranged symmetrically on either
side of the median axis of the egg, its origin from three quad-
rants could not be bilaterally symmetrical. It is quite certain
that, in Planorbis at least, no secondary mesoblast arises from
the posterior quadrants, unless at a very much later period of
development.

Several cases have been pointed out by Lillie, among accounts
of the embryology of the lamellibranchs, where the figures of the
authors show strong evidence of the existence of secondary meso-
blast. The figures of Cyclas by Ziegler and Stauffacher, of
Teredo by Hatschek, of Anodonta by Goette and by Schierholz,
of Ostrea by Horst, show mesoblast cells in the early stages of
gastrulation that could scarcely have arisen from the pole cells.
In all these genera, cells are figured in front of, as well as
behind, the blastopore. There are also similar cases in papers
on other groups of mollusks. Kowalevsky’s figure of a sagittal
section of the larva of Dentalium shows a large mesoblast cell
in the blastocoel at either end of the gastrula. And similar
indications of secondary mesoblast are shown in Fol’s figure of
a sagittal section of the larva of Firoloides.

The case of Paludina vivipera is an interesting one in this
connection. It is one of the few points of agreement, among
those who have worked on the form, that mesoblastic pole cells
do not occur. Tonniges finds that, in this form, mesoblast is
produced from certain cells lying in front of the blastopore. If
we accept Tonniges’s account, the formation of the mesoblast
in Paludina would seem to correspond to the formation of the
secondary mesoblast in other forms.

The researches of Eisig and Wilson on the development of
annelids suggest that the occurrence of secondary mesoblast
may be typical for both annelids and mollusks, and indicate
another striking point of agreement to the many which exist
between the methods of cleavage of these groups. At the same
time they serve to connect more closely the cleavage of anne-

Non2s\) LARLY DEVELOPMENT OF PLA NORSIS. 413

lids and mollusks with that of the polyclades, in which the
mesoderm has a radial origin from one or more quartettes of
micromeres. Until more is known, however, of the origin, and
especially of the fate, of the cells which have been called sec-
ondary mesoblasts, it must remain uncertain whether there is
any true homology between these cells and the mesoblast of
the polyclades, although such a comparison naturally suggests
itself. The subject is one of considerable interest from the
standpoint of phylogeny, and the reader may be referred for
suggestive discussions of the problem to the papers of Conklin
(27), Wilson (98), and Eisig ('98).

The Entomeres.

The egg of Planorbis is peculiar among the eggs of mollusks,
in that the entomeres are of small size and undergo numerous
divisions before the beginning of invagination. The fourth
quartette consists of cells which greatly exceed in size the four
small cells at the vegetal pole. The three cells of this quartette
which form entoderm, 4a, 40, and 4c, divide horizontally when
the egg contains about fifty-six cells. The six cells resulting from
this cleavage are arranged in the form of a horseshoe about the
four cells in the center, the opening between the ends of the
curve being on the posterior side of the egg. The next cleay-
age occurs when the egg contains about ninety cells. Each of
the six cells of the fourth quartette divides in a nearly radial
direction. These divisions are, however, slightly oblique, and
are bilaterally symmetrical with respect to the median plane of
the egg. The divisions of the cells on the right side of the egg
are slightly laeotropic, while those on the left side are slightly
dexiotropic. These divisions are followed by a cleavage of the
three small cells, A, B, and C, at the vegetal pole, forming a
fifth quartette. The cell D, which is the smallest of the group,
does not divide; nor have I been able to observe its cleavage at
any subsequent stage, although it could be recognized after the
process of gastrulation had made some progress. It is quite an
exceptional fact that the cell D, which, in many forms, is the
largest cell of the egg, should, in Planorbis, be the least in size

AI4 HOLMES. [Vor. XVI.

of allthe cells. From the twenty-eight-cell stage until the period
of gastrulation it has remained without a single division. The
cleavage of the central cells is bilateral; the cell B divides
radially ; the cleavage of A is laeotropic; that of C dexiotropic.
These divisions are followed by an equatorial cleavage of the
cells of the fourth quartette. The derivatives of 44 divide first,
but the cleavage of the cells in the other two quadrants soon
follows. There are, after these divisions are completed, twenty-
four cells of the fourth quartette and three of the fifth; these,
with the four small cells at the vegetal pole, make a total of
thirty-one entomeres. The entoderm cells at this stage are of
small and about equal size, and are easily distinguished from
the surrounding ectoderm cells by their yellow color. Their
number is increased somewhat before gastrulation has pro-
gressed very far, but I have not attempted to follow their
cleavage beyond the point just described.

Small spheres of an albuminous substance gradually accumu-
late in the entoderm cells and become quite numerous near the
period of gastrulation. These spheres stain very darkly in
haematoxylin and make observation of the nuclei very diffi-
cult. At their first appearance, these dark bodies are found
also in the ectoderm; but, as development proceeds, they
become more and more confined to the entodermic cells. The
staining reaction of these bodies is much like that of the sur-
rounding albuminous matter in which the embryo floats, and it
is very probable that they are simply masses of albuminous
substance that has been ingested by the cells and has not been
chemically transformed. After invagination certain of the ento-
dermic cells increase enormously in size, becoming filled with a
transparent, yellowish substance that is little affected by stains.
Rabl has shown that in the end of the cells turned toward the
enteron there are masses of deeply staining substances which
he regards as material which has been absorbed by the cells,
but not completely transformed into the yellowish substance
which fills the greater part of the cell. It is doubtless the same
material that forms the darkly staining bodies in the egg.

The ingestion of albumin occurs in a similar manner in the
egg of Limax, and has been described by Meisenheimer, whose

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NO22.)) (LARLY DEVELOPMENT, OF PLANORBIS, AI5

figures show very clearly the history of the process. The deeply
staining bodies occur in all the cells at an early period, even in
the sixteen-cell stage, and increase in number as the develop-
ment proceeds. The cells of the ectoderm take in the albumin
more rapidly than in Planorbis; but, finally, this function is
transferred entirely to the cells of the entoderm. The process
of digestion of albumin, by which the egg is nourished and
enabled to grow, is carried on at first with equal facility by all
the cells of the egg. This process soon predominates in the
entomeres, although, for a time, it is carried on more rapidly
by all the cells. Finally, the ingestion of albumin is relegated
to the cells of the enteron, which have become specialized for
digestive purposes, and the other cells of the egg no longer
share this function.

The Rudiments of the Cerebral Gangha and Eyes.

The two portions of the cross, which are separated by the
median apical plate, form the regions which give rise to the
cerebral ganglia and eyes. These two rudiments, in eggs
stained with silver nitrate, appear as dark masses surrounded
on every side by large and very transparent cells. The tip
cells of the lateral arms, and the cell lying immediately above
them, do not enter into the formation of these masses, but
increase in size and go into the head vesicle. With the excep-
tion of these two cells in each arm, all the cells in the lateral
arms of the cross, the cells of the anterior arm, except the tip
and basal cell, and the central region of the cross, except the
four apicals and the two cells lying in front of them, enter into
the formation of these two rudiments. The composition of
these two patches of cells when they are definitely marked off
may be seen in the following table:

Lerr RUDIMENT. RiGHT RUDIMENT.
Tqrt2-2 [ghe2:l1.L.2 [chsl-2:2 [Gte2-2-1-1
1gi-l-22 {Hll-2.2 [ohe2-1-1.2 [Gle2-2-1.2
Tgqhe2--1.2 [ht-2-1.2 2.1 Teh2-1-2.1 TGle241-2.1.2
[gh2-1-2-1 [Gl-2-1.2.2.2 [ch 2-1-2.2 [Ge21-2-1.2
[qte2+1.2.2 1Gl-2-2-2-1 Tcl-21eLLd tghhen

Jqi-2-11.¥.1 [ Gl:2.2-2.2 Toh2 111.2 Iq '1-2.2

416 HOLMES. [VoL. XVI.

There are the same number of cells in each of the two rudi-
ments which, as far as the size and position of their component
cells is concerned, show a perfect bilateral symmetry. Yet the
derivation of the cells on the two sides does not exactly corre-
spond. The right rudiment contains cells from three quadrants,
while the left contains cells from but two. This is because
the intermediate cells fromthe d@ quadrant lie to the right of
the median axis of the egg. The posterior arm of the cross,
which is composed of cells of this quadrant, takes no part in
the formation of these structures. The two products of the
cleavage of the intermediate cell and the apical 1d@°™’, which
enters the apical plate, are the only cells of the first quartette
in the @ quadrant which do not go to form the head vesicle.

The cells forming the cerebral rudiments multiply with great
rapidity. The areas become thickened and a proliferation of
cells occurs which gives rise to the cerebral ganglia. The
early appearance of these rudiments was observed by Rabl,
who designated them a bilobed apical plate (Scheitelplatte).
The further history of the fundaments of the cerebral ganglia
may be followed in Rabl’s paper. The areas above described
are not exclusively employed in the formation of the cerebral
ganglia and eyes. The cells become so numerous before differ-
entiation begins that it is impossible to trace the cell origin of
the different structures arising from them. As the eyes arise
at the outer sides of the cerebral ganglia, it is quite certain
that they are formed from cells derived from the lateral arms
of the cross. Whether this can be said also of the tentacles is
uncertain.

The Apical Plate.

The term ‘apical plate’’ has been applied by Conklin to a
median belt of large, clear cells in Crepidula extending from
the apical sense organ to the prototroch. It is composed of
seven cells which become covered by fine cilia and remain for
a long time undivided, while the neighboring cells rapidly mul-
tiply. In Planorbis there is a median belt of large cells extend-
ing from the head vesicle to the prototroch, and which, from its
similarity to the apical plate in Crepidula, I have designated by

No: 2.)) BLARLY DEVELOPMENT OF PLANORBIS. 417

the same name. The peculiar apical sense organ in Crepidula,
I am satisfied, does not occur in Planorbis. In the former
genus the four cells which form this organ remain of small size,
acquire a tuft of long cilia, and become united later with a pair
of nerve cords which arise from the cerebral ganglia on either
side. The existence of such a structure in a molluscan larva
is a striking mark of relationship with the annelid trochophore.
The presence of this organ in the larva of Planorbis might
naturally be looked for, but the four apical cells which form
this organ in Crepidula become in Planorbis very much enlarged
and thinned out and form a part of the apical plate. The cells
which compose this plate are six in number, the four apical
cells just mentioned and a pair of cells lying in front of these.
Of the origin of this pair of cells I am not entirely certain, but
I think they are the cells 10°"*" and 1c*"*’, These cells arose
from the divisions of the intermediate cells lying in the angles
between the arms of the cross. The cell lineage of the apical
plate in Planorbis may be expressed as follows :

apieancelisita” 10, Ics 74, Laan
intermediate cells Ua rcrht

The anterior end of the apical plate is limited by the upper
median cell of the prototroch, and its posterior boundary is
formed by the basal cell of the posterior arm of the cross.

The Cell Lineage of the Head Vesicle.

Owing to the fact that the cells composing the head vesicle
in Planorbis are few in number and of large size, I have been
able to determine the exact lineage of all the components of
this structure. The head vesicle may be said to first appear
when the posterior trochoblasts and the cells of the posterior
arm of the cross begin to enlarge. This enlargement, which
begins before the 100-cell stage, causes the upper pole to move
toward the anterior side of the egg. The tip cells of the lateral
arms of the cross enlarge rapidly, and, at a later period, the
cells 1a**’, 1c**’, lying just above the tip cells, also enlarge. All
of these cells, owing to the anterior rotation of the upper pole

418 HOLMES. [VoL. XVI.

of the egg, come to lie behind the masses of cells which form
the rudiment of the cerebral ganglia.

The cells composing the head vesicle are given in the follow-
ing table:

Iq ':2-t

i Id 1.2,1,2
Cells of the posterior arm of the cross

Id? I
I qd 22
Posterior trochoblasts

Iq! 2.2
Pes tp
Cells of the lateral arms of the cross | ih

The number of cells in the head vesicle is twelve, of which
nine belong to the first and three to the second quartette. The
area of the head vesicle, when it reaches its maximum size, is
fully equal to that of the rest of the embryonic body. The
cells composing it are very thin and transparent and of rela-
tively enormous size. All of the cells which make up the head
vesicle were present in the egg after the division of the cells
1a™’, 16°’, etc., which occurs at about the 64-cell stage, and
only one cell which is destined to form a part of the structure,
viz., td***, undergoes division after this period. The cells,
indeed, become so exceedingly thin in later stages that it is
difficult to see how their division could be effected. What
becomes of these cells when the head vesicle disappears is
uncertain.

The Cell Lineage of the Prototroch.

It has been long known that the velum in the pulmonate gas-
teropods is a rudimentary structure. It was first noticed by
Vogt, and has been described since in various pulmonates by
Rabl, Fol, and Lankaster. The velar lobes, which are such a
characteristic feature in the so-called veliger stage in other mol-
lusks, are absent in the pulmonates, and all that can be said to
correspond to the velum is a double row of ciliated cells extend-

No.2) LARLY DEVELOPMENT, OF PLANORSIS. 419

ing from the ventral side of the body, immediately in front of
the mouth, towards the dorsal side of the embryo. This row of
cells has been called the prototroch, from its similarity to that
structure in the annelid trochophore. Owing to the marked
resemblance of this structure in the larvae of two such distinct
groups as mollusks and annelids, considerable interest is natu-
rally attached to a comparison of its cell origin in these forms.

The cell origin of the prototroch was first determined in the
annelids by E. B. Wilson, who discovered that in Nereis the
four cells, 1a’, 10°, etc., which he called the trochoblasts, gave
rise to this organ. Later, the origin of the prototroch in sev-
eral annelids has been carefully studied by Dr. A. D. Mead, with
the result that the cell origin of this organ was shown to be
identical in every case. In Amphitrite and Clymenella the cell
lineage of the prototroch was carried out in detail to a late stage,
but the other forms studied agree with these as regards the
protoblasts of this organ as far as their cleavage was observed.
In both Amphitrite and Clymenella the prototroch arises from
the four trochoblasts, which divide twice, forming sixteen cells,
and three of the cells of the second quartette in each of the
three quadrants, a, 6, and c. Thus three out of the four cells
of the second quartette in each of these three quadrants go to
form the prototroch. Mead argues that in Nereis, also, the pro-
totroch arises in the same way, although Wilson’s derivation of
this organ differs from Mead’s as regards the fate of the upper
cells of the second quartette. Child’s account of the formation
of the prototroch in Arenicola agrees, point for point, with
Mead’s, and Mr. Treadwell’s observations in Podarke indicate
a similar origin of the prototroch in that form (Child, '97;
Treadwell, ’97).

So far as known, the prototroch in the Mollusca arises in a
manner which is strikingly similar to its origin in the annelids.
Blochmann’s derivation of the velum in Neritina from the pecul-
iar granulated tip cells of the lateral arms of the cross is doubt-
less incomplete, since these cells form only a part of this organ
in other mollusks, as in annelids. In Crepidula the velum at
first consists of a double row of cells, the dorsal ends of which
become “indistinguishable from the surrounding cells.” ‘The

420 HOLMES. [Vou. XVI.

median portion of the first row,” says Conklin, “arises from the
cells which lie just beyond the ventral end of the apical plate.
These cells are in all*probability 10°**"? and 16°***? [inserting
the correction in Conklin’s note, p. 204]. One of these cells
is shown dividing in Fig. 71. In Fig. 72 a transverse row is
formed, which is plainly the first row of velar cells.”” The por-
tion of the first velar row lateral to these six cells is evidently
derived from the anterior turret cells, Ia2* and 10°. The ante-
rior turret cells divide bilaterally in a nearly horizontal plane,
and these, with the median cells just mentioned, form a row of
cells, extending around the anterior half of the egg, connecting
the tip cells of the lateral arms. The tip cells of the lateral
arms divide, forming a transverse row of four cells, which forms
a further continuation of the first row of velar cells as far as the
undivided posterior trochoblasts and tip cells.

Regarding the second row, Conklin says: ‘It is probable
that the mid-ventral portion of the second velar row, V7’, is
derived from the cell which I have identified provisionally as
26°’, and which lies just beyond the median cells of the first
row (Figs. 56, 69, and 70). I have not been able to determine
whether any part of the second row arises by subdivision of the
cells of the first ; if not, this row may include a few cells of the
third quartette (3a""" and 30°"', Fig. 56) at the points opposite
the anterior turrets. . .. Thus the preoral velum is composed
of a few cells of the first quartette, many of the second, and
possibly a few of the third.”

In Planorbis the cells composing the prototroch are few in
number and are arranged in a double row. The products of the
division of the tip cell of the anterior arm of the cross go to
form, as in Crepidula, a part of the upper row of cells. The
tip cell divides, as far as I can determine, but once, and the two
daughter-cells become pushed apart by the cell 16°*"", which
forms the median cell of the upper row. These cells extend
to the anterior trochoblasts on either side, but, in later stages,
they may sometimes be separated from them by cells which
wedge in from below. The anterior trochoblasts, which origi-
nally lay, the one above the other, became shifted by the ante-
rior rotation of the upper pole of the egg, so that they come to

Now2in 2ARLY) DEVELOPMENT OF PLANOREIS. 421

lie at nearly the same horizontal level, the originally upper cell
lying in front. The tip cells of the lateral arms lie immediately
behind the anterior trochoblasts, but do not, as I formerly sup-
posed, form a part of the prototroch, but enter into the forma-
tion of the head vesicle. They mark, in fact, the transition
between these two structures, and might be considered as
greatly enlarged velar cells. The chief differences between the
first row of velar cells in Planorbis and Crepidula are that in
the former the lateral tip cells become greatly enlarged and do
not divide, and the anterior tip cell divides but once, the daugh-
ter-cells becoming pushed apart by one of the cells of the first
quartette.

The lower row of cells in the prototroch is derived from the
second quartette. Conspicuous among these are the two large
cells lying below the median portion of the upper row. They
are symmetrically placed on either side the median line. The
two cells originally lying above these, 24°*" and 26*"', have
been forced aside by the forward rotation of the upper pole,
so) that the tip cells, 20°°" and 2d'"”, come to lie ‘next to) the
cells 25°** and 26*"*, The cells of the lower row at the sides
are small, and are added to the prototroch at a later stage,
when the number of cells in the regions from which they arose
was so great that it would be very difficult to trace their lineage.

The Shell Gland and the Foot.

The shell gland makes its appearance some time after the
closure of the blastopore. A very early stage in the develop-
ment of this structure is shown in Pl. XXI, Fig. 51. It is
located a short distance behind the tip of the posterior arm of
the cross, in a region which is formed from the cells of the
second quartette. It is derived, doubtless, from derivatives
of 2d’ and 2d*". It forms a tolerably deep invagination, the
cavity of which becomes almost entirely obliterated.

The foot arises as a protuberance behind the mouth. The
two halves are separated by a rowof clear cells extending back-
ward some distance from the definitive mouth —a fact which
may or may not indicate a double origin of this organ. A very

A422 HOLMES. [Vou. XVI.

similar group of cells is seen in the middle portion of the foot
in Crepidula (Conklin, '97, p. 143). Both Lillie and Conklin
derive the foot from cells of the second quartette. It seems
probable that, in Planorbis, cells from the third quartette also
enter into its formation. The cells immediately behind the
blastopore are derived from the third quartette, and it is not
unlikely that the median portion of the anterior end of the foot
is derived from some of these cells.

The Larval Kidney.

The larval kidney in Planorbis has the form of a V-shaped
tube, situated on either side of the body behind the head. At
the junction of the two arms is a large perforated cell, the
so-called giant cell, the lumen of which communicates with the
canals of the arms. The inner arm is directed toward the head;
it is formed of a row of perforated cells, and the lumen is cili-
ated and provided with a ciliated opening near the end. The
outer arm is directed downward and is provided with an opening
to the exterior. The movements of the cilia lining the arm of
the larval kidney may easily be seen in the living embryo.

The larval or head kidney of the pulmonates has been observed
several times by the older writers on the embryology of these
forms (Stiebel, Ganin, Gegenbaur, Stepanoff), and it has been
mistaken for the rudiment of the nervous system, and also for
the oesophagus. The giant cell discovered by Biitschli rather
strangely had escaped the notice of Fol, who has given an
otherwise quite accurate description of this organ.

The development of the larval kidney of pulmonates has been
studied by Fol, Rabl, and Wolfson, all of whom came to very
different conclusions regarding its origin. According to Fol,
the larval kidney in Planorbis makes its first appearance as
a small ectodermal pouch. ‘La poche s’approfondit dans la
direction du dos, de telle fagon qu’elle finirait par rencontrer
celle du cété opposé sur la ligne dorsale. Mais la croissance
dans ce sens s’arréte de bonne heure, l’organe se recourbe a
peu prés a angle droit et s’allonge maintenant dans la direction
de la bouche.” The larval kidney, therefore, according to Fol,

NOI 2); ZARLY DEVELOPMENT, OF PLANORSIS. 423

is entirely of ectodermal origin. In a paper on the development
of the fresh-water pulmonates, Rabl (75) described this organ,
which he mistook for a part of the nervous system, as arising
in a somewhat similar way to that described by Fol.

According to Wolfson’s account of the larval kidney in
Lymnaea, a large velar cell on either side of the embryo is
pushed inward, becomes perforated by a canal, and gives rise
to a hollow outgrowth which is directed anteriorly and opens
by a ciliated mouth. Wolfson holds, in opposition to Fol, that
the Urniere are unicellular organs, basing his opinion on the
study of a large number of sections.

The account of the origin of the larval kidney given in Rabl’s
later paper, “‘ Ueber die Entwicklung der Tellerschnecke”’ ('79),
differs radically from the foregoing descriptions. According to
Rabl’s later account, the origin of the larval kidneys can be
traced back to a large cell lying in the mesoblastic bands.
These large cells, according to Rabl, result from the first divi-
sion of the mesoblastic teloblasts, though it is more probable
they arose from the second (see p. 407). By the teloblastic
budding of the two middle cells, two rows of smaller cells are
produced which carry forward the large cells, v,, v,, at their
ends. These large cells also bud off small cells anteriorly, like
the teloblasts. Each mesoblastic band comes thus to consist
of a large cell with a row of smaller cells in front, followed by
another large cell with a similar row in front of it. The cells
v, and uv, become perforated by a canal and come to be elongated
and bent, forming the giant cell of the larval kidney. Then
some of the cells lying in front and behind the giant cells also
become perforated and form the anterior and lower arms. The
larval kidneys, therefore, according to Rabl, are multicellular
organs and entirely mesodermic in origin. The teloblastic
budding of the protoblasts of the larval kidneys is a fact of
much interest. It recalls the behavior of the nephroblasts
described by Whitman (’78) in Clepsine, only in Planorbis the
two teloblastic series are placed end to end, instead of side by
side. Such a difference might easily be produced by a varia-
tion in the direction of one of the divisions of the teloblasts.

In view of the contradictory accounts of the origin of the

424 HOLMES. [VoL. XVI.

head kidneys, and the important theoretic bearing of the sub-
ject, I was led to attempt to verify, if possible, Rabl’s descrip-
tion of the development of these organs. I have not followed
the subject in detail, but have carried it far enough to satisfy
myself of the essential correctness of Rabl’s results. The large
cells, v,, v, of Rabl are very conspicuous elements of the meso-
blastic bands, especially in later stages, as they increase consid-
erably in size. They may frequently be seen perforated by a
canal; and one case was found in which the canal did not
extend entirely through the cell, but was narrowed to a point
near the posterior side. The shape of the canal would seem to
indicate that it arose by a sort of invagination at the anterior
end of the cell. It may be, however, that the perforation arises,
as has been shown in other cases, by the coalescence of a series
of intracellular vacuoles. The mesodermic origin of the giant
cell of the head kidney is a matter about which, I believe, there
cannot be any doubt. Whether a portion of the external canal
is ectodermic in origin, as Erlanger ('92) found in Bythinia, is
uncertain. The principal portion of the structure, however,
undoubtedly arises from the mesoderm.

There can be little doubt, when we study the structure and
development of the larval kidneys of the pulmonate gasteropods,
that these organs represent true nephridia. As the definitive
renal organs of the gasteropods are regarded as nephridia also,
there occur in the pulmonates two pairs of these organs. Lar-
val kidneys similar to those of the pulmonates have been
observed in the embryos of Oncidium and some _lamelli-
branchs. There are sac-like mesodermic larval kidneys in
Paludina and Bythinia, which may be the homologues of the
above structures. Whether or not the two pairs of renal
organs in Nautilus represent nephridial structures, it is quite
probable that the existence of the two pairs of nephridia should
be regarded as typical for the Mollusca. Whether this indi-
cates that the molluscan body is composed of two segments,
is a question which need not here be discussed.

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No.2) LARLY DEVELOPMENT, OF PLANORBIS: 425

Gastrulation,; Fate of the Blastopore.

The beginning of invagination may be observed when the
egg contains about 175 cells. The egg at this time is a hollow
blastula with comparatively thin walls, especially on the upper
side. The apical pole at this time has been pushed forward
through an angle of about 45°, and the rotation continues dur-
ing the process of gastrulation. For some time before invagi-
nation begins the egg flattens and the lower pole loses its
convexity. The invagination is first seen in the center of the
vegetal pole; the four central cells and the cells of the fifth
quartette first sink in, forming a small, round concavity; as
this pit becomes deeper, the cells of the fourth quartette are
involved in the process. All of the yolk-laden cells become
invaginated, and the stomatoblasts may be seen at the edges of
the depression. Doubt has been expressed as to whether the
cells called entodermic and ectodermic in the early cleavage of
mollusks really prove to be so by their history. In Planorbis,
at least, the edge of the blastopore marks quite sharply the
boundary between the protoplasmic and yolk-laden cells. The
invaginated area is at first nearly circular in outline, but, as the
depression deepens, it assumes an elongated form; and, finally,
the mouth of the gastrula becomes reduced to a narrow, slit-
like orifice. The length of the elongated blastopore becomes
reduced by closure from behind. The anterior end of the
blastopore is a comparatively fixed point, and is situated just
behind a pair of small cells of the second quartette. Pl. XX,
Fig. 46, shows a stage in which the blastopore is reduced to a
minute slit lying between two cells. Another preparation
showed these two cells in contact, so that the blastopore in
this species may be said to close, though it is only for a brief
period. The oesophageal invagination occurs very soon after
the blastopore closes, and at exactly the same place. Its orifice
is small and nearly circular, and becomes surrounded by cilia.
It gradually deepens and gives rise to a diverticulum on the
ventral side, which becomes the pouch of the radula.

The gastrulation in Planorbis is purely embolic. The cells
of the ectoderm do not slip over the edges of the entomeres, as

426 HOLMES. [Vor. XVI.

in many other gasteropods, even in the least degree. In the
species of Planorbis studied by Rabl the blastopore is said to
become the mouth. Whether this difference is merely a spe-
cific one, or whether Rabl failed to observe the blastopore dur-
ing the short time it is closed, is uncertain.

Part II. GENERAL CONSIDERATIONS.
Reversal of Cleavage and Reversed Asymmetry.

The fact that in certain gasteropods, with sinistral or reversed
shells, the cleavage is also reversed, was first pointed out by
Mr. Crampton (94). Crampton studied the cleavage of two
closely related genera of fresh-water pulmonates, Physa and
Lymnaea, the former of which has a sinistral shell, while in the
latter the shell is of the normal or dextral type. The cleavage
of Physa was found to agree, point for point, up to the stage at
which the primary mesoblast is formed, with that of Lymnaea ;
but with this exception, that the direction of every cleavage is
reversed. The reversal was observed in the cleavage which led
to the four-cell stage, and was shown to give rise to a different
position of the cross furrow in the two forms. Crampton also
points out that Planorbis, according to Rabl’s paper, affords
another instance of reversed cleavage. The possibility of a
causal connection between reversed cleavage and a reversal of
the shell was pointed out, but further discussion of the subject
was not attempted. Haddon’s figure of the eight-cell stage of
Janthina ('82) apparently shows, as Crampton observes, that the
cleavage of this form is reversed. This would form the only
exception to the rule that the cleavage is dexiotropic in all unre-
versed forms. It seems not unlikely, however, that Haddon’s
figure is misleading on this point.

The cleavage of another species of Physa, P. fontznalis, was
found to be reversed by Wierzejski, and Brooks (’79) figures a
four-cell stage of Planorbis parvus, which, according to his
statement concerning the origin of the two upper cells, affords
another instance of reversal of cleavage. There are, therefore,
two species of Physa and three of Planorbis in which the cleav-

Nov 2] LARLY DEVELOPMENT, OF FPLANORSIS: 427

age is known to be reversed.! Planorbis is not usually described,
however, as having a sinistral shell; in fact, the shell may be
markedly dextral. In the celebrated series of Planorbis shells
found in the deposits at Stannheim there is every gradation
between shells which are coiled in one plane and shells which
are as markedly dextral as those of Littorina or Paludina. Many
recent species, P. albus, complanatus, and nitzdus, for example,
have a shell with a more or less decided dextral coil. Never-
theless Planorbis is a reversed form, whatever the direction of
the coil of its shell may be. The anal and genital orifices and
the opening of the mantle cavity lie on the left instead of the
right side. This is the essential point; the direction of the
coil of the shell is a secondary matter. The shell of Planorbis
belongs to the type which Lang calls “ pseudo-dextral,”’ and has
no necessary connection with the essential features of the asym-
metry of the animal.

The fact that in five cases a reversed asymmetry of the ani-
mal is associated with a reversal of cleavage, while in all dextral
forms whose cleavage is known with any degree of accuracy
the cleavage is dexiotropic or unreversed, certainly affords a
strong presumption in favor of the view that there is some
causal relation between the nature of the asymmetry of the
body and the type of cleavage of the egg. And there are facts
which render this conclusion more or less probable a pvior7.
Conklin found that the beginning of asymmetry in Crepidula
could be traced back to the cleavage of a single entodermic cell.
The time and direction of the cleavage of this cell were found
to give the initial bending of the entodermic area in a direction
which determined the direction of the coil of the embryo. If
we suppose a reversal of cleavage to occur in Crepidula, leading
to a corresponding division on the other side of the body, it is
not improbable that there would result a reversal of the asym-
metry of the animal.

An interesting case in relation to this problem is afforded by
the “inverse embryos”’ of Ascaris, described by Zur Strassen
(96). In the normal embryos of Ascaris certain cells are

1T have recently found reversed cleavage in another sinistral gasteropod,
Ancylus rivularis Say. (See the American Naturalist, November, 1899.)

428 HOLMES. [Vou. XVI.

arranged in an asymmetrical but perfectly definite manner ;
but Zur Strassen found that in exceptional cases —in one egg
out of about forty —this asymmetry was reversed ; that is, the
arrangement of cells, normally occurring on one side of the
embryo, was found on the opposite side, one embryo being
the mirrored image of the other. As the adult Ascaris is, in
certain respects, asymmetrical, Zur Strassen was led to ascer-
tain the proportion of reversed specimens in thisform. It was
found that, out of 125 individuals, four were reversed. Reversed
adults occur, therefore, in about the same proportion as reversed
eggs. As reversed eggs were seen, even in advanced stages of
development, in an apparently normal condition, Zur Strassen
concludes that they develop into reversed adults. We have
here a reversal of cleavage occurring as an exceptional varia-
tion in eggs of the same species and probably giving rise to a
reversal of some features of the structure of the adult form.

In Planorbis I have been unable to trace the origin of asym-
metry to the cleavage of a single entodermic cell, as Conklin
did in Crepidula. Immediately before gastrulation begins the
entoderm is composed of a nearly circular patch of small ento-
meres, which are quite numerous (over 30), and of nearly equal
size. Iam quite certain that, before invagination, the entoderm
gives no hint of the direction of the coil of the adult animal,
nor does it manifest any appreciable asymmetry until a long
time after the gastrula stage. In the gastrula shown in PI.
XX, Fig. 46, the bilateral symmetry is almost perfect. This
gastrula contains several hundred cells; yet, if one carefully
examines the figure, which is an exact camera drawing showing
the outline of every cell, it will be found that for nearly every
cell on one side of the body a corresponding cell can be found
on the other side. The only deviation from bilateral symmetry
that could be observed in this gastrula was a slight torsion in
the cells of the head vesicle. The torsion can be observed in
most gastrulae, but whether it has any connection with the final
asymmetry of the animal could not be ascertained. Attention
has been called to the fact that the arms of the cross exhibit a
slight twist in a laeotropic direction, and that the direction of
the twist is doubtless connected with the reversed cleavage of

No. 2.] LARLY DEVELOPMENT OF PLANORBIS. 429

this form. This twist is slight, and can no longer be detected
when the cross comes to be mainly resolved into two isolated
patches of small cells. It is possible that the torsion of the
cross is never really lost, and that it is in some way connected
with the beginning of the reversed asymmetry of the animal.
If this were true, the reversed asymmetry of the adult would be
shown to be connected with the reversal of cleavage of the egg.
The egg, however, passes through stages of development in
which it exhibits a well-nigh perfect bilateral symmetry, so that
it seems scarcely possible to connect directly these two phe-
nomena. There must, however, be some structural basis for
the asymmetry of the adult in the stages which exhibit such
marked bilateral symmetry. The effects of reversed cleavage
may be seen in the asymmetrical arrangement of certain cells
forming the cross in the first stages of gastrulation. It does
not seem improbable that this asymmetry persists in stages in
which it can no longer be observed, and that it forms the struc-
tural basis of the reversed asymmetry of the adult.

The Relation between Reversed Cleavage and the Direction
of the First Cleavage Plane.

If we compare the four-cell stages of the eggs of Lymnaea
and Planorbis in respect to the relation of the first cleavage
plane to the median axis of the future animal, the fact will
become manifest that, in the two cases, this median axis is cut
by the first cleavage plane at a different angle. In Planorbis
and Physa the first cleavage plane makes with the median axis
a negative angle of about 45°; while in Lymnaea it cuts this
axis so as to form with it a positive angle of 45°. It is obvious
that, while in both cases the first cleavage plane is oblique to
the median axis of the future animal, the first cleavage plane in
the sinistral form is at right angles to this plane in the dextral
form. As the second cleavage plane is always at right angles
to the first, is it not reasonable to suppose that, in the sinistral
gasteropods, the first cleavage furrow corresponds with the sec-
ond in the other forms? Has there not been, in the reversed
forms, simply a reversal of the order in which the first two

430 HOLMES. [VoL. XVI.

cleavage planes make their appearance? And is not this the
circumstance that determines the different direction of spiral
cleavage in the reversed gasteropods?

It has been discovered by Conklin that the first cleavage in
Crepidula is prospectively spiral and dexiotropic, as indicated
by the rotation of the nuclei in the two-cell stage from left to
right. I have not convinced myself that there is an opposite
rotation of the nuclei in the two-cell stage of Planorbis, but the
second cleavage is clearly dexiotropic even in its first stages.
There is doubtless some structural basis for the different char-
acter of the second cleavage in Planorbis in the two-cell stage.
The second cleavage of Crepidula is laeotropic, and that of
Planorbis dexiotropic; and it seems probable that this difference
is connected with the different relations of the first cleavage to
the future longitudinal axis of the animal. If we suppose that
in Crepidula a preformed longitudinal axis is cut obliquely by
the first cleavage plane, it may help us to account for the rota-
tion of the nuclei in the two-cell stage, and consequently the
laeotropic second cleavage. As this axis in the sinistral forms
would be cut at the opposite angle, we may have, in this cir-
cumstance, an explanation of the different direction of the spiral
cleavage that is manifested in the second, if not somehow in
the first division of the ovum. Certain it is that, if the longi-
tudinal axis of the embryo is in any sense preformed in the egg,
it is cut at planes approximately at right angles to each other
in the dextral and sinistral forms. An oblique cleavage in
relation to the bilateral organization of the egg might cause a
certain amount of torsion that would manifest itself either at
the end of that division or at the beginning of the next. Given
two eggs in which this bilateral organization is cut at opposite
angles by the first cleavage plane, it is probable that the torsion
in the two cases would take place in opposite directions.

General Considerations on Spiral Cleavage.

If we glance over the literature on spiral cleavage, we shall
find that, in different forms, the first cleavage is said to stand
in different relations to the future longitudinal axis of the ani-

NO. 21) LARLY) DEVELOPMENT OF PLANORBIS: 431

mal. In most cases the first cleavage plane has been found to
be oblique to this axis, and in many forms the median axis is
found to lie approximately midway between the first two cleav-
age planes (Neritina, Physa, Planorbis, Clepsine, Discocelis).
In some instances, however, the first cleavage plane is said to
be transverse to the median axis of the embryo (Nereis, Um-
brella, Teredo). The instances in which the first cleavage
plane is said to be oblique are by far the most numerous, and
it will be well to devote some attention to the purported excep-
tions to this rule, with a view to determine whether these
exceptions are not more apparent than real. The first cleavage
furrow, in advanced stages of cleavage, comes to follow a very
crooked path, and the upper portion may run in a quite differ-
ent direction from the lower. Since the entomeres are usually
of large size, the portion of the first cleavage furrow lying be-
tween these cells has generally been taken to indicate the direc-
tion of the first plane of cleavage, while the upper part of this
furrow lying between the ectomeres has been disregarded. It
is this circumstance, as will appear, that gives rise to the dif-
ferent accounts of the axial relations of the first cleavage plane.
In the ectodermic portion of the egg the axial relations of the
first two cleavage planes are remarkably constant in all forms
with spiral cleavage. The different quartettes of ectomeres
have exactly the same relative arrangement in annelids, mol-
lusks, and polyclades; and cells of the same origin in these
forms have almost identical axial relations. In both annelids
and mollusks certain cells become arranged in the form of a
cross, the arms of which have very constant relations to the
embryonic axes. The arms of the cross in mollusks, which are
always made up of cells of similar origin, are always anterior, pos-
terior, right and left. In the annelids the cross is mainly formed
of cells corresponding to those lying between the arms of the
molluscan cross, and the arms lie at an angle of about 45° with the
median axis of the embryo. As the upper portions of the first
two cleavage furrows pass between cells of corresponding origin
in all these forms, they maintain almost as constant axial rela-
tions as the arms of the cross themselves. J/¢ may be satd that
the longitudinal axis typically bisects the angle between the upper

432 HOLMES. [Vou. XVI.

portions of the first two cleavage furrows. As Conklin ob-
serves, ‘no exception is known, either among mollusks or
annelids, to the rule that the second and fourth quartettes
lie in the future median and transverse planes, and that the
first, third, and fifth quartettes lie midway between these planes.
The axial differences, therefore, of the first two cleavage planes,
which have been mentioned, are differences merely in the axial
relations of the four primary entoderm cells, and do not affect
the axial relations of the other cells of the ovum, which are
always the same among annelids and mollusks.”

The behavior of the first cleavage furrows in Crepidula is
very instructive, for it proves that the coincidence of the lower
portion of the first cleavage furrow with the transverse axis is
not indicative of the axial relations of the first cleavage plane
at the time of tts formation. In Crepidula, before the formation
of the fourth quartette, the two parts of the first cleavage fur-
row lie approximately in the same plane. Now the longitudinal
axis of the future embryo is definitely marked out at this period
by the direction of the anterior and posterior arms of the cross.
The first and second cleavage planes at this stage are oblique
to these arms of the cross, and hence to the future longitudinal
axis of the embryo. Thus, as far as can be ascertained, the
original direction of the first cleavage plane is oblique; subse-
quent shiftings of the cleavage furrows are of no concern. It
is to be noted that it is the upper parts of the first two furrows
that retain very nearly their original direction in relation to the
‘median axis, while the axial relations of the lower portions
change. The shifting of the lower portions of the first cleav-
age furrows in no wise alters the fact that the original direction
of the first two cleavage planes is oblique. The transverse
direction finally taken by the lower portion of the first cleavage
furrow in Crepidula is, therefore, due to the fact that it has
become shifted in relation to the future median axis of the
embryo.

In Nereis, according to Wilson, the first cleavage plane is
transverse, and the second coincides, approximately, with the
future median plane. It is evident, as Mead has pointed out
(97, p. 301), that Wilson uses only the portion of the first

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NOZ.)) LARLY DEVELOPMENT OF PLANORBIS, 433

cleavage plane lying between the entomeres as the basis of
orientation. In Amphitrite, according to Mead, “if the second
cleavage furrow is followed around the whole egg, its course is
found to be an irregular zigzag, but its general direction is at a
considerable angle to the sagittal plane.” In both Nereis and
Amphitrite the portions of the first two cleavage furrows lying
between the ectomeres have essentially the same axial relations.
It is only in the portions of these furrows lying between the
entomeres that the axial relations vary to any marked degree,
and it is probable that in Nereis, as in Crepidula, the transverse
direction of the lower portion of the first cleavage furrow is
due to the shifting of the entomeres in relation to the median
axis.

It is very probable that if the lower portion of the first cleav-
age furrow were not shifted in relation to the upper, the whole
cleavage plane would be, in every case, oblique to the future
median axis of the embryo. In fact, it may be said that, when-
ever the lower portion of the first cleavage furrow has a direction
different from that of the upper portion, tts direction has been
altered in relation to a part of this furrow which lies at a
remarkably constant angle to the future median axis of the en-
bryo, this ts consequently tantamount to becoming shifted in
relation to the median axis itself. In such cases the lower por-
tion of the first cleavage furrow can no longer be considered
indicative of the direction of the first cleavage plane.

It is apparently, therefore, a universal characteristic of forms
with spiral cleavage that the first cleavage plane is oblique to
the future longitudinal axis of the embryo. As far as is known,
the first cleavage plane, in forms with normal or unreversed
cleavage, always makes a positive angle with the future median
axis, while it makes a negative angle with this axis in forms
whose cleavage is of the reversed or sinistral type. These
facts, I believe, are not devoid of significance in relation to the
general theory of spiral cleavage. In the previous section the
suggestion was made that the difference in the direction of the
first spiral cleavages in Crepidula and Planorbis might be due
to the circumstance that the first cleavage plane cuts a pre-
formed longitudinal axis in one form at a positive, and in the

434 HOLMES. [VoL. XVI.

other at a negative, angle. We are naturally led from this sup-
position to the view that spiral cleavage, in general, may be due
to the oblique direction of the first cleavage in relation to a
preformed longitudinal axis of the ovum. The possibility is
open, on the other hand, that the direction of the first cleavage
is not predetermined in the ovum, and that the median axis is
determined only during cleavage. The evidence, however, is
apparently against such a view, although it is still far from
complete. The fact that the isolated blastomeres of the gas-
teropod egg exhibit the phenomenon of “ partial development ”
(Crampton, ’96) in a marked degree affords a very strong reason
for regarding the median axis of the embryo as determined at
the time of the first division. It does not necessarily follow
that this conclusion is to be extended to all forms with de-
terminate spiral cleavage; yet Crampton’s experiments may
rightly be held, I think, as affording no little support to this
generalization.

If the first division were always of a spiral character, as it is
in Crepidula, the second cleavage would, as a consequence, be
also more or less oblique, and the spiral character of the suc-
ceeding cleavages may be regarded as simply a consequence
of Sach’s law, that the direction of every cell division tends
to be at right angles to the preceding division. The second
division in forms with determinate spiral cleavage is, I believe,
always a spiral one. In Planorbis, as in Physa (Crampton, '94),
Limax (Kofoid, '95), Crepidula (Conklin, '97), and Amphitrite
(Mead, '97), the spiral character of this cleavage is manifested
by the inclination of the spindles before a very marked constric-
tion of the cytoplasm takes place, and cannot, therefore, be
regarded as a consequence of the shifting of the blastomeres.
It seems not improbable that the spiral character of the second
cleavage is a consequence of a spiral tendency of the first cleav-
age — that the agencies that cause the rotation of the nuclei
and spheres in Crepidula after the first division are present in
other eggs also, although they produce no visible effect until
the second cleavage. In both annelids and mollusks, spiral
cleavage is soon superseded by cleavage of the bilateral type,
and there appears no reason to doubt that bilateral cleavage in

No.2.) LARLY DEVELOPMENT OF PLANORBIS. 435

these forms is correlated with the bilaterality of the future
animal. May not the delayed appearance of bilaterality be due
to the circumstance that the oblique direction of the first cleav-
age started the divisions in a spiral direction, which is only
overcome later by the tendency to bilateral cleavage?

It is worthy of note, in this connection, that in many eggs
the first cleavage plane is oblique to the axis of elongation.
This is the case, according to Conklin, in Urosalpinx, and,
according to Fol (75), the first cleavage furrow in Cymbulia
is oblique to the line connecting the two attraction spheres.
Among the Rotifera, Tessin (86) found that in Eosphora the
first cleavage furrow is oblique to the long axis of the egg and,
therefore, to the median axis of the animal. In Callidina,
according to Zelinka (91), the first cleavage plane is oblique
to the long axis of the egg, but by a shifting of the cells it
comes, finally, to be transverse. And in Asplanchna, Jennings
found that the first cleavage amphiaster “lies at first somewhat
oblique to the longitudinal axis of the egg, but before cleavage
takes place the spindle swings into coincidence with it.” The
nucleus in the larger of the two cells subsequently rotates to
the right, so that a line joining the two nuclei would cut the
first cleavage plane at an oblique angle. The possibility sug-
gests itself that the rotation of this nucleus may be due to the
same circumstances that caused the oblique direction of spindle
before cleavage. The behavior of the egg of Asplanchna would
seem to indicate that it tends to divide obliquely, as in Callidina
and Eosphora, but that this tendency is overcome by the tend-
ency to divide at right angles to its longest diameter, and only
manifests itself at the beginning and at the end of cleavage.
The first cleavage of Asplanchna recalls that of Crepidula in
that the phenomenon of nuclear rotation is manifested after
the completion of division. It is quite evident that among
these rotifers other factors besides the shape of .the egg influ-
ence the direction of the first cleavage. According to Hert-
wig’s law, it would be expected that the first cleavage plane
would uniformly lie at right angles to the long axis of the egg.
The fact that the first cleavage is oblique to this axis in Calli-
dina and Eosphora, and manifests a tendency to obliquity in

436 HOLMES. [VoL. XVI.

Asplanchna, points to the conclusion that the egg possesses
a certain degree of cytoplasmic organization, which tends to
determine the cleavage in a certain direction, even in opposi-
tion to Hertwig’s law. And this naturally leads one to suspect
that the first cleavage plane may be predetermined in eggs like
those of most mollusks and annelids in which no means of
orienting it are apparent.

The observations of Blochmann ('g2) on Neritina show, if
correct, that in this form it is very probable that the longitu-
dinal axis of the embryo is determined before the first division.
Blochmann found that in the two-cell stage the granules which
later go into the tip cells of the lateral arms of the cross were
aggregated into two small patches on the two sides of the egg.
If these groups of granules existed, as seems very probable,
before the first cleavage, it would show that the axial relations
of the embryo were determined in the undivided egg. “Aus
dem Umstand,” says Blochmann, “dass die hellen Kornchen
schon auf dem Stadium der Zweitheilung vorhanden sind (Fig.
39), kann man wohl folgern, dass die beiden Anhaufungen auch
schon in dem eben in die Furchung eingehenden Ei vorhanden
waren, und dass sie die Enden eines Durchmessers einnahmen,
also eine Achse bestimmten.’’ And Blochmann adds, in a foot-
note: “ Bei unbefruchteten Eiern habe ich manchmal nach dem
Austritt der Richtungsblaschen um den animalen Pol eine An-
haufung von solchen hellen Kérnchen beobachten konnen (Fig.
23 bis 26), wage jedoch vor der Hand nicht zu entscheiden, ob
dieselben hierher zu beziehen sind.” It is unfortunate that
Blochmann’s observations on this latter point were not more
decisive. Could the chief axes of the ovum be determined
before fertilization, it would manifestly exclude the possibility
that the entrance path of the spermatozodn determines the
longitudinal axis of the future embryo. If in any eggs with
determinate spiral cleavage the direction of the first cleavage
plane and, consequently, the median axis of the future embryo
is determined only after fertilization, as is claimed by Mead in
the case of Chaetopterus, it may still be true that the spiral
character of the first cleavages is determined by a bilateral
organization of the egg substance, which is developed between

No. 2.] ZARLY DEVELOPMENT OF PLANORBIS. 437

the period of fertilization and the occurrence of the first divi-
sion of the ovum. The direction of the first cleavage plane and
the direction of the spiral tendency of the first cleavage (whether
to the right or the left) are two different things and may well
be determined at different times. Roux has discovered that, in
the frog’s egg, the plane of inclination of the egg axis, as well
as the direction of the first cleavage, is determined by the en-
trance path of the spermatozoon. Although in Rana esculenta
the axis of the eggs is oblique before fertilization, it was found
by Roux that the inclination of the primitive egg axis stands in
no constant relation to inclination of the axis of the fertilized
egg, which is determined by the entrance of the sperm. There
is thus in the frog’s egg a redistribution of some of the egg
substances after fertilization and before the first cleavage, with
reference to the future plane of symmetry, if there be not a
differentiation also of the cytoplasm. That the period between
fertilization and cleavage is a period of active differentiation in
the eggs of many forms is a conclusion to which many facts
point. Further investigations will have to be made, however,
before it can with certainty be determined whether, in eggs
with spiral cleavage, the chief axes are established before the
first division of the ovum by any sort of differentiation of the
egg substance.

In eggs with bilateral cleavage it is the rule that the first
cleavage plane and the median axis of the embryo coincide.
There are several exceptions to this rule, however, but they
occur in forms in which the cleavage is not of a highly deter-
minate type, as is shown by the variations in the cleavage of
different eggs of the same form. Where the cleavage is defi-
nite and determinate, the cell divisions occurring with regularity
and precision, with little individual variation in the eggs of the
same species, the direction of the first cleavage plane, in eggs
with bilateral cleavage, appears to mark accurately in almost
every case the direction of the median axis of the embryo.
This is notably the case in the cleavage of the ctenophores
(Metschnikoff, Agassiz, Chun). In the cephalopods bilateral
cleavage is again beautifully illustrated (Kolliker, Bobretzky,
Vialleton, Ussow, '81; Watasé, '91); though subject to some

438 HOLMES. [Vou. XVI.

individual variation (Watasé, '91), the early cleavage is quite
definite, but soon the divisions become too irregular to follow
in detail. In the tunicates we find bilateral cleavage of a
most conspicuous and determinate type, the median axis of the
embryo, as in the preceding forms, being marked out by the
first cleavage furrow (Seeliger, van Beneden and Julin, Castle,
'96). Among the vertebrates there are no cases of that regu-
larity and. determinateness of cleavage which characterize the
early development of many invertebrates. The cleavage is
quite regular in some forms until a certain period, after which
the divisions follow no apparent order. In most forms whose
cleavage has been carefully studied there has been found con-
siderable variation in the cleavage of different eggs of the same
species. To the extent, however, that the cleavage of the eggs
of vertebrates follows any definite plan, it may be said to belong
to the bilateral type. The first cleavage plane often coincides
with the median axis of the embryo, though in some forms it
may apparently form any angle with this axis. In Amphioxus,
although the form of cleavage is exceedingly variable, exhibit-
ing cases of radial, spiral, and bilateral divisions in different
eggs, there is a predominant tendency to bilaterality in the
early cleavage (Wilson, ’93), and the first cleavage marks the
future longitudinal axis. The cleavage of fishes, after the first
few divisions, usually becomes quite irregular. In Batrachus,
according to Miss Clapp, the first cleavage plane may form a
considerable angle with the median axis of the embryo. The
early cleavage of this form is, nevertheless, quite markedly
bilateral, though, as Miss Clapp informs me, it is subject to
considerable individual variation, and the plan of cleavage
becomes very irregular in later stages. Morgan found, also,
in Fundulus that, while the cleavage up to a certain stage was
of the bilateral type, the first cleavage bears no constant rela-
tion to embryonic axes. While in the frog and some other
Amphibia the first cleavage plane and the median axis of the
embryo commonly coincide, the first cleavage plane in Diemyc-
tylus (Jordan) and Triton (Hertwig) is usually transverse to this
axis. The axial relations of the first cleavage in case of the
frog, however, are known to be largely influenced by gravity

No.2.] EARLY DEVELOPMENT OF PLANORBIS. 439

(Pfliiger), and in Diemyctylus, Jordan found that the first
cleavage plane often deviated considerably (45-50°) from the
transverse axis.

In Amblystoma, Jordan and Eyclescheimer found that the
first furrow became so irregular that they considered it improb-
able that it should ever come to separate, exactly, the right and
left halves of the embryo; and the same conclusion may well
be drawn from the cleavage of many other vertebrates. Where,
in eggs with bilateral cleavage, the first cleavage plane forms an
oblique angle with the median axis, this angle varies in differ-
ent eggs, z.e., the first cleavage plane does not stand at a con-
stant oblique angle to the median axis. When the first cleavage
plane has covstant¢ axial relations, it is either median or, more
rarely, transverse (Polychoerus, Gardiner, '95). Determinate
bilateral cleavage may readily be conceived to occur in which
the first cleavage furrow stands at a certain constant oblique
angle to the median axis, the second at right angles to the first,
and only the third or fourth meridional cleavage furrow coin-
ciding with the median plane of the embryo. But the course
of bilateral cleavage does not run in this manner. Where, as
in the toadfish, the first cleavage plane may form a considerable
angle with the median axis, and the cleavages, up to a certain
period, are symmetrical in relation to this plane, it is obvious
that, unless an extensive shifting of the blastomeres occurs, the
cleavage cannot be of the determinate type ; cells of correspond-
ing lineage cannot have the same fate in different eggs. In
cases like this the form of cleavage can hardly be held to
express a bilateral organization of the egg substance and can-
not have much morphological significance. Even in eggs devoid
of bilateral organization the cleavage might form, according to
Sach’s law, a more or less definite pattern, whose form would
be largely dependent on the amount of yolk in the egg, extrin-
sic conditions, etc.; or the organization of the egg may be such
as to exert no influence on the early cleavage. But in cases
where bilateral cleavage is determinate, where cells of the same
lineage always have the same fate in different eggs, the first
cleavage plane apparently always coincides with either the
median or the transverse axis of the embryo. Where the first

440 HOLMES. [VoL. XVI.

cleavage plane does not coincide with either of these axes,
bilateral cleavage, when it occurs, is of the indeterminate type.
The converse proposition also appears to hold good, vzz., where
the cleavage is determinate and the first cleavage plane coincides
with the median or transverse axis, the cleavage is of the bilat-
eral type. If these rules have exceptions, they are sufficiently
general not to be devoid of significance. The cleavage of the
ovum is influenced by a large number of factors, both internal
and external. Of these factors the degree of organization of
the egg and the direction of the first cleavage plane with refer-
ence to the planes of symmetry of this organization play, I
believe, an important part.

Reversal of Cleavage and Cell Homologies.

Detailed study of the early developmental stages of annelids
and mollusks has brought to light numerous and striking points
of resemblance between the cleavage of various members of
these groups; and the cleavage of the polyclades, as shown by
Lang’s work in Discocelis, is so similar to that of the above
forms that it may properly be considered as belonging to the
same general type. Professor Wilson, in his paper on the
«Cell Lineage of Nereis,’ has called attention to the close
resemblances of the cleavage of the annelid Nereis to that of
the gasteropods Crepidula and Neritina and the polyclade Dis-
cocelis as follows: “Up to a late stage in the spiral period
(twenty-eight cells) every individual blastomere and every cell
division is represented by a corresponding blastomere and a
corresponding cell division in the embryo of the polyclade, and
in that of the gasteropod. In all three the first two cleavages
and the upper and lower cross furrows have the same relations.
In all, three groups of four micromeres each are successively
separated from the macromeres, —the first group in a right-
handed spiral, the second in a left-handed spiral, and the third
in a right-handed spiral, like the first. The micromeres of the
second and third groups alternate with one another so as to
form an outer belt of eight cells that surrounds the four primary
micromeres.”’

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Professor Wilson also pointed out that in all three groups the
first cleavage of the first and third quartettes occurs in the same
direction, and that the cleavage of the gasteropods and the anne-
lid is characterized by an additional point of agreement, in that
the primary mesoblast cell, 4d, is given off in both cases in a
left-handed spiral, from the left posterior macromere D.

It was found later by Lillie (95) that the cleavage of Unio,
one of the lamellibranchs, agrees with that of the gasteropods
and annelids, not only in the features mentioned, but in several
other points of undoubted morphological significance ; and Conk-
lin has since added many other striking resemblances which
characterize the cleavage of annelids and gasteropods. The
discovery made by Conklin that the turret cells in Crepidula
have the same origin, position, and, in the main, the same fate
as the trochoblasts of the annelids, and that the prototroch, in
both groups, is completed by certain cells of the second quar-
tette, affords one of the most notable points of similarity in
the cleavage of these forms.

Resemblances such as these would seem to indicate, as Conk-
lin strenuously insists, that cleavage has a much greater mor-
phological significance than has usually been assigned to it.
Yet, while recognizing all these wonderful similarities between
the cleavage of different classes of animals, does it follow that
the form of cleavage has any really fundamental connection
with the process of development? While it is true that there
are numerous cases in which cells of corresponding origin and
position have the same fate in widely separated groups, there
are other instances in which cells of the same origin have a
very different fate in forms which are much more closely allied.
(Compare the fate of the larval mesoblast cell in Unio with that
of the same cell in Planorbis.) And there are also cases in
which it has been found that cells which have the same fate
have a quite different origin, even in the same class of animals ;
instance the origin of the secondary mesoblast in Crepidula and
Planorbis. It is hard to reconcile these facts on the view that
the process of cleavage has any fundamental connection with
the homology of organs. Neither can the similarity in the
cleavage of different groups be accounted for by attributing it

442 HOLMES. [VoL. XVI.

to extrinsic mechanical conditions. The resemblances between
the cleavage of mollusks and annelids are too numerous and
too close to be explained in this manner, or even, as I believe,
by the principle of “parallel precocious segregation.” But
while similarity of cleavage in different groups, provided it is
long continued and close, may be held to indicate genetic affin-
ity, the converse of the proposition, that differences in the form
of cleavage imply lack of relationship, does not always follow.
It is well known that the form of cleavage may vary even in
eggs of the same species. The summer and winter eggs of
daphnids have entirely different forms of cleavage, yet both
develop into the same kind of embryos. In many vertebrates
the particular manner in which the egg is divided appears to be
a matter of little moment as regards its future development.
In Renilla, Wilson found that the early cleavage of the egg
presented great variations which were without any apparent
influence on the end result.

It is obvious that no general rule can be drawn regarding the
phyletic significance of cleavage.: In some groups cleavage has,
doubtless, a high degree of ‘systematic worth”’; in others it
may have very little. Similarly, as a mark of affinity between
the different groups of animals, as in the case of gasteropods
and annelids, cleavage may bea character of considerable value ;
or again, as in the case of the gasteropods and cephalopods, its
evidence may be of little weight. It seems probable that simi-
larities of cleavage should be regarded as an zuczdental and not
a necessary expression of genetic affinity. Whether or not the
relationship between different classes of animals expresses itself
in the early cleavage of the ovum may depend largely upon
external conditions, or upon the amount of yolk in the egg, or,
perhaps, upon the degree of cytoplasmic differentiation that
has been reached before cleavage begins. It is not my pur-
pose, however, to attempt to discuss what may be the reason
for the varying morphological significance of cleavage forms in
different groups of animals. The fact I would emphasize is
that mere cell genealogy stands in no xzecessary relation to the
genealogy of organs. This conclusion, which is supported by
a variety of considerations, receives a strong confirmation — if

No.2.) LARLY DEVELOPMENT OF FPLANORSIS. 443

not a demonstration — through the facts of reversal of cleav-
age. It can readily be seen that, by virtue of reversed cleavage,
the relative positions of certain cells become different from those
they would occupy if the cleavage were of the normal or unre-
versed type. For example, the position of the cells of the third
quartette in each quadrant is, in the dextral forms, to the right,
and, in the reversed forms, to the left of the cells of the second
quartette. Similarly, the trochoblasts lie to the right of the
apical cells in the dextral forms, and to the left of these cells
in the sinistral forms. In the dextral forms the cells in the
two anterior quadrants of the third quartette are 3a and 34,
and in the posterior quadrants 3c and 3d. In the reversed
forms 34 and 3c are anterior, and 3a and 3d posterior. In the
dextral forms the trochoblasts are Ia’, 16’, anterior, and Ic’, 1a’,
posterior; while in the reversed forms the anterior trocho-
blasts are 10°, 1c’, the posterior, 1a’, 1d. In both reversed and
unreversed forms the corresponding arms of the cross are
derived from the same quadrants.

The anterior trochoblasts in both Crepidula and Planorbis go
to form the prototroch, and the posterior ones go into the head
vesicle. The cells which have a similar position in the two
forms have the same fate, although they have a different origin.
The cells which go into the prototroch in Crepidula are 1a* and
10°, while in Planorbis the cells which have this fate are 16° and
Ic’. Conversely, cells of the same cell origin have different
fates, vzz., 1a* goes into the prototroch in Crepidula, while in
Planorbis it forms a part of the head vesicle. Although the
cell 1° goes into the prototroch in both forms, it forms a part
of the rzght side of this structure in Crepidula and a part of the
left side in Planorbis. It certainly appears that, in a certain
sense, the fate of a cell is a function of its position. It has
been remarked by Driesch that, if the blastomeres of an egg
could be shifted about at will, their development would take
place in accordance with their relative positions. While in
reversed cleavage nature has performed an experiment for us
in the shifting of blastomeres, and while the results show that
the fate of the cells is in accordance with their position, and
not their genealogy, the experiment differs considerably from

444 HOLMES. [Vor. XVI.

the hypothetical shifting process of Driesch. The blastomeres
are not shifted about promiscuously, but cells occupying simi-
lar positions to those in the unreversed eggs contain cytoplasm
derived approximately from the same portion of the ovum;
and it may be for this reason, and not on account of the mere
fact of position, that they come to have the same destiny. Cor-
responding portions of the egg cytoplasm develop along similar
lines, whose direction appears to be independent of the precise
form of cleavage, even when the cleavage is of a highly deter-
minate type. The direction of every cell division may be
reversed up to a late period of cleavage without altering the
fate of the cells having the same position in the egg.

Reversal of cleavage, like the pressure experiments of Driesch
on echinoderm eggs, and of Wilson on the eggs of Nereis,
shows that the immediate causes of the differentiation of cells
lie, not in the nucleus, but in the cytoplasm. While certain
cells have the same position and fate in reversed and unreversed
eggs, the nuclei of these cells have entirely different lines of
descent ; and, conversely, nuclei having the same origin come
to lie in entirely different portions of the embryo. Thus, in
perfectly normal development, the fate of cells appears to be
entirely independent of the origin of their nuclei. How this
fact can be reconciled with the view that the differentiation of
blastomeres is mainly the result of qualitative nuclear divisions,
I cannot understand, unless we suppose that there is some com-
plex mechanism for the proper sorting of nuclear material to
provide for the contingency of reversed cleavage.

82

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94

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22.) LARLY DEVELOPMENT OF PLANORSBIS. 445

BIBLIOGRAPHY.

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CLAPP, CORNELIA M. Some Points in the Development of the Toad-
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ConkKLIN, E.G. The Embryology of Crepidula. Journ. of Morph.
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CRAMPTON, H. E. Reversal of Cleavage in a Sinistral Gasteropod.
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@. zool. Stat. Neapel. Ba. xiii.

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446 HOLMES. [Vor. XVI.

"75

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’80
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"96

94
"95

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"74

62
"95

"81

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448
92
93
194
795

'96

98
91

'96

HOLMES.

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2. The Cell in Development and Inheritance. New York.

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WISTINGHAUSEN, C. von. Untersuchungen iiber die Entwicklung
von Nereis Dumerilii. J7itthezl. a. d. zool. Stat. Neapel. Bd. x.

ZUR STRASSEN, O. Embryonalentwicklung der Ascaris megalocephala.
Arch. f. Entwickelungsmechantk ad. Organismus. Bad. iii.

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EXPLANATION OF PLATE XVII.

Fic. 1. Egg mass of Planorbis trivolvis.

Fic. 2. Undivided egg.

Fic. 3. 2-cell stage, showing the second cleavage spindles and the torsion of
the dividing cells.

Fic. 4. 4-cell stage viewed from the side, showing the laeotropic division
of ZB.

Fic. 5. The same egg seen from the upper pole, showing the laeotropic incli-
nation of the spindles. ;

Fic. 6. 8-cell stage seen from the side.
Fic. 7. The same egg seen from the animal pole.
Fic. 8. Apical view of the 24-cell stage.

Fic. 9. Same egg seen from the side.

Fic. 10. Same egg seen from the vegetal pole, showing the division of D
which produces the primary mesoblast cell D‘ or JZ.

Fic. 11. View of the lower pole of the 33-cell stage, showing the large meso-
blast cell lying partly in the cleavage cavity.

Fic. 12. Lateral view of the same egg, showing the cleavage of the lower
tier of the second quartette.

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EXPLANATION OF PLATE XVIII.

Fic. 13. Apical view of an egg in the 49-cell stage.

Fic. 14. Lower side of same egg, showing the mesoblastic pole cells, 47' and
M?, lying partly in the cleavage cavity.

Fics. 15, 16, and 17. Lateral views of the same egg.

Fic. 18. Egg of about 54 cells seen from the vegetal pole. The cells of the
fourth quartette are seen in process of division.

Fic. 19. Lateral view of same egg, showing dexiotropic cleavage of 2a':",
By an error the same figure is repeated in Fig. 20.

Fic. 21. Apical view of an egg of about 64 cells, showing a division of two
of the basal cells of the cross.

Fic. 22. View of posterior side of the same egg. The portion of the meso-
blastic pole cells, 17" and JZ, exposed at the surface of the egg, has become
considerably reduced.

Fic. 23. Lateral view of an egg of about 80 cells, showing the division of the
upper pair of cells of the third quartette in the a@ quadrant.

Fic. 24. Vegetal pole of an egg of about the same stage. The mesoblastic
pole cells have entirely disappeared from the surface.

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EXPLANATION OF PLATE XIX.

Fic. 25. Apical view of an egg of 104 cells. The trochoblasts have increased
in size, become clearer, and have apparently compressed the arms of the cross
which stand out in greater contrast to the cells between them than in the earlier
stages of development.

Fics. 26, 27, 28, and 29. Lateral views of the same egg. It will be seen that
the cells of the second quartette in the 4 quadrant have not kept pace.with the
divisions of those of the other three quadrants. In Fig. 29, 3a"? has divided,
while its fellow, 32%", remains entire; the corresponding divisions have both
occurred in the @ quadrant.

Fic. 30. Lower pole of same egg. The cells of the fourth quartette have
undergone a second cleavage.

Fic. 31. Apical pole of an egg of 130 cells. The tip cells of the cross have
enlarged and become clear, the arms show a marked laeotropic twist, and the split-
ting of the arms is begun by a transverse division of one of the cells of the anterior
arm. ‘The apical pole has begun to rotate forward, and the posterior trochoblasts
have become slightly larger than the others.

Fic. 32. Left side of same egg.

Fic. 33. Anterior side of same egg. The third quartette in the 6 quadrant
consists of a double row of four cells. In the c quadrant the divisions of the third
quartette are somewhat more advanced. 3c??? has just divided, while the corre-
sponding cell, 3c", is entire. 3c":?:? has divided, and its fellow, 3c*-*?, is undergo-
ing division. The upper cells of the second quartette are beginning to become
clear and to take on the character of the neighboring trochoblasts.

Fic. 34. Right side of same egg; the forward rotation of the apical pole may
be seen here as in Fig. 32.

Fic. 35. Posterior side of same egg.

Fic. 36. Vegetal pole of same egg. A fifth quartette has been formed, and
the cells of the fourth quartette in the 46 quadrant have undergone a division at
right angles to the previous one; the corresponding cells in the a and ¢ quadrants
remain undivided.

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EXPLANATION OF PLATE XxX.

Fic. 37. Apical view of an egg of about 150 cells. 26"-' is in process of divi-
sion, although this change had taken place in the earlier stage shown in Fig. 31.
A longitudinal splitting has taken place at the base of the lateral arms of the
cross.

Fic. 38. Vegetal pole of the same egg. The lower side is flattened and
slightly depressed in the center. In the 4 quadrant two of the secondary meso-
blast cells have lost connection with the surface, and two others, 36771? and
3071-12, have still a small portion of their surface visible at the outside of the
egg.

Fic. 39. Anterior view of an egg of about the same stage as the preceding.
The tip cells of the anterior arm of the cross have become clear, and are being
pushed by each other by the forward rotation of the apical pole. The lower cells
of the second quartette are somewhat dislocated by the same process.

Fic. 40. Apical view of a later stage, showing the two upper cells of the
second quartette of the anterior side of the egg now lying side by side.

Fic. 41. Vegetal pole of an egg of about the same stage. The depression
of the entoderm is deeper than in Fig. 38. A division has apparently occurred
in 247+? and in 167", The middle cells of the anterior quadrant of the second
quartette, 26711, 2471-7, 25%-?-7, and 2417, have been dislocated by the rotation of
the apical pole so that they lie in a transverse line. The secondary mesoblast
cells no longer appear at the surface of the egg. The stomatoblasts in the a, 4,
and ¢ quadrants still remain undivided, while the corresponding cell of the d quad-
rant is crowded away from the entomeres by the cells of the third quartette, which
come to meet in the middle line (cf Figs. 30, 36, 38, and 41).

Fic. 42. Apical pole of a later stage. The anterior rotation of the apical
pole is increased, and the cells of the middle of the cross are beginning to enlarge
and become clear.

Fic. 43. Right side of same egg.

Fic. 44. Lower pole of same egg; mouth of gastrula reduced to a slit.

Fic. 45. Posterior side of a gastrula, showing the large head vesicle.

Fic. 46. Ventral side of the same gastrula, showing the blastopore reduced
to a minute slit lying between a pair of oblong cells. Prototroch shown by a band
of clear cells in front of the blastopore. Rudiments of the cerebral ganglia shown
by two patches of dark cells separated by a median band of large, clear cells, the
apical plate 4.P.

Fic. 47. A later stage, showing the prototroch, apical plate, 4.P., blastopore,
/., and the rudiment of the right cerebral ganglion. The cell boundaries shown
in the figures in this plate are not diagrammatic, but represent accurately the out-
lines of every cell. In eggs stained with silver nitrate these boundaries are as
clear as shown in the figures.

Fic. 48. View of anterior portion of a gastrula, showing the cell 14%???
pushed forward until it comes in contact with the prototroch.

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EXPLANATION OF PLATE XXI.

Fic. 49. Mesoblastic pole cells, showing the small cells budded off at the
anterior end.

Fic. 50. Optical section of an egg of about the stage shown in Fig. 41.
P.M., primary mesoblast; 5.47., secondary mesoblast.

Fic. 51. Posterior side of a young embryo, showing the large head vesicle,
h.v., and the beginning of the shell gland, s.g.

Fic. 52. Optical section of a slightly later stage. JZ, mouth; s.g., shell gland;
¢.g., rudiment of cerebral ganglion which is proliferating cells; 2., prototroch; ¢.c.,
entodermic cells that have become gorged with albuminous matter; mes., cells of
the mesoblastic band; ¢.c., giant cell that has become partially perforated.

Fic. 53. Surface view of the same stage. J, mouth; %., prototroch; c.g.,
rudiment of cerebral ganglion. The patch of clear cells around the mouth gradu-
ally narrows posteriorly until it becomes a narrow band of clear cells separating
the two halves of the foot.

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Volume XVI. August, I900. Number 3.

JOURNAL

OF

PO EL Oly Gx

CHE DEVELOPMENT OF THE COXAL (GLAND,

BRANCHIAL CARTILAGES, AND GENITAL
DUCTSVORY LIMULUS: POLY PIE MUS:

WM. PATTEN ano ANNAH PUTNAM HAZEN.

CONTENTS.

PAGE

TPN TROD UL CMO Nyseetaseretcence rte re sack ea ICE BU PGES AD ek SI eeu nag 460

NTMI VTi RETO) She peecstee is mien ste Std Le NE oe ee be Eek Big LM eee 460

PEO TAT pd © RUST CAT RCE: VDE Weer neta a ee ce Lh Ut be re saa et Sean ates 9 Cee ne dea eo ELE 461

IV. FORMATION OF MESOBLASTIC SOMITES AND BLOOD SINUSES.........-.. 463

MeO EVEEORMENE OF THE (GENITAL DUCTS (..32.0..20- cece sun( tone eee ee 465
VI. DEVELOPMENT OF THE BRANCHIAL CARTILAGES, MUSCLES, AND

IN GGRRAV IE SUS ee seen entn cise ccteaattaaet neucasaaantaace feateci evanaseenseat sneha eaten ene eee CIC)

lel mlnrnio, NE PETRIC (GTA ND! )scee ear SE ee a eee eve 472

A. Development-of the Nephridial Lobes .1..-2..250). ee 472

B. Structure of the Nephric Gland in the Adult... 476

“TULA Usa Ui mais 020 Cont Bake: 5 Mea aa a RE NT DERE OR PREY RETA AE ALND ANIC 479

AnmMevelopment Of ther Nephric) Duct ck vrescastrtes eae eee ee 479

PES MAD OBESE SAC Ys Seon Vide EK BSA Le oe ee ea 482

GC, iheMephric Duct of the Adult. .i..005. 0a eee 483

PX CELLS FOR MOOUBTEUL,) SIGNIFICANCE | :ccsc. csc e aie eee este ee 484

PReaE SU MMAR Vilinses seen hare, eke AU Peek ats OU i Ae SOW Ante Uae SG

459

460 PATTEN AND HAZEN. [VoL. XVI.

I. INTRODUCTION.

THE work described in the following pages was mainly done,
during the year 1895 and 1896, in the Biological Laboratory
of Dartmouth College, and was continued through the summer _
season of 1896 in the Marine Biological Laboratory at Woods
Holl.

The problem we had in mind at first was the development of
the branchial cartilages, but it was finally deemed advisable to
work out, in connection with this problem, the development
of the genital ducts and the ‘coxal gland,” or nephridia, as all
three of these organs are closely associated with one another
during development.

We were in a certain measure prepared for the independent
origin of the nephric lobes and nephric duct by the discovery
that in the adult kidney, as seen by the aid of injections, a
distinct nephric duct is present, which could hardly be anything
else than the tube seen by other investigators in the embryos.

This left the development of the nephric lobes to be accounted
for in some other way than as a modification of the embryonic
duct.

We are indebted to Professor Whitman for the privileges of
the Marine Biological Laboratory at Woods Holl and to Dr.
William A. Redenbaugh for assistance in working out the
course and origin of the nerves found in the region of the
nephridial lobes.

II. MeETHOoDs.

The embryological material was killed either in Perenyi’s
fluid, picro-nitric acid, picro-sulphuric acid, corrosive sublimate,
or formalin. The most satisfactory results were obtained with
Perenyi’s fluid. The embryos remained in it overnight. In
the morning the much distended membranes could be easily
removed, and the eggs placed at once in large quantities of
95% alcohol, which was changed often to prevent the yolk from
swelling and cracking. Low grades of alcohol must be avoided
in the preliminary stages of hardening.

No. 3.] LIMULUS POLYPHEMUS. 461

The embryos were stained zz foto with Delafield’s haema-
toxylin or with borax carmine, followed by Lyon’s blue on the
slide. The larvae and young Limuli were treated with Dela-
field’s haematoxylin, and picro-acid fuchsin, or eosin.

The xephric duct may be injected by forcing a canula into its
external opening at the base of the fifth leg, or it may be liga-
tured into the end of the duct, just below the shell.

Starch masses, or a thick celloiden mixed with lamp black,
were used for injections. After celloiden injections are hardened
in 80% alcohol for several days, the connective tissue may be
dissected away from the duct, and its course followed without
much difficulty. Complete casts of the duct may be obtained
by maceration in strong hydrochloric acid ; but in all cases the
casts which-were made showed a good deal of shrinkage. A
good starch, asphalt, or celloiden mass will completely fill the
duct and penetrate deeply into the nephridial lobes.

III. CritTicAL REVIEW.

The Nephridia of Limulus were first described by Packard
in 1875, who concluded that they were renal organs, compa-
rable with the green glands of Crustacea. In 1880, he com-
pared them with the shell glands of the Entomostraca.

Lankester described them in 1880, and later ('g2) discussed
their histological structure in detail, comparing them with the
coxal glands of scorpions and Mygale. Both Packard and Lan-
kester saw only the lobes and the longitudinal stolon of the
adult, and entirely overlooked the large thin-walled duct and its
external opening. Packard tried to find a duct by injecting the
gland, but failed. In 1895 Tower discovered the opening of
the gland in the adult in the interarticular membrane on the
posterior side of the base of the fifth leg. It is situated, as he
correctly states, on a papilla, readily seen with the naked eye,
and surrounded by a dark gray ring.

Gulland (85) and Kingsley (85 and '93) gave an incomplete
description of the embryonic duct and its relation to the fifth
coelomic cavity. They were deceived, however, in mistaking
the developing duct and its end sac for the developing nephric

462 PATTEN AND HAZEN. [VoL. XVI.

lobes, while the nephric lobes themselves were entirely over-
looked. They were further misled by not knowing the exist-
ence of the adult duct with its permanent external opening,
and other characters, which clearly distinguish it from the
nephric lobes.

Kingsley (93) supposed that the four nephridial lobes of one-
half of the body arose from a portion of the coelom of the
fifth somite, which had retained its position on the ventral side
of the embryo after the other coelomic cavities had emigrated
toward the dorsal surface. The fate of the dorsal portion of the
fifth somite was not determined, but he states that it persists
as a perfectly distinct cavity with epithelial walls on either side
of the heart. From its position and from its posterior termina-
tion he is inclined to think that this portion of the coelomic
epithelium is finally converted into the reproductive organs
(p. 200). ‘Fora considerable time,” he says, the ventral portion
of this coelomic cavity “shows no change worthy of remark.”
At length, however, it “‘begins to elongate and to become bent
upon itself like the letter U, the rounded portion being directed
anteriorly.” An ingrowth of ectoderm unites with this tube to
form its outer opening, situated on the posterior side of the
coxa of the fifth leg. He also states that “‘in the neighborhood
of the coxal gland at this stage may be seen numerous lacunae
in the mesoderm, which is rapidly assuming the trabecular con-
dition characteristic of the later stages”’ (p. 203). He was
unable to trace any connection between these lacunae and the
coelomic cavity, and is strongly of the opinion that none exists.
Again he states (p. 204) that there is a “formation of trabeculae
of mesodermal tissue which invade the cavity, and, passing from
wall to wall of the proximal portion of the organ, tend to sub-
divide it and give it an anastomosing character.” In the older
stage he finds a fenestration in the region of the end sac, which
is “the beginning of the anastomosing condition of the adult,
while the proximal (internal) limb is thrown into a series of four
outwardly directed diverticula, which are segmentally arranged
and occupy somites 2-5” (p. 205). Kingsley suggested “that
the whole organ of the adult is derived from the coelom of
somite V, and that the apparently metameric lobes figured by

No. 3.] LIMULUS POLYPHEMUS. 463

Packard . . . are not the remnants of the Nephridia of the
corresponding somites, but are rather the derivatives of the
diverticula of the duct” (p. 205). He maintains that, “ besides
an increase in the size of these lobes, all that is necessary to
convert his reconstruction into the ‘coxal gland’ of the adult
are closures of the external opening, more or less complete
fusion of the two limbs of the duct, accompanied by an increase
in the anastomoses, the result being to convert coelom and duct
into the spongy tissue of the adult.” Mr. Gulland’s account
does not differ materially from this, but is not as complete.
He speaks of septa, which grow inside the tube and divide it.
In one place he found ‘the tube continuous with connective-
tissue spaces, which everywhere surround the gland.”

Our own observations have shown that there is no division of
the fifth somite into a dorsal and a ventral portion. It is very
clear, also, that the nephric duct is not a transformation of the
ventral portion of this somite, but a special epithelial outgrowth
from it, and that the rest of the somite persists as the end sac
only. There are no segmental diverticula of the duct, and no
part of the duct is converted into the glandular tissue of the
nephric lobes. It remains practically unchanged, except in
length and in the number of its convolutions, as the perma-
nent nephric duct of the adult. The nephridial lobes are
derived from segmental clusters of cells which arise independ-
ently of the duct from the somatic layer of the second, third,
fourth, and fifth thoracic somites.

It is obvious that the comparisons and conclusions of our
predecessors in this line of work were based on incomplete
anatomical and embryological data. We must still wait a more
thorough study of the anatomy and development of the green
glands, coxal glands, and shell glands of other arthropods before
their relations to one another can be satisfactorily determined.

IV. ForRMATION OF THE MESOBLASTIC SOMITES
AND BLoop SINUSES.

The Thoracic Somttes.— Soon after the formation of the
thoracic appendages, and before the abdominal ones have

464 PATTEN AND HAZEN. [VoL. XVI.

appeared, a solid mass of mesoderm cells is seen at the base
of each thoracic appendage. Each mass of cells gradually
extends in a lateral direction to form a transverse band quite
distinct from those on the opposite side of the body. Beneath
the mesoderm, and enclosing the yolk, is a thin non-cellular
membrane (Pl. XXIV, Figs. 35 and 36), which gradually sep-
arates from the central portion of each mesodermic band, but
remains continuous with it around its margins. At the same
time nuclei appear to migrate from these margins into the
membrane. In this way an imperfectly closed sac, the somzte,
is formed with a very thick outer wall, the somatic, and a thin
inner one, the splanchnic layer (Pl. XXIV, Figs. 37 and 38).

Each somite now grows rapidly in a lateral direction, making
with its mate on the opposite side almost a half circle. The
concave side of the posterior thoracic somites is directed back-
wards, that of the anterior somites forwards.

As the appendages grow in length, a space is formed between
the ectoderm, forming the apex of the leg and the thick somatic
mesoderm at its base. A few scattered mesoderm cells remain
attached to the inner surface of the ectoderm, and they gradu-
ally form the walls of a spacious cavity, which later is converted
into the blood channel that passes through the center of each
leg. We could not certainly determine whether this space is
to be regarded as a part of the coelom, as in the abdominal
sinuses, or not.

The further history of the thoracic somites, except the fifth,
has not been carefully studied.

The abdominal somites develop in a different way and are
much more clearly defined. They separate from the paired
bands of mesoderm formed by the primitive streak as hollow
masses, with distinct and continuous walls of nearly uniform
thickness. Each abdominal somite is at first quite separate
from all the others, and its cavity remains closed for a long
time. Figs. 2-8, Pl. XXII, are longitudinal sections showing
the first three abdominal somites and a part of the primitive
streak in an embryo in which the abdominal appendages are
beginning to appear.

In this series the median ends of the somites are thin-walled

No. 3.] LIMULUS POLYPHEMUS. 465

tubes ; towards their lateral ends their somatic walls are much
thickened to form the muscular tissues of the appendages.

The chelarial segment, it will be observed, contains a well-
defined abdominal somite, although it is smaller than those in
front or behind it. This fact shows conclusively that the che-
laria are true appendages, having the same morphological value
as the other appendages of the body.

The opercular somite is the largest, the following ones
gradually diminish in size from before backwards.

Figs. 8 and 9, Pl. XXII, represent longitudinal sections
through an older specimen. They show four fully formed
abdominal somites, with a fifth one just separating from the
anterior end of the primitive streak.

The somites grow over the surface of the yolk in a postero-
lateral direction, till they unite in the median dorsal line. The
circular bands of mesoderm thus formed become smaller and
smaller in diameter from the opercular segment towards the
posterior end of the body.

At an early period the distal ends of all the somites become
continuous with a thickened band of proliferating cells that
forms a well-defined margin extending round the whole embry-
onic area (see Patten, '90, p. 373).

The somites of the opercula and gill-bearing segments persist
as the well-defined blood sinuses leading from the abdominal
appendages to the pericardium.

V. Tue DEVELOPMENT OF THE GENITAL Ducts.

The genital ducts arise as diverticula from the median ven-
tral side of the opercular somite, and extend towards the median
line along the base of the opercular cartilages. They lose (?)
their connection with the somites as soon as two or three gill
leaves are formed on the first branchial appendage, and then
remain in a very rudimentary condition until after the second
larval stage. The distal ends of the diverticula finally unite
with shallow invaginations of the ectoderm, while their proxi-
mal ends unite in a manner not yet determined with the genital
organs.

466 PATTEN AND HAZEN. [Vou. XVI.

The first traces of the genital ducts were found in embryos
having three abdominal somites (Pl. XXII, Figs. 1-8). In this
series the sections begin near the median line and extend
towards the lateral ends of the somites. Near the median line
are a few loose mesoderm cells. Those near the surface of the
yolk are undergoing degeneration. Their nuclei contain large
granules that stain deep red in borax carmine, and the nuclear
membrane seems in some cases to have ruptured.

The lumen of each somite increases in size as one moves
away from the median line. At the same time the somatic
layer increases rapidly in thickness, till just beneath the append-
ages it is many cells deep. The splanchnic layer contains only
a few isolated nuclei. The lateral ends of the somites have no
lumen and consist of mesoderm cells not arranged in distinct
layers.

About midway between the median and lateral ends of the
opercular somite the genital duct appears as a long diverticulum,
extending from the coelom into the somatic layer (Pl. XXII,
Fig. 6, g.d.). A similar diverticulum, although not as marked,
is shown in the first gill in Pl. XXII, Fig. 5. It was not deter-
mined whether this was a rudimentary genital duct or the begin-
ning of the branchial cartilage.

In Pl. XXII, Figs. 2 and 3, a body resembling an extra
somite is present between the operculum and the first gill. Its
median end was hollow, while its lateral extremity was united
with the mesoderm of the first gill (Pl. XXII, Figs. 2-4, SO’).
Such a structure was not seen in any other series of sections
of this age.

In the next stage five abdominal somites are present. They
are shown in Pl. XXII, Figs. 9-20, which represent a series of
longitudinal sections beginning near the median line and extend-
ing almost to the lateral ends of the somites.

In the opercular and first gill segment the somites have moved
towards the yolk, leaving behind at the base of each appendage
a thick ring of mesoderm, derived from the somatic mesoderm
(Pl. XXII, Figs. 12-20, SO'-SO’). As the appendage grows
in length, a space is formed between its ectoderm and the ring
of mesoderm at its base; and in these spaces and around the

No. 3.] LIMULUS POLYPHEMUS. 467

somites are found numerous blood corpuscles, but free cells are
never found at this stage inside the somites.

The chelarial somite is large and distinct near the median
line (Pl. XXII, Fig. 9, SO’), farther away from the median line
it becomes constricted and nearly disappears (Pl. XXII, Fig.
11, SO’), and still farther away it again becomes more dis-
tinct (Pl: XXII, Figs: 12: and 13, SO’).

The genital duct at this stage is considerably enlarged.
It forms a diverticulum on the ventral wall of the opercular

Fic. 1,— A diagram showing the relative positions and shapes of the cartilage, the genital duct,
and the somite in the operculum. The somite is a large cavity at the base of the oper-
culum, closed at the median side and extending on the surface of the yolk beyond the
outer ectodermic margin of the appendage. On its ventral margin the somite pushes
down into the space in the appendage and at the same time bends towards the median line,
making a closed diverticulum. The diverticulum lengthens and becomes relatively
smaller, and breaking free from the somite it finally becomes the genital duct (g.d.). On
the ventral side of the diverticulum is a band of cells which form the cartilage rod (c.r.).
Around the base of the operculum and on the ventral side of the somite is a ring of
mesoderm cells. On its median and lateral sides it is continuous with the ectoderm.

somite, with its blind end directed towards the median line.
By comparing the different sections of this series it will be
seen that the opercular somite is swollen at its median end
(Pl. XXII, Fig. 9, SO’), and reduced to a narrow tube on the
median side of the point where the genital duct arises from it
(Pl. XXII, Figs. 11 and 12, SO’). On the lateral side of this
point the somite again enlarges to form a spacious chamber
(Pl. XXII, Figs. 16-20, SO’).

The relations of the genital duct, appendage, mesodermic
ring, and cartilage to the opercular somite are shown in the
accompanying diagrammatic cut (Fig. 1).

468 PATTEN AND HAZEN. [Vou. XVI.

In the next series of sections (Pl. XXIII, Figs. 21-24) the
genital duct is seen to open into the opercular somite, just
median to a transverse plate of mesoderm cells that will ulti-
mately develop into the opercular cartilage.

In a still later stage, where one gill leaf has formed on the
posterior surface of the first branchial appendage (Pl. X XIII,
Figs. 25-29), the ectodermic portion of the appendage extends
a considerable distance beyond the ring of mesoderm around
its base. Slender processes extend from the ectodermic cells
of one wall to those on the opposite side. In the space beyond
the mesoderm, blood corpuscles are frequently found. Pl. XXIII,
Fig. 25, represents one of the sections from this series near the
median line, showing the genital duct (g.d@.) directly under the
base of the cartilage. The cartilage has now grown out from
the ventral wall of the somite in a median direction over the
genital duct, and it also extends some distance ventrally into
the appendage. Following this series of sections laterally, the
somite is next seen at the posterior side of the genital duct
(Pl. XXIII, Fig. 26) with a band of muscle cells (#.) upon its
dorsal wall. The partition between the somite and the duct
becomes thinner, until finally the duct and somite unite, as
shown in Pl. XXIII, Fig. 27.

In the next stage to be described two or three gill leaves
have formed on the first branchial appendage (Pl. X XIII, Figs.
30-32). The operculum is greatly elongated, and its opposite
walls are united by numerous outgrowths of the ectoderm. The
remnants of the mesodermic ring are still plainly visible at its
base. The genital duct is relatively smaller than in the preced-
ing stage and extends in a transverse direction along the dorsal
edge of the opercular cartilage. Its proximal end now loses its
connection with the opercular somite and remains for some time
in a rudimentary condition. In the second larval stage it can
be found at the base of the opercular cartilage as a short tube,
still unconnected with either the ectoderm or the coelom.

In specimens half an inch long it was well developed and
united at its distal end with an infolding of the ectoderm.
The manner in which its inner end becomes united with the
genital organs has not been determined.

No. 3.] LIMULUS POLYPHEMUS. 469

VI. THE DEVELOPMENT OF THE BRANCHIAL CARTILAGES,
MUSCLES, AND NERVES.

The cartilages in the abdominal appendages of Limulus first
appear as solid outgrowths of the ventral wall of the somites.
The following descriptions and drawings produced as proof of
this statement are based in the main on what takes place in
the operculum. The cartilages in the other abdominal append-
ages develop somewhat later, but in a very similar manner.

In an embryo of three abdominal segments, as shown in
Pl. XXII, Figs. 1-8, where the genital duct is seen as a
diverticulum on the median ventral wall of the somite, there
is no trace of cartilage in the thick somatic layer of mesoderm.
But when five abdominal somites are developed the cartilage
can be easily distinguished (Pl. XXII, Figs. g-20, cv.). At this
time the somite has moved toward the yolk, leaving a large part
of the somatic layer behind, as the ring of mesoderm around
the base of the appendage. At the same time a transverse
plate of cells is formed subtending the ring. This plate of
cells is the “ Anlage”’ of the opercular cartilage. Its ventral
edge hangs freely into the cavity of the appendage; its median
and lateral ends are continuous with the cells forming the
mesodermic ring, while its dorsal edge is continuous with the
ventral wall of the somite. The opercular cartilage is therefore
derived from the ventral wall of the somite and lies just lateral
to the point where the genital duct opens into it. At this
time the cartilage cells do not differ in histological appearances
from those in the surrounding mesoderm.

The cartilage of the first gill-bearing appendage develops in
a similar manner, but more slowly than that in the operculum,
as seen in Pl. XXII, Figs. 12-16, SO*. There are openings
in the anterior wall of the first gill somite that appear to afford
a normal communication between the coelom and the surround-
ing blood spaces. The posterior wall of the gill contains a mass
of ectoderm cells in active division, evidently preparing for the
formation of the gill leaves which appear there at a later period.

The relations of the mesodermic ring, cartilage, genital duct,
and somite are shown in cut I.

470 PATTEN AND HAZEN. [VoL. XVI.

In the next stage, where one gill leaf is developed on the
first branchial appendage (Pl. XXIII, Figs. 25-29), the oper-
cular cartilage has grown rapidly; its ventral edge projects
into the mesodermic ring, but the median and lateral sides are
still continuous with it.

In the preceding stage (Pl. XXII, Figs. 15-20) the cartilage
extended only from the point of union of the genital duct with
the somite to the lateral margin of the mesodermic ring. In
this stage (Pl. XXIII, Figs. 25-29) it has grown in a median
direction along the ventral wall of the genital duct, so that the
two structures are for a short distance completely fused with
each other (Pl. XXIII, Figs. 25 and 26, cv).

The cartilage cells now begin to show the features that char-
acterize them so clearly in the later stages; vzz.: (1) they are
larger than the surrounding mesoderm cells and have distinct
cell walls; (2) they are arranged in rather regular order; and
(3) the protoplasm stains very lightly in borax carmine.

The first gill cartilage shows a very similar condition to that
in the operculum.

In the next stage, with three gill leaves on the first branchial
appendage (Pl. XXIII, Figs. 30-33), the cartilages form long
flat plates that extend some distance beyond the ring of
mesoderm into the appendage (Pl. XXIII, Fig. 33). The car-
tilage of the first gill is attached to the anterior wall of its
appendage and extends from there to the somite at its base.
A section nearer the lateral line would show a similar attach-
ment of the opercular cartilage to the anterior wall of its
appendage. The cartilage cells are of uniform size and have
a characteristic appearance and arrangement in rows. The cell
walls are sharply outlined against the clear homogeneous proto-
plasm. The nuclei are large and oval and deeply stained. The
cartilage is surrounded by a thick hyaline membrane, which is
continuous with the outer wall of the somite.

In the early Trilobite stage (Pl. XXIII, Fig. 34) the carti-
lages differ but little, except in size, from those in the adult.
Each abdominal somite has now become a venous sinus bounded
by a thin membrane. The dorsal wall of the sinus is composed
of small oval cells, which take a very deep stain. On the ven-

No. 3.] LIMULUS POLYPHEMUS. 471

tral side the cells are larger and gradually merge into the cells
composing the branchial cartilages.

The ectodermic cells on the dorsal side of the embryo are
long and slender and arranged in rows, with their nuclei near
their outer ends. At the dorsal extremity of the cartilage there
is a point where the boundaries between these ectoderm cells,
the cartilage, and muscles merge together, and it is impossible
to distinguish between them (Pl. XXIII, Fig. 34, x).

A perichondrium is now visible, composed of a layer of spin-
dle-shaped cells, apparently derived from the mesodermic ring,
and not from a transformation of the peripheral cartilage cells.
The latter, as we have shown, are formed from the mesothelium
of the somatic walls.

The distal ends of the branchial cartilages finally fuse with
the ectoderm that forms the anterior wall of the appendage.
At these points the cartilage and ectoderm are so completely
united that it is not possible to distinguish their original bound-
aries. Thus, an appearance is produced that might easily mis-
lead one into believing that the cartilages were growing as
inward proliferations from the ectoderm. But, aside from the
fact of their union at these points, there is no evidence that
such is the case.

The spaces in the distal ends of the appendages are crossed
by fibrous columns ariSing from the ectodermic walls. At the
base of each column are several nuclei, as though the columns
were formed by the union of several cells. No mesoderm
extends into the appendages beyond the distal ends of the
cartilages.

The Abdominal Muscles. — Distinct muscles are first seen on
the dorsal wall of the somite of the operculum and the first gill
in specimens in which one gill leaf is developed (Pl. XXIII,
Figs. 25-29, m.). They increase rapidly and grow into the
yolk, forming the dorso-ventral muscles of the adult. There
are also stout bands of longitudinal muscles which extend from
the anterior end of one somite to the posterior end of the pre-
ceding somite. All the mesoderm cells of the mesodermic
ring are apparently converted into muscles attached to the
base of the abdominal appendages.

472 PATTEN AND HAZEN. [Vor. XVI.

The Bvranchial Nerves. —In studying sections of young
embryos the mesodermic ring is often found fused with the
ectodermic wall of the appendage, so that it is impossible to
distinguish between them. In some of these places were found
what appeared to be large ganglionic or sensory cells, no doubt
derived from the overlying thickenings of the ectoderm. They
differed in general appearance and coloring from the surrounding
cells, and were most numerous at the posterior margin of the
appendages, between the mesodermic ring and the ectoderm.
In older specimens nerve cells and fibers were found in corre-
sponding positions (Pl. XXIII, Figs. 25 and 26, z.).

In a later stage (Pl. XXIII, Figs. 30 and 31) the nerves are
clearly outlined in both the operculum and the first gill. Ina
still older embryo (Pl. XXIII, Fig. 33), on the posterior side of
the first gill, is a proliferation of nerve cells and fibers resem-
bling a sense organ. It represents the same proliferation seen
in Pl]. XXIII, Figs. 25-27. From it is formed the first gill leaf.
The nerve fibers in question are the large ventral nerves that
supply the abdominal appendages. They appear to develop,
therefore, as fibrous outgrowths (of the central nervous sys-
tem ?) that receive at various points along their course fibers
and cells from the overlying ectoderm, and especially from
those points where the gill leaves are about to appear.

VII. THe NeEpuric GLAND.

A. The Development of the Nephridial Lobes. — The nephrid-
ial lobes are formed from loose clusters of cells, situated near
the base of each of the six thoracic appendages. The cells
show a distinctly segmental arrangement, and are derived from
what appears to be the median somatic wall of the mesoblastic
somites. The nephridial cells of the first and sixth appendages
degenerate and disappear after the second larval stage. Those
in the second, third, fourth, and fifth appendages form the
nephridial lobes of the adult.

The nephric duct arises in quite a different manner, as a
tubular diverticulum of the somatic wall of the fifth thoracic
somite; the nephridial cells of this segment do not appear till

No. 3.] LIMULUS POLYPHEMUS. 473

long after the duct, and the lobes in the other thoracic seg-
ments are clearly differentiated.

Nephridial cells are first seen in the fourth segment at about
the time the somite appears. They appear a little later in the
third segment, and then in the second, first, sixth, and fifth in
the order named.

The nephridial cells may be seen before the abdominal
appendages are formed, or before the somites are clearly out-
lined, on the dorsal wall of the mass of mesoderm at the base
of the appendages (Pl. XXIV, Fig. 35, .c.). This mass of meso-
derm corresponds to the mesodermic ring of the abdominal
appendages, and later forms the somatic wall of the somite.
The splanchnic layer is at this period represented by a thin
non-nucleate membrane next the yolk.

The nephridial cells may be recognized by their larger size,
their sharp nuclear stain, and by their lighter colored, trans-
parent protoplasm. They are best developed near the center of
the mesodermic mass at the base of the appendage, and from
that point they gradually merge in all directions into the sur-
rounding mesoderm.

In the next stage (Pl. XXIV, Fig. 36) the nephridial cells
are larger and more numerous, and some of the oldest ones
contain a few small granules.

In the next stage (Pl. XXIV, Fig. 37) they have increased
greatly in size and numbers, and are very conspicuous, owing
to the deep blue color they take when treated with Lyon’s
blue. The oldest cells have developed slender pseudopodia-like
processes.

The membrane over the yolk contains distinct cells, forming
the splanchnic layer of the somites, and the latter are as dis-
tinctly differentiated as at any time in their development. They
do not at any time form closed sacs like the abdominal somites.

The section shown in Pl]. XXIV, Fig. 38, passes through the
posterior margin of the fourth leg of the same embryo, and
shows the reduction in size and grade of development of the
cells on the periphery of the future nephric lobes.

Pl. XXIV, Fig. 39, represents a section through the middle
of the fourth nephric lobe in a still older stage. A few cells

474 PATTEN AND HAZEN. [VoL. XVI.

appear to be leaving the central mass and moving along the
walls of the somite towards the dorsal side of the egg. Some
of them may possibly give rise to the nephric cells, which at a
much later period are found around the pericardium.

The above description applies to the nephridial cells of the
fourth leg; but a similar series of changes has also taken place
in the chelicerae and the second and third legs. At a corre-
sponding place in the fifth leg the duct of the nephridia has
been developing. The true nephridial cells do not appear in
that appendage until later. A longitudinal section (Pl. XXIV,
Fig. 40), a little to one side of the median line, shows these
bunches of developing nephridial cells. In the fifth leg the
section passes through the end sac of the nephridial duct. In
the sixth leg there is a cavity similar to the end sac, but it is
not in the plane of the section.

The nephridial cells of the fourth and fifth leg are shown
on a much larger scale (x 400), in a longitudinal section, in
Pl. XXIV, Fig. 41. Here the cells are crowded with large
spherical granules that stain an intense blue in Lyon’s blue.
The cell walls are often very faint and easily overlooked. A
few of these cells are seen in the fifth leg on the ventral wall
of the end sac. The walls of all the thoracic somites have
broken down and disappeared, except those of the fifth somite,
which is now more clearly outlined as the end sac of the nephric
duct (e.5.).

In a longitudinal section of an embryo, just before the
Trilobite stage (Pl. XXV, Fig. 43), the segmental arrangement
of the nephric cells is still clearly shown. The cells are rela-
tively larger than before, and the granules are breaking up into
smaller ones (Pl. XXIV, Fig. 41). During the Trilobite stage a
marked change occurs in the character of the nephridial cells.
The oldest ones have elongated and become irregularly cylin-
drical, the coarse granules have disappeared, and the finely gran-
ular protoplasm has collected around the periphery. The nucleus
has also taken up its position just inside the cell wall (Pl. XXV,
Fig. 45, #.c. and g.z.c.). These hollow elongated cells then unite
end to end, forming a loose network of branching tubules
(Pl. XXV, Fig. 47,.9c.):

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No. 3.] LIMULUS POLYPHEMUS. 475

These changes begin first in the center of each segmental
mass of nephric cells, so that for a time each set of tubules is
unconnected with the tubules in the adjacent segments.

Some nephric cells are still present like those described in
the earlier stages (Pl. XXVIII, Fig. 80, ¢c and d). On the
periphery of the lobes are small granular cells that stain deeply
in Lyon’s blue, and which appear to be blood corpuscles (s.7c.).

The formation of nephric tubules now begins to extend for-
ward and backwards, forming at first a few slender chains of
cells uniting the ventral ends of the nephric lobes with one
another. As these connecting cells become canalated, the
branching tubules in each lobe are united into one system.
The various kinds of cells seen in the nephridial lobes are
shown in /PE XXVIII) Figs. 74, 75; 77, and. 70,

In a crab about one inch long a cross-section in the region
of the fifth leg (Pl. XXV, Fig. 48) shows that the nephridial
lobes now consist of several distinct layers or strata, composed
of cells in different stages of development. The end sac
(e.s.) on the median side opens through numerous tubules into
smaller ones, which again break up in the loose tissue of the
gland. The tubules in the region of the end sac are large, and
open with such wide mouths into the end sac that it is not
possible to determine just where the sac ends and the tubular
tissue begins. The layer of large tubules nearest the end sac
is lined with pavement cells filled with coarse granules. These
tubules are evidently formed from the hollow cell chains of
the previous stage by the multiplication of the nuclei and
the breaking up of the peripheral protoplasm into separate
cells. Thus, the intracellular chain of vacuoles is changed
into the intercellular lumen of a duct lined by many flattened
cells.

The next layer is composed of chains of cells with finely
granular protoplasm and conspicuous nuclei. Most of the cells
are hollow, and united end to end to form a network of intra-
cellular tubules like those seen in the second larval stage
IX XV, Fig. 47).

The outer layer or cortex of the lobe consists of many small
cells with very conspicuous nuclei. Among them are some

476 PATTEN AND HAZEN. [VoL. XVI.

enormous cells filled with refractive spherules. One large
spherule usually occupies the center, surrounded by many
smaller ones, so numerous as to completely hide the nucleus
(Pl, XXVEIT Pig.377):

The division of the nephric lobes into concentric layers is
not a sharp one, still it is clearly evident on careful examina-
tions of the sections with moderately high powers. Pl. XXV,
Fig. 48, was drawn on too small a scale to show these different.
layers well. There can be no doubt that the nephric lobes from
now onward increase in size exogenously, and that the stratified
appearance of the lobes in section is due to a succession of
different stages of development that begins with indifferent
mesoderm cells on the periphery and ends with the fully
formed intracellular tubules in the center of the lobes.

The nephridial lobes of the second, third, fourth, and sixth
leg resemble those in the fifth but the stratification of the
layers in the latter is more clearly marked. Cross-sections of
the stolons uniting these lobes also show very clearly the con-
centric strata.

The difficulty of following the history of the nephridial cells
is much increased by the granules, which come and go, and
which change the appearance of the nephridial cells so much
that it is hard to recognize their various phases. They appear
to accumulate in the cells until the canalization of the latter
and their union to form a system of connecting tubules afford
the necessary means for their discharge into the end sac, and
from there to the exterior. It is certain that the granules begin
to diminish in numbers about the time the nephric duct acquires
an opening to the exterior.

B! Structure of the Nephric Gland in the Adult.— In young
Limuli about two or three inches long the nephridial cells form
compact masses of tissue easily distinguished from the sur-
rounding organs. The cells at the base of the first and sixth
appendages have disappeared; those at the base of the four
remaining appendages form the four permanent lobes of the
kidney.

In the adult the lateral surface of each lobe, except the first
(Pl. XXVIII, Fig. 83), is flattened and lobulated, with a roughly

INO.3.] LIMULUS POLYPHEMUS. 477

slipper-shaped outline. This is the growing surface of the lobe,
and contains just beneath the outer layer the finest tubules and
capillaries. The median side of the lobe is somewhat wedge-
shaped, the coarsest ducts being nearest the apex of the wedge.
At the ventral ends of the lobes the wedge-shaped surfaces
gradually widen, as the coarser tubules diverge to meet those
forming the stolon uniting the four lobes. The network of
coarse longitudinal ducts of the stolon empty into the end sac
situated in the middle of the fifth lobe (e.s.), and from there
the secretions pass to the exterior through the long nephric
duct.

The size and outline of the different lobes, especially the first
one, vary a good deal in different individuals.

The nephric gland lies deeply imbedded in the muscles
around the base of the legs, and can be readily recognized in
the fresh condition by its brick-red color. In some specimens
the surface is a pale yellow, or is mottled with red patches.
The inside of the lobes, however, was always brick red. Each
nephridial lobe had two ear-like lobules attached to its median
ventral end near the stolon. On the first lobe they were large,
massed one above the other, and entirely covering the collect-
ing tubes. On the remaining lobes they were much smaller,
and were connected with them by a slender stalk.

A short distance from where the pedal arteries leave the
circum-oral ring a large blood vessel arises which extends lat-
erally along the posterior ventral margin of each nephric lobe.
It passes directly through the median portion of each lobe to
supply the muscles and other tissue beyond. Before entering
the nephric lobes many branches are given off, the anterior
ones supplying the nephric lobes, the posterior ones the adja-
cent muscles. These blood vessels form a rich mass of capil-
laries round the nephric tubules.

Nerves. —Two sets of nerves pass close to or through the
nephric glands. The haemal, or integumentary, nerves of
the third, fourth, and fifth thoracic neuromeres pass through
the stolon, and between the nephric lobes, to the sides of the
carapace, without apparently giving off any branches to the
gland (Pl. XXVIII, Fig. 83, zz¢.z., and Pl. XXVI, Fig. 49, z.).

478 PATTEN AND HAZEN. (VoL. XVI.

Eight smaller nerves arise from the roots of the pedal nerves
and supply the coxal muscles at the base of the coxite. There
are two of these nerves to each lobe, one on either side (PI.
XXVIII, Fig: 83; e¢-207 yi Lhe ‘third, fourth, and wsevenem
nerves pass directly through the stolon; the second, fifth,
sixth, and eighth nerves pass over its dorsal, and the first
over its ventral, side.

No branches could be found running from these nerves
into the lobes, although sections show the presence of numer-
ous fine nerve bundles ramifying through the lobes in all
directions.

In sections of the adult gland one may distinguish five con-
centric layers, each layer containing nephridial tissue in differ-
ent stages of development. Beginning at the center of the lobe,
we have in order: (1) large collecting tubes (Pl. XXVI, Fig.
54); (2) small clear-walled tubules, Fig. 53; (3) tubes lined
with granular cells, Fig. 52; (4) chains of vacuolated cells,
Fig. 51; (5) large granular cells, Fig. 50.

The large granular cells are very numerous on the ventral
and dorsal sides of the nephric lobes (Pl. XXVI, Fig. 49, g.c.).
Under a higher power two or three nuclei are sometimes seen
in a single cell. In borax carmine and Lyon’s blue, or in
Delafield’s haematoxylin and eosin, the cell wall takes a dark
stain and appears as a fine thread among the unstained gran-
ules. Wedged in between them were occasional bunches of
from five to ten or more small dark-colored cells, probably
blood corpuscles (Pl. XXVI, Fig. 50, 0.g.¢.).

Small bundles of nerve fibers are abundant in the granular
tissue, especially on the median dorsal side of the nephridial
lobe (Pl. XXVI, Fig. 50, .). In fresh specimens this tissue
has a dull orange color and resembles adipose tissue. A layer
of loose connective tissue forms an indistinct boundary between
the cells just described and the true nephridial cells.

The latter form the layer marked Zc. in Pl. XXVI, Fig. 409.
It consists of small cells with large dark nuclei (Pl. X XVI, Fig.
51). The innermost cells are vacuolated, and have fine granu-
lar protoplasm on the periphery, and some have united end
to end to form delicate intracellular tubules like those seen in

No. 3.] LIMULUS POLYPHEMTS. 479

the early Trilobite and second larval stages. These cells and
tubules represent the peripheral terminations of the system of
tubules leading into the end sac. This layer nearly surrounds
the lobe. It is thickest at its apex, becomes thinner on the
median ventral side, and disappears entirely on the median
dorsal side, where the longitudinal collecting tubes unite the
lobe with ».ne another. Within this layer is one formed of
tubules, lined with large granular cells, as shown in Pl. XXVI,
Fig. 40, gt. and Pl. XXVI, Fig. 52. They are surrounded by
a loose connective tissue, containing nuclei larger than those in
the granular cells. There are two kinds of nuclei in the walls
of the tubules, one small, dark, and homogeneous, the others
larger, and showing clearly the chromatin granules.

Inethe next layer(Pl XX VI, Fic. 49, 2p: and Pl XxxXVE
Fig. 53) the cells have lost their granules and have flattened
out to form a thin endothelial lining to the tubules. The
tubules are large, and really form a meshwork of spaces
separated by vacuolated connective tissue. Blood channels,
containing large granular blood corpuscles, are abundant in
the connective tissue surrounding the tubules. The large col-
lecting tubules are best developed in the center of the lobe
and on the dorsal surface at their median ends. The endothe-
lium of these tubes (P]. X XVI, Fig. 54) stains more deeply, and
is vertically striated on its surface farthest from the lumen of
the tubes, next the very distinct basement membrane. The
tubes are widely separated by a spongy connective tissue,
richly supplied with blood vessels.

Vit Dae Neruric Duer:

A. The Development of the Nephric Duct.— The nephric
duct develops as an evagination of the somatic mesoderm of
the fifth leg. The duct cells appear before the nephridial cells
of that segment, and before the boundaries of the somite are
clearly defined, as an oval plate of columnar cells, easily recog-
nized by their large size and clear protoplasm (Pl. XXVI, Figs.
55-57). At the edges of the plate they pass gradually into the
undifferentiated mesoderm that covers the yolk. Beneath the

480 PATTEN AND HAZEN. [Vou. XVI.

plate is a noncellular membrane forming the boundary of
the yolk. On the median side of the center of the plate is
a shallow outfolding (Pl. XXVI, Fig. 56, ed.) that marks the
beginning of the tubular portion of the duct.

In the next stage this outgrowth (Pl. X XVII, Figs. 59-64)
has formed a short tube, with its solid distal end growing
towards the median line and meeting the ectoderm at the base
of the fifth leg; the margins of the original plate now form the
funnel-shaped opening (nephrostom ?) into the underlying space.

Fic. 2. — Diagrams representing two stages in the development of the nephridial lobes and the
duct. They represent the left halves of the nephridia seen from the neural side. Clusters
of the nephridial cells are seen in the chelicera, the 2d, 3d, and 4th legs. The end sac and
nephridial duct are in the sth leg. At this time the nephridial cells are large and granular.
The end sac is a closed cavity with a few granular nephridial cells appearing on its ventral
surface.

The lips of the funnel have gradually united with the membrane
over the yolk, and at the same time nuclei migrate into the mem-
brane. Thus, aclosed sac is formed which we have called the exd
sac. The entire ventral wall is formed by the nephric plate, which
represents the somatic layer of the fifth somite. The end sac
apparently represents the coelomic cavity of that somite, and the
dorsal wall is formed from the splanchnic layer of the somite.

No. 3.] LIMULUS POLYPHEMUS. 481

A proliferation of the ectoderm on the posterior median side
of the fifth leg is seen in Pl. XXVII, Fig. 63, ect.pg., mark-
ing the beginning of the ectodermic infolding, which in the
following stage unites with the distal end of the nephric
duct.

In the next stage (Pl. XXVII, Figs. 65-70) the distal end of
the duct has united with the ectodermic infolding (Pl. XXVII,
Fig. 71). The nephric plate is still visible as the flaring mouth |
of the duct (Pl. XXVII, Figs. 65 and 66), the lateral lip being
much longer than the median one.

Fic. 3 A. — Diagram of the 2d larval stage, showing nephridial cells in the chelicera, the 2d, 3d,
4th, 5th, and 6th legs. The end sac is at the base of the sth leg, surrounded by nephridial
cells. The duct extends forward to the 2d leg, and backward to the 6th. The proximal
arm is much coiled, the distal arm is nearly straight.

Fic. 3 B.— Diagram representing the adult condition. The median ends of the lobes have
grown forward and backward and united to form the stolon. This portion of the nephridia
is composed of large collecting tubules, which carry the excreta to the end sac in the 4th
lobe; from there they pass into the nephridial duct.

The relative size and positions of these parts are shown in a
little earlier stage in Pl. XXVI, Fig. 58.

In the next stage the duct elongates very rapidly, and as each
end is fixed a f-shaped tube is formed with the loop reaching
forward to the middle of the fourth leg.

The ectodermic portion of the duct is very short (Pl. XXVII,
Fig. 73), and may be readily distinguished by its small deeply
stained nuclei and by its delicate internal lining of chitin.

482 PATTEN AND HAZEN. [VoL. XVI.

During and after the Trilobite stage the lateral arm of the
duct becomes convoluted and a second loop is formed near its
proximal end, directed backwards and medianly, and lying dorsal
to the median arm of the first loop (cuts 3 A. and 3 B.). The
median arm of the first loop becomes considerably dilated, and
apparently acts as a reservoir for the secretions of the gland.
It remains a straight tube throughout life. The anterior end
of the lateral arm (/.2.) is smaller and somewhat convoluted,
the foldings increasing in number and extent towards the pos-
terior loop.

The structure of the nephric duct during the Trilobite stage
is shown in Pl. XXV, Fig. 45. The cells lining the duct now
have no distinct cell walls, although the walls are easily seen in
the preceding and in the following stages. u

The anterior loop of the duct now extends as far forward
as the second leg. The following parts may be distinguished
(cut 3 A.); vzz.: (a) the short ectodermic portion ; (4) the dilated
median arm of the anterior loop; (c) the slightly coiled lateral
arm of the anterior loop; (¢@) the much coiled posterior loop;
(e) the end funnel; (/) the end sac.

B. The End Sac.—The early stages in the formation of
the end sac out of the fifth thoracic somite have already been
described.

Before the Trilobite stage a longitudinal section shows the
presence of a few enlarged finely granular cells in the walls
of the end sac. These granules increase in size and numbers
till the cells present the appearance shown in Pl. XXIV, Fig.
42, ¢.5. They now resemble those cells from which the nephrid-
ial tubules develop.

In the Trilobite stage (Pl. XXVIII, Fig. 80) the coarse gran-
ular protoplasm has nearly disappeared, and the sac is lined with
a delicate layer of protoplasm, with here and there a nucleus.
Numerous finger-like evaginations of the wall of the sac have
developed, the walls of which have the same structure as those
of the sac itself.

It was not possible to determine the exact manner in which
these evaginations were formed. A careful study indicates that
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No. 3.] LIMULUS POLYPHEMUS. 483

evaginations of the walls of the sac, and that the outgrowths
were subsequently increased in length by the addition of ne-
phridial cells to their distal ends. These cells in turn become
hollowed out and united with the cells forming the nephric lobes.
In this way the system of tubules in the nephric lobes becomes
continuous with those leading into the end sac.

There seem to be two sets of tubules opening into the sac;
one set arises from its anterior wall and leads into the longitu-
dinal tubules of the stolon, and hence to the three anterior
nephric lobes; the others lead into the tubules of the fourth
lobe. On its median side the sac opens through a small neck
into the nephric duct.

Finally, in the adult, the end sac becomes so irregular through
the formation of the numerous large tubes opening into it that
its original boundaries cannot be distinguished.

C. The nephric duct of the adult lies along the edge of the
plastron dorsal to the nephric lobes, and extends backwards
from the base of the second leg to the anterior side of the
sixth. The thin-walled transparent tube is easily torn, and,
unless injected, it is very difficult to trace out its various con-
volutions. On careful dissection the course of the duct is seen
to be as follows: At the distal end of the duct, just before it
opens to the exterior, is the ectodermic portion (Pl. XXVIII,
Fig. 83, ect. and cut 3 B.). It is sharply marked off from the
rest of the duct by its thick walls lined with chitin. From
this point the duct turns at right angles and extends in a
dorsal direction, till it reaches the plastron, along the lateral
edge of which it extends as far as the first nephric lobe (Pl.
XXVIII, Fig. 83). It then bends directly backwards, dimin-
ishing rapidly in size up to the angle of the second loop, which
in Pl. XXVIII, Fig. 83, is seen on the median side of the
fourth nephric lobe. From here on the calibre of the tube
remains about the same. It now turns forwards, parallel to
the dorsal limb of the loop, as far as the posterior margin of
the third lobe, and then backwards to form a large mass of
coils, lying a little behind and dorsal to the fourth lobe.
From this coil the proximal end of the duct issues and passes
forwards and ventrally to the end sac, buried in the interior of

484 PATTEN AND HAZEN. [Vou. XVI.

the fourth lobe. From the end sac many tubes lead forward
into the stolon. The latter consists of a coarse network of
anastomosing tubes, from which branches are given off that
extend along the median dorsal face of each lobe, diminishing
in size as they go. The entire substance of the lobes may
be colored a deep red by injecting red gelatine into the main
duct.

Along the walls of the duct are here and there short, blunt
evaginations or pockets ending blindly. In some cases the
pockets of one tube may unite with those of another, thus form-
ing communications between the separate coils (Pl. XXVII,
Figs. 81 and 82, go. and c.u.t.). They are most numerous in
the extensive coil lateral to the fourth nephric lobe.

IX. CELLts oF DousBTFUL SIGNIFICANCE.

During the Trilobite stage certain cells appear on the
nephridial lobes, which may be readily recognized by pecul-
iarities of shape and coloring (Pl. XXV, Fig. 45; Pl. XXVIII,
Figs. 74 and 80, s.~c.). They are dark purple when stained in
Lyon’s blue and borax carmine. These cells were found only
in the vicinity of the hollow nephridial tubules, with which they
were often so closely connected that it was impossible to ascer-
tain with certainty whether they were inside or outside the
tubules. Occasionally they were on the outer margin of the
tubules, and it would then appear as if they were about to
separate from them (Pl. XXVIII, Fig. 74, c.).. In Pl. XXVIII,
Fig. 80, at the dorsal side of the end sac, about a dozen were
collected, which suggested a point of proliferation either by cell
division among themselves or from the nephridial tubules or
sac. At the left (v.c.) one of the cells is much larger than the
others and shows vacuolations in the protoplasm. A few of
these cells were found in the second larval stage, as shown in
Pl. XXV, Fig. 46, ~c., and also in the region of the heart (PI.
XXVIII, Fig. 76, 7.c.), after which they entirely disappeared. A
large number of granular cells appear at this time, and it seems
probable that they are different conditions of the same cells,
although no convincing proof of it could be found.

No. 3.] LIMULUS POLYPHEMUS. 485

The last-named cells may be found in the second larval stage
distributed throughout the nephridial lobes (Pl. XXV, Fig. 47,
g.c.), and they occur in large masses along their lateral margins.
These cells are round or oval, and are filled with great num-
bers of coarse granules, which usually completely conceal the
nucleus. They are well shown in Pl. XXV, Fig. 46, g.c., and
Pix OV LIT, Big. 75,02.

In larvae three-quarters of an inch long the cells on the
lateral margins of the lobes are enormous (Pl. XXV, Fig. 48,
and Pl. XXVIII, Fig. 77). Similar cells were found through-
out the body, from the proventriculus to the first gill, In the
anterior sections they are most numerous on the dorsal and
lateral sides of the proventriculus. They also extend laterally
on the ventral side of the body close to the ectoderm. Pos-
terior to this they are less abundant around the alimentary
canal, but are thickly massed around the base of the legs. In
the sixth leg and in the operculum they are more numerous
than in any other place. Similar cells are found in the region
of the heart. The origin and fate of these cells were not
determined with certainty. They agree in some respects with
the granular cells seen in the early stages of the nephric lobes,
and which, as we have seen, subsequently cleared up and
formed the nephric tubules.

During the Trilobite and second larval stage, cells are found
in the pericardial region that closely resemble nephridial cells.
They are most abundant on the dorsal side of the pericardium
in the sixth thoracic segment and over the proventriculus. Many
cells are hollow and united end to end, forming loose-branching
tubules like those in the nephridial lobes of the Trilobite stage
(Pl. XXVIII, Fig. 76, 4.c.2.). Among these cells are a few of
the large granular ones (g.c.’), and some of the small dark red
cells (7.c.) like those seen in the nephridial lobes. All these
cells probably arose from the nephridial “‘Anlagen”’ at the base
of the legs, and were carried to their present position by the
growth of the somites over the dorsal surface of the egg.

The same kind of cells are also found in the chelicerae and
in the sixth leg. Those in the cheliceral segment (Pl. XXIV,
Fig. 40) disappear early. Those in the sixth leg appear before

486 PATTEN AND HAZEN.

the Trilobite stage (Pl. XXV, Fig. 43, 2.c.°). These cells first
become granular, then vacuolated, and then united end to end
(Pl. XXVIII, Fig. 79). In Limuli about three-quarters of an
inch long the cells were still present, but they were not united
with the permanent nephric lobes, and appeared to be degen-
erating.

X. SUMMARY.

1. Branchial Cartilages.—A thick ring of somatic mesoderm
forms at the base of each abdominal appendage. The gill
cartilage arises as a plate of somatic mesoderm attached by its
dorsal end to the ventral wall of the somite, and continuous on
either side with the ring of mesoderm. The ventral end of
the cartilage finally extends through and beyond the mesoder-
mic ring and becomes attached to the anterior wall of the
corresponding appendage.

2. Theventral ends of the abdominal somites persist as venus
sinuses.

3. The genztal ducts arise as diverticula of the median
ventral side of the opercular somite. They remain in a rudi-
mentary condition until after the second larval stage.

4. Nephric Duct.—A nephric plate is formed from a single
layer of columnar cells of the somatic mesoderm on the median
side of the fifth somite. The plate is gradually evaginated to
form a funnel, opening by a wide mouth into a thin-walled end
sac that represents the fifth somite; the opposite end unites
with a shallow ectodermic invagination at the base of the fifth
leg. The tube becomes much convoluted, and is converted
directly into the adult nephric duct. Finger-like outgrowths
of the end sac finally unite with the hollow cell chains of the
adjacent nephric lobes.

5. Zhe Nephric Lobes.—A mass of nephric cells arises inde-
pendently of the duct from the median dorsal portion of the
somatic layer of each of the six thoracic somites. The cells
become enlarged and filled with coarse granules. The granules
become smaller or disappear, and a vacuole appears in each
cell. The latter elongate and unite end to end to form irregu-
lar masses of branching intracellular tubules. The cell masses

LIMULUS POLYPHEMUS. 487

in the second, third, fourth, and fifth legs form the four lobes
of the adult organ. Offshoots extend forward and backward
from the median end of each lobe, that unite with each other
and with the end sac to form the longitudinal ducts of the
stolon. The nephric cells of the first and sixth somite disap-
pear. In the stolon, and in the larger tubules on the dorsal
side of each lobe, the nephric cells become flattened, and by
repeated divisions are finally converted into a pavement epi-
thelium, thus changing the intracellular lumina into intercel-
lular ones. New tubules are formed throughout life by the
transformation of indifferent cells on the ventral surface of
each lobe.

488

95

"85

B85

'92

'93

90

"81

'82

84

85

"72

athe

80

90

96

oo

PATTEN AND HAZEN. [VoL. XVI.

BIBLIOGRAPHY.

GoopricH, E. S. On the Coelom, Genital Ducts, and Nephridia.
Quart. Journ. Micr. Sct. Vol. xxxvii. 1895.

GULLAND, G. L. Evidence in Favor of the View that the Coxal Gland
of Limulus and Other Arachnids is a Modified Nephridium. Quzart.
Journ. Micr. Sct. Vol. xxv. 1885.

KINGSLEY, J. S. Notes on the Embryology of Limulus. Qvzart.
Journ. Micr. Sct. Vol. xxv. 1885.

KINGSLEY, J.S. The Embryology of Limulus. /ourn. of Morph.
Vol. vii. 1892.

KINGSLEY, J. S. The Embryology of Limulus. /ourn. of Morph.
Vol. viii. 1893.

KISHINOUYE, K. On the Development of the Araneina. /ourn. Coll.
Sct. Univ. Japan. Vol.v. 1890.

LANKESTER, E. R. Limulus an Arachnid. Quart. Journ. Micr. Sct.
Vol. xxi. 1881.

LANKESTER, E. R. On the Coxal Glands of Scorpio, hitherto unde-
scribed, and corresponding to the Brick-Red Glands of Limulus.
Prac. Roy. Sct. Vol. xxiv. \Wiee2.

LANKESTER, E. R. On the Skeleto-trophic Tissues and Coxal Glands
of Limulus, Scorpio, and Mygale. Quart. Journ. Micr. Sci. Vol.
xxiv. 1884.

LANKESTER, E. R. A New Hypothesis as to the Relationship of the
Lung Book of Scorpio to the Gill Book of Limulus. Quart. Journ.
Micr. Sct. Vol. xxii. 1885.

MILNE-EpwaRpbDs, H. Recherches sur l’anatomie des Limulus.
Ann. Sct. Nat. Vol. xvii. 1872. Also Miss. Sct. Mex. 1873.
PACKARD, A. S. An Undescribed Organ in Limulus, Supposed to be

Renal in Nature. Amer. Nat. Vol. ix. 1875.

PACKARD, A. S. The Anatomy, Histology, and Embryology of
Limulus Polyphemus. Jem. Boston Soc. Nat. Hist. 1880.

PATTEN, W. On the Origin of Vertebrates from Arachnids. Quvart.
Journ. Micr. Sct. Vol. xxxi. 1890.

PATTEN, W. Variations on the Development of Limulus Polyphemus.
Journ. of Morph. Vol. xii. 1896.

TOWER, R. W. The External Opening of the “ Brick-Red” Gland in
Limulus Polyphemus. Zool, Anz. 18 Jahr., pp. 471, 472.

No. 3.] LIMULUS POLYPHEMUS. 489

INDEX LETTERS TO PLATE XXII.

ap. = anal plate. ps: = primitive furrow.
£&.c. = blood corpuscles. pS. = primitive streak.

ch. = chelaria. SO° = unexplained cavity appearing
¢.y. = opercular cartilage. only in this series.

ect. = ectoderm. SOs = somite of the chelaria, opercu-
g. = first gill. lum, and first to third gills,
gd. = genital duct. respectively.

op. = operculum.

EXPLANATION OF PLATE XXII.

All the sections were outlined with a camera and drawn to the same scale.

Fics. 1-8 were drawn from a series of longitudinal sections through the
region of the chelaria (c4.), operculum (o/.), and the first gill (g.’) of an embryo in
which the abdominal appendages were beginning to show in surface views. Borax
carmine, I5 mu.

Fic. 1. Section No. 1, through the median line of the embryo. A few
scattered mesoderm cells lie between the ectodermic layer and the yolk. There
were also a very few large nuclei in the yolk, and others from which chromatin
granules were escaping, as if they were beginning to degenerate. x 200.

Fic. 2, Section No. 2. The median ends of the somites are shown by four
bunches of cells. .SO' is the somite of the chelaria; SO?, that of the operculum;
SO°, one which disappeared shortly after this stage ; SO%, somite of the first gill
about to separate from the primitive streak (f.5.). 200.

Fic. 3. Section No. 3. The somatic cavities are distinct ; SO° merges with
the ectoderm on the ventral side of the somite. 200.

Fic. 4. Section No.6. S0O', SO?, and SO? are larger than in the previous
sections. The cavity of SO° has disappeared, and in its place are a few mesoderm
cells which disappear in the next section. x 200.

Fic. 5. Section No. 11. The operculum and the first gill are much larger
than formerly, and are now filled with mesoderm. Their somatic cavities remain
distinct. X 200.

Fic. 6. Section No. 13. SO? shows a diverticulum, the beginning of the
genital duct (g.d). x 200.

Fic. 7. Section No. 18. SO? remains large. SO? has almost closed. x 200.

Fic. 8. Section No. 23. SO? is the only abdominal somite remaining ; its
lumen disappears a few sections farther toward the lateral side. X 200.

Fics. 9-20 were drawn from a series of longitudinal sections through the region
of the chelaria, operculum, and the first gill of an embryo somewhat older than
the one in the preceding series. In this embryo there were four perfect abdominal
somites ; the fifth was just breaking free from the primitive streak. The series

490 PATIEN AND HAZEN.

begins with Fig. 9 near the median line and extends laterally almost to the outer
edge of the appendages. Borax carmine and Lyon’s blue, Io u.

Fic. 9. Section No. 1, near the median line, showing five abdominal somites,
SOs. SO is attached to the primitive streak (#.5.); the point of separation is
indicated by a slight furrow on the dorsal side. The enlargement at the anterior
end of the primitive streak will form the sixth somite. x 200.

Fic. 10. Section No. 3. SO* extends in a lateral direction beyond the point
of attachment to the primitive streak. SO'4 are distinct and free from the
mesoderm at the base of the appendages. x 200.

Fic. 11. Section No.6. The median end of the genital duct (g.¢.) may be
seen in the operculum. SO? is small and lies close to the surface of the yolk.
X 200.

Fig. 12. Section No.7. The genital duct is larger than in the previous section,
and is nearer SO”. X 200.

Fic. 13. Section No. 12. The genital duct and SO? are in contact. In the
first gill the mesoderm cells are arranged in a row, extending down into the mass
of mesoderm from the ventral side of the somite to form the cartilage (c.7.) of the
first gill. x 200.

Fic. 14. Section No.13. The cavity of SO? and the genital duct are separated
by a thin membrane only. X 200.

Fic. 15. Section No. 14. SO? and g.d. have united. A deep furrow on the
anterior side indicates the point of union. x 200.

Fic. 16. Section No. 17. A line of cells on the ventral margin of the opercu-
lar somite shows the median limit of the opercular cartilage. x 200.

Fic. 17. Section No. 20. The opercular cartilage is free from the mesoderm,
and consists of a single row of cells on the ventral side of SO*. x 200.

Fic. 18. Section No. 22. The end of the cartilage rod is broader than before,
and a thin membrane connects it with the adjacent mesoderm. x 200.

Fic. 19. Section No. 26. The opercular cartilage has disappeared. The ven-
tral margin of the somite connects with the posterior bunch of mesoderm cells.
The somites show a tendency to bend in a posterior direction. x 200.

Fic. 20. Section No. 32. X 200.

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LIMULUS POLYPHEMUS. AQI

INDEX LETTERS TO PLATE XXIII.

' B.c. = blood corpuscles. m. = nerve.
Ch. = chelaria. 2.p. = proliferation of nerve cells
¢.r. = Cartilage rod. and fibers.
cu. = cuticular membrane. o.p. = operculum.
ec.p. = ectodermic proliferation. SO' = somite of the chelaria.
ect. = ectoderm. SO* = somite of the operculum.
end. = endothelium. SO? = somite of the first gill.
g- = first gill. v.s. = venous sinus.
gd. = genital duct. x. = fusion of cartilage, ectoderm,
m. = muscle cells. and mesoderm.
ms. = mesoderm. yk. = yolk.

EXPLANATION OF PLATE XXIII.

FIGs. 21-24 represent longitudinal sections through the region of the chelaria,
operculum, and first gill of an embryo a little more advanced than the one from
which Figs. 9-20 in Plate XXII were drawn. The sections were 15 4 thick and
stained in borax carmine and Lyon’s blue.

Fic. 21. Section No. 1, near the median line. The somites of the operculum
and the first gill have extended so far in a lateral direction that the somite of the
chelaria could not come in the same longitudinal sections with them, but would be
found in the sections nearer the median line. The somites of the operculum (o..)
and the first gill (g.’) are large and distinct. The genital duct (¢.d.) shows at the
base of the operculum. The lumen seen here, ends in a solid mass of mesoderm
in the preceding section. x 200.

Fic. 22. Section No. §. The genital duct is larger than in the preceding sec-
tion, and has approached SO?. x 200.

Fic. 23. Section No.10. The genital duct and SO? have united. A few car-
tilage cells (c.r.) are seen on the dorsal wall of the somite. x 200.

Fic. 24. Section No. 10. Both SO? and SO? show a tendency to bend in a
posterior direction. The opercular cartilage (c.v.) is still present. x 200.

Fics. 25-29. Longitudinal sections through the region of the operculum and
the first gill from an embryo with two gill leaves on the first gill. The sections
were 15 uw thick and stained with borax carmine and Lyon’s blue.

Fic. 25. Section No. 1, near the median line. The opercular cartilage and
genital duct extend toward the median line some distance beyond the somite.
The opercular cartilage is attached to the genital duct between bunches of meso-
derm at the base of the operculum. The first gill cartilage is now visible. In
the spaces between the ectoderm and mesoderm of the appendages are a few
blood corpuscles (B.c.). Some of the mesoderm cells on the yolk show muscular

492 PATTEN AND HAZEN.

striations (#.). At the posterior side of the first gill are nerve fibers beneath
the place where the first gill leaf is forming (z.). x 200.

Fic. 26. Section No. 5. The genital duct and opercular cartilage are larger
than in the preceding drawing. SO? shows at the posterior side of the operculum.
SO} is greatly enlarged. xX 200. |

Fic. 27. Section No. 8. The genital duct (g.d.) and SO? are separated by a
thin membrane. X 200.

Fic. 28. Section No. 10. The genital duct and SO? have united. The oper-
cular cartilage remains on the dorsal side of the somite. SO? and SO? show a
tendency to extend in a posterior direction. X 200.

Fig. 29. Section No. 19. The somites extend a long distance laterally and
posteriorly as closed cavities. x 200.

Fics. 30-33. Longitudinal sections through the operculum and first branchial
appendage of an embryo in which the third gill leaf had commenced to form.
The embryo appears older than that of the preceding series as the appendages
have lengthened considerably.

Fic. 30. Section No. 1, near the median line. Shows the median end of the
genital duct at the base of the operculum. As the somite has grown laterally and
the genital duct toward the median line, they no longer appear in the same longi-
tudinal sections. Nerve fibers (7.) are found at the posterior side of the base of
the operculum and the first gill. x 200.

Fic. 31. Section No. 3. Shows the genital duct with a small lumen and the
median edge of the opercular cartilage. x 200.

Fic. 32. Section No. 10. Shows the lateral end of the genital duct connected
with the base of the opercular cartilage. The genital duct is relatively much
smaller than in the preceding series and separate from the somite (SO*). The
first gill has a well-formed cartilage. x 200.

Fic. 33. Section much farther from the median line. It shows the long,
slender cartilage plates; that of the first gill is attached to the ectoderm on the
anterior side of the appendage. A similar condition would be found in other sec-
tions in the operculum. The cartilage cells are placed in rows and show a charac-
teristic appearance, and take a lighter stain thanthe mesoderm. The cartilages
are surrounded by athick membrane. Each somite has been transformed into a
large venous sinus, and extends from the base of the cartilage through the yolk
to the dorsal side of the embryo. A number of nerve fibers and cells (#.) are
shown in the gill leaf ing.’ x 200.

Fic. 34. Longitudinal section through the operculum and the first gill of a
specimen with five gill leaves on the first gill, showing the opercular cartilage
attached to the anterior wall of the operculum and to the venous sinus at the base
of the appendage. The cartilage is surrounded by a membrane, the perichondrium
(fc.). At the apex of the leg slender processes reach from one ectodermic wall
to the other. The outer wall is covered by a thin cuticular membrane. xX 200.

LIMULUS POLYPHEMUS. 493

INDEX LETTERS TO PLATE XXIV.

ap.>° = the second to the sixth thoracic m.r. = marginal ring.
appendages. n.c. = nephridial cells.
é.c. = blood corpuscle. n.c.*-> = nephridial cells of the first to the
ér. = brain. fifth appendages, respectively.
6.5. = blood space. ped.n. = pedal nerve.
6.v. = blood vessel. J.c. = sensory cells.
che. = chelicera. SO = somite.
ect. = ectoderm. sop. = somatopleure.
e.5. = end sac. sp.c. = spinal cord.
g.m.c. = granular nephridial cells. spl. = splanchnopleure.
mes. = mesoderm. yk. = yolk.

EXPLANATION OF PLATE XXIV.

FIGS. 35-39 were drawn from cross-sections through corresponding regions of
the third and fourth thoracic appendages of embryos of varying ages. The sec-
tions are all arranged so that the median line is at the upper margin of the plate.
The sections of the younger embryos are 3 » to 5 u thick, the older ones Io up.

FIG. 35. Cross-section through the middle of the fourth appendage of an
embryo in which none of the abdominal appendages had been formed. On the
dorsal margin of the mass of mesoderm lying near the median side of the base of
the appendage may be seen four or five larger cells, the “ Anlage” of the nephrid-
ial lobe (7.c.). X 200.

Fic. 36. Cross-section through the middle of the third appendage of an
embryo slightly older than that in Fig. 35. The nephridial cells are more numer-
ous, larger, and have faint granulations. A thin non-cellular membrane covers the
yolk at the base of the appendage. A space is formed between the mesoderm and
the apex of the appendage, which later develops into the blood space of the legs.
X 200.

Fic. 37. Cross-section through the middle of the fourth appendage of an em-
bryo older than that in Fig. 36. The somites are imperfectly formed in the
thoracic appendages. The somatic layer is several cells thick; the splanchnic
layer is represented by a thin membrane with a few nuclei. The nephridial cells
are larger than in the preceding figures. They possess slender pseudopodia,
have become finely granular, and take a deep stain in Lyon’s blue. X 200.

Fic. 38. Drawn from the same series and the fourth section back of that in
Fig. 37. It shows the nephridial cells smaller than at the middle of the base of
the appendage and without pseudopodia. The somite extends out laterally asa
closed cavity: x 200.

Fic. 39. Cross-section through the middle of the fourth appendage. Large
granular cells with long processes are shown on the dorsal margin of the meso-

494 PATTEN AND HAZEN.

derm. The largest of these cells lie beyond the lateral base of the appendage.
X 200.

Fic. 40. A longitudinal section from an embryo of about the same age as
that in Pl. XXIII, Figs. 30-32. The somites of the thorax have disappeared,
except the one in the fifth appendage, which remains as the end sac to the nephrid-
ial duct. Bunches of nephridial cells are found in the chelicerae and in the second,
third, and fourth appendages. Nephridial cells appear later in the sixth append-
age. X 100.

Fic. 41. Longitudinal section through the fourth and fifth appendages. The
nephridial cells are filled with large granules, among which the larger nuclei are
visible. Often the cell boundaries were very indistinct or else entirely invisible,
giving the appearance of several nuclei in the same cell. In the fifth appendage
the end sac shows as a closed cavity, with a few of the larger nephridial cells on
its ventral wall. x 4oo.

Fic. 42. Longitudinal section through the fourth and fifth legs. It shows a
bunch of large, granular, nephridial cells at the base of the fourth leg. In the
fifth leg the end sac is lined with granular cells which are similar to those in the
fourth leg, except that they are smaller. There is a blood space between
the appendages and at the apex of the appendages. x 300.

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Journal of Morphology Vot.Av.

Lith Werner S Winter, Frankfort UM.

LIMULUS POLYPHEMUS. 495

INDEX LETTERS TO PLATE XXV.

ap.>* = the second to the sixth ap- 7.m.c. = longitudinal section through
pendages, respectively. nephridial cells.
ant.n.d. = anterior arm of the nephric n.c.-° = nephridial ‘cells in the first
duct. to the sixth append-
br. = brain. ages, respectively.
6.5. = blood space. 2.c. = nephridial cell.
6.uv. = blood vessel. 2.a. = nephric duct.
¢.m.c. = cross-section through ne- | #./3,#./.4= third and fourth nephridial
phridial cell. lobes.
é.5. = end sac. z.¢, = nephric tubules.
ex.n.d. = nephric duct near the external oe. = oesophagus.
opening. pél. = plastron.
gc. = granular cells. post.n.d. = posterior arm of the nephric
g.m.c. = granular nephridial cells. duct.
gt. = tubules lined with granule Ga Tedecellss
cells. a.¢. = areolar tissue.

EXPLANATION OF PLATE XXV.

Fic. 43. Longitudinal sections through the second to the sixth legs of an
embryo a little younger than the Trilobite stage. The chelicera have remained
near the median line, so that they are not included. Bunches of nephridial cells
are seen at the base of the second, third, fourth, and sixth legs, and the nephric
duct in the fifth leg. x 100.

Fic. 44. A transverse section through the region of the second, third, and
fourth legs of the Trilobite stage. Nephridial cells are seen at the base of the
third and fourth legs. The duct has grown anteriorly nearly as far as the second
leg, and both the distal and proximal limbs are shown in the figure. x Ioo.

Fic. 45. Enlarged drawing through the dorsal region of the fourth and fifth
legs from the same series as Fig. 44, showing the nephric duct and the nephridial
cells in various stages of development. x 400.

Fic. 46. Horizontal section through the fourth and fifth legs of a specimen in
the second larval stage. As the section was cut near the external opening of the
duct, only one portion of itisseen. The nephridial lobes form a definitely marked
area, and the portions in the second, third, fourth, and fifth legs have united with
one another. The nephridial lobes are composed of a lacuna tissue, in which are
large granular cells of varying sizes. X 200.

Fic. 47. Cross-section through the fourth leg of a younger specimen of the
second larval stage than the preceding one. The dorsal side of the section is on
the right. The nephric duct is much coiled, and several sections through it are
shown. Cylindrical cells with fine granules around the periphery are uniting end

496 PATTEN AND HAZEN.

to end, forming long, narrow tubules. These tubules are found on the median
and dorsal side of the nephridial lobes. A number of small cells with large gran-
ules (g.c.) show on the lateral side of the lobes. x 300.

Fic. 48. Cross-section through the fifth leg of a crab about one inch long.
The dorsal side of the drawing is on the right and the median at the lower margin
of the page. It shows sections through the nephridial duct and the end sac with
its long, branching diverticula. The structure of the lobe differs in different places.
Near the end sac it is composed of long, branching tubules, lined with cells con-
taining coarse granules. Outside this layer the tubule cells are smaller ; on the
lateral side of the lobe the tissue is aerolated. On the lateral margin are large
cells filled with very coarse granules. x 100.

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of Morphology Vol.XVI.

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LIMULUS POLYPHEMUS. 497

INDEX LETTERS TO PLATE XXVI.

a. = anterior. m. = median.
art. = artery. m.art. = main artery.
c. = cells filled with fine granules. mes. = mesoderm.
cap. = capillaries. m.r. = Marginal ring.
c.tis. = connective tissue. m.y. = membrane on the yolk.
c.t. = collecting tubules. nm. = nerve.
é.n.d. = evagination of nephric duct. 2.¢. = nerve cord.
é.s. = end sac. u.a. = nephridial duct.
ex.op. = external opening of the nephric 2.f. = nerve fibers.
duct. p- = posterior.
fg. = fine granular cells. p.n.d. = nephric plate.
gc. = granular cells. s.d. = striated layer.
g-t. = granular tubules. so. = somite.
A.c. = hollow cells. z. = tubules.
int.n. = integumentary nerve. zp. = tubular portion.
/. = lateral. yk. = yolk.

EXPLANATION OF PLATE XXVI.

Fic. 49. Longitudinal section through the middle of the second lobe of an
adult nephridium. The section was 5 uw thick and was stained in borax carmine
and Lyon’s blue. The median side of the lobe is at the right. The lobe is com-
posed of four distinct regions or layers, each of which has a characteristic structure.
x 16. (1) The outer portion is formed of very large granular cells (g.c.) (see
Fig. 50) most abundant at the lateral end of the lobe on both dorsal and ven-
tral sides. Two smaller groups are shown on the median side. Small nerve
fibers penetrate this tissue. (2) 4.c. A dark layer which surrounds the lobe,
except for a short distance on its median ventral side. It is composed of small
cells with fine granules around the periphery. They are probably hollow cells
(Z.c.) which are uniting end to end (see Fig. 51). (3) g.¢. A faintly stained layer
inside of %.c. and surrounding the entire lobe. It is composed of small tubules
which are lined with large granular cells (see Fig. 52). (4) The larger part of
the lobe is contained in this tubular portion (¢.f.) (see Fig. 53). (5) ¢.¢. The col-
lecting tubular portion is similar to (4), except that the tubules are larger. They
connect the small tubules of each lobe with the collecting tubes of the stolon. It
is shown on the dorsal side of the figure and near the median end of the lobe
(see Fig. 54).

Fic. 50. An enlarged drawing through the large cells which surround the
lateral end of Fig. 49 (g.c.). They are filled with small granules, which did not
stain in either borax carmine and Lyon’s blue, or Delafield’s haematoxylin and
eosin. Several nuclei are often found in the same cell. Nerve fibers penetrate

498 PATTEN AND HAZEN.

throughout the tissue. A bunch of small, round cells, with fine granules around
their periphery (/.g-c.), is seen, apparently within the large cell on the left. x 270.

Fic. 51. Some of the hollow cells which nearly surround the nephridial lobe
(Z.c., Fig. 49). It shows cells with a finely granular periphery, apparently uniting
end toend. xX 270.

Fic. 52. Enlarged section through the tubules lined with large granular cells.
X 270.

Fic. 53. Enlarged section of the tubular part (2..) of the lobe. The tubules
are surrounded by a loose connective tissue, in which not infrequently were large,
round cells with granular protoplasm (c.). The tubules form an anastomosing
network throughout the central portion of the lobe. They have a cellular lining
which is separated by a dark membrane from the connective tissue (c.¢is.). x 270.

Fic. 54. Enlarged portion of the longitudinal collecting tubules (c.z.). The
tubules are large and branching. They have a heavily striated lining, which is
separated bya dark membrane from the connective tissue. Nerves and capillaries
are shown in the connective tissue, and also a few large granular cells, similar to
those in Fig. 53. x 270.

Fic. 55. Cross-section through the anterior part of the fifth appendage of a
specimen the same age as that in Pl. XXIV, Fig. 36 (5 u, haematoxylin). The
section shows the nephric plate of mesoderm cells at the base of the appendage.
It is continuous on both the lateral and median sides with a cellular membrane
which lies upon the yolk. x 200.

Fic. 56. The fourth section posterior to Fig. 55, showing an evagination of
the nephric plate to form the nephric duct. x 200.

Fic. 57. The third section posterior to Fig. 56, showing the posterior margin
of the nephric plate. x 200.

Fic. 58. Reconstructed outline of the nephridial duct and end sac, made from
a specimen of the same age as Pl. III, Fig. 40. x 400.

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A LIMULUS POLYPHEMUS. 499

INDEX LETTERS TO PLATE XXVII.

ap = fifth appendage. mes. = mesoderm.
ect.p. = proliferation of ectoderm. m.r. = Marginal ring.
e.n.@. = ectodermic portion of nephrid- #.c. = nerve cord.
ial duct. m.a@. = nephric duct.
é.5. = end sac. p.n.d. = nephric plate.
g.m.c. = granular nephridial cells. J5.m. = sphincter muscle.

/.n.d. = lip of the nephric duct. 50. = somite.

EXPLANATION OF PLATE XXVII.

Fics. 59-64 were drawn from a series of cross-sections through the fifth ap-
pendage of an embryo of the same age as that in Pl. XXIV, Figs. 37 and 38.
The sections were cut 8 uw thick and stained with Delafield’s haematoxylin.

Fic. 59. Section No. 1, showing a mass of mesoderm cells at the base of the
appendage, in the middle of which are a few larger and lighter-colored cells which
mark the anterior margin of the nephric duct. x 200.

Fic. 60. Section No. 2, showing the nephric plate folded on itself, making a
double layer of large, clear cells, which extend toward the ectoderm. The somite
is a closed cavity dorsal to the nephridial duct. x 200.

Fic. 61. Section No. 4, showing the nephric duct extending out to the ecto-
derm on the median margin of the appendage. x 200.

Fic. 62. Section No. 6. The nephric duct and somite are much reduced in
size, but retain the same relative position as before. x 200.

Fic. 63. Section No. 7, showing the somite reduced to a long, narrow space
on the surface of the yolk. In place of the nephric duct is a row of large
mesoderm cells with a slight outward projection. x 200.

Fic. 64. Section No. 10. The nephric duct is represented by a row of large
cells, continuous on the median and lateral sides with the yolk membrane. The
mesoderm at the base of the appendage and the somite have entirely disappeared.
X 200.

Fics. 65-70 are drawn from a series of cross-sections through the fifth
appendage of an embryo somewhat older than the one in the preceding series.
The proximal end of the duct has grown away from the median line, changing its
general direction somewhat.

Fic. 65. Section No. 1 shows the anterior margin of the nephric plate. x 200.

Fic. 66. Section No. 3 shows the mouth of the duct opening with a broad
lateral lip on the ventral side of the somite. x 200.

Fic. 67. Section No. 5 shows cross-section through the middle of the duct.
X 200.

Fic. 68. In Section No. 9 the lumen of the duct has disappeared. x 200.

Fic. 69. Section No. 10, showing the distal end of the duct. x 200.

500 PATTEN AND HAZEN.

Fic. 70. Section No. 11, showing the union of the ectodermic invagination
with the mesodermic portion of the duct. x 200.

Fic. 71 is a longitudinal section through the middle of the fifth appendage in
the early Trilobite stage, showing the opening of the nephric duct to the exterior.
X 200.

Fic. 72. Section on the lateral side of the base of the fifth appendage in a
late Trilobite stage. It shows the end sac, which extends beyond the base of the
appendage on the surface of the yolk. Granular cells (g.c.) are developing on its
ventral margin. X 200.

Fic. 73. Longitudinal section through the fifth appendage of a specimen
somewhat older than that of the preceding figures. It shows the distal arm of the
nephridial duct and its opening at the posterior side of the appendage. The ecto-
dermic portion is characterized by numerous small cells, which at the very end
seem to be forming a sphincter muscle around the opening. The mesoderm cells
are large, with a faintly colored protoplasm. In many specimens a lumen was
continuous throughout the mesodermic and ectodermic portions. x 300.

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Journal of Morphology. Vol.XV1-

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LIMULUS POLYPHEMUS. 501

INDEX LETTERS TO PLATE XXVIIL

c.m.t. = cross-section through a nephrid- Z.r.c. = large red cells.
ial tubule. mus. = muscle.

c.¢, = connecting tubes. z.l.-4 = nephridial lobes.
ec.p.=ectodermic part of nephric p.a. = pedal artery.

duct. pl. = plastron.

é.5. = end sac. p.m. = pedal nerves.
é.s.t. = tubule of the end sac. po. = pockets in the walls of the
ex.0. =external opening of the nephrid- duct.

ial duct. r.c. = red cells.

g.c. = granular cells. v.c. = vacuolated cells.

f.c.t. = hollow cells forming tubules. S.7.c. = small red cells.

EXPLANATION OF PLATE XXVIII.

Fic. 74. A fewcharacteristic nephridial cells of the Trilobite stage. @. Longi-
tudinal section through a cell with finely granular protoplasm around the periphery.
As the section passes near the surface of one end of the cell the granules show a
reticulated arrangement. 4. A cross-section through two cells similar toa. The
nucleus adheres to the side of the cell. c. Shows a small red cell on the surface
of 4. d. Red cells, with a clear protoplasm which took a deep stain in Lyon’s
blue. X 475.

Fic. 75. Sections through the nephridial cells from one of the older specimens
of the second larval stage. a. Longitudinal section through a tubule. 4. Cross-
section through a tubule. ¢andd. Cells in which large, dense-looking granules
cover the nucleus. X 475.

Fic. 76. Cross-section through the region on the dorsal side of the heart of
Limulus in the second larval stage, showing hollow cells similar to those in the
nephridial lobes. Several cells have united end to end to form branching tubules.
Other cells are present filled with large granules (g.c.), while others of the same
size have only a few granules in them. X 300.

Fic. 77. Section through a granular cell of a young Limulus three-fourths of
an inch long. (See Pl. XXIII, Fig. 48.) It shows one enormous granule in the
center with smaller ones around it. X 475.

Fic. 78. Section through nephridial cells, from the younger specimens of
the second larval stage. a. Longitudinal section through tubules which show a
granular periphery. 6. Section through two cells which have united. c. Cross-
section through a tubule. d. A small cell with the nucleus surrounded by
granules. X 475.

Fic. 79. Section of cells in the sixth leg of a specimen in the second larval
stage. X 475. a. Long hollow cells uniting, similar to those in the nephridial
and pericardial regions of the same age. 4. Large triangular cells uniting.

502 PATTEN AND HAZEN.

c. Cross-section through a hollow cell. d. Section through a granular cell.
e. Section through a triangular cell filled with granules.

Fic. 80. Cross-section through the end sac of a specimen in the Trilobite
stage. The section is posterior to the point where the nephridial duct opens into
the end sac. The end sac is lined with small and finely granular cells. One large
cell is shown on the dorsal side of the sac, filled with small granules, and with
pseudopodia extending out from its free margins. At the lateral side the end sac
shows a projection similar to both the wall of the end sac, and to the long hollow
cells which are characteristic of this age. Ventral to this sac is another of similar
structure, which unites with the end sac in the second section posterior to this.
These projections are either outgrowths from the wall of the end sac, or nephrid-
ial tubules united with it. Scattered among the nephridial cells were a number
of small red cells. Some of them had a faintly granular protoplasm, others were
vacuolated, and still others in which nothing but the cell walls could be distin-
guished outside the nucleus. X 400.

Fic. 81. Drawing of the injected nephric duct of an adult Limulus from the
dorsal side. The main part of the duct is coiled and folded upon itself many
times, the distal arm alone remaining straight, running from a point in front of
the anterior transverse process of the plastron along the edge of the plastron as
far as the fourth nephric lobe. It then passes between the muscles through the
median end of the last nephridial lobe to the exterior. Along the free margin of
the duct, slight projections, or pockets (/o.), are found. In other places small
connecting tubes (c.¢.) unite different portions of the duct.

Fic. 82. In this case the duct has been dissected apart along its entire length.
In many places small tubes were found, connecting one fold of the duct with
another which lay either beneath or beside it. In most cases these connecting
tubes had to be cut in order to free and unfold the duct. <A few of them are left
untouched (c.z.¢.). In other places small evaginations or pockets in the wall of
the duct were found along its free margin (Zo.). The nephric duct lies below the
outer edge of the plastron, deeply imbedded in muscle, the genital organs, and in
the hepatic caeca. The fourth lobe of the Nephridia shows from below.

Fic. 83. Drawing of the nephric region of an adult Limulus, showing the
nephric duct, nephridial lobes, and the blood vessels and nerves.

The four nephridial lobes lie at the base of the second, third, fourth, and fifth
legs. They are connected along their median dorsal ends by a band of collecting
tubes —the stolon. A short distance from the oral ring small branches arise from
the pedal arteries of the second, third, fourth, and fifth legs, and pass along the
ventral surface of each of the nephridial lobes, supplying the muscles beyond.
During its course, each artery sends off alternate branches to the muscles and to
the lobe. The arteries which pass to the lobes break up into small branches,
which fill the nephridia with a network of vessels.

Large integumentary nerves (z#¢.7.), arising from the haemal side of the brain,
pass out between each nephridial lobe to the sides of the carapace. On either side
of each lobe a smaller nerve arises from the haemal side of the pedal nerve which
supplies the tergo-coxal and the plastro-coxal muscles on the posterior and anterior
sides of the base of the coxite. The nephric duct lies near the lateral dorsal side
of the lobes. The distal arm passes through the posterior median end of the
fourth lobe to the external opening at the base of the fifth leg. The ectodermic
portion of the duct extends from the external opening to the fourth lobe,

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DHE SEMBRY OLOGY. OF). A) TERMITES
Eutermes (Rippertit 2).

FIRST PAPER.

(Including a contribution to the discussion as to the primitive type of development, and the
origin of embryonic membranes (amnion), and of the mesoderm, in the Insecta.)

HENRY McELDERRY KNOWER.

CONTENTS.
PAGE , PAGE

GND LOGIC HOM ss ereetes ee eek eee 1 | Changes in amnion and mesoderm
TNS OUTIL ee eer ta a Pe eo eee eg 2 during this elongation.................... 23
General description of egg................ 3 | Remaining development up to
“TODAS (0) Uae eee One eee ee 6 hatching e .cties. tee sesetsseee eee 25
Segmentation and formation of the

first rudiment of the embryo........ i) GENERAL.
@risinvof they mesoderm. --:ere--7.-- 16 | Conclusions as to the primitive na-
Origin of the amnio-serosal fold...... 18 ture of this type of development.. 29
Growth of the amniotic and meso- Origin of the mesoderm of insects .. 32

Glermaliruciim Cmts: ccs tek esse ences 1g | Origin of the amnion of insects........ 36
Elongation of embryonic rudiment Bxplanatony: mOte sess: tereenesecsseeeneee 53

UP CONSCSMEN tatlOM en serene ctsesrennane 2E7) |) Bibliography. sn.cccccssssesrecesesreeeeeenee 55

INTRODUCTION.

TuHouGH the Termites must be ranked among those insects
which have best preserved ancestral morphological traits, their
development has not been studied up to the present time.
Indeed, it is only within the last few years that we have been
enabled to judge of the details of development of any of the
primitive forms of insects. The technical difficulties which
the investigator of these forms must meet are very great, much
time being required to obtain few results, especially in dealing

1 This paper was accepted as a thesis for the degree of Doctor of Philosophy
in the Johns Hopkins University, May, 1896. (See explanatory note at the end.)

595

506 KNOWER. [VoL. XVI.

with early stages. As Wheeler (26) has suggested, no doubt
this has deterred many from working on such material.

Having had the good fortune to secure Termite eggs in
ample quantities, I have devoted considerable attention to the
development of these interesting insects, in the hope that such
a study might throw light on some vexed questions of insect
embryology. The present paper must be confined chiefly to
the earlier stages of development, the later changes being left
to be described at another time in the near future. The eggs
were obtained in Jamaica at different times, by myself, and
through the kindness of Dr. Hough, Dr. Sigerfoos, and the late
Dr. Conant. I am also indebted to Mr. Taylor, of Jamaica,
for assistance in obtaining material, and to Dr. William Patten
for valuable suggestions as to technique. It is a great pleasure
to express my appreciation of the constant kindness and useful
criticism and advice of Professor W. K. Brooks, during my
work at the Johns Hopkins University.

TECHNIQUE.

My material was fixed with hot water, with cold picro-
sulphuric acid (alcoholic), and with hot alcoholic picro-sulphuric,
the latter giving the most satisfactory histological results when
the acid was promptly washed out after fixation. Specimens
fixed in hot water and transferred into 70 per cent alcohol are
quite satisfactory.

As Wheeler found in working with Orthoptera, sections
through these eggs are very difficult to obtain. Though
tedious, Heider’s (13) method of painting each section before
cutting gives good results. A modification of Dr. William
Patten’s ingenious method of orienting small objects for cut-
ting is most satisfactory in sectioning these eggs. I feel that
my success in handling this material is largely attributable
to Dr. Patten’s suggestions as to technique and accuracy in
methods of study.

In sectioning early stages of the Termite germ-disc, I break
off most of the yolk in clove oil, with very sharp needles, under
the dissecting microscope, and stick the particle to be sectioned

No. 3.] THE EMBRYOLOGY (OFA) TERMITE. 507

on a piece of tracing paper smeared with a thin layer of collo-
dion fixative, and ruled with parallel lines by a needle point.
The paper is now dipped into xylol and then placed in the
paraffine bath. After imbedding, the paper may be stripped
off, leaving lines on the surface of the paraffine which give the
proper direction for cutting. It is possible in this way to obtain
especially thin sections of quite early discs, in any desired plane.
Enough yolk adheres to the disc, when dissected off, to make
plain its relations to the interior of the egg. In later stages
the entire egg is satisfactorily sectioned by the same method,
care being taken to prick the side which will lie uppermost in
the paraffine bath, to insure penetration. To prevent the yolk
from becoming brittle, eggs which are to be sectioned should
not be kept in clove oil any longer than is necessary to clear
thoroughly.

In studying the egg as an entire transparent object, I have
found it best to prick each specimen with a sharp needle under
the dissecting microscope; then stain in Grenacher’s borax
carmine for two days; wash out in cold 7o per cent alcohol,
acidified with 20 drops of nitric acid to every 100 cc. alcohol,
for three or four days or longer, until the stain remains only in
the nuclei; transfer gradually to absolute alcohol, and finally
into xylol, which gives whiter and clearer specimens than
clove oil.

Eggs for sectioning are usually stained in this way before
cutting, though it is best not to wash these out so much, and
as I have said, clove oil is then used to clear.

Good surface views of the germ-band at various ages are also
obtained by staining the specimen, dissected off from the yolk,
in rather strong Delafield’s haematoxylin for a very short
time.

GENERAL DESCRIPTION OF THE EGG.

There are no special chambers for nurseries, or for the queen,
in the nests of this species of Termes. (This species has been
described from Jamaicaas Eutermes Rippertiz. I shall question
this for reasons which will appear at another time. Hence the
interrogation mark after Rippertii in the title.) Oncutting

508 KNOWER. [VoL. XVI.

open a nest, the passages which traverse it in all directions
are seen to have no regular arrangement. The walls of the
inner passages are thinner than those at the surface. The
queen is generally found in the interior of the nest, near its
base, surrounded by numerous workers and larvae, and not far
from the eggs. The eggs have been collected into heaps, by
the workers, as they were laid, and piled up without any appar-
ent system in the passages near the queen. The older larvae
also assist in caring for the eggs. It is an easy matter to col-
lect quantities of eggs, since they are quite moist and adhere
together in masses, in which all stages of development are to be
found, from unsegmented ova to larvae emerging from the egg-
membranes. Considerable time must be subsequently devoted
to picking out stages from such mixed material.

The unsegmented egg is about 0.5 mm. long by 0.22 mm. in
the shorter diameter. It is elongated, larger at one end, and
markedly convex on one side. This shape makes it easy to
determine the planes of symmetry from the start, since the
enlarged or mycropylar end is found to be the posterior pole,
while the convex side is the ventral surface. In the course of
its development the embryo changes its position by a remark-
able process of “revolution,” like that described for the Libel-
lulids and certain primitive Orthoptera. This must be kept in
mind in speaking of the anterior and posterior poles, and of the
dorsal and ventral surfaces. As used above, the micropylar
end of the egg is the definitive, as well as the primary, posterior
pole, and the convex micropylar side becomes the final ventral
surface.

It will appear that the embryonic rudiment when first estab-
lished, and until shortly after the closure of the amniotic cavity,
lies entirely on the convex ventral surface of the yolk. At this
period the entire embryo occupies but a small area on this sur-
face, just beneath the micropyles, at its extreme posterior limit,
the hind end of the germ-band reaching the posterior pole of
the egg.

In the last stages of development the same relative position,
lost during intermediate changes, is reéstablished by ‘revolu-
tion,” so that the posterior end of the embryo comes again to

Now3:] THE EMBRYOLOGY OF A TERMITE. 509

lie on the convex surface at the micropylar pole of the egg,
while the head coincides with the anterior limit of the same
surface.

Dr. Hagen (12) described the micropyles of the eggs of this
species as follows: “The micropyles of Termes eggs have not
before been known. Near the upper pole of the egg, on each
side, there are four to six flat impressions; viewed in profile
they are similar to a soup dish. In the middle of this shallow
funnel is a tube of smaller diameter going through the yolk-
membrane in the direction of the egg pole.”

This description must be modified somewhat; since I have
found that the funnels are more numerous, and are grouped on
the ventral surface. The chorion is the only membrane pene-
trated by these funnels. The micropyles are arranged in a
semicircle, on the convex face of the egg, near the posterior
end. This semicircle is composed of from 12 to 18 funnels,
which vary considerably in arrangement. They may be strung
out into a single line, curving over the ventral surface, just
above the place where the embryonic disc first appears, and
extending up on either side toward the dorsal surface. Often
the funnels at the two ends of the line are crowded together,
while the ventral median ones form a single row (Pl. XXIX,
Fig. 1). In some eggs, on the other hand, the funnels of the
median ventral row are gathered together, while the lateral
openings extend in a line on either side. In surface prepara-
tions the rims of the funnels at the mouths may be seen to be
slightly corrugated.

Sections of the chorion above the germ-disc, as represented
in Pl. XXXI, Fig. 31, cut the micropyles through the wide
outer opening, the penetrating canal, or the inner opening.

Dr. Hagen (12) thought he could “see distinctly a bunch of
filiform spermatozoa inside the micropyles in some eggs,” and
that in one case he had succeeded in bringing them out by
pressure. I have never found any spermatozoa. Filiform
bunches are often seen beneath the chorion. On examination
such appearances have proved to be due to various objects, but
never to spermatozoa. In surface views of alcoholic specimens,
the folds of the vitiline membrane and protoplasmic threads

510 KNOWER. [Vou. XVI.

attached to the chorion, at times might be mistaken for bunches
of filiform bodies. When such specimens are crushed, the
membrane wrinkles up and looks like bunches of filaments.
The eggs from one nest had been attacked by fungus to such
an extent that the hyphae, entering the micropyles, had rami-
fied in the yolk and used it up, destroying the embryos. Dr.
Hagen could hardly have confused such large objects as these
hyphae with spermatozoa. As sperm failed to appear in any
of my stained specimens, I am inclined to think that folds in
the membranes or strands of protoplasm were mistaken for
spermatozoa.

hHE VY ODK:

My observations on the yolk were made on preserved
material. The yolk-mass, thus studied, is composed of a lot
of polygonal bodies, which are vesicles containing a homoge-
neous coagulable fluid. These bodies stain deeply in haema-
toxylin and carmine, and very considerably in size and shape
(Pl. XXXI, Figs. 30-37, yé.). The other constituent of the yolk-
mass is an oily fluid distributed in small globules throughout
the egg, and often found collected in one or two quite large
drops. When alcoholic specimens are crushed, the oil globules
flow together and run out, leaving the yolk-bodies for the most
part intact. In such cases the character of the vesicles can be
made out. They are found to vary in structure, part of them
being filled entirely with homogeneous coagulable substance
quite elastic under pressure.

Many of the bodies, however, differ from this. They contain
the homogeneous stainable substance, but also little oily drops,
of which there may be only a few or a great number in a
vesicle. The greater the collection of these drops, the less is
the homogeneous material, so that it is often reduced to a
porous substratum for the drops. It is possible to crush such
vesicles so that the oil drops escape through the membranous
walls. Examined in clove oil or in sections the yolk has quite
a different appearance. In these specimens the oily matter is
all dissolved out of the yolk, even from the interior of some of
the vesicles, Treatment with oils and subsequent heating dur-

No. 3.] THE EMBRVOLOGY (OF Ay TERMITE: Sit

ing imbedding often cause numbers of the yolk-bodies to fuse
together into large bodies. This may be very marked, large
spaces being left in the yolk by the solution of the oily fluids.
In sections single yolk-bodies are frequently found riddled with
holes, left by the solution of the oil drops formerly imbedded
in their substance (Pl. XXXI, Figs. 30, 31, 34, 37, p. y0.).

I should derive the yolk-bodies in which oil drops have col-
lected from the homogeneous vesicles. The homogeneous
(albuminous) coagulable fluid of these bodies apparently be-
comes transformed into soluble substances which, first forming
in isolated drops, finally fill the whole vesicle. In this way the
oil globules and free oily fluids of the yolk would appear to
arise from the (albuminous ? ) yolk-bodies, to furnish the growing
embryo easily assimilable nutriment. The sections (Pl. XXXI,
Figs. 30, 33, and 34) show finely fragmented yolk-bodies lying
beneath the embryonic disc. Other sections do not show a
similar fragmentation, the solution having been more complete.
When yolk-bodies in which drops have collected are prepared
for sectioning by the usual methods, chemical changes take
place which result in homogeneous fused masses of stainable
(albuminous ? ) substance, and in the solution and removal of the
fatty matters.

There is apparently no definite arrangement of the different
yolk elements. There is no peripheral layer of protoplasm
distinguishable before the formation of a blastoderm. I have
found no “segmentation of the yolk”’ during the early stages
of development.

SEGMENTATION AND THE CHANGES IN THE BLASTODERM
LEADING TO THE ESTABLISHMENT OF THE FIRST
RUDIMENT OF THE EMBRYO.

The first sharply marked rudiment of the embryo is, as in
the case of certain of the Orthoptera (Stenobothrus, Stagmo-
mantis, Gryllus, and Oecanthus), a relatively small disc of
closely crowded cells at one pole of the egg. Since the
history of the origin of these embryonic discs has not been
worked out, I have studied, with special care, the segmentation

Sy 2 KNOWER. [VoL. XVI.

and those changes in the blastoderm cells which result in the
formation of the embryonic rudiment on the surface of the
yolk.

I have endeavored to answer the following questions: Is the
disc formed immediately during the segmentation, by cells wan-
dering ‘directly to the point on the surface where the disc is
to appear? Is a blastoderm over the entire yolk surface first
formed as a result of segmentation, and then the disc from its
cells? Again, if this latter is the method, is the embryo a
result of a simple multiplication of the cells of a restricted area
of the blastoderm, or is there some other factor present in the
formation of the disc ?

The answer to these questions requires considerable atten-
tion to the earliest stages, but is of much importance to an
understanding of the origin of the “under-layer” and of the
amnion.

The position of the polar-bodies marks the dorsal pole of the
shorter axis of the egg. I have not studied the formation of
the polar-bodies from the nucleus, as the few eggs which were
at the proper stage did not show the process distinctly enough.
To separate from the polar-bodies, the segmentation nucleus
moves from the center of the yolk to the center of the dorsal
surface. After their formation, the polar-bodies appear as two
little rod-like masses of chromatin surrounded by a small quan-
tity of protoplasm, and lie at about the middle point of the
flattened dorsal surface of the yolk-mass (Pl. XXIX, Figs. 2
and 3). Later the chromatin breaks into fragments, but the
little collection remains visible for a number of divisions.

The segmentation nucleus, on returning from the dorsal
surface, lies, just previous to the first division, in the center of
the yolk at the intersection of the shorter and longer axes of
the exe (PU OIE ia 12).

The first spindle lies at right angles to the shorter axis, so
that one of the cells arising from the first division wanders
toward the enlarged posterior pole, where the embryo will first
appear. The other cell remains near the position that the
mother nucleus held (Pl. XXIX, Fig.3). At the start, then, there
is a decided proliferation toward the future embryonic area.

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NO.:3:] THE EMBRYOLOGY OF A TERMITE. 513

This is brought out better in the following stage, which exhibits
two cells in the enlarged end, one on the shorter axis, and one
in the small end; that is, there are three nuclei nearer the
posterior than the anterior pole (Pl. XXIX, Fig. 4). The cleav-
age becomes irregular with the eight-cell stage, one or more
nuclei dividing before the time for a typical rhythm of divisions.
For several divisions there is a slight preponderance of cells in
the larger end of the egg. For instance, one egg has four
nuclei in this end, one on the shorter axis, and three anteriorly ;
while another has five in the posterior, and four in the other end
(Pl. XXIX, Fig. 5). Generally, during the early stages of
cleavage, there are three or four more cells in the larger than
in the smaller end of the egg. After five or six divisions, the
resulting nuclei have taken positions at about equal distances
apart through the yolk. The nuclei are each surrounded by a
little mass of protoplasm, and may consequently be spoken of
as cells. As far as can be determined, there is no proto-
plasmic continuity between these cells at this early period.
Later, when the embryonic disc begins to appear, continuity
is established between its cells; but even then a connection
between the blastoderm cells of other regions, or between these
and the yolk-cells, is not made out with any degree of cer-
tainty. A view of the ventral surface of an egg at this stage
shows very well the equal distribution of the nuclei on that
side, and the same is found to be true of the nuclei on the
remaining surface of this egg (Pl. XXIX, Fig.6). (Refer to
end of paper, to the explanation of Figs. 4 and 5, in regard to
cleavage.)

Most of the cells have now reached the surface, there being
only a few in the yolk which lie at equal distances apart. In
properly prepared material, the changes that follow and lead to
the appearance of the embryonic disc can be most distinctly
traced in entire, transparent eggs studied in clove oil, cedar oil,
and balsam. The following description refers chiefly to speci-
mens studied in this way and to sections through certain stages.
I have already stated that the various stages are mixed together
indiscriminately when collected. The series illustrating the
growth of the disc had to be picked out from a great mass of

514 KNOWER. [Vor. XVI.

material. There can be little doubt, however, that a typical
series is here figured, for the figures are based on an examina-
tion of a great many specimens, and the chief stages are well
marked.

Since the first rudiment of the embryo is formed from
surface cells alone, the few yolk-cells may be neglected in
the description.

Pl. XXIX, Figs. 7-102, represent successive changes on the
surface of older eggs. The nuclei are found at all points on
the surface in the act of dividing, or in pairs just subsequent to
division. In the posterior half of the egg this activity becomes
especially pronounced, while the nuclei of the anterior half are
comparatively inert.

Three surfaces of a somewhat older egg are shown in PI.
XXIX and Pl. XXX, Figs. 11-115 (ventral, dorsal, and lateral
views). As compared with the preceding figures and with the
following ones, it is evident that the number of cells in the
anterior half of this egg has reached a maximum, which remains
constantly about the same in older specimens. The nuclei in
this half are few and widely separated. The opposite end, on
the other hand, is the seat of active multiplication and change.
This is true of the whole posterior end, but it is evident in the
three views of the egg before us that the dorsal (Pl. XXX,
Fig. 112) and lateral (Pl. XXX, Fig. 11>) surfaces of this half
are less crowded with nuclei than is the ventral side. The
ventral surface (Pl. XXIX, Fig. 11) exhibits an extensive area of
rather closely crowded nuclei, stretching to the extreme limits
of the surface posteriorly and laterally. A side view (Pl. XXX,
Fig. 11>) shows a considerable lateral extension of this area,
relatively crowded as compared with the rest of the surface.

The posterior half of the surface represented in Pl. XXIX,
Fig. 10, exhibits an activity in division and a distribution of
nuclei of about the same intensity in its entire extent, forward
to the shorter diameter of the egg. A line drawn through the
shorter diameter of this figure divides rather sharply an ante-
rior half, with but few widely separated nuclei, from a posterior
half, in which the nuclei are more numerous and lie compara-
tively close together down to the line just drawn. Near the

No:3/] THE EMBRYOLOGY OF A TERMITE. 515

posterior pole this area is slightly more crowded than near
the shorter diameter; but there is very evident activity here,
contrasting sharply with the inertia of the cells on the anterior
side of the line.

Drawing a similar line across the middle of the older egg
(Pl. XXIX, Fig. 11), we find no change anterior to the line.
In the region just posterior to this line, extending as far back
toward the pole as a second line drawn parallel through the
anterior end of the dotted pointer ca., there are fewer nuclei
than in a corresponding region of the younger egg (Pl. XXIX,
Fig. 10) —by actual count, nearly one-third less than in the
earlier stage, or 26 to 36 nuclei. On the other hand, in area
ca., Pl. XXIX, Fig. 11, a decided increase in the number of
cells is evident, as compared with the preceding stage. The
nuclei here are not only one-third more numerous (about Io1
to 157), but are much more closely crowded together.

Such a comparison indicates strongly that, in addition to a
special activity in cell division within the area ca. of Pl. XXIX,
Fig. 11, certain cells have actually wandered into this area from
more anterior portions of the surface.

If the number of cells in the region anterior to ca., down to
the line through the shorter diameter, had remained the same
as in the preceding younger stage (Pl. XXIX, Fig. 10), there
would have been reason to conclude that this constant number
had been maintained, in spite of a multiplication of cells, by a
migration back into ca. One-half of the product of the divi-
sions of the nuclei might have wandered back into ca. from the
more anterior region, without disturbing the relations existing
in Pl. XXIX, Fig. 10. As it is, the evidence of a migration
back into the area ca. is much stronger, since an actual decrease
in the number of nuclei anterior to ca. has been shown; while
the increase in the cells of ca. is sufficient to allow for this addi-
tion from without, as well as for that from a multiplication of
the cells already within its limits.

Similar results are obtained from a comparison of dorsal
surfaces.

It may be claimed that this method is inconclusive, since the
specimen from which Pl. XXIX, Fig. 11, was drawn cannot be

516 KNOWER. [Vou. XVI.

proved to have certainly passed through a stage like that of
Pl. XXIX, Fig. 10, having been selected from a lot of eggs in
which all stages were mixed indiscriminately. The condition
shown in Pl. XXIX, Fig. 11, may have been reached without
migration by a more active multiplication of cells in the area
ca. from the first, the blastoderm anterior to this region remain-
ing comparatively inert. In other words, the center of activity
may have been placed more anteriorly in Pl. XXIX, Fig. 10,
than in PIP XX DS Pies 11, from the start:

In spite of this possibility of error, I believe the figures do
represent successive stages, and that the area ca. on the sur-
face of the egg in Pl. XXIX, Fig. 11, etc., has been established,
not only by a multiplication in that region, but also by the addi-
tion of cells migrating into it from without. This conclusion
seems justified by a similar examination and comparison of
many eggs in these stages.

Pl. XXX, Fig. 12, is a slightly older ventral surface showing
a like extension of the area ca., where more nuclei are now
found. Note especially the rather short intervals between the
nuclei in the posterior and lateral regions.

Pl. XXX, Figs. 13 and 14, exhibit in ventral and lateral views
a further result of the processes just studied.

Comparing Pl. XXX, Fig. 13, with the younger stages in
Pl. XXIX, Fig. 11, and Pl. XXX, Fig. 12, the number of nuclei
in regions anterior to the area ca. is seen to have remained
constant, in spite of a multiplication of cells there being
demonstrable. Within the former area ca. there has been a
great increase of nuclei, especially near the center. This is
undoubtedly due in part to continued cell division here; but
also, as the above observation makes plain, there is evidence of
an addition of migrating cells resulting from multiplication in
more anterior regions.

Comparing Pl. XXX, Figs. 13 and 14, still closer with Pl.
XXX, Fig. 12, additional and striking evidence is found of a
further migration of cells from the boundaries toward the
center of the former area ca.

On rolling the egg figured in Pl. XXX, Fig. 12, the area ca.
stands out more sharply from the surrounding surface than is

No. 3.] THE EMBRYOLOGY OF A TERMITE. ise y/

shown in the figure. Near the lateral and posterior boundaries,
as well as in the center, the nuclei are about equally distributed
and lie rather close together. Turning to the older stage (PI.
XXX, Fig. 13), it is evident that the nuclei in the lateral por-
tions of the same area are fewer than in the younger egg, and
nearly twice the distance apart. The egg (Pl. XXX, Fig. 14),
being rolled slightly on one side (though not nearly so much so
as Pl. XXX, Fig. 11, with which it must not be compared),
shows this better than Pl. XXX, Fig. 13, in which the convexity
of the surface makes it impossible to give an accurate idea of
the distribution of the nuclei at the sides. The letters /4.d.
indicate a like region in both figures (Pl. XXX, Figs. 13 and
14). It is the portion of the surface lying outside of (lateral to)
the position marked by these letters that shows a diminution in
the number and a wider separation of the nuclei, as compared
with the previous stage.

These changes within the limits of the posterior half of the
ventral surface, between the stages of Pl. XXX, Figs. 12 and
13, resulting in a perceptible diminution in the number of nuclei
laterally, with an increased crowding toward the center, appar-
ently necessitate an active migration of cells centripetally,
cooperating with cell multiplication, to establish the embryonic
disc.

Pl. XXX, Fig. 15, is an example of an older egg, showing an
extreme concentration of the embryonic disc.

In Pl. XXX, Fig. 18, which represents the ventral surface at
a much later stage, the embryonic region, now appearing as a
conspicuous and sharply defined circular disc of nucleated
protoplasm, hardly occupies one-half of the area formerly
marked ca. The surrounding cells are few and widely scat-
tered, while the comparatively broad, crowded area in the
earlier figures (Pl. XXX, Figs. 12 and 13) has contracted to the
smaller, densely crowded, circular embryonic rudiment. There
is a marked concentration in the germ-dis¢ visible in passing
from the stage shown in Pl. XXX, Fig. 17, to that of Pl. XXX,
Fig. 18. Note the concentric crowding of the nuclei along the
sides of the disc in Pl. XXX, Fig. 18, as compared with the
preceding figure.

518 KNOWER. [VoL. XVI.

A study of sections of eggs passing through these stages
apparently confirms what is learned from surface views.

In its early stages the embryonic disc is in cross-section a
comparatively broad, flat plate of protoplasm formed by the
fusion of its cells, the neighboring cells of the blastoderm
being connected rather loosely with the edges of this area
(Pl. XXXI, Fig. 30). In reaching its final restricted size in
Pl. XXX, Fig. 18, the broad plate of protoplasm, whose bound-
aries were well defined in an earlier section, has become much
reduced in extent. The section of the completed disc (Pl.
XXXI, Fig. 31) shows the plate contracted to a decidedly
shorter diameter. (The two sections (Pl. XXXI, Figs. 30 and
31) are drawn to the same scale.)

The manner in which the mesoderm arises (described further
on), partly by a crowding of cells below from the embryonic
area as it becomes defined, is another argument in support of
the view here advanced for the formation of the first rudiment
of the embryo.

The area of the blastoderm, the origin and gradual concen-
tration of which we have thus traced, will be henceforth spoken
of as the embryonic area or germ-disc. Though it might be
so called at an earlier stage, it hardly merits the term before
reaching the definiteness of outline shown in Pl. XXX, Fig. 18.

The facts here reviewed appear to me to prove that the
embryonic disc is not formed directly in the segmentation by
cells wandering toward a predetermined point. The evidence
indicates also that the disc is not the result of simply active
cell multiplication in a restricted area of the blastoderm. The
truth seems to be that segmentation results in the establish-
ment of a blastoderm of cells scattered over the entire surface
of the yolk, and that then, as these cells increase in numbers,
a process of concentration draws many of them together to
form an area on the ventral surface, which is the first rudiment
of the embryo, the germ-disc. This is shown in the entire series
of stages figured, and is brought out vividly by a comparison
of Pl. XXX, Figs. 12 and’ 13, with Pl) XXX, Fig. 73. /IneBk
XXX, Fig. 12, the embryonic area spreads over the whole of the
posterior half of the ventral surface of the yolk. In Pl. XXX,

No. 3.] THE EMBRYOLOGY OF A TERMITE. 519

Fig. 13, the limits of this diminishing area have drawn well in
toward the center and away from the lateral margins of this
portion of the ventral surface. In Pl. XXX, Fig. 18, the germ-
disc hardly covers one-half of its extent in Pl. XXX, Figs. 12
or 3.

The appearances are not at all what would be expected from
a simple cell multiplication in a restricted area. In such a
case the growing disc should, it seems, be formed from the
coalescence of several areas multiplying around separate cen-
ters, or should spread out on all sides as its cells multiply
around a single center. As the figures show, the disc is here
formed by a steady contraction of a primarily extensive area
toward a central point.

The fact that, at even so late a stage as one showing the
amnio-serosal fold, the nuclei of the disc are of the same size as
those in the surrounding blastoderm, perhaps lends some sup-
port to the above contention ; since we should expect a rapid
multiplication within a restricted area of the blastoderm to
produce a mass of cells in that region of smaller size than on
the surface elsewhere. In the Termite, during this period, the
nuclei of the blastoderm in the whole posterior half of the egg
appear to divide with about the same rapidity. The process of
concentration, which draws the cells together to form the disc,
is accompanied by a steady multiplication of the cells about to
be incorporated in it, but the nuclei of the rest of the blasto-
derm divide also. The position of the embryonic disc is conse-
quently not marked by nuclei smaller than those elsewhere on
the blastoderm, in the stages we are considering.

The first rudiment of the embryo is certainly not formed
around a number of discrete centers, as is claimed for some
decapod crustacea and certain insects. The concentration lead-
ing to its first formation is, from the start, most apparent in the
posterior portion of the disc. The posterior border becomes
sharply defined at an early stage, as the cells draw together in
concentric rows from the posterior pole. The lateral edges are
next involved ; but much later, when the disc is otherwise well
outlined and its cells are quite closely crowded, the nuclei of the
anterior end have not yet drawn together (Pl. XXX, Figs. 18 and

520 KNOWER. [VoL. XVI.

1g). When the amnion is about to close over, the cells of this
end have drawn together and become incorporated in the disc.

I cannot determine whether the concentration, in the early
stages, is accomplished by the migration of independent amoe-
boid cells toward the embryonic area, or whether the blastoderm
outside this area is from the first a continuous membrane of
loosely connected cells which contracts toward the center of
the germ-disc. I believe, however, the blastoderm cells beyond
its limits to be independent, to a late stage in the formation of
the disc.

A less marked concentration of the surface cells has been
observed in other insects in similar stages, resulting in a closer
approximation of the cells of the embryonic area. Refer to
Patten (21), Figs. 1 and 2 of Pl. XXXVI (A), and Fig. 5 of
Pl. XXXVI (B), and Wheeler (25), Figs. 63, 64, 66, and 68.

In the Termite’s egg, where the embryo is a comparatively
small disc when completely established, the concentration to
establish this disc is an especially notable process.

McMurrich (18) has discovered a similar method of the for-
mation of the embryonic rudiment in Isopods. His figures,
17-19 and 50-52, show the formation of the germ-band in
these crustacea by a concentration of the surface cells toward
the ventral side of the egg. He finds an intimate connection
between this phenomenon and the formation of an “ under-
layer,’ and my observations on the Termite’s egg lead me to a
similar conclusion for it. Hence the detail in which I have
described the early stages.

ORIGIN OF THE MESODERM.

I have studied the origin of the under-layer with especial care,
on account of the recent conflicting results of Wheeler (26) and
Heymons (14) in regard to its formation in the Orthoptera.

In the Termite there is no gastrula invagination. The
under-layer begins to appear at an early stage in the formation
of the disc, somewhat earlier than Pl. XXX, Fig. 14, when its
cells first begin to be crowded. During this period, at irregu-
lar points in the embryonic area, lateral as well as median,

No. 3.] THE EMBRYOLOGY OF A (TERMITE, 521

some of the cells are pushed below the surface by the concen-
tration of the blastoderm. Other cells are separated toward
the under surface of the ectoderm, by tangential divisions of
its nuclei, at various scattered points (Pl. XX XI, Fig. 30).

As these processes continue, the under-layer constantly
gains in bulk. Its formation is to be traced back to the con-
centration of the cells of the disc, and when this has reached the
stage represented by Pl. XXX, Fig. 18, the under-layer cells
have for the most part collected into a plug projecting into the
yolk. From the surface this plug appears as a darkened area
of crowded nuclei near the center of the disc.

Preparations of a series of discs, after the under-layer has
become thus crowded into a plug, illustrate the growth of this
collection of cells. Pl. XXX, Figs. 16-19%, show, in surface
views, the gradual extension of the plug, up to the time when
the amnio-serosal fold has grown well forward over the disc.

Sections through these stages and those just preceding and
immediately following, taken in connection with what has been
learned from surface views, give interesting data as to the for-
mation of the under-layer and the amnion.

Pl. XXXI, Fig. 30, gives a cross-section of the single-
layered disc at a stage somewhat older than Pl. XXX, Fig. 13,
when it is first definitely outlined from the surrounding blasto-
derm. There is a crowded appearance of the cells, and some
of the nuclei are displaced from the surface and seen wedged
below. At various points in the surface layer, at the sides as
well as near the middle, nuclei are also found in the act of
dividing toward the lower surface, thus adding to the number
of cells adhering in the lower layer of the disc.

A cross-section (Pl. XX XI, Fig. 31) of the embryonic area
through the region of the plug at the stage (Pl. XXX, Fig. 18),
when compared with Pl. XXXI, Fig. 30, cutting the same
region of a younger disc, shows that the plug has grown con-
siderably by the gradual addition of cells from the ectoderm
and their subsequent multiplication. The mesodermal plug is
still in close continuity with the ectoderm.

A sagittal section of a disc of this age (Pl. XXXI, Fig. 32)
shows the plug quite distinctly.

522 KNOWER. [VoL. XVI.

Both surface views and sections of these stages agree in
exhibiting no gastrular groove. On the contrary, it is as I
have stated — the under-layer arises at all points in the germinal
disc, as a result of the concentration of this area and of the
tangential divisions of its cells. The formation of a mesoder-
mic plug is apparently a further outcome of the concentration.
(Consult McMurrich (18) on the formation of the under-layer
in Isopods.)

A discussion of the general bearing of these facts on the origin
of the mesoderm in insects will be found further on.

ORIGIN OF THE AMNIO-SEROSAL FOLD.

I have devoted much attention to the early history of the
embryonic membranes, on account of the general interest their
presence excites.

When the amnio-serosal fold is first clearly defined as a fold
in sections, it appears from the surface (Pl. XXX, Figs. 19
and 192) as a semilunar fold along the posterior border of the
embryonic disc, extending forward on either side toward the
anterior end. Sagittal sections of this stage make plain that
the inner or amniotic layer of the fold is not distinguishable
from the ectoderm of the germ-disc, except by its position
(Pl. XXXI, Fig. 33). It is of the same thickness as the ecto-
derm, and its nuclei are arranged in the same layers, inverted.
The outer or serosal portion of the fold, on the other hand, is
quite different (Pl. XX XI, Fig. 33). This is a thin membrane
of much flattened cells with nuclei far apart. This membrane
resembles the rest of the extra-embryonic blastoderm of which
it is a continuation. (This evident distinction between amnion
and serosa ts tmportant, as will appear further on.)

Figs. 19 and 33 of Pls. XXX and XXXI, though represent-
ing the amnion when first appearing as a completed fold, do
not exhibit the earliest stage in the formation of the amnio-
serosal fold of the Termite.

Several stages before a fold can be made out in sections, its
position is outlined on the surface of the disc. When the under-
layer plug first appears in surface views, the embryonic disc

No. 3.] THE EMBRYOLOGY OF A TERMITE. 523

is quite sharply marked out, especially on its posterior border
(Pl. XXX, Figs. 16 and 17). It is along this border that the
amnion is to appear. Pl. XXX, Fig. 18, with the two figures
just referred to, shows that, as concentration of the embryonic
area proceeds, the nuclei at the posterior end draw together into
the disc in concentric rows, which results in a closely crowded
semicircle of cells that becomes quite conspicuous in surface
views. In Pl. XXX, Fig. 18, this semicircle has become a
band of nuclei, much darker than the region of the disc just in
front of it, where the nuclei are not so densely crowded.

Sagittal sections of discs in these stages (Pl. XXXI, Fig. 32),
younger than that illustrated by Pl. XXXI, Fig. 33, teach that
the posterior margin, corresponding to the dark semicircle on
the surface, differs from the rest of the disc only in a some-
what greater thickness of the ectoderm. There is as yet no
fold in sections.

It is evidently the posterior thickened margin of Figs. 18 and
32, which has folded over in Figs. 19 and 33, to become the
amnion.

It will be noted then, in reference to the origin of the amnion,
that it is formed with the disc in the same process of concen-
tration, and that it is, at first, evidently merely a specialized por-
tion of the disc before folding forward to become the amnion.

This agrees essentially with the figures which Bruce (6)
gave for Mantis (Pl. IV, Figs. 42 and 43); with Patten’s (21)
description and figures of the Phryganid; with Will’s (27)
account of the Aphids; and with the results of most observers,
though all do not agree in regarding the amnion as a part of
the embryonic rudiment.

T have reserved a final section of this paper for a general dis-
cussion of the origin of the mentbranes tn insects.

CoNTINUED GROWTH OF THE AMNIO-SEROSAL AND MESODERMAL
RUDIMENTS TO THE CLOSURE OF THE AMNIOTIC CAVITY.

Preparations of eggs illustrating successive stages in the
closure of the amniotic cavity show that this is accomplished
by the single semilunar fold growing forward from the posterior

524 KNOWER. [VoL. XVI.

end of the disc. There are no separate lateral folds, nor is
there any ‘“‘head-fold.”’ Ina series of specimens represented in
Figs. 19-24, Pls. XXX and XXXI, the membranes are found
extending further and further anteriorly over the disc. In
Pl. XXX, Fig. 23, the amniotic cavity remains open in only a
single spot at the anterior extremity of the disc, the closure of
which opening, in Pl. XX XI, Fig. 24, completes the process.

A series of sagittal sections, like that shown in Pl. XXXI,
Figs. 32-35, gives a better idea of what has just been pointed
out in the surface figures. (The nuclei in the resting stage in
this series of figures are represented in solid black for the sake
of clearness. They resemble those in Pl. XXXI, Figs. 30 and
31, being large, vesicular, and containing fragmented masses of
chromatin.)

Pl. XX XI, Fig. 32, already referred to in a previous section,
exhibits the appearance and relations of the amnio-serosal and
mesodermal rudiments when first well established. The meso-
dermal collection of cells lies under the anterior half of the
embryonic disc. It does not extend beneath the extreme
anterior end, and is still rather intimately associated with the
ectoderm from which it arose. Behind this mesodermal plug,
and between it and a posterior thickening of the ectoderm
(already indicated as the first rudiment of the amnion), is a
thinned region of the disc with only one layer of nuclei,
corresponding to the lighter portion of the surface view in a
like position. Note the immensely enlarged yolk-cell nucleus
as compared with one of the mesoderm.

In Pl. XXXI, Fig. 33, asection of the stage(Pl. XXX, Fig. 19),
except for an increase in the size of the rather loose mesodermal
plug (due partly to a continued migration from the ectoderm,
as indicated by the direction of the spindle of the dividing
ectoderm nucleus anteriorly, and by the crowding of the cells
in the lower layers of the ectoderm), the most striking change
is a bending forward of the thickening, marked amnion in the
preceding stage, to form a fold. The bend takes place in the
thin, single-layered portion of the disc. The serosal cell pos-
teriorly is much flattened, and is drawn forward by a very
slender thread of protoplasm. It is interesting to observe, in

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No. 3.] THE EMBRYOLOGY: OF A) TERMIPE: 525

this section and the following ones, fine protoplasmic processes
running out from the ectoderm. In some instances I have
traced such threads out to the chorion and into the micropylar
funnels.

As the cells of the amniotic fold have multiplied, it has bent
well forward in the next figure (Pl. XX XI, Fig. 34). Its cells
form a thick mass and are arranged in two layers. Posteriorly
it passes into the ectoderm through the thinned region pointed
out in the former stage. The flat serosal cells lie superficially
drawn forward with the amnion. The mesodermal plug is
more sharply defined from the ectoderm, its cells lying loosely
together in the former position and dividing in places.

When the amniotic cavity is finally closed completely (PI.
XXXI, Fig. 35),as in Pl. XXXI, Fig. 24, from the surface, the
resemblance between the amnion and the ectoderm is most strik-
ing. The cells of both are arranged in two layers and divide in
a similar manner. The serosa is nowa very thin membrane of
large, flat cells, stretching over the embryo and enclosing the
yolk. Its nuclei are found, from now on, in resting condition,
with one or more nucleoli and granular looking chromatin. They
divide seldom. The mesoderm is now sharply separated from
the ectoderm, and from this time the separation appears to be
maintained. A few mesoderm cells have pushed back to the
extreme posterior end of the embryo. At the anterior end the
former relations remain unchanged. The yolk-cell nuclei are
of remarkable size and have apparently remained undivided
from an early stage.

GROWTH OF THE Disc-SHAPED EMBRYONIC RUDIMENT INTO
AN ELONGATED GERM-BAND UP TO THE TIME
OF ITS SEGMENTATION.

Comparing Pl. XXXI, Fig. 24, with figures of earlier stages, it
is evident that considerable change has taken place in the
shape of the embryo. The disc has now grown larger. It is
about twice as long as broad, and while the posterior end is
enlarged and rounded, the anterior extremity is rather pointed.
The cells of this disc and of the amnion have become much

526 KNOWER. [Vou. XVI.

smaller by repeated divisions, while those of the serosa are now
comparatively very large, having before this practically ceased
to divide. This transparent egg also shows the few large
yolk-cells, seen better in sections.

The growth of the embryo, from the time when the amniotic
cavity is completely closed, is chiefly at its posteriorend. The
hind end of the embryonic band pushes back over the posterior
end of the yolk-mass, just beneath the serosa (Pl. XX XI, Figs.
25, 27, and 29), while the head end remains fixed. (In some
exceptional eggs the embryo is found out of its usual position,
slipped forward or backward.) This growth continues for some
time over the posterior pole, no marked change being apparent
superficially, except an increase in length and breadth. The
anterior end, however, becomes gradually less pointed.

A germ-band slightly older than that shown in Pl. XXXI, Fig.
24, while not yet one-half the length of that in Pl. XXXI, Fig.
26, would have already acquired a square, broad anterior end, as
in the later stage.

The embryo in Pl. XX XI, Fig. 26, is not in the usual position
at this period, some few eggs thus exhibiting the germ-band
entirely on the ventral surface, and giving its shape and rela-
tions better than can be shown by drawing an embryo dissected-
off from the yolk. Pl. XXXI, Fig. 27, represents in side view
this same stage, as it is found usually, with the few exceptions
just noted.

The germ-band now continues to push back around the yolk-
mass, until about one-third up on the flattened dorsal side of
the egg, when the embryo forms a U-shaped figure, lying over
the enlarged end of the yolk (Pl. XXXI, Fig. 29). At this time
the band is still unsegmented. Posteriorly it terminates in a
rounded extremity. The anterior end has in the mean while
undergone considerable change. From beinga narrow-pointed
tip to the band (Pl. XX XI, Fig. 24), it first gradually widened into
a square end (Pl. XX XI, Fig. 26, and stages between this and PI.
XXXI, Fig. 24), and finally spread out over the yolk anteriorly
and laterally, until now (Pl. XXXI, Figs. 28 and 29) this region
has become the most prominent part of the embryo. Anteri-
orly, just in front of the point where the mouth is to appear,

No. 3.] THE BM BEVOLOGY OFA TERMITE yey

the cephalic region is slightly emarginated. On either side it
extends up on the yolk as a broad lobe with rounded borders.
Such is the appearance of the embryo just before segmentation.
(See also next section for a description of Pl. XX XI, Fig. 28, of
this stage.)

CHANGES IN THE MESODERM AND AMNION DURING THE
ELONGATION OF THE GERM-BAND BEFORE
ITS SEGMENTATION.

Pl. XXXI, Fig. 36, is a sagittal section through a stage in
the elongation of the embryo, slightly older than that of PI.
XXXI, Fig. 24, when the anterior end has broadened and
become square, as in Pl. XXXI, Fig. 26. Compared with
Pl. XXXI, Fig. 35, this whole embryo is decidedly longer.
The amnion appears thinner, its cells are becoming arranged
in a single layer, especially at the anterior end. As the germ-
band has grown posteriorly, the mesoderm has multiplied by a
division of its own cells and followed back, not quite so rapidly
as the ectoderm, becoming a flattened pad of cells beneath
this layer. (The mesoderm cells are well seen as a flat layer
beneath the entire width of a germ-band of this age dissected-
off and stained for a surface view.) The mesoderm extends
no further forward than in section, Pl. XXXI, Fig. 35, but the
ectoderm of the anterior end of the embryo has pushed out in
front to a slight degree.

Pl]. XXXI, Fig. 25, is a side view of an egg of about the
same age as that sectioned in Pl. XXXI, Fig. 36. The embryo
occupies a peculiar position for one of this stage, ordinarily
being found on the ventral surface as shown in the younger
egg (Pl. XXXI, Fig. 24). It appears to have slipped back into
the exceptionally large space between the chorion and yolk.
It gives a good idea of what is shown in the section, Pl.
XXXI, Fig. 36, just described. Note the inflated amniotic
cavity. The amnion is seen partly in optical section where it
passes into the ectoderm posteriorly, and anteriorly where it is
drawn out into a thin membrane. On its surface the cells
form a mosaic. The mesoderm cells lie loosely beneath the

528 KNOWER. [Vou. XVI.

thick ectoderm and, in this case, form an especially large mass
under the posterior end of the band.

Turning to Pl. XXXI, Fig. 37, we find several important
changes. It is a section of the stage in Pl. XXXI, Fig. 27,
before the appearance of cephalic lobes. The embryo now
forms an elongated band bent over the posterior pole of the egg.
The mesoderm has followed the growing posterior end and has
become arranged in a thinner layer. Its anterior cells appear
to have retained their primary position, as in the preceding
stage, but the greater mass of mesoderm has been carried back
with the elongating ectoderm, leaving only a single layer
beneath the middle of the embryo. This growth of the meso-
derm is, I believe, accomplished independently of the ectoderm,
by a multiplication and rearrangement of its own cells. There
is still a sharp division between the two layers. The growth
seems to be more active at the posterior end, while the middle
region appears to be pulled out, as it»were, the anterior end
remaining stationary. The size of the yolk-cells still precludes
a later origin of entoderm from these. There is no trace of
entoderm up to the time of the segmentation of the germ-band.
The ectoderm just in front of the anterior limit of the meso-
derm has grown further forward than in the preceding section
(Pl. XXXI, Fig. 36). This anterior extension of the ectoderm
will continue in later stages, and give rise to the cephalic lobes.

The effect of the backward elongation of the germ-band on
the amnion, whose cells are now apparently multiplying but
seldom, is well shown in the section before us. Posteriorly it
still retains to a slight degree the character of the ectoderm,
though much thinned out. Anteriorly the amnion has been
stretched out by the pull from behind into a very thin mem-
brane of flattened cells. I have found but few dividing nuclei
in the later stages of the amnion, the membrane appearing to
be stretched rather than to actively grow. This is beautifully
seen in surface preparations, where the amniotic cells, now
much larger than those of the more rapidly multiplying ecto-
derm, stand out in bold relief, lying closer together posteriorly.

The oldest stage of the germ-band just before segmentation
is dissected-off from an egg like that in Pl. XXXI, Fig. 29,

No. 3.] PRE NEMBRVOLOGVVOF Al TERIAITE. 529

and drawn in Pl. XXXI, Fig. 28. It is flattened out with the
under (or yolk) surface uppermost.

This embryo exhibits a uniform ectoderm, with cells some-
what more closely crowded in the cephalic lobes. Along the
borders of these expansions this crowding is greatest. At the
extreme front end of the band, in the median line, wedged in
between the lateral lobes, there is a small triangular area of
ectoderm, in some preparations much more distinctly shown.
Cells of the amnion are seen at the edges of the germ-band.
The under, mesodermal layer is shown in such preparations very
beautifully. Its cells being differently shaped from those of
the ectoderm, lying more loosely, and at the same time staining
rather more intensely, the entire layer stands out with remark-
able distinctness. A larger collection under the posterior end
of the band is apparent, as was shown in sections of the younger
embryo (Pl. XXXI, Fig. 37). Passing anteriorly the cells be-
come more scattered. Only two or three cells have wandered
forward into the cephalic lobes — the anterior end of the meso-
derm being fixed at the base of this region. Here there is a
little collection, on either side, under the posterior ends of the
cephalic lobes.

Graber’s (9) preparations of the germ-bands of Stenobothrus
vartabtlis, removed from the yolk in like manner, make a simi-
lar picture. His Fig. 76 of Taf. VI represents a stage which
may be compared with my Pl. XXXI, \ Fig.) 28,: for) the
Termite, though the cephalic lobes are not so broad in Steno-
bothrus. In the Termite the mesoderm does not lie so evi-
dently along the middle line, but forms a flat layer extending
nearly to the edges of the band. The earlier germ-bands of
the Termite have a shape somewhat different from those of
Stenobothrus (Graber (9), Figs. 74 and 75), and here again the
mesoderm is not so markedly on the middle line.

GENERAL SKETCH OF THE DEVELOPMENT FROM THE FIRST
APPEARANCE OF SEGMENTS UP TO HATCHING.

Before proceeding to a discussion of the phenomena which
have been described, I shall trace the remaining course of

530 KNOWER. [VoL. XVI.

development briefly, referring to the. series of diagrams on
Pl. XXXII for the general characteristics necessary to an under-
standing of this special study. A complete series of figures
of the later stages will be published in the near future.

The first traces of segmentation and appendages appear,
suddenly, a little later than the last stage described, where the
germ-band had become a U-shaped cap over the posterior end of
the yolk-mass (Pl. XX XI, Fig. 29). At this stage the antennae
have just become evident as backward processes of the cephalic
lobes, post-oral in position. The first maxillary and first tho-
racic are more distinct than the other anterior segments, which
are however outlined. The “tail-piece” is long and unseg-
mented. The anterior segments through the first thoracic have
therefore arisen almost simultaneously. There are no macro-
somites described by Graber (8) and (9) for Stenobothrus and
other forms.

Later embryos exhibit a progressive increase in the length
and complexity of the germ-band.

When the hind end of the band has pushed forward along
the dorsal surface of the yolk almost to the anterior end of the
egg, three additional segments have been added. These are
the two posterior thoracic segments and the first abdominal,
and they are added successively from before back; since I have
embryos in which the first thoracic is the last segment dis-
tinguishable, others with an indistinct second thoracic behind
this, and yet a third lot with three distinct thoracic and an
indistinct abdominal segment. In older embryos more ab-
dominal segments are added behind. A “tail-piece”’ of un-
specialized (ecto- and mesoderm) material is found at the end
of the band during this process, the abdominal segments being
successively differentiated from its anterior edge. (See final
section of this paper and final plate.)

Graber’s (9) beautiful figures of the development of the
Orthopteran, Stenobothrus, would serve fairly well, in most
respects, to illustrate the general features of the growth of the
germ-band of the Termite from a disc-like rudiment to an
elongated, segmented embryo at the period of “revolution.”
This process was not observed by Graber in Stenobothrus. It

No.3.) THE EMBRVOLOGY OF A TERMITE: 531

should be remembered, though, that the Termite’s germ-band
exhibits no ‘‘macro-somites’”’ of Graber, and that the disc lacks
the prominent gastrula groove of Stenobothrus.

As a whole, the resemblance between the Orthopteran and
the Termite during the embryonic stages is striking.

A stage corresponding to that figured by Brandt (3), Fig. 11,
for Calopteryx is reached, with the appearance of the mouth
and the labrum, and the subsequent folding of the head up
from the surface of the yolk. At the same time the segments
and appendages have become more prominent.

The embryo, unlike the Libellulid, is not immersed in the
yolk. (See Pl. XXXII, this paper, also Korschelt and Heider
(17), figures on pp. 774, 776, and 777.)

In the Termite, when the germ-band has grown along the
dorsal surface of the yolk to the anterior end of the egg, the
posterior portion of the abdominal region sinks slightly into
the yolk. As the embryo continues to elongate, this bend in
the abdominal region becomes more marked, the tail-end of the
band coiling ventrally into nearly a complete circle. (See dia-
grams, Pl. XXXII.)

This caudal flexure is a very characteristic phenomenon. It
occurs in many insects and is much like that of the Libellulid.
(See Korschelt and Heider (17), figures on pp. 774, 776, and
777.) I cannot explain it. It certainly appears to take place
here (as in the Libellulid), without being necessitated by any
combination of mechanical forces that can be stated.

The formation of this flexure has furnished me with a warn-
ing, and a good example of what at first sight appears to be a
simple mechanical process, but proves to be a phenomenon not
so readily dismissed. In many specimens, a very plausible
explanation of it seems to be the resistance offered to the
posterior end of the elongating germ-band by the chorion,
lying at right angles to its course at the anterior end of the
egg, This will not serve as an explanation, however, since in
many preparations the flexure occurs before the anterior end of
the egg is reached (as in the Libellulids). It is clear in one
instance, at least, that the tail end of the embryo might grow
back on the surface of the yolk around the anterior pole, as in

532 KNOWER. [VoL. XVI.

some insects. There was no caudal flexure in this specimen,
the hind end of the band turning part way over the pole.

At the completion of the elongation of the embryo the
appendages have become quite long. The head is enlarged
and globular. The first maxillae are tri-lobed, and the second
pair less markedly so. In the abdominal region ten well-marked
segments have become established, each with a distinct pair of
appendages. No appendages are figured by Brandt on the
abdomen at the corresponding stage of Calopteryx, and none
of the figures of Graber to which I have referred exhibit such
well-marked rudiments in that region.

From this stage until “revolution” the embryo undergoes
but little change externally, though the sides of the band grow
dorsalward, and the appendages elongate considerably.

“Revolution” is accomplished as described and figured by
Brandt (3) (also see Korschelt and Heider (17), figures on
p. 777) for the Libellulid. The amnion and serosa fuse into
a single membrane at one point, only to tear open over the ven-
tral side of the embryo and retract dorsally, to finally form the
“dorsal-organ”’ at the back of the head (stages O and P,
Pl. XXXII). The head of the embryo now slips up along the
ventral surface of the yolk to the anterior end of the egg,
while the tail end comes to lie beneath the micropyles at the
opposite end (see diagrams, Pl. XXXII). The ventral surface
of the embryo is now entirely on the micropylar, ventral side
of the egg, as was the case until after the closure of the
amniotic cavity. The embryo has, therefore, returned to the
orientation of its first rudiment, the germ-disc.

The remaining processes, up to hatching, consist in the
closure of the body along the dorsal mid-line, the completion
of the appendages, and the continued development of the
internal organs. In the stages following ‘revolution,’ the
embryo increases so greatly in bulk that, just before it leaves
the egg, this has become distended to a remarkable size as

oo?
compared with the unsegmented egg.

—————

No. 3.] THE EMBRVOLOGY OF A TERMITE. 3

GENERAL CONCLUSIONS AS TO THIS TYPE OF INSECT
DEVELOPMENT.

Korschelt and Heider’s recent text-book (17) contains an
argument for a modification of Will’s and Wheeler’s well-known
theory of a connection between the “invaginate,” “immersed ”’
type of development exhibited by the Libellulids and some
Hemiptera, and the type followed by myriopods.

On p. 775 (17) we find: “The invaginate type is best seen
in the Libellulids, which represent the direct connecting link
(Anschluss) with the phenomena exhibited by the myriopods,
and hence must be regarded as the more primitive type.”
Again on p. 787 (17): “Wir haben oben gesehen dass bei den
Myriopoden bei fortschreitenden Liangenwachsthum des Keim-
streifs derselbe in seiner Mitte eingeknickt und in das Innere
des Eies versenkt wird. In dieser Einsenkung, welche wir uns
zunachst durch das raumliche Missverhaltniss zwischen dem
langestreckten Keimstreif und der rundlichen Eiform ent-
standen zu denken haben, werden wir (wie dies schon Graber
andeutete und Will neuerdings ausfiihrlicher begriindet hat)
den Ausgangspunkt fiir die Entwicklung des invaginirten
Keimstreifs der Libelluliden zu suchen haben. Wir werden
demnach fiir die Entwicklung des Insecten Keimstreifs die
Form der Invagination als die urspriingliche betrachten.”
This account is apparently based on Heider’s (13) discussion
of the subject in his monograph on Hydrophilus. It is a
modification of Will’s (27), also Wheeler’s (25) theory, against
which in its original form Graber (9), in a more recent paper
than the one referred to before, brought forward strong
objections.

Since the publication of the text-book of these two authori-
ties on insect embryology, further investigation has shown,
that besides Oecanthus which is mentioned in it, a number of
Orthoptera, as well as the Termite (which is strikingly orthop-
teran), exhibit developmental phenomena similar to those of the
Libellulids. It now seems evident that there are no grounds
whatever for regarding the method of development followed by
this latter group as at all more primitive than that observed for

934 KNOWER. [Vo. XVI.

Oecanthus, Gryllus, or the Termite. These forms should be
looked to for a connecting link, if one exists (on this question
refer to the discussion of the origin of the amnion in insects in
the last division of this paper), between the phenomenon of
« doubling-up,”’ exhibited by the myriopod embryo, and the
formation of an amnion in the Pterygota.

There is much reason for believing the development of
the Libellulid to be secondary, since the embryo is of the ‘‘im-
mersed”’ type.

A. A superficial germ-band is generally characteristic of
Arthropods, and when we find one sunken into the yolk, there
is cause to believe this position has been assumed secondarily.
Among the insects, most forms (and especially the Orthoptera
and Termites) agree in having superficial embryos. The excep-
tions are rather marked, and are found among the Lepidoptera,
Hemiptera, and Libellulids. In the Lepidoptera, as in the
Myriopoda, the ‘immersed ”’ position is admitted to have been
secondarily derived from the superficial for protection, nutri-
tion, or some other unknown cause. It appears to me most
probable that the same is true for the Libellulids and the
Hemiptera, with inner germ-bands.

Hence I should regard the superficial embryos of the Orthoptera
and the Termite as more typically primitive for insects.

B. A striking character of the development of the Termite
is the small size of the first rudiment of the embryo, the germ-
disc, when compared with the definitive length of the embryonic
band. The primary rudiment must elongate through the whole
length of the egg, and add successively all the segments of the
body before the embryo is fully formed. This is equally notice-
able in the case of some of the Orthoptera, but is less pronounced
in most insects, particularly among the more specialized forms
of the group. In these there is a tendency toward a formation
of the embryonic band in its full extent from the start.

Now zt seems to me that the Termite and the Orthoptera, with
a superficial embryo beginning in a disc which must elongate
considerably to attain the definitive number of segments, have
adhered most nearly to the typical method of development for
Arthropods, and probably best represent the embryonic develop-

No. 3-] THE EMBRYOLOGY OF A TERMITE. sels

ment of the ancestral insects. The facts of the development
of the Crustacea, Palaeostraca, the Arachnids, and the Myrio-
pods (see Korschelt and Heider (17), p. 741) show a similar
disproportion in size between the primary rudiment and the
definitive segmented adult. This may be illustrated from the
Arthropods by referring to the growth of a Nauplius into its
adult form. A similar method of growth is found in the develop-
ment of the Annelid from the Trochophore, where also growth
is uncomplicated by the presence of yolk. Ido not mean to
raise any question of homology between the primary disc-shaped
rudiment of the insect embryo, and either the Nauplius or the
Trochophore, but to point out that a certain few insects (Ter-
mites, etc.), otherwise primitive, have retained a methodof growth
(see closing paragraphs of this paper) fundamentally similar to
that followed by other segmented forms. In most insects, and
particularly in the more specialized forms, the formation of a
segmented embryo is more direct, a rather long germ-band being
established from the first (and, as I take it, precociously), of
more nearly the definitive length of the embryo. (WVote that
Graber's (9) classification of germ-bands 1s not here accepted.)

C. These primitive forms (Orthoptera and Termite) are also
characterized by another peculiarity of interest in the present
discussion. The amnion arises very early and completely covers
the embryo soon after its appearance as a small disc. We do
not know with certainty to what need of the embryo the amnion
responds, but we are not surprised to find it in its most primi-
tive condition in the very forms under consideration, which are
primitive in so many other morphological characters. I believe
this is the case, and that zzsects, in which the membranes become
prominent and cover over the embryo comparatively late in its
growth, represent a secondary condition. If, as is generally
supposed, the amnion arose as a protection for the germ-band
against mechanical injury or too rapid evaporation, or as a sac,
to receive accumulated waste products, as Wheeler (25) suggested,
it would have been a great advantage for it to appear in the
ancestral Pterygota at the earliest possible moment in the
growth of the embryo. This moment occurs when the first
rudiment of the embryo, the germ-disc, is established and about

536 KNOWER. [VoL. XVI.

to grow into the elongated segmented embryo. From this time
a superficial germ-band would be constantly exposed to the
dangers mentioned. Hence the invagination, at this period, of
a part of the disc, resulting in the formation of the amnio-
serosal fold.

The Termite and some of the Orthoptera (Stenobothrus,
Gryllus, etc.) have best retained this method of the formation
of the amnion.

In other Orthoptera, the Libellulids, some of the Hemiptera,
and many other insects, the ancestral history is not so well
preserved. In these the amnion no longer closes over at the
earliest possible stage. Wheeler’s figures of the germ-bands
of Blatta and Doryphora (25), Graber’s of Lina (9), Heider’s of
Hydrophilus (13), and Weismann’s of Chironimus (24) illustrate
its usual late closure.

The Libellulids and some of the Hemiptera retain to a
decided degree ancestral characters, but the much-retarded
closure of the amniotic cavity, and the presence of the so-called
secondary ‘“head-fold,” together with the marked secondary
“immersed”’ position of the germ-band, render these forms
less typical examples of the probable primitive method of
development. (Refer to the discussion of the origin of the
amnion in insects, in the last division of this paper, for further
consideration of these questions.)

THE ORIGIN OF THE MESODERM IN INSECTS.

Recently the origin of the under-layer in what are regarded
as the most primitive insects, the Orthoptera, has been care-
fully studied by two well-known investigators who have reached
quite contradictory results.

Wheeler (26), in his “ Contribution to Insect Embryology,”
has devoted considerable space to a review of the question. His
conclusion is expressed in these words: “It follows from the
observations here recorded, fragmentary as they are in many
respects, together with Graber’s observations on Stenobothrus,
that the Orthoptera can no longer be regarded as hors de ligne,
so far as the formation of their germ-layers is concerned. In

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No. 3.] THE EMBRYOLOGY OF \A TERMITE, 537

all the families of the order, save the Phasmidae, an invaginate
gastrula has been found, and there can be little doubt that the
investigator who is so fortunate as to study embryos of this
family will find in them essentially the same process of germ-
layer formation. The view is now pretty generally held that
in the Insecta both mesoderm and endoderm arise from a
median longitudinal furrow (the former layer throughout
nearly the entire length, the latter only in the oral and anal
regions of the germ-band), and that vitellophags, or cells left
in the yolk at a time when the remaining cleavage products are
traveling to the surface to form the blastoderm, take no part
whatsoever in the formation of the mesenteron, but degenerate
2m situ and finally undergo dissolution.”

I have been unable to obtain a copy of Heymons’s study of
the germ-layer formation of Orthoptera and Dermaptera (14),
but his conclusions have appeared in abstracts and are as fol-
lows: The yolk-cells take no part in the formation of the
embryo. There is no true gastrulation process, but the under-
layer arises from all parts of the embryonic area. When what
is usually regarded as a typical gastrula invagination occurs, as
in most insects, it is to be explained, not as gastrulation, but
as a simple mechanical process caused by an aggregation of
cells at one point. The layer generally known as the mesento-
derm is in reality only mesoderm, the endoderm appearing rela-
tively late and arising from the ectoderm of the stomodeal and
proctodeal invaginations.

My results agree with Heymons’s conclusions as to the origin
of the mesoderm of insects primitively tn a collection of cells
arising diffusely from the ectoderm, but I must differ from him
and agree with Wheeler in the latter's interpretation of the
tnvaginate groove, from which the endoderm and mesoderm arise
in most tnsects, as a true gastrula.

The Termite, which is certainly as primitive as any other
insect hitherto described, exhibits no gastrula invagination. I
have shown that the under-layer begins to appear at all points
in the embryonic rudiment at an early stage of its formation.
The plug of lower-layer cells, which becomes so prominent
as the germ-disc grows more distinct, is apparently largely

538 KNOWER. [Vou. XVI.

the outcome of concentration of the cells of the disc toward
the center. The relation of such a manner of formation of the
under-layer to that generally described for insects is interesting
to consider. This process does not appear to me to be derived
from an invagination as a slurred gastrula. It is rather a |
method of delamination, where there is a further tendency in
the lower-layer cells to collect toward a middle point.

A similar method has been described for Crustacea, Arach-
nids, and Myriopods, and all of these facts, taken together, lend
weight to Heymons’s contention that an indefinite migration
below is the more primitive method of forming the under-layer
in insects.

Heymons’s explanation of the gastrula groove commonly found
in insects, however, requires examination.

He does not attribute to such invaginations the significance
of a process of gastrulation. From his standpoint the invagi-
nate groove (which, as Wheeler points out, is so universally
present among insects, and so essentially involved in the estab-
lishment of the under-layer) is a mechanical process and inde-
pendent of the formation of mesoderm or endoderm.

I do not see the strength of this position.

In so far as this author finds the diffuse method of the origin
of the mesoderm in certain Orthoptera the primary one, and
offering a favorable basis for the origin of an invaginate gas-
trula, he seems justified.

I cannot, however, take the further step with him and dis-
miss the invaginate gastrula, found so universally among insects,
as no gastrulation, but as simply a result of the crowding of an
aggregation of cells at one point. Though we still have such
an aggregation in the Termite, it has not in this group led to
invagination as a mechanical necessity. In the place of invagi-
nation there is simply a crowding of certain ectoderm cells,
arising at irregular points, below into a solid plug extending
down into the yolk.

As far as our understanding of mechanical forces and their
necessary results goes, the reason is not clear, without further
addition, why the mesoderm came in other forms to arise in a
groove instead of continuing to wander below in a solid mass.

No. 3.] THE EMBRYOLOGY OF A TERMITE. 539

That the under-layer is formed most easily and efficiently by
a process of invagination seems evident, from the almost uni-
versal appearance of the gastrula groove in insects. Given
first the more primitive, diffuse method of forming this layer
still persisting in the Termite and, as Heymons claims, in
other primitive insects, we may attribute to Natural Selection
its improvement until an invaginated gastrula groove has
become the common and readiest means of attaining the end.
When we use Natural Selection as the agent of this change,
we of course mean that the primary organic structure (in this
case the mesodermal cell rudiment arising diffusely from all
points of the ectoderm) was forced to respond to a further com-
bination of forces in the environment which we cannot define
in more exact physical terms.

From this point of view the usual method of forming the
mesoderm in insects, by a well-marked gastrula groove, is not
an independent or accidental phenomenon, but has been derived
from a more primitive method of migration already established
in the earlier insects, not as a direct and necessary result of
apparent and readily stated mechanical conditions, but as a
response to additional forces, compelling an important change
in the older but less direct process which is still efficient in
some primitive insects. These “additional forces ’’ (mechani-
cal, chemical, or what not), included under the general term
‘‘adaptive,” did not “necessarily”’ disturb in the Termite the
primitive habit established in their ancestors. In other insects,
when new conditions (mechanical or others) made it possible
and more desirable, invagination arose as a response.

A study of the origin of the ‘‘lower-layer” in the Termite
shows a very close connection between this and the establish-
ment of the first rudiment of the embryo by a concentration of
the blastoderm cells toward a certain area (as in the case of
Isopods discovered by McMurrich). This more general phe-
nomenon must be first explained before attempting the special
problem of the exact mechanical nature of the origin of the
mesoderm, which is too intimately bound up with the solution
of the former question to be considered alone.

As to the entoderm of the Termite, I must say that it

540 KNOWER. [Vou. XVI.

appears late, after the segmentation of the germ-band. The
yolk-cells (as both Heymons and Wheeler claim) can take no
part in the formation of this layer; since at an early stage,
before the closure of the amniotic cavity, they have become
very large and unlike the cells which later form the entoderm.

The fact that this layer arises so constantly among insects
with the mesoderm at the two ends of the invagination, termed
«‘gastrula”’ (see Wheeler (26) ), is a strong point against Hey-
mons’s assumption of the independent, accidental character of
this groove.

I shall be obliged to defer to another time the discussion of
the method of the origin of the entoderm, its exact relation
to the mesoderm and to the gastrula groove, when this occurs,
as well as its association with the stomodeal and proctodeal
invaginations.

THE ORIGIN OF THE AMNION IN INSECTS.

The discussion as to the cause of, and the primitive method
of origin of, the embryonic membranes of insects has at least
developed some extremely interesting ideas.

At present, opinions seem to halt between, first, the Ryder-
Wheeler (26) hypothesis of a purely mechanical and independ-
ent origin of the amnio-serosal fold among the winged insects ;
and, second, the theory of Will (27), Wheeler (25), and Korschelt
and Heider (17), recently championed by Heymons (15), which
associates the formation of embryonic membranes in insects,
more or less closely, with a certain phenomenon exhibited
by the myriopod embryo. Wagner’s (23) views I shall put, for
convenience, in the first category ; while Willey’s (29) recent
contribution, though in some respects agreeing with the sec-
ond, will have to be considered alone.

A, Examining first the Ryder-Wheeler theory, we find that
Wheeler (26) has adapted Ryder’s (22) ‘‘mechanical explana-
tion”’ for the origin of the amnion of vertebrates to the insect
amnion. Of course the term “mechanical’’ is here used in its
narrower sense, referring the question to immediate antecedent
causes, which alone are claimed to necessitate the result. The

NOT 3.4 THE EMBRYOLOGY OF A TERMITE. 541

question whether the origin of organic structures is ultimately
purely a problem of mechanics, as a first cause, is not raised.
Here the contention is that certain evident and simply stated
conditions of pressure and mechanical strain are alone sufficient
to force the amnio-serosal fold to arise.

Wheeler (26) advocates this idea concisely, as follows: ‘The
amnio-serosal fold is a mechanical result of a local induplica-
tion of the blastoderm, due to rapid proliferation in a single
layer of cells.” ‘There is the vesicular one-layered blasto-
derm filled with yolk, and the germ-band arising by rapid
proliferation at one point. The resistance of the yolk being
less than the external resistance of the tightly fitting chorion
and vitelline membrane on the one hand, combined with the
peripheral resistance of the extra-embryonal blastoderm on
the other, the germ band is forced to invaginate. This invagi-
nation is favored by the displacement of yolk during its lique-
faction and absorption by the growing embryo. We may
suppose that this invagination, which results in the formation
of the amnio-serosal fold, assumed a definite and specific char-
acter in different groups of insects.”

Similar mechanical conditions are appealed to as the cause
of certain invaginations in other forms ; the Cestode head in
Cysticercus ; the Nemertine in the Pilidium; the formation of
the young Spatangid in the Pluteus; the development of the
amnion and serosa in vertebrates ; and the imaginal discs of
insects.

a. 1. Even if we admit the presence of just such a com-
bination of forces as is enumerated above, they seem to be
subsidiary and insufficient alone, without a further cause, to
explain the origin of the membranes for the following general
reasons:

It must be recalled that no amnion results in the similar
rapidly proliferating areas of crustacean eggs, that such a
membrane is lacking among the myriopods and apterygote
insects (in spite of Heymons’s (15) claim, which requires fur-
ther and more convincing proof, as we shall see later), and
that it is not formed in certain of the higher insects. It
should also be remembered that similar membranes are want-

542 , KNOWER. [Vou. XVI.

ing in anamniote vertebrates, where the mechanical conditions,
as far as this theory goes, appear to be much the same as in
amniote vertebrates.

Apparently similar conditions of pressure and mechanical
strains would be brought to bear on the embryonic areas of the
myriopods, the apterygota, or crustacea, as are claimed to neces-
sarily force the formation of the amnion of insects, but no
amnion appears in the former groups. The invaginations
which do occur (to form the eyes, the digestive tract, etc.) in
some of the rapidly proliferating areas of the decapod blasto-
derm would be generally thought to necessitate something
more than such an enumeration of mechanical strains to
explain them.

In those highly specialized insects that entirely lack an
amnion, its failure to appear is even more marked. Here,
within the same group, there are forms which, in the face of
the forces above stated as sufficient to produce an amnion,
have none.

The effort to apply such a simple mechanical explanation
to the origin of various organic larval structures may seem
plausible at first sight; but, carried to its logical limit, not
so much so. Why stop at the structures mentioned? Might
not the germ-layers, the central nervous-system, as well as
other such rapidly proliferating areas, be as readily included ?
Heymons, as I have shown, has already attempted an affirma-
tive answer for the origin of the gastrula groove.

a. 2. Turning from such general considerations to my own
special results, the formation of the caudal flexure of the Ter-
mite seems a case in point.

This ventral flexure of the tail end of the embryo, as I have
pointed out, at first seems just as reasonably to be ascribed,
solely and directly, to a necessary result of pressure or me-
chanical strain as the instances referred to by Wheeler. A sin-
gle unusual specimen proved beyond doubt such a conclusion to
be false, and that what might appear superficially to be a neces-
sary method of growth could be accomplished in an entirely dif-
ferent manner. It was certainly proved to be independent of the
resistance of the chorion, which seemed so determinative at

No. 3.] THE EMBRVOLOGY (OF A TERMITE: 543

first sight. Here was another case of the nearest explanation
not necessarily being the true one.

It seems hardly necessary to say that the fact that such
invaginations can be watched step by step sometimes, and can
be actually observed to encounter resistance at every stage,
is no proof that such resistance causes the process.

a. 3. My study of the formation of the embryonic rudiment
and of the origin of the amniotic fold of the Termite indicates
forces of a very different nature from those formulated by
Wheeler; in fact the very reverse.

As the germ-disc becomes sharply defined, the area of the
blastoderm occupied by it is distinguished by the closer crowd-
ing of its cells, while the surrounding cells become flattened
and pulled apart into a thin membrane. There appears to
be a contraction toward the embryonic area, as is observed
in the formation of the embryonic rudiment in other insects
and other arthropods. At any rate, the extra-embryonal blas-
toderm may be said to be stretched and kept so by the changes
taking place in the embryonic area.

Before the amnion arises it is clearly differentiated as a
special thickened area of the germ-disc. When the embryonic
rudiment doubles-up, and this posterior portion of it folds over
to become the amnion, the extra-embryonal blastoderm is
pulled forward and further stretched.

It seems correct to speak of the tension of the serosa as due
to the activities tn the embryonic area, rather than to reverse
the case and explain important changes tn this area as a result
of such tension.

In studying the growth of the germ-disc, I can find no indi-
cations of a rigid resistance to its growing edges claimed to
be offered by the rest of the blastoderm. The cells around
the rapidly proliferating area do not seem to be fixed, im-
movable points; and the membrane they form does not
appear to be more resistant to this more active area than
is the yolk.

Another important point is the fact, as I have shown, that
the amnion is not a derivative of the extra-embryonal blasto-
derm, as Wheeler (26) concludes in his latest paper.

544 KNOWER. [Vou. XVI.

If the Ryder-Wheeler mechanical theory were correct, the
most natural place to expect the fold would be just at the junc-
tion of the rapidly proliferating germ-disc with what is claimed
as a rigid, resistant, extra-embryonal region. We would look
for the weakest point here. The fold does not, however, occur
here in the Termite or other amniote insects.

My own observations, and a general review of the question,
lead me to believe that the embryonic membranes of insects
are adaptive structures, which arose in the winged insects as a
response to some definite need of the embryo. I do not think
the exact combination of physico-chemical forces, codperating
to bring about this result, can be stated at present. The eggs
of the anamniote apterygota are, to all appearances, as far as
mechanical conditions go, similar to those of winged insects.
The physical constitution of the egg was already favorable
to the origin of the amnion in the ancestors of the latter
forms; but before one arose, certain additional forces were
necessary, which must be associated with some necessity of
covering over the embryo at an early stage. Whether this
necessity (physico-chemical, no doubt) was one of protection,
prevention of evaporation, better nutrition, or to furnish a
depository for waste products, may not be decided ; but any
one of these suggestions, or all together, would be reasonable
cause.

When forms arose among the higher insects, as adaptations
to special new conditions, the early completion of the process
became less important ; and in a few cases the amnion ceased
to appear, being no longer needed. (If it is any more precise,
we may say that the amnion was no longer maintained by the
physico-chemical forces which originated it.)

B,. I must refer to Wagner’s (23) comprehensive theory of
the origin of insect embryonic membranes and other organic
structures, as another example of a simple, clear-cut mechanical
explanation of such problems, which also illustrates the diffi-
culty of correctly estimating and balancing forces, and their
necessary effect on organized matter.

In a few words, his idea is as follows: Think of the similar
cells of a uniform epithelium as an organic molecule, so built

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No. 3.] CHE EMBRYOLOGY OF A TERMITE. 545

up together that a certain homogeneous reciprocal relation is
attained. Now, when certain cells of this layer become altered
in nature under the influence of some special forces, the recipro-
cation with neighboring cells is likewise altered, resulting in so
changing the relations with these latter that a separation of the
changed cells from the layer of similar, unchanged cells must
take place.

The mechanical basis of the theory is what happens when a
foreign, inorganic particle is introduced into a fluid or viscid
layer (‘“‘ Haut’) whose elemental drops codperate reciprocally
to forma uniform sheet. The foreign particle would be thrown
out as a result of purely mechanical tensions.

So in the case of the origin of the germ-layers, by invagina-
tion or immigration ; the sub-epithelial muscle cells of Medusae ;
gland cells; central nervous-system; sense organs; Cestode
head in the cysticercus; the extra-embryonal and embryonic
cells; embryonic membranes ; imaginal discs, etc. Whenever
two kinds of cells occur in an epithelial layer, one sort is
thrown out, so to speak, by invagination or immigration. Cases
where this has not taken place represent the early stages of
the process (as certain epithelial gland cells or muscle cells).
In all these cases the common and necessary cause of invagina-
tion and immigration is claimed to be the sharp differentiation
of certain cells, physically and chemically, to such an extent
that they must move from their primitive position.

It seems hardly necessary to observe that, though this theory
is strictly logical and far-reaching, Wagner does not explain
the fundamental question why certain cells rather than all are
modified; and that he overlooks an important and essential
difference between the living modified cell in the uniform cell
layer, on the one hand, and the foreign, inorganic, dead par-
ticle in the homogeneous fluid layer on the other. The theory
must collapse when we reflect that, instead of being obliged by
the supposed necessity to immigrate as an inorganic particle,
the modified living cell could accommodate itself to its old
neighbors and remain with them. Of course the inorganic
particle would have no such power of adaptation resident in liv-
ing protoplasm. Possibly this adaptability of living substance

546 KNOWER. [VoL. XVI.

is the reason Wagner finds gland cells, muscle cells, etc., not
wandering out of the otherwise uniform layer. When a migra-
tion of specialized cells does take place, we shall have to look
further than to such a simple statement of inorganic physics
for the explanation.

C. I shall not examine Willey’s (29) hypothesis at length,
since, in as far as it refers to the origin of the amnion, it
appears to be largely a statement of Heymons’s (15) views,
which will be considered further over.

Willey’s main thesis seeks to prove, by reversing a theory of
Hubrechts’s, that the extra-embryonal blastoderm of the insect
egg (2.e., the serosa and amnion) is a secondary cellular mem-
brane, derived in a curious roundabout manner from a more
primitive, extra-embryonal trophic membrane, “the tropho-
blast’’; which, “as it is preserved to us in the embryo of
Peripatus novae-britanniae, arose in adaptation to a viviparous
habit acquired by the terrestrial descendant of an aquatic
ancestor; and that it became transformed, whether directly or
by substitution, into the serosa, in correlation with the second-
ary deposition of yolk-laden eggs.”

The following fundamental assumptions seem to me inad-
missible: That the viviparity of Peripatus is primitive; that
“lecithality and deposition of the eggs of insects are both
secondary’”’; that this application of the idea of substitution in-
volving the reverse of Hubrechts’s idea is reasonable; that the
amnion is a derivative of the extra-embryonal blastoderm in
insects; or that the serosa of the insect egg has any such indi-
rect phylogenetic history, believing it as I do to be directly
comparable to the inactive extra-embryonal surface cells (Deck-
schicht) of other yolk-laden eggs.

D.1. I have already discussed, in the division of this paper
headed General Conclusions as to this Type of Insect Develop-
ment (page 29), certain aspects of the theory presented in
Korschelt and Heider’s text-book. Reference must be again
made to that section of my paper, where the original sources
and criticisms of the theory are quoted. This theory, which
originated with Will and Wheeler (25), was later modified by
Heider.

No. 3.] THE EMBRYOLOGY OF A TERMITE. 547

The origin of the embryonic membranes of insects is referred
to the peculiar phenomenon of “ doubling-up ” exhibited by the
myriopod embryonic band.

It was originally claimed by Will that the invaginations in
the two cases are genetically connected to such an extent that
some of the posterior segments of the elongated myriopod
band, on bending forward, were directly transformed into the
amnion in the ancestral insect embryo. In this way the adult
insect came to have fewer posterior abdominal segments than
the myriopod.

Judging from Heider’s remarks in his monograph on Aydro-
philus, no genetic relationship is now meant to be implied
between the “doubling-up”’ of the myriopod embryo, and the
invagination to form an amnion in the insects.

The idea now advanced is, that the resemblance in the two
cases is of sufficient importance to intimately connect the
phenomenon presented by the elongating myriopod embryo
with that observed in Libellulids, which are said to best exhibit
what is termed the primitive, invaginate type. The similarity
in the two instances, however, is only claimed to be the result
of the action of a common cause of the invagination.

Will’s idea of a transformation of a part of the segmented
body of the embryo of the myriopod into an amnion for the
insect embryo has been abandoned since Graber’s (9) criticism.

Heider in his monograph (13), and in the text-book with
Korschelt (17), points out the similarity between the amnion
and the rudimentary ectoderm of the embryonic band from
which it arises; and in this is in agreement with what has been
observed and figured by most investigators, as I have already
noted in another connection.

D. 2. Recently, Heymons (15) has studied the interesting
apterygote Lepisma saccharina. Asa result, he claims to have
furnished us with a convincing intermediate stage between the
phenomenon of doubling-up of the myriopod embryo, and the
formation of an amnion and amniotic cavity in winged insects ;
which he thinks proves that the latter process is directly derived
from the former.

In describing the embryonic rudiment the author speaks

548 KNOWER. [VoL. XVI.

of the entire extra-embryonal region as a serosa, before the
doubling-up takes place. As this happens, he says (in a para-
graph on page 587 of his paper) that the cells of the edges of
the embryonic band become pulled out into a thin cellular
membrane, the amnion. None of his figures, however, give
proof of such a process of transformation of a part of the

Fic. 3. Fic. 4.

Diagrammatic figures (sagittal sections), comparing the “ primary ventral flexure’’ (“‘ doubling-
up,” or invagination) of the myriopod and apterygote embryos with the amniotic fold
of the winged insect. (1) Myriopod (Julus) embryo, at the first appearance of the
ventral flexure; (2) similar stage of the embryo of Lepisma (apterygote); (3) early
amniotic fold (doubling-up, or invagination) of the unspecialized embryonic rudiment
of the Termite; (4) later stage of the Termite embryo, after the closure of the amniotic
cavity —a stage similar to that of the two types in 1 and 2, but the “‘tail-piece”’ is straight
here. ex. extra-embryonal blastoderm; ¢.Z. posterior, unspecialized ectoderm; wz.2.
posterior, unspecialized mesoderm ; a.s. segmented region anterior to unspecialized tissues
of “‘tail-piece ”; ck. chorion; y. yolk; in 3, Z.e. primitive, unspecialized ectoderm of germ-
disc ; @7z. amniotic fold in undifferentiated ectoderm; in 4, se. serosa; a. amnion.

embryonic, rudimentary ectoderm into the amnion, as observed
in the winged insects. The figures show no more as to this
question than that, as the embryonic band sank into the yolk,
the extra-embryonal cellular membrane attached to its edges
was pulled along.

The result, as figured, is a band doubled-up like that of the
myriopods, and not differing from this except in lying deeper in

No. 3.] THE EMBRYOLOGY OF A TERMITE. 549

the yolk, into which some of the extra-embryonal membrane is
dragged down. (See my Text-Figure 2.)

Comparing the diagrammatic text-figures here copied, — one,
Fig. 1, after Heathcote (31), Fig. 14, showing the doubling-up
of the myriopod (Julus) embryo, and another, Fig. 2, after
Heymons (15), for Lepisma, — we find the thick germ-band in
each case enclosing a cavity. In the myriopod egg the two
ends of the embryo are on the surface, and there pass into
the thin extra-embryonal membrane. In the Lepisma egg the
embryo lies more internally. Except for this, there is no essen-
tial difference apparent in the relations of the extra-embryonal
blastoderm to the embryo or in the nature of the open cavity.
Compare these two figures with the third text-figure of the
Termite, at a stage before the closure of the amniotic cavity,
and with the fourth figure of the closed amniotic cavity of the
Termite. (All eggs are represented as spherical for better
comparison.)

Does the so-called amniotic cavity of Lepisma constitute any
nearer approach to the true amniotic cavity than the one pre-
viously found between the “doubled-up ” body of the myriopod
embryo? I think not, without a further and more convincing
series of figures of some stages in the formation of the so-called
amnion, which would prove it to be any more truly an amnion
than the part of the blastoderm external to the embryonic band
of the myriopod, or in any way different from this, except in
being pulled down into the yolk.

Instead of being an important intermediate stage between
the phenomenon exhibited by the myriopod embryo and the
formation of a true amnion, and amniotic cavity, in the winged
insects, there is nothing in the figures (nor does the single
descriptive paragraph convince without further figures) to give
reasonable grounds for the claim that there is any such differ-
ence between the phenomenon exhibited by the myriopod and
that shown by Heymons for Lepisma.

The gap between the open cavity in the doubled-up myriopod
embryo and the true, closed amniotic cavity of winged insects,
seems just as wide as before Lepisma was studied from this
standpoint; except in as far as the apterygota have been

550° KNOWER. [Vou. XVI.

shown to exhibit this phenomenon similar to the myriopods —
an important point in itself, indeed, if the amniotic fold of the
winged insect is to be derived from an earlier invagination.

Willey differs with Heymons as to interpreting the ventral
flexure of the embryo of Lepisma as comparable with the later
caudal flexure of insect embryos. Without admitting his
theory of the trophoblast, I must agree with Willey in this
distinction.

In many respects the development of Lepisma bears a close
resemblance to that of the primitive Orthoptera and the Ter-
mite. It is interesting to find the germ-disc originating at the
posterior pole of the egg as in the Termite. The absence of a
gastrula groove, in connection with the origin of the meso-
derm, is also in agreement with what Heymons has found in
some Orthoptera, and with the results here submitted for the
Termite.

£. The conclusions reached from the above general review
of the question before us, in the light of my own special obser-
vations, and again referring to my views, expressed in a pre-
vious section of this paper, as to the primitive type of insect
development, may be summed up as follows :

é. I. The amniotic fold did not arise as a necessary result of
any combination of purely mechanical forces which has been
formulated up to the present time.

é. 2. The amnion and amniotic cavity of insects are adaptive
structures, which, as far as our knowledge now goes, arose first
in the winged insects as a response to some definite need of
the developing embryo.

é€. 3. The amnion is primitively a derivative of the rudimen-
tary embryonic ectoderm.

é. 4. An ‘¢invaginate’’ type of development is the more
primitive one for insects. Irrespective of its relation to the phe-
nomenon of doubling-up of the myriopod or apterygote embryo,
it has been shown to be associated with the more primitive
insects, and the most primitive (probably) method of mem-
brane formation outlined in paragraph 5 below.

It must be added to this, that in the light of researches of a
more vecent date than that of the publication of the text-book of

No. 3.] THE EMBRYOLOGY OF A TERMITE. 551

Korschelt and Hetder, tt is found that the Termite and certain
Orthoptera with superficial embryos, as is explained in this
paper, represent the mvaginate type of development there sug-
gested better than do the Libellulids, with embryos “immersed ”’
in the yolk and other secondary characters. Other methods
of origin of the amniotic fold are most probably derived from
that best exhibited by the Termite and certain Orthoptera.

e. 5. It became important for some reason (whether for
protection, better nutrition, accumulation of waste products,
etc.) associated with a new habitat or mode of life, that the
superficial embryos of the ancestors of the winged insects should
be completely covered over. The forms we may now consider
primitive for a number of reasons exhibit a relatively small,
superficial disc as the first rudiment of the embryo. Here
was an especially favorable condition for the earliest possible
appearance of the membranes, at a time when they might be
particularly needed. Only a few forms have retained this
process in a near approach to its primitive form. (I believe
that the amnion is formed from the rudimentary ectoderm by
essentially the same method, on the similar germ-discs of the
Termite and certain Orthoptera, though in the Termite the
fold is more evident at the posterior end.) Changed conditions
have led to a disappearance of the membranes in a few insects.

é. ©. The ventral flexure of the first rudiment of the embryos
of the invaginate type which forms the amniotic fold has not
been proved to be of a phylogenetic significance.

e. 7. Even if it can be shown conclusively, in the case of
the apterygote egg, that the open cavity is a somewhat nearer
approach to the amniotic cavity of the winged insect than that
found in the myriopod egg (or, in other words, what Heymons
speaks of as amnion is a derivative of the rudimentary ecto-
derm, as in the Termite, and not simply a part of the blasto-
derm comparable to that lying outside the limits of the embryo
in the myriopod egg), it must be remembered that the open
invaginations of the myriopod and apterygota may not even be
due to causes similar to those calling for the closed amniotic
cavity of winged insects. These may be entirely distinct phe-
nomena with very different significance.

552 KNOWER. [Vou. XVI.

There is, however, undoubtedly a resemblance between this
invagination and the phenomenon exhibited by the myriopod
embryo, which is strengthened by the appearance of the same
condition in the apterygote egg. This suggests strongly a
common cause (the general adaptive nature of which I have
suggested in agreement with others already quoted) ; but
Korschelt and Heider’s ((17), pp. 734 and 787) further idea,
that this cause is associated necessarily with the resistance
offered by the spherical chorion to the growth of the elongat-
ing germ-band, does not seem convincing, since these authors
themselves suggest an objection in the different behavior of
some myriopods, see (17), p. 735 ; since similar conditions do
not necessitate a like invagination in certain insects, or in the
elongating band of the arachnid or in that of a fish, for reasons
I have suggested in another place ; and since the amniotic
fold of the Termite arises on the nearly circular disc, before
such conditions would be effective.

e. 8. The possibility of a connection with the invagination
(‘‘doubling-up ’’?) of the myriopod embryo seems sufficiently
strong to warrant a new statement of how a fundamentally simi-
lar invagination, in the primary embryonic rudiment of the
myriopod-like ancestors of winged insects, may have formed a
starting point for the formation of an amnion.

As has been pointed out, some theory associating the two
invaginations has seemed probable to a number of investigators.

Will (27), basing his theory on a study of the Hemipteran
embryo, first insisted on a derivation of the amnion from a
region of the myriopod body.

Wheeler (25), at about the same time and independently of
Will, advocated much the same idea, though he simply quotes
Will in regard to the degeneration of segments into an amnion.

Graber (9) justly criticised the idea of a disappearance of
certain posterior abdominal segments of the myriopod-like
ancestors of the insects, by a degeneration into an amnion and
a forward migration of the anus.

Finally Heider (13), and later Korschelt and Heider (17),
presented a modification of the theory I have outlined, which,

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No. 3.] THE EMBRYOLOGY OF A TERMITE. 553

accepting a fundamental common cause connecting the two
invaginations, abandoned further comparison.

Heymons (15) has quite recently claimed to have carried this
a step further, in a manner which I have already considered.

If we proceed from the assumption that some like necessity
of removing the embryo from surface insults, or of furnishing
it with better conditions of nutrition, etc., caused an invagina-
tion of the embryo of the myriopod (or apterygote) and of the
superficial rudiment of the ancestral winged insect, it is pos-
sible in the case of the Termite embryos at an early stage, just
before the closure of the amniotic cavity, to make a comparison
of a somewhat different nature from what has hitherto been
suggested.

The condition found in the Termite permits us to see how
we may retain an essential feature of Will’s idea (also Wheeler’s)
of a derivation of the amnion from a portion of the ancestral
myriopod’s embryonic tissue, in association with a process of
invagination, without involving the further idea of a transforma-
tion of definitively organized tissue, with the disappearance
of segments and the migration of the anus. It will, however,
be found that the following is not an effort to trace the amnion
in a phylogenetic sense back to the myriopod.

Referring back to the text-figures, we find practically the
same condition in the three first diagrams—a doubled-up, com-
paratively thick embryonic band, enclosing a cavity which opens
on the surface of the egg. In the Termite this opening in
Text-fig. 3 closes, and the outer wall of the cavity, which is
a portion of the ectoderm of the first rudiment of the embryo,
becomes the amnion. (See fourth text-figure.)

It is evident that if the invagination of the winged-insect
embryo is to be derived from that in the myriopod (or aptery-
gote) egg, the amnion of the insect most probably arose from
some portion of the thickened, unspecialized (striped in the
text-figures) ectoderm of the myriopod (or apterygote) ancestor.

My idea is that, since it has been shown that the amniotic
fold of the Termite is a specialized portion of particularly the
posterior ectoderm of the embryonic rudiment, at a very early
stage, before elongation begins and before the appearance of

554 KNOWER. [Vou. XVI.

segments or the anus, the comparison with the embryonic,
invaginating rudiment of the apterygota and myriopod should
be made with the ectoderm alone, and at a correspondingly
early stage in its differentiation.

My effort ts not to derive the amnion from a portion of the
myriopod body in a phylogenetic sense; but to explain how, in
association with a fold similar to that of the myrtiopod-like
ancestor, but appearing sooner, tt may represent an early special-
zzation of the undifferentiated tissue (ectoderm) of a primary
embryonic rudiment common to the two arthropods (see B, pp. 30-
31); and how thts folding off of the amnion need not prevent the
usual continuation of the development into an elongated embryo
comparable to the myriopod.

Such a comparison may be readily made by referring back to
the diagrammatic text-figures.

These figures of course represent actual stages in the devel-
opment of the three forms. The two upper figures illustrate
the first appearance of the ventral flexure (doubling-up) of
a myriopod, Text-fig. 1 (Julus, after Heathcote), and of a
wingless insect (the apterygote Lepisma, after Heymons),
Text-fig. 2.

Following Heathcote (31) in his description of the myriopod
development, we find in the first text-figure that the ventral
flexure occurs here comparatively late in the ontogeny, after a
few anterior segments have been formed from both layers. The
important fact to note is that the bending takes place just
behind the last segment differentiated, and in a region that
Heathcote speaks of as unspecialized tissue, commonly termed
the ‘‘tail-piece.”” I have indicated in the diagrams the usual
sharp distinction between the early ectoderm and mesoderm in
this posterior region, Text-fig. 1 (see Heathcote’s Fig. 30).
(Note that the as yet undifferentiated ectoderm is striped in
the diagram, while the similar mesoderm is a simple black line.)

Examining Heymons’s results for Lepisma, as represented
in the second text-figure (Text-fig. 2) to the right, we find
essentially the same conditions as in the myriopod. (See his
Fig. 1, (1§), for the sharp separation of primary ectoderm from
mesoderm.)

No. 3.] THE EMBRYOLOGY OF A TERMITE. 555

Turning finally to the two lower text-figures of the develop-
ing Termite embryo, we recall that the germ-disc, when the
amnion first folds up and before the closure of the amniotic
cavity, is in a very undifferentiated state. (The suggestion is
made in a previous section of this paper (2, p. 30) as to the
comparatively primitive nature of this small rudiment.) I have
indicated in the diagram illustrating this stage (Text-fig. 3) that
the entire rudiment, ectoderm and mesoderm, is quite unspe-
cialized. The upper layer is striped, as is the ectodermic tissue
of a similar early stage in differentiation in the “ tail-piece”’ of
the other figures. The lower iayer is also an unspecialized
mass.

The condition of the tissues is just what was found in a
much later stage of the myriopod (or apterygote), in the partic-
ular region where the ventral flexure occurs (Text-figs. 1 and 2).

A first difference is, that though the flexure takes place at
a corresponding stage in the differentiation of the tissues, it
occurs at a much earlier period in the development of the
winged insect ; in fact, at what I have pointed out is the earli-
est possible stage for the origin of an amnion. Another and
second point is that only the ectoderm is here concerned in the
flexure. Thirdly, if a posterior portion of the primary unspe-
cialized ectoderm becomes amnion, what will be the effect on
the further development of the embryo?

The first point of difference, the relatively very early appear-
ance of the flexure in case of the insect, may be unimportant ;
since the two invaginations before us develop at a like stage
in the differentiation of corresponding tissue (see text-figures).
We have suggested apparently good reasons for an especially
early folding, or invagination, of the superficial rudiment of the
ancestral winged insect. Text-fig. 3 shows this taking place
before any segments have been differentiated.

As to the second point, in regard to the ectoderm, it must
be first recalled that Heathcote’s Fig. 30 for Julus indicates a
special participation of the ectoderm in the flexure, when first
beginning. Further, in both the myriopod and the apterygote
on the one hand, and the winged insect on the other, there is
a marked separate though associated development in the upper

556 KNOWER. [VoL. XVI.

(ectodermic) and lower (mesodermic) layers when once estab-
lished. Each layer develops certain structures peculiar to it
(text-figures). The primitive ectoderm alone would, on a priori
grounds, be expected to be the layer to differentiate a protective
structure, as the amnion has been thought to be. Finally, in
the Termite the doubling-up to form the ectodermic amniotic
fold takes place distinctly before the mesoderm has spread
beneath the posterior region, where the process is inaugurated
(Text-fig. 3).

The third point suggested was the effect on further develop-
ment of the early formation of an amnion from the unspecial-
ized posterior ectoderm. It is interesting to observe, as the
fourth diagram (as well as the final plate in this paper) shows,
that after the formation of the amnion as one of its organs, the
ectoderm, as well as the mesoderm beneath, continues to grow
posteriorly, carrying the amnion behind and budding anteriorly
the ectodermic portions of the segments of the body until,
finally, we reach a stage identical with that of the myriopod or
apterygote. The unspecialized tail-piece of this stage was
formed in the usual manner from the original, undifferentiated,
posterior tissue of the primary rudiment, from which the amnion
arose at an earlier stage.

In a sense the formation of the ectoderm of the tail-piece, in
this later stage of the winged-insect embryo, may be thought of
as a regeneration of the lost terminal material which went into
the amnion; just as a piece of the ectoderm of a developing
hydra (worm, or other form) might be removed at an early
stage, without disturbing the further development of parts from
the ectoderm, since the ectoderm remaining would supply the
loss. This statement must, however, be accepted as an illus-
tration of regeneration from undifferentiated tissue, only in so
far as such a process is comparable to normal growth following
the differentiation of an organ from unspecialized tissue.

From this point of view the amnion ts not a substitute for, or
a transformation of a posterior region of the myriopod body. It
7s not derived from any previous structure. It is a specialized
structure folded off in the winged-insect embryo, for some
adaptive reason similar to those causing the doubling-up of the

No. 3.] THE EMBRYOLOGY OF A TERMITE. SO/,

myriopod embryo, at an especially carly period, from the primitive
unspectalized ectoderm of an ancestral disc-shaped rudiment. It
arises espectally in a posterior region of the carly ectoderm of the
Termite. A similar primitive origin may, however, be associated
with the sinking of the embryonic rudiments of other insects
(especially in the forms I have taken as primitive), where the
folding occurs in other portions of the early ectoderm, for it must
be remembered that such ectoderm can produce lateral as well as
serial organs.

The less number of segments in the winged-insect or aptery-
gote, as compared with the myriopod, was attained, as far as
the embryology shows, by an arrest in a primitive method of
growth common to arthropods and similar to budding, which
was continued for a longer time in the many segmented
ancestors of insects.

The reason for the shorter duration of this process in the
later group is not known, but must be sought in such related
fundamental problems of growth as regeneration of lost parts,
metamerism, and the cleavage of the ovum.

e. 9g. As has been said, the ventral flexure of myriopod
embryos of the present time may be proved later to be con-
nected in no sense with an amniotic fold. Even if such turns
out to be the case, the above comparison will then have served
a good purpose, in calling attention to a plausible interpretation
of the amniotic fold as originating primarily by invagination in
winged-insects, independently, and not traceable to any previous
similar phenomenon.

é. 10. If the above view is applied to the vertebrate amnion,
the participation of both primary and, at the point of origin of
the fold, undifferentiated layers of the body-wall would be under-
stood in a sense similar to the formation of other early organs,
in which both primary layers codperate.

EXPLANATORY NOTE.

This paper was accepted as a thesis, May, 1896. It was
abstracted in the Johns Hopkins University Circulars, Vol. XV,
June, 1896. Unavoidable delay in publishing and a renewed

558 KNOWER. [VoL. XVI.

study of some additional and better material have rendered the
present revision advisable. I have hence included a consid-
eration of two recent papers, that of Heymons (15) and that
of Willey (29). It is a pleasure to here thank Professor
C. O. Whitman for many courtesies extended to me during
two summers’ work at the Woods Holl Marine Biological
Laboratory.

AvuGusT 16, 1899.

No. 3.] TRAE EUBKVOLOGY (OF A LERMITE: 559

Io.

II.

12.

14.

15.

16.

17.

18.

19.

SPECIAL REFERENCES TO BIBLIOGRAPHY.

AYERS, H. On the Development of Oecanthus Niveus and its Para-
site Teleas. Jem. Boston Soc. Nat. Hist. Vol. iii. 1884.

BoBRETZKY, N. Ueber die Bildung des Blastoderms und der Keim-
blatter bei Insecten. Zeztschr. f. wiss. Zool. Bd. xxxi. 1878.

BRANDT, A. Beitrage zur Entwicklungsgeschichte der Libelluliden
und Hemipteren. J7ém. Acad. St. Pétersbourg (7). Tome xiii.
1869.

BRAUER, FR. Systematisch-Zoologische Studien. Sztz. Berl. Akad.
Wiss. Wien. Bd.xci. 1885.

BRAUER, A. Beitrage zur Entwicklungsgeschichte des Skorpions.
Zettschr. f. wiss. Zool, Bd. lvii. 1893. Bd. lix. 1895.

Bruce, A. T. Observations on the Embryology of Insects and
Arachnids. A Memorial Volume. Baltimore. 1887.

GRABER, V. Die Insecten. Munchen. 1877. In Die Naturkrafte.
1879.

GRABER, V. Ueber die primare Segmentirung des Keimstreifs der
Insecten. Morph. Jahrb. Bd. xiv. 1888.

GRABER, V. Vergleichende Studien am Keimstreif der Insecten.
Denkschr. Akad. Wiss. Wien. Bd. lvii. 1890.

GRABER, V. Vergleichende Studien uber die Keimhiillen und die
Riickenbildung der Insecten. Denkschr. Akad. Wiss. Wien.
Bd. lv. 1888.

GRASsI, B., and SANDIAS, A. Costituzione E. Svillupo della Societa
dei Termitidi. (The embryology was not studied.) Catania. 1893.

HUAGEN; El. 2706. Boston Soc: Wat. Hist. Vol. xx, Dec Sy s7oe
p-121.

. HaGen, H. Monographie der Termiten. Lz. Entom. Bde. x-xii.

HEIDER, K. Die Embryonalentwicklung von Hydrophilus piceus L.
Jena. 1889.

Heymons, R. On the Development of Orthoptera and Dermaptera,
with special reference to germ-layer formation. Adstract in Journ.
Mic. Soc. 1894.

Heymons, R. Entwicklungsgeschichtliche Untersuchungen an Le-
pisma saccharina. Zez¢tschr. f. wiss. Zool. Bd. |xii. 1897.

KISHINOUYE, K. The Development of Limulus longispina. Unzver-
sity of Tokyo. 1891.

KORSCHELT und HEIDER. Lehrbuch der vergleichenden Entwick-
lungsgeschichte der wirbellosen Thiere. Jena. 1890.

McMoraricu, J. P. Embryology of the Isopod Crustacea. Journ.
of Morph. Vol. xi. 1895.

METSCHNIKOFF, E. Untersuchungen tiber die Embryologie der
Hemipteren. Zeztschr. f. wiss. Zool. Bd. xvi. 1866.

560
20.

21.

22.

24.

25.

26.

27

28.

29.

30.

Bue

KNOWER. [VoL. XVI.

MULLER, Fr. Beitrage zur Kenntniss der Termiten. Jen. Zettschr.
jf. Naturwiss. Bd. vii. 1873.

PATTEN, Wm. The Development of Phryganids, with a preliminary
note on the development of Blattagermanica. Quart. Journ. Micr.
Sct.) VOle xxiv.) WOod

RYDER, JOHN A. The Origin of the Amnion. Amer. Nat. Vol. xx,
pp. 179-185. 1886.

WAGNER, J. Observations on the Formation of the Germ-Layers,
Yolk-Cells and Embryonic Membranes of Arthropods. Bzo/. Cen-
tralbl. Vol. xiv, No. 10. 1894.

WEISMANN, A. Die Entwicklung der Dipteren im Ei nach Beobach-
tungen an Chironimus sp. musca vomtaria und Pulex canis. Zeztschr.
f. wiss. Zool. Bad. xiii. 1865.

WHEELER, Wm. The embryology of Blatta germanica and Dory-
phora decemlineata. Journ. of Morph. Vol. iii. 1889.

WHEELER, Wm. A Contribution to Insect Embryology. Journ. of
Morph. Vol. viii. 1893.

WILL, L. Entwicklungsgeschichte der Viviparen Aphiden. Sfengel’s
zool. Jahrb., Abth. f. Anat. und Ont. Bd. iii. 1888.

VIALLANES, H. Sur quelques points de l’histoire du développement
embryonnaire de la Mante religieuse. ec. Biol. du Nord de la
France. Tomeii. 1889, 1890.

Also

WILLEy, A. Trophoblast and Serosa. A contribution to the Mor-
phology of the embryonic membranes of insects. Quar. Journ.
Micr. Sct. Vol. xli. 1899.

KNowWER, H. McE. The Development of a Termite [Eutermes (Rip-
pertii?)]—a preliminary abstract. /ohus Hopkins University Cir-
culars. Vol. xv, No. 126. June, 1896.

HEATHCOTE, F. G. The Early Development of Julus terrestris.
Quar. Journ. Micr. Sct. Vol. xxvi. 1886.

No. 3.] THE EMBRYOLOGY OF A TERMITE. 561

EXPLANATION OF FIGURES.

All figures drawn with aid of camera. From Figs. 1-29 inclusive, about the
same magnification. Figs. 1-24 drawn with Zeiss oc. 4. Olject. A. magnifying 97
times. Figs. 24-29 inclusive, drawn with Bausch & Lomb’s tube 160 mm., oc. 25
mm., ofject. 17 mm., magnifying 96 times. Figs. 30-37 inclusive, drawn to same
scale with Bausch & Lomb’s oc. 25 mm., odyect. 4.2 mm., magnifying 450 times.
Tube 160 mm.

562 KNOWER.

EXPLANATION OF PLATE XXIX.

(The posterior ends of all eggs are placed uppermost.)

Fic. 1. Ventral surface of egg tipped up somewhat to show micropyles at
posterior end. 4., anterior; /., posterior; 1-g on each side the micropylar
funnels.

Fic. 2. Optical section of egg with one nucleus, segmentation nucleus, in
center. Egg stained in borax carmine and viewed as a transparent object in clove
oil. Yolk bodies not shown in figure. The chromatin of the nucleus is seen in
the center of a small mass of lightly stained protoplasm: £.é., polar bodies.

Fic. 3. Optical section of egg with two nuclei: £.4., polar bodies; d.x.,
dividing nucleus, in which the chromatin is separated into two masses.

Fic. 4. Optical section of egg with four nuclei. The nuclei are not all in the
same plane. A line connecting the two posterior nuclei is in a-plane at right
angles to one joining the two anterior nuclei lying in the plane of the paper. To
reach this position, the axes of the spindles of the two dividing nuclei of the last
stage must have rotated in opposite directions. See McMurrich on Isopods for a
similar phenomenon (18). All the nuclei are dividing, @.z. The chromatin of the
polar bodies has become much fragmented, £.é.

Fic. 5. Optical section of egg with nine nuclei. Nuclei scattered in yolk.
Axes of dividing nuclei of last stage have rotated, as before, to make angles with
one another. An odd nucleus shows irregularity in divisions : 9.., polar bodies.

(All the remaining figures of this and the next plate are surface views.)

Fic. 6. Ventral surface of older egg. The cells are at equal distances apart.

Fic. 7. Surface view of right side of egg with more nuclei than the last.
The nuclei somewhat more numerous in the posterior half : 4.4., polar bodies.

Fic. 8. Ventral surface. Nuclei dividing, d.~., everywhere on surface.
Numerous pairs of just separated nuclei, s.7., show division anteriorly as well as
posteriorly.

Fic. 9. Ventral surface of egg with twice the nuclei of last. More cells in
posterior half, due to movement that way and to multiplication.

Fics. to and 10a. Ventral and dorsal surfaces of egg with double the nuclei
of last stage. The nuclei of the posterior half of the egg are more numerous
than on the other end. They lie rather close together, nearly as far forward as
the smaller diameter.

Fics. 11, 118, and 11>, Ventral, dorsal, and lateral surfaces of an older egg.
In Fig. 11 of ventral surface, note that the anterior limit of the area ca. of rela-
tively closely crowded nuclei of last stage has drawn nearer the posterior pole,
away from the smaller diameter of the egg.

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JOURNAL OF MORPHOLOGY VOLUME XVI.

Hi. Mo, E. Knowzr, DEL.

Fig. 2.

Fig. 3.

Fig. 4.

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PLATE XXIx.

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564 KNOWER.

EXPLANATION OF PLATE XXX.

Fic. 112. Dorsal surface. The cells at posterior end of this surface crowded
closely together to form posterior border, 7.4.d. (posterior limit of disc) of the
area ca. of ventral surface. The collection of nuclei forming this border are in
sharp contrast to those scattered over this surface.

Fic. 11b. Side view, giving better idea of how markedly the cells on the sur-
faces of the egg have crowded back to the region marked ca. in figures: /./.d.,
posterior limit of cap or disc well shown ; a./.¢. anterior limit of disc ; 4. and P,,
anterior and posterior.

Fic. 12. Ventral surface of older egg with disc of nuclei forming area ca.
about equally distributed to its borders.

Fic. 13. Further contraction of disc ; all of its boundaries now well within
limits of ventral surface: /.4.d., lateral border of disc.

Fic. 14. Ventral surface of last, seen slightly on one side to show outlines of
contracting area.

Fic. 15. Posterior end of egg tipped up to show an especially marked concen-
tration of embryonic disc.

Fic. 16. Ventral surface of older germ-disc torn off with a piece of the
chorion: c., chorion ; z/.f., under-layer plug.

Fic. 17. Ventral view of egg showing germ-disc with crowded posterior mar-
gin: am.t., amnion thickening, later to fold forward ; w/.f., under-layer plug more
distinct.

Fic. 18. Ventral surface showing am.t., amnion thickening, at its maximum of
crowding before folding over.

Fic. 19. Older egg, somewhat on side to show amnion fold, am. The w/.f.,
extensive under-layer plug. Cells of serosa, and middle thin region of disc, faint.

Fic. 192. Same stage dissected off to show details. Amnion fold reaches for-
ward on sides anterior to w/.p. Anterior limit of disc, a./.¢d. Note dividing
nuclei, straight black rods.

Fic. 20. Side view of amnio-serosal fold, am., soon after its origin. Cells of
embryo not shown: sc., serosa cells posterior to fold ; em.d., embryonic disc ; sc.a.,
serosa cells on yolk anterior to embryo ; c#., chorion.

Fic. 21. Side view of amniotic fold (partly in optical section) half covering
the disc: am., amnion, of same appearance as ectoderm of disc, being several
layers thick; am.s., side view of amnion; my., two micropyles in distended,
wrinkled chorion, ch.

Fic. 22. Optical section from side of closing amnion; o.amc., opening from
exterior into amniotic cavity; sc., serosal cells, large and faint on surface of yolk;
ye, yolk-cells deeply stained and lying within the yolk. Cells of embryo and
amnion not shown.

Fic. 23. Same stage as foregoing seen from surface. Lettering as before.

om ;

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YURNAL OF MORPHOLOGY VOLUME XVI. I
Jo! f PLATE Xxx.

Fig. 20.

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H. Mo, E. Knowen, Det a

THE EMBRYOLOGY OF A TERMITE. 565

EXPLANATION OF PLATE XXXI.

Fic. 24. Ventral surface of egg, just after closure of amniotic cavity: ae.,
anterior end of embryo; yc., yolk-cells intensely stained; sc., serosal cells, large
and of light color.

Fic. 25. Side view (optical section) of slightly older embryo, slipped back out
of its usual position. The black ectoderm appears thicker in such an optical
section than it actually is: y., surface of yolk-mass; a.e., anterior end of embryo;
ch., chorion ; am., amnion ; z/. (mes.) under-layer, or mesoderm, extending forward
beneath ectoderm.

Fic. 26. Ventral view of germ-band two or three stages older than Fig. 24,
unsegmented and without cephalic lobes. The germ-band at this age is usually
placed as in the next figure.

Fic. 27. Embryo like that in the last figure, in its usual position. Seen from
side: a.e., anterior end; ch., chorion; am., amnion; /.¢., posterior end.

Fic. 28. Unsegmented germ-band with cephalic lobes, just before the appear-
ance of segments. Same stage as that of next figure, Fig. 29. The embryo is
dissected off from the yolk, and drawn with lower (yolk) side uppermost : am.,
amnion cells, along edges of band; a.¢., anterior triangular area, between cephalic
lobes. The mesoderm cells are large black masses.

Fic. 29. Unsegmented germ-band in same stage as last. Side view, to show
position on yolk. The amnion is faintly seen as a row of small dots beneath the
chorion.

Fic. 30. Cross-section through middle of germ-disc, at about the age of Fig.
13, perhaps slightly older: z/.1. (mes.) under-layer or mesoderm nucleus crowded
below the surface; d.z., nucleus dividing to separate a cell below; yc., yolk cells ;
ybs., yolk bodies of all sizes, and two perforated with holes left by solution of oil
drops. (0.003 mm. thick.)

Fic. 31. Cross-section through region of under-layer plug, w/.p. (mes.), the
mesodermal rudiment, at the stage of the germ-disc shown in Fig. 18; a.x.,
nucleus dividing to separate cell below; my.z., inner opening of micropyle; mzy.c.,
penetrating canal of micropyle through chorion ; my.o., outer opening of micro-
pylar funnel; c#., chorion; 7.véd., perforated yolk bodies. A large yolk-cell lies
under the middle of section. Yolk bodies are large. (0.003 mm. thick.)

The following six sections form a series, illustrating the growth of the germ-
disc and the mesoderm; also the origin and growth of the amniotic fold. Nuclei
diagrammatic, except the dividing ones.

Fic. 32. Median sagittal section of disc at stage in Fig. 18: am.t., amniotic
thickening of ectoderm ; w/.p. (mes.), under-layer or mesodermal plug; a.ec., ante-
rior end of ectoderm of disc; yc., yolk-cells; yd., yolk body. Four dividing
nuclei. (0.004 mm. thick.)

566 KNOWER.

Fic. 33. Median sagittal section of disc, Figs. 19 or 198; sc., serosal cells;
am.f., amniotic fold; 2/.f. (mes.), under-layer or mesodermal plug ; @.ec., anterior
ectoderm ; yc., yolk-cell. Seven nuclei in various stages of division. (0.004 mm.
thick.)

Fic. 34. Median sagittal section of an embryo slightly younger than that in
Fig. 22: sc., serosal cells; y., yolk-mass of finely fragmented bodies beneath
embryo ; am.f., amniotic fold ; o.amc., opening into amniotic cavity ; a.ec., anterior
ectoderm ; #/.f. (mes.), anterior end of mesodermal plug ; yc., yolk-cell; f.yd., per-
forated yolk body. Six dividing nuclei are seen. (0.004 mm. thick.)

Fic. 35. Median sagittal section of embryo at stage Fig. 24: ch., chorion;
am. amnion; am.c., amniotic cavity, now completely closed; sc., serosal cells;
a.ec., anterior ectoderm; yc., yolk-cells; 2#/.f., mesodermal plug; mes., mesoderm.
Seven dividing nuclei in different phases. (0.004 mm. thick.)

Fic. 36. Median sagittal section of embryo, between Figs. 24 and 26, with
anterior end square: sc., serosal cells; s., serosa; am., amnion; am.c., amniotic
cavity; a.ec., anterior ectoderm; mes., mesoderm; ye., yolk-cell; yd. yolk body.
Ten dividing nuclei in different stages. (0.004 mm. thick.)

Fic. 37. Median sagittal section of embryo in Fig. 27 (or 26): am.f., amnion
at posterior end; am.a., amnion, thinned out at anterior end; amc., amniotic
cavity; mes... mesoderm under posterior end; mes., mesoderm in the middle
region; mzes.a., anterior limit of mesoderm ; ec¢.cp. (anterior ectoderm as in previ-
ous figures a.ec.), now ectoderm of cephalic region; #.yé., perforated yolk body ;
yc, yolk-cells, nuclei in large masses of protoplasm. Nine dividing nuclei. (0.004
mm. thick.)

JOURNAL OF MORPHOLOGY VOLUME XVI.

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PLATE XXX!
Fig. 28.
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H. Mc, E. Kyower, DEL.

THE EMBRYOLOGY OF A TERMITE. 567

EXPLANATION OF PLATE XXXII.

The outlines of the figures on this plate were drawn with the aid of a camera.
They are magnified 55 diameters by Bausch & Lomb’s oc. 50 mm., odyect. 17 mm.

The series represents, diagrammatically, in side view and partly in optical
section, the principal stages in the development of the Termite embryo, from the
complete establishment of the germ-disc to the dorsal closure of the body-walls.

The origin and history of the embryonic membranes are particularly emphasized.
The general relations of the embryo to these membranes, to the yolk-mass, and
to the axes of symmetry of the egg are also well brought out. Note the position
of the micropyles, on the primary (and definitive) ventral surface of the posterior
end of the egg, in studying the remarkable changes in position which the embryo
passes through.

STAGE A. Germ-disc when first established, em.; extra-embryonal blastoderm,
ex.; yolk, y.; chorion, c#.; micropyles, my.
« B. Germ-disc with posterior amniotic thickening, a.¢., and mesodermal
plug.

C. Amnioticfold. Amnion a part of disc,am.; serosa, extra-embryonic, s.

« D. Amniotic fold just before closure of amniotic cavity. Amniotic cavity

open anteriorly, amc.; serosa, s.

£. Immediately after closure of amniotic cavity, amc.

« F. Early stage in the posterior elongation. The amnion begins to be
stretched.

« —G. Elongating germ-band before appearance of cephalic lobes. Amnion
not fully stretched posteriorly. Mesoderm a flat under-layer.

“ #H. Unsegmented germ-band with cephalic lobes, c./.

Z. First appearance of segments. Antennae, az¢.; mandibular segment,
md.; first maxillary, #ax.'; second maxillary, max.?; first thoracic,
th.*; tail-piece, Za.

“XK. Further elongation. Addition of second and third thoracic segments,
th.2 and ¢#.3; and an indistinct first abdominal, aé.', from anterior
portion of tail-piece. Appearance of labrum, /., and stomodeum,
st. Folding of head up from yolk.

“« Z. First stage of caudal flexure. Cephalic and thoracic appendages well
marked.

“MM. Caudal flexure pronounced. Abdominal segments established. Proc-
todeum, g7., well developed. Anterior appendages prominent.

«“ —W. Just before “revolution.” Head globular and standing off from yolk.
Maxillae tri-lobed. Anterior appendages long and beginning to
segment. Abdominal appendages prominent. Stomodeum and
proctodeum long.

“« 0. “Revolution.” Head slipping up along ventral surface to the anterior
pole of the egg. Embryonic membranes, especially serosa, contract-
ing dorsally. Proctodeum, /y., a long tube. Second maxillae
moved inward and not seen.

1568 KNOWER.

Stace . Completion of “revolution.” Dorsal growth of body-walls. Tracheal
stigmata, ¢r. Dorsal organ, d.o., the retracted remnants of embryonic
membranes.

“ — R. Closure of body-walls on the mid-dorsal line complete. Dorsal organ
has disappeared within yolk-mass, which is now enclosed in mid-
gut. Ventral ganglia shown. As compared with the preceding
stage, P, this is more truly an optical section, not showing the
body-walls except along the boundaries of the body.

TI °
JOURNAL OF MORPHOLOGY VOLUME XVI. PLATE XXXII

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THE GASTRULATION OF AMPHIOXUS.
T. H. MORGAN anp ANNAH PUTNAM HAZEN.

MATERIAL for a study of the process of gastrulation of
Amphioxus was collected in 1895 at Faro, Sicily, and at the
Stazione Zoologica in Naples.1 A part of the material was
stained at once and surface preparations made; another part,
after staining, was imbedded in paraffine, and the remaining
eggs were preserved in alcohol.

Kowalevski’s and Hatschek’s account of the gastrulation left
many points still unsettled, and Lwoff’s description of the proc-
ess was so different from those of his predecessors that the
entire problem appeared in a new light. Since our work
began, no less than three papers have appeared dealing with
the gastrulation of Amphioxus. It might seem, under these
circumstances, that further work would be superfluous ; yet the
more we have studied the process in Amphioxus the more diffi-
cult has the problem appeared, and none of these authors seem
to us to have reached a satisfactory conclusion,

Methods.

The eggs were preserved in several ways. Corrosive-acetic
preparations are best for surface views, and show very clearly,
both in surface views and in sections, the dividing and the
resting nuclei. Hermann’s fluid blackens the embryo so that
it cannot be used for surface preparations. The yolk granules
come out very clearly in sections, and the cell boundaries are
generally very well shown. Flemming’s solution — the stronger
formula — gives nearly the same results as Hermann’s fluid.
Embryos fixed by the two latter solutions do not need subse-
quent staining, although iron haematoxylin will bring out clearly
the nuclei, especially those in process of division. After the

1 For further details, see Morgan (’96).
569

570 MORGAN AND HAZEN. [VoL. XVI.

corrosive-acetic solution the embryos stain well in picro-lithium
carmine. Without using the highest powers of the microscope,
very little can be made out of the cell-structure of the embryo.
Almost all our work, therefore, has been done with a Zeiss
immersion 2 mm.

Gastrulation.

One of the great difficulties in following the changes that
take place during gastrulation is due to the absence of land-
marks. Our attention has been largely directed toward the dis-
covery of points of orientation of the early gastrula. Hatschek
relied mainly on the form of the embryo in optical section, and
we have also found this, under certain circumstances, a valuable
means of orientation. Wilson noticed that the pore at the
vegetative pole, which is sometimes left at the end of cleavage,
persists occasionally throughout the early period of invagina-
tion, and by this means he showed that the vegetative pole was
brought into contact with the animal pole; in other words,
that the invagination was radially symmetrical. We hoped at
first, by using this opening as a fixed point, to determine the
later changes that take place, but all traces of the pore soon
vanish, except in very abnormal cases.

It is of importance to determine at as early a stage as
possible the orientation of the gastrula; and here we have been
more successful, since we have been able to distinguish the
dorsal and ventral lips of the blastopore at the beginning of
the gastrulation process. The gastrula is not perfectly sym-
metrical, and the same is probably true of the blastula,
although more difficult to demonstrate. During invagination
the vegetative pole is brought near the animal pole, yet the
endoderm turns in such a way that the dorsal side can be
distinguished from the ventral. A careful study of the yolk
granules, and their appearance in the cells, has aided greatly in
orientation. Certain cells, that are turned in at one side, contain
relatively fewer and lighter yolk granules, and these cells can
be traced from the first stages of invagination until the closing of
the blastopore. They mark the dorsal side. As this is a con-
stant feature, it gives a definite means of orienting the embryo.

No. 3.] THE GASTRULATION OF AMPHIOXUS. 571

We have also studied the number and position of the cells
surrounding the blastopore at different periods, in the hope of
discovering some region of more rapid growth. Finally, the
karyokinetic phenomena of the embryo have been carefully
examined, for on this process Lwoff has mainly relied. It has
been necessary to take all these factors into consideration in
order to follow the changes that take place during gastrulation.

Kowalevski and Hatschek have shown that the blastula is
composed of larger and smaller cells, and several more recent
authors have observed the same fact. MacBride makes the
surprising statement that the cells of the blastula wall are all
of equal size. He has probably confused cross-sections of the
blastula with longitudinal ones, otherwise it is difficult to see
how such a mistake could have been made.

The yolk granules almost completely fill the cells around the
vegetative pole and gradually diminish in number toward the
animal pole.

The gastrulation begins by a flattening of ‘the vegetative part
of the egg (Pl. XXXIII, Fig. 1). The lower hemisphere then
turns in and slowly obliterates the segmentation cavity (PI.
XXXIII, Fig. 2). The invagination is not entirely radially
symmetrical, for the inturned cells bend over more toward one
side of the embryo than toward the other (Pl. XX XIII, Fig. 2).
At this time the segmentation cavity almost disappears at one
side, which becomes subsequently the dorsal side of the embryo,
but a portion of the segmentation cavity is left around the
remaining part of the inturned cell plate, and, in general,
is largest exactly opposite the dorsal side, and in this way a
dorsal and a ventral side of the gastrula are distinguishable at
an early stage of gastrulation. The outline of the embryo may
also be used, as Hatschek demonstrated, to determine points of
orientation, as shown in Pl. XXXIII, Fig. 2. The outline of
the dorsal side is less rounded than that of the ventral side.
This seems to be constant; but, if the sections do not pass
dorso-ventrally, this difference cannot be made out.

A further point of orientation is found in the appearance of
the inturned cells of the dorsal side. These cells are somewhat
smaller than the other invaginated cells, and they resemble

572 MORGAN AND HAZEN. [Vou. XVI.

closely the cells of the outer surface of the dorsal side
(Pl. XX XIII, Fig. 1). On the other hand, at the ventral lip
there is rather an abrupt transition between outer and inner
cells (Pl. XX XIII, Fig. 2). MacBride has called attention to
these differences, but it is not improbable that in the early
stages, at least, he has confused the dorsal and the ventral
sides of the gastrula.

Although the embryos can be oriented, as stated above, yet
it is still very difficult to determine how the closure of the
blastopore is brought about. While the blastopore becomes
smaller, the embryo is, at the same time, changing its shape,
so that it is not possible to assume that any one point is fixed
in relation to the others. Moreover, the possibility of cell-
migration must also be kept in mind. MLwoff has considered -
the presence of karyokinetic division as a criterion of growth;
but it must not be forgotten that growth need not follow
unless after division the cells increase in size. Furthermore,
cell migration is known to take place without cell division.1

As stated above, we had hoped to make use of the vegetative
pore, and by this means to determine how the closure of the
blastopore takes place. The pore is most often present in
series of eggs that do not develop normally, and hence there
is a certain amount of risk in using such a feature to deter-
mine the changes that take place in the normal egg. The
pore seems generally to disappear in the later stages. More-
over, other openings are sometimes found in the endoderm, due,
in some cases at least, to changes in shape of the cells at the
time of division. Pl. XXXIV, Fig. 17, shows a cross-section
of an egg in which the vegetative pore is large, and lies near
the highest point of the invaginated cells.

Occasionally a gastrula is found with the cells around the
vegetative pore turned outward (Pl. XXXIV, Fig. 16); and the
cells remain in this position even during later stages, as shown
in Pl. XXXIV, Fig. 19. This embryo throws some light on
the way in which the blastopore closes (Pl. XXXIV, Fig. 19).
As it was found among embryos in which, on an average, the

1 In the sea-urchin the archenteron is formed by cells pushing into the seg-
mentation cavity,

No. 3.] THE GASTRULATION OF AMPHIOXUS. 573

blastopore was nearly closed, the figure shows the relative
growth of the dorsal and ventral walls. It will be noticed
that the ventral wall is longer than the dorsal, and this is
confirmed by other results.

In one case that we have met with, an ectodermal pore was
present at the animal pole (Pl. XXXIV, Fig. 18), yet the pres-
ence of this pore has not prevented the invagination of the
larger cells. If, as seems probable, the opening was present
during the gastrulation period, the embryo shows that the
process of gastrulation may take place even when a large pore
is present in the wall. That this is possible is also shown when
the vegetative pore is present, and yet invagination takes place.
Therefore, whatever mechanism is invoked to explain the proc-
ess of gastrulation, the process is of such a nature that it does
not demand a closed blastocoel space.

At first the outline of the blastopore is oval (Pl. XXXIV,
Fig. 11). A large number of cells bound the opening, but, as
there is no sharp line of demarcation between the outer and
the inner invaginated cells, and since some of the cells at the
edge are partly within and partly without the rim of the blasto-
pore, the exact number and the shape of the boundary cells
cannot be accurately determined. In Pl. XXXIV, Fig. 11,
about forty-two cells form the rim of the blastopore. At a
later stave the number is smaller. In) Pl XXXIV Pic) 12;
about thirty-four cells are around the margin. When the blas-
topore has further closed (Pl. XXXIV, Fig. 13), twenty-eight
cells were counted. Finally, when the blastopore is reduced
in size, as shown in Pl. XXXIV, Fig. 14, only ten cells were
present.

How can we picture to ourselves the gradual reduction in
number of the cells as the blastopore becomes smaller? It
appears at times that, as the rim of the blastopore diminishes,
certain of the cells continue at the edge, but that others are
left behind in the general movement toward the center. In the
latest stage the boundary cells are elongated, and as a result a
larger number of cells surround the reduced blastopore than
would be the case if the cells all retained their earlier form. If
we judge by the shape alone of the blastopore, the closing takes

574 MORGAN AND HAZEN. [VoL. XVI.

place at nearly the same rate from all points; at least, until the
blastopore is much reduced in size. The outline of the blasto-
pore is generally oval, with the long axis connecting the dorsal
and ventral sides. At the time when the embryo is flattened
on the dorsal side to form the nerve plate, the blastopore
changes its shape, so that it becomes somewhat elongated from
side to side (Pl. XXXIV, Figs. 13 and 14).

Karyokinetic figures are found sometimes parallel to the
margin (Pl. XXXIV, Fig. 12), sometimes at right angles to it.
There is great irregularity in the distribution of the dividing
cells. In some embryos a large number of cells are in process
of division, in other embryos nearly all the cells are in a resting
stage. Sometimes groups of cells may be found dividing and
others resting, but such occurrences are exceptional, and, as a
rule, the karyokinetic figures are scattered irregularly over the
surface.

Lwoff has claimed that at a certain period cell division is
more rapid on the dorsal side of the blastopore than elsewhere,
and, in consequence, the ectoderm turns in and forms the dorsal
wall of the archenteron. A careful examination of a large num-
ber of embryos at all stages of development gives no support to
this view.

It is important in comparing the changes in the shape of the
embryos of different stages to use the same series preserved
by the same methods. It is also important that sections in
the dorso-ventral plane and in oblique planes be carefully
distinguished.

The early gastrula is shallow and saucer-shaped (Pl. XX XIII,
Fig. 2). The lips are then brought nearer together, so that the
embryo is longer, but correspondingly narrower (Pl. XX XIII,
Fig. 3). The embryo continues to narrow dorso-ventrally and
also laterally, and at the same time it grows longer. This
process continues, and for a time the growth of the dorsal
and ventral lips seem to be about equal. In still later stages
(Pl. XXXIII, Fig. 5) the ventral wall seems to grow more
rapidly ; it bends inward toward the dorsal surface, and extends
farther posterior than does the dorsal lip.

Since there are no definitely fixed points, it is very difficult

No. 3.] THE GASTRULATION OF AMPHIOXUS. 575

to determine in what region the bending in of the wall takes
place. The method of closing cannot, therefore, be determined
by any one criterion, but all the observed results must be taken
into account.

It should be noticed that in these figures the dorsal (d.) and
the ventral (v.) lips of the blastopore are, throughout the early

33

:

OD)

KIX

©

Fics. I-XX,

stages, equally distant from the point of greatest curvature (a).
If, on the other hand, the bending took place nearer the dorsal
or the ventral lip, one side would be shorter and the other
longer; and this is not the case.

Hatschek figures one side — the ventral —as longer than
the other side, —the dorsal, — but the dorsal subsequently
grows in length until it is approximately the same length as

576 MORGAN AND HAZEN. [VoL. XVI.

the ventral; the ventral wall increasing very little, if at all,
during the period of closing. A comparison of our Figs. 2, 3,
and 5, Pl. XX XIII, shows unmistakably that both the ventral
and dorsal walls are longer in the older stages.

A comparison of Hatschek’s Fig. 24 with our Pl. XXXIII,
Fig. 2, will show that what Hatschek supposed to be the dorsal
side is in reality the ventral one.

It is important to keep in view the different ways in which
the gastrulation might be interpreted, and we have tried to
illustrate in the accompanying text-figures the different possi-
bilities. The first series, I-V, represents a symmetrical closure
of the blastopore. The final closure at f is opposite the ante-
rior end c. The primary axis of the blastula and of the gastrula
corresponds with the antero-posterior axis of the embryo. The
closure of the blastopore is shown in V, and is seen to take
place equally from all points. Kowalevski supposed this to be
the way in which the blastopore closes.

The second series, VI-X, represents an unsymmetrical clo-
sure of the blastopore. The ventral lip at a is supposed to be
fixed in VI, and in the successive figures, VII-IX, the dorsal
lip is represented as bending over and elongating to close the
blastopore. The closure of the blastopore takes place over the
dorsal side of the embryo. Fig. X shows the successive stages
in the closure of the blastopore. If we compare Figs. V and X,
we see that when the blastopore is turned upward toward the
observer the point of greatest bending of the wall is opposite
the blastopore in V, while in X the point of greatest bending
is anterior to the center of the blastopore (see also Fig. VIII).
This series, VI-X, represents Hatschek’s idea as to the way in
which the blastopore closes.

The reverse method of closing is shown in series XI-XV.
Here the dorsal lip is represented as fixed, and the ventral as
bending over to close the blastopore. Hence the closing is on
the ventral side of the embryo.

It will also be noticed that in the last two series (VI-X and
XI-XV) the point of greatest bending shifts gradually around
the anterior end of the embryo as the closing takes place ; while
in the first series the point of greatest bending remains fixed.

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No. 3.] THE GASTRULATION OF AMPHIOXUS. 577

There are, in fact, two ways in which we may think of the
changes shown in Figs. VI-X and XI-XV as taking place.
The wall that grows backward to close the blastopore may
either simply elongate as a result of cell growth, and the
bending at the anterior end remain fixed, or the growth back-
wards might be due to a gradual shifting of the part of
greatest bending along the opposite side; in consequence of
this the dorsal wall would increase in length at the expense
of the ventral in one case, and the ventral at the expense of
the dorsal in the other. It is also possible that both a change
in the bending and cell growth might take place at the same
time.

In a fourth series we have tried to represent our own idea of
the method of closure of the blastopore. In the first figure of
this series, XVI, the gastrula is represented as being somewhat
unsymmetrical. The dorsal lip is at ¢c, the ventral at a. The
point of greatest bending of the gastrula is near the center, but
a little toward the ventral side. Ata later stage, XVII, both
dorsal and ventral sides of the gastrula have come nearer
together, and the embryo has, in consequence, become longer.
The dorsal and ventral sides are of about equal length, and the
point of greatest bending is opposite the blastopore. In a later
stage, XVIII, the walls grow longer and the dorsal side flattens
somewhat. The opening of the blastopore is still opposite the
point of greatest bending. In the last stage, XIX, the ventral
lip grows faster and the blastopore opens more on the dorsal
side. Fig. XX shows the method of closure as seen from
the posterior side. The series of concentric circles represent
the successive stages. The final stage is excentric, owing
to the more rapid growth of the ventral lip.

If instead of a dorso-ventral series we had made use of a
lateral one, that is, one from right to left, to illustrate our
idea of the method of closure of the blastopore, we would have
given a series exactly like that drawn in I-V. Even in the
dorso-ventral series our idea of the method of closure corre-
sponds more nearly to that of the first series than to any other ;
the points of difference being: first, the unsymmetrical gas-
trula at the earliest stage (XVI); second, the flattening of the

578 MORGAN AND HAZEN. [VoL. XVI.

dorsal side in XVIII; and, third, the more rapid growth of the
ventral wall in the final stage of closure.

Cell Division during Gastrulation.

Cell division occurs throughout the entire period of gastru-
lation both in the ectoderm and in the endoderm. We have
examined a large number of preparations, both sections and
surface views, to see whether cell division is more frequent in
one part of the embryo than in another. Although cell division
is present at all times and in all stages, yet we have found, in
general, no region of more rapid cell multiplication.

Great individual differences exist, for while one preparation
may show more karyokinetic figures on the dorsal or on the
ventral lip of the blastopore, other preparations of the same
age may show other regions of cell multiplication. Hence,
with a preconceived idea as to the place at which more rapid
growth occurs, one could easily find preparations confirming
such a view. But an unprejudiced examination shows that
cell division is general and not restricted to any special region.
Text-figs. XXI-X XVIII show in what regions cell division is
taking place in certain embryos of various ages. The drawings
are diagrammatic reconstructions of serial sections. The dorsal
side is indicated by the parallel lines. One figure of each pair
shows, therefore, the dorsal, and the other the ventral side.
The dividing nuclei in the ectoderm are represented by solid
dots, and those in the endoderm by circles.

Fig. XXI a, 0 represents the embryo at midnight, corre-
sponding in age with the embryo shown in Pl. XXXIII, Fig. 2.
Only two ectodermal cells are dividing, while many endodermal
cells are in process of division. Another embryo of the same
age is shown in Fig. XXII a, 4. Here the number of cells
dividing in ectoderm and endoderm is about the same. The
dividing cells are scattered nearly equally throughout the
embryo.

The next stage, Figs. XXIII and XXIV a, 4, represent the
embryo at 3.30 A.M. Both show cells dividing around the blas-
topore, and there is also a region of division at the anterior end.

No. 3-] THE GASTRULATION OF AMPHIOXUS. 579

In one of these the ectoderm cells on the dorsal side and in
the other on the ventral side are dividing more rapidly. Other
preparations of the same age were found in which the cell divi-
sion is not more common around the blastopore than elsewhere.

At 4.30 A.M. the embryo has closed in further (Figs. XXV
Gy Gand XXVG a, 6). In the first; of. these) figures itis

YOCVIIE @
Fics. XXI-XXVIII.

noticeable that cell division is very frequent in the ectoderm
of the ventral side. In the other very little cell division is
present, and that mostly around the ventral lip of the blastopore.

Figs. XXVII a, 6 and XXVIII a, 6 at 6 a.m. show, in one
case, several endoderm cells and few ectoderm cells in process
of division, In the other embryo the reverse is true.

580 MORGAN AND HAZEN. [VoL. XVI.

After this stage the blastopore closes in more rapidly from
the ventral side, and cell division seems to be more rapid on
this side, both in ectoderm and endoderm.

Distribution of Yolk in the Embryo.

At the time when the blastula wall first begins to flatten we
have attempted to discover whether the yolk is equally distrib-
uted throughout the endodermal plate. In many cases we
have found that the embryos preserved in Flemming’s or in
Hermann’s fluid show at one end a region of small cells bear-
ing fewer yolk granules, and these granules are lighter in color
than those of the rest of the inturned cells! (Pl. X XXIII,
Fig. 1). At the opposite end of such a section there is an
abrupt demarcation between the large yolk-bearing cells that
are turned in, and the smaller and lighter cells outside. This
difference in the two ends of the section is shown by many
series. In other series, cut more or less at right angles to the
preceding, the difference is not seen, of course, in the mid-
dle sections of the series, but generally at one end of the
series the lighter cells can be found, and at the opposite end
the yolk-bearing cells extend to the margin of the blastopore.

This difference in the endoderm we have been able to trace
throughout all the subsequent periods of development, and we
have been able to show that the region of clearer cells be-
comes the dorsal wall of the archenteron. Hence the dorsal
lip of the blastopore is identified even at the time when the
first beginning of gastrulation is evident. It is, therefore,
highly probable that even in the spherical blastula there is a
bilateral symmetry already present. We have laid more stress
on this means of orientation than on any other, and have deter-
mined the dorsal and ventral sides in this way. _

A later stage in the gastrulation is shown in Pl. XXXIII,
Fig. 2. At this time the endodermal plate has bent upward
and almost touches the ectoderm. The section is in a dorso-

1 It is difficult to determine whether the lighter granules are in reality lighter
than those elsewhere, or whether it is only an optical effect of the light passing
through fewer granules.

No. 3.] THE GASTRULATION OF AMPHIOXUS. 581

ventral plane and shows the difference in the ectoderm at the
dorsal and ventral lips of the blastopore. At the dorsal lip
the endodermal cells are, as before, lighter and contain less
yolk and differ little from the ectodermal of the outer surface;
in fact, there is really no histological difference between the
cells just outside and just within the lips of the blastopore
over the dorsal side. It is to be noticed that there are more
of the lighter cells on the dorsal wall than in the preceding
stage. This might result either by a continued rolling in of
outer cells, or by a multiplication of cells already inside, or by
both processes combined, or even by differentiation of the cells
anterior to the clear region. It is certain that the endodermal
cells continue to divide during development, and we have seen
no evidence of an actual inrolling. Whether the division of
the cells in this region is, in itself, sufficient to account for
the increase in the number of cells is a point almost impossible
to determine.

The same section (Pl. XX XIII, Fig. 2) shows also the char-
acteristic shape of the embryo at this stage; over the dorsal
side the embryo is less curved than over the ventral side. The
deepest part of the invaginated endoderm is somewhat toward
the ventral side. The effect produced is that the ventral part
of the endoderm is thrown over toward the ventral side of the
embryo. Surface views of entire embryos show the same
asymmetry in the gastrula. On the ventral side of this sec-
tion (Pl. XX XIII, Fig. 2) there is an abrupt transition between
the large yolk-bearing cells and the smaller cells that continue
out into the ectoderm. Whether these smaller cells that are
partly turned into the blastopore belong to the endoderm or to
the ectoderm can only be determined by their fate. They will
be considered later.

There is generally present on the ventral edge a dilatation
of the segmentation cavity that is characteristic for this period ;
on the dorsal side the ectoderm and the endoderm are almost
in contact.

An older stage is shown in Pl. XXXIII, Fig. 3. The
embryo is now cup-shaped and the archenteron much deeper
than before. The deepening is, in part, the result of the

582 MORGAN AND HAZEN. [VoL. XVI.

approach of the sides of the embryo, so that it is now narrower
dorso-ventrally and also from side to side. As a result, the
blastopore becomes smaller (Pl. XXXIV, Figs. 11 and 12) and
at the same time the saucer-shaped embryo (Pl. XXXIII,
Fig. 2) becomes elongated into a cup-shaped form. The cells
are smaller and more numerous in the older embryo (PI.
XX XIII, Fig. 3), but there is no evidence that the change in
form of the embryo is the result of cell activity in any partic-
ular region. The dorsal wall of the archenteron is covered by
lighter cells, and this region is longer than in the preceding
stage. The clear cells of the dorsal wall are now larger than
the ectodermal cells outside the blastopore, although just at
the lips of the blastopore the transition between inner and
outer cells is gradual. At the ventral lip the yolk-bearing
cells come nearly to the lip of the blastopore and are followed
by a few small, rounded cells. The largest endodermal cells
bearing the greatest number of yolk granules are found at the
innermost part of the archenteron ; the cells decrease gradually
in size, and in the number of granules contained in them,
toward the ventral side.

Around and within the ventral lip of the blastopore, during
the early gastrula stages, there are frequently found, as just
described, small cells, that contain much less yolk than the
large endodermal cells, and closely resemble the ventral ecto-
derm in appearance (Pl. XXXIII, Figs. 2 and 3). Even in
later stages these small cells are often found. We have care-
fully examined many different series of sections to see if the
spherical form of these cells is the result of cell division, but
cell division does not seem to be more frequent here than
elsewhere, and the nuclei of the cells are often in the resting
stage. It is difficult to determine whether any of these cells
come to lie eventually in the archenteron, or whether in later
stages, as the yolk-bearing endodermal cells multiply, these
cells turn out into the ectoderm.

The dorsal side of an embryo (Pl. XXXIII, Fig. 4 A) some-
what older than the last is shown in Pl. XXXIII, Fig. 4 2.
The lighter cells of the dorsal wall are shown also in the figure,
and their similarity to the ectoderm outside is evident. The

No.3.] THE GASTRULATION OF AMPHIOXUS. 583

yolk granules in the ectoderm are fainter and are beginning to
disappear. The granules grow fainter in that part of the ecto-
derm which corresponds to the animal pole. Around the blas-
topore they persist longer; the cells outside the dorsal lip
containing granules similar to those in the cells of the dorsal
wall of the archenteron.

An older stage, two hours later than the last, is drawn in
Pl. XXXIII, Fig. 5. The embryo has greatly elongated, the
blastopore is reduced in size and now opens somewhat on the
dorsal surface at the posterior end of the body. The cells
over the dorsal wall of the archenteron are broader than those
elsewhere ; they are also clearer and contain fewer and lighter
yolk granules. As much as two-thirds of the dorsal wall is
formed by these cells, which pass gradually into the more
anterior cells containing more yolk. The cells of the ventral
wall are smaller than those at the anterior end and are also
smaller and darker than those over the dorsal wall.

The cells of the ectoderm over the dorsal surface are very
similar to the endoderm cells over the dorsal wall. The more
anterior of those ectodermal cells are smaller, while those near
the dorsal lip of the blastopore are almost identical with those
within.

On the ventral wall the ectodermal cells are smaller than else-
where, but they grow larger as they approach the ventral lip of
the blastopore. At the sides of the blastopore, also, there are larger
cells, so that a ring of large cells surrounds the blastopore.

In the later stages the cells over the dorsal and dorso-lateral
walls of the archenteron continue to grow clearer and the yolk
in them to disappear. A cross-section of an embryo at 8 A.M.
is shown in Pl. XX XIII, Fig. 6. Here the difference between
the dorsal and ventral wall is clearly seen. The dorsal surface
of the embryo is slightly flattened to form the medullary plate.
This section is taken at about the level of the posterior third
of the embryo. Sections of the same series taken at the
extreme anterior end show that the cells on the dorsal wall
contain more yolk and resemble those on the ventral wall.

In the later stages changes take place in the endoderm,
bringing about a rearrangement of the cells and also involving

584 MORGAN AND HAZEN. [VoL. XVI.

a change in the character of the cells themselves. The ante-
rior end of the archenteron becomes larger, and at the same
time the cells covering this portion of the cavity become
smaller (Pl. XX XIII, Fig. 7 A). This region was at first sur-
rounded by the large dark yolk-bearing cells (Pl. XXXIII,
Fig. 5), but these cells seem to shift posteriorly ; at any rate,
the large yolk-bearing cells are now found on the ventral and
ventro-lateral walls of the archenteron at the posterior end of
the body. p

About this same stage, or a little later, a curious change
takes place that has greatly puzzled us. Yolk granules begin
to appear in all the cells of the body, and what is most sur-
prising is that the yolk in the large endodermal cells does not
seem to decrease in amount. We cannot offer, therefore, any
explanation as to the meaning of this phenomenon. It might
seem that the change was brought about by a decrease in the
volume of the cells, and that they contained the same absolute
amount of yolk as before, the change being apparent rather
than real. Measurements of the embryo show, however, that
it is increasing in size, and the body-walls are as thick or
thicker than before. The cells are, it is true, higher and nar-
rower than before, but we do not see how this alone could pro-
duce the effect. However, even in the cells of the ectoderm
yolk granules reappear in later stages. A longitudinal section
of an embryo at 11 A.M. is drawn in Pl. XXXIII, Fig. 7 A.
A detailed and more magnified part of the dorsal wall (at x,
in Fig. 7 A) is shown in Fig. 7 5. The ectoderm that forms
the nerve plate contains more yolk granules than in the earlier
stages, and the endoderm of the dorsal wall (ezd.) is now also
filled with yolk granules. Another part of the same embryo
anterior to the last (at y in Fig. 7 A) is shown in Fig. 7 C.
In this region the ectoderm contains almost no yolk, but the
endodermal cells are filled with it. A section through the ven-
tral wall (at ¢ in Fig. 7 A) is shown in Fig. 7 D. Here the
ectoderm contains very little yolk, while the endodermal cells
are tall, closely packed together, and filled with yolk granules.

A later stage (Pl. XX XIII, Fig. 8) shows that the yolk gran-
ules reappear even in the ectoderm. At this time the first

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No. 3.] THE GASTRULATION OF AMPHIOXUS. 585

eight body cavities are given off; the cells that form the noto-
chord, and the mesodermal pouches are filled with yolk gran-
ules, and the ectoderm is also. This reappearance of the yolk
in the later stages occurs after the blastopore has been closed,
and at a time when all the regions of the body are easily dis-
tinguishable. What we have stated in regard to the earlier
stages, throughout which the yolk has been gradually disap-
pearing from certain cells and remaining constant in others, is
not effected by these later changes. We feel, therefore, no
less certain of our results, based, as they are, largely on the
distribution of the yolk and the histology-of the cells during
the early stages.

Irregularities in the Endoderm.

In looking for the vegetative pore as a means of orienting
the gastrula stages, we soon found that, at other places,
depressions occurred in the endoderm resembling the point
at which the vegetative pore closes (Pl. XXXIII, Fig. 9).
We had, therefore, to abandon this landmark as a means of
orientation. Often we have found that irregularities in the
endoderm are due to the rounding up of cells during the final
stages of division. In other cases, as in Pl. XXXIV, Fig. 20,
at x, the nuclei are in a resting condition, but this may be
interpreted to mean that after the division the nuclei have
become spherical, but the cells have not yet lost their rounded
contour. This view is supported by the fact that two such
cells are generally found together. In the ectoderm, too, we
have often seen two dividing cells showing the same tendency
to become spherical during division. However, cells in all
phases of division may be found that have not rounded up as
described, yet even in these cases the inner free ends of the
cells are rounded, thus breaking the even contour of the
endoderm.

Oblique Sections through Blastopore.

It has been pointed out that the early gastrula is somewhat
asymmetrical (Pl. XXXIII, Fig. 2), the invagination being

586 MORGAN AND HAZEN. [VoL. XVI.

deeper on the ventral side, making the outline of the embryo
more rounded there. A somewhat similar effect is sometimes
produced by an oblique section through a slightly elongated
embryo. Sections similar to text-fig. XXIX d are some-
times obtained, but it is unsafe to judge from such a section
alone that the more rounded side is the ventral one. The
entire series of sections must be known, a—/ During the
first stages of gastrulation there is an extraordinary amount of
variation in the shape of the embryos. Forms with a some-
what elongated blastopore are not infrequent, and an oblique
section of such an.embryo produces the effect shown in the
figures. Even in the later stages, the same asymmetry may
be found as shown in Pl. XXXIII, Fig. 4 A. Asa matter of
fact, the dorsal side of the embryo is in this case the shorter

664996

one, but other embryos of the same age may be cut in such a
way that the shorter side is the ventral one. It would there-
fore be easy to arrange a series of these forms in such a way
as to make it appear that the closure of the blastopore is over
the dorsal side. Only after a careful and prolonged examina-
tion of both sections and surface views have we been convinced
that this condition is the result of an oblique section of a some-
what elongated embryo. If, for instance, the series of sections
to which XXIX c belongs, be examined, it will be found that
at one end of the embryo the sections are completely closed,
a-b; but at the other end of the series the opening of the
blastopore enlarges, as shown in e, f.

Sections of the kind, with the dorsal side shorter than the
ventral, might seem to confirm Hatschek’s view of the closing
of the blastopore over the dorsal side, but we hope we have
shown this view to be erroneous. Without other means of
orientation the shape of the section may be very misleading,

—— >.

No.3.] THE GASTRULATION OF AMPHIOXUS. 587

especially when such great variation is present. Sections of
embryos preserved in corrosive acetic will not show the differen-
tiation of the dorsal wall upon which we have mainly relied,
and after repeated examination of series of sections preserved
by this fluid, we have been obliged to give up all hope of orient-
ing with certainty the early gastrula stages. In the later
gastrula stages the shape of the embryo is sufficiently char-
acteristic to determine its orientation, but, in the early stages,
while in many cases the shape of the embryo might seem to be
sufficiently characteristic to determine the dorsal and ventral
sides, yet such a criterion alone is very unsafe. We do not
hesitate to say that many of the sections figured by some of
the more recent authors are in all probability oriented wrongly.

Historical Review.

Kowalevski gave a very brief account of the process of gas-
trulation in Amphioxus. A radially symmetrical invagination
is described; the gastrula grows longer, and the small blasto-
pore lies at the posterior end. Later the blastopore shifts some-
what to the dorsal side. More recent writers have inferred
from Kowalevski’s brief account that the gastrula axis corre-
sponds to the embryonic axis, and while such a conclusion is
probably» true, yet Kowalevski’s description is so very brief
that we can only infer this to be his meaning.

Hatschek claimed to have been able to distinguish a bilateral
symmetry at the time when the invagination is completed.
We have shown that even at an earlier stage the bilaterality is
present and is shown by the differentiation in the flat plate
that is subsequently turned in. MHatschek noticed that the
large yolk-bearing cells around the vegetative pole of the
blastula, that subsequently form the endodermal plate, occupy
only about one-third of the circumference of the blastula wall,
and hence are, at first, too small to fill the entire inner surface
of the cap-shaped gastrula. He supposes that increase in the
volume of the endodermal cells takes place during invagination,
and this increase, he suggests, is brought about by absorption
of the fluid of the blastocoel space; yet his figures show more

588 MORGAN AND HAZEN. [VoL. XVI.

large yolk-bearing cells in the endoderm after invagination
than in the flattened endodermal plate of the blastula. Lwoff
points out the insufficiency of the mechanism proposed by
Hatschek and claims that other cells beside the large yolk-
bearing ones are also turned in, and in this way he accounts for
a sufficient number of cells to fill the archenteric cavity. We
have also tried to show that, at the dorsal side, cells poor in
yolk are invaginated, although we prefer to speak of these
cells as endodermal and not as ectodermal, as Lwoff has
done.

Hatschek’s idea of the method of closure of the blastopore
is illustrated by our series of text-figures, VI-X. The dorsal
lip is supposed to bend around and meet the ventral lip, thus
closing the gastrula mouth along the dorsal side of the embryo.
Hatschek offers this view, not in a dogmatic spirit, but simply
as more in line with his own observations, admitting, however,
that Kowalevski’s view may be the correct one. Hatschek
noticed the early asymmetry of the gastrula, but a comparison
of his Fig. 24 with our Pl. XX XIII, Fig. 2 shows that what he
has identified as the dorsal (anterior) lip of the blastopore is, in
our estimation, the ventral lip. The older stages, however, are
oriented in the same way as are our own. MHatschek noticed
that the transition from ectoderm to endoderm is sharpest on
the ventral side, and at that point in the later stages he located
the two historic pole cells.

It is not entirely clear to us how Hatschek imagined the
backward growth of the dorsal lip of the blastopore to take
place. His figures lead us to suppose that the result is, in
part at any rate, produced by an increase in the number of
cells of this region. The ventral wall remains unchanged, and
the ventral lip bends around only so far as it takes part in the
reduction of the blastopore.

Lwoff draws a sharp distinction between ectoderm and endo-
derm ; the latter cells being characterized by their size and the
amount of yolk contained in them, and he believes that the
difference is present even in the blastula stage, so that “ehe
die Einstiilpung beginnt, dass also die Sonderung der zwei
primaren Keimschichten—des Ektoderms und Entoderms —

No. 3.-] THE GASTRULATION OF AMPHIOXUS. 589

hier als Resultat der Furchung zu betrachten ist.”! He be-
lieves the difference has an important practical meaning, for
when the invagination is completed, not only the endoderm,
but also a portion of the ectoderm is turned in, so that, in
the gastrula of Amphioxus, not all the cells that line the
archenteron are to be designated as endoderm.

Lwoff dissents from Hatschek’s statement that at the end of
cleavage and before the gastrulation begins all division ceases.
On the contrary, Lwoff points out that cell division continues
throughout the gastrulation period,— as we have also found, —
and that the ectodermal cells divide more frequently than do
the endodermal. Lwoff further differs from Hatschek as to
the way in which gastrulation takes place. Hatschek states
that the endodermal cells are turned in to form the inner layer
of the cap-shaped stage and line the entire inner cavity of the
cap. Lwoff thinks that the invagination continues during the
period of closure of the blastopore. The increase in the ecto-
derm cells is, according to Lwoff, a most important factor in
the gastrulation process. ‘Auf den Langsschnitten durch die
Gastrula sieht man Mitosen iiberall im Ektoderm, am zahlreich-
sten aber sind sie an der Seite, die spater zur Riickenseite der
Larve wird und am dorsalen Umschlagsrande zu bemerken.
Man sieht Mitosen auch in den Zellen der dorsalen Wand der
Hohle, die sich als eingestiilpte Ektodermzellen erweisen. Die
Langsschnitte zeigen, dass die Ektodermzellen an diesem Um-
schlagsrande umbiegen und nach innen wachsen. Das Anwach-
sen der Zellen muss an dieser Stelle sehr bedeutend sein, weil
auf Medianschnitten der Gastrula sich eine Anhaufung von Zellen
oftmals bemerken lasst ; die Zellen verlieren hier den Charakter
des einschichtigen Epithels und sind unregelmassig zweischich-
tig gelagert. Manchmal lassen sich an der dorsalen Wand der
Hohle Unebenheiten bemerken und einzelne Zellen lésen sich
sogar aus dem Zellverbande los und erscheinen als rundliche Zel-
len, die neben den iibrigen Zellen liegen. Ich muss hervor
heben, dass die Umbiegung der Ektodermzellen und deren
Einstiilpung nur am dorsalen Umschlagsrande sich bemerken
lasst, am ventralen Umschlagsrande dagegen eine scharfe Grenze

1 Lwoff, p. 5.

590 MORGAN AND HAZEN. [VoL. XVI.

zwischen den Entoderm- und Ektodermzellen sichtbar ist. Man
koénnte freilich den Einwand erheben, dass alle eingestiilpten
Zellen als Entodermzellen zu bezeichnen sind; aber dieser
Einwand konnte nur auf der vorgefassten Meinung beruhen,
dass alles, was nach innen gelangt, als Entoderm zu bezeichnen
ist. Ich habe schon oben in Uebereinstimmung mit Hatschek
angegeben, dass der Unterschied zwischen den Ektoderm- und
Entodermzellen schon im Blastulastadium, also vor der Ein-
stiilpung, sich bemerken lasst ; dieser Unterschied kann seine
Bedeutung nicht verlieren, wenn es sich ergiebt, dass die Ekto-
dermzellen sich an der Einstiilpung auch betheiligen. Die
aktive Rolle der Ektodermzellen bei der Einstiilpung und die
Betheiligung derselben an der Bildung der dorsalen Wand der
Hohle kann auf solche Weise keinem Zweifel unterliegen.”’?

In regard to the mechanism of the invagination, Lwoff again
dissents from Hatschek’s view. He shows that Hatschek’s
account of the enlarging of the endodermal cells is an insuffi-
cient explanation in itself, and, moreover, the inturned cells,
instead of enlarging, are reduced in size by cell division. We
have found that it is difficult to tell how much the endodermal
cells increase in volume after each division. A very slight
increase in the size of each cell would be sufficient to greatiy
increase the area of the cell plate, even if the cells themselves
do not after division assume their original size. Lwoff’s state-
ment that during gastrulation the endodermal cells almost cease
to divide is certainly incorrect. Dividing cells are to be found
throughout the entire gastrulation period in the endoderm.
Some preparations even show only the endodermal cells divid-
ing, and others only the ectodermal. Lwoff has observed the
latter only, but an examination of a large number of embryos
shows that division takes place in both layers. The number of
ectodermal cells is larger than that of the endoderm, so that
cell division might be found somewhat more often in the outer
layer; but from this it would not follow that any cell of the
ectoderm divided more often than any cell of the endoderm.
Further, Lwoff’s statement that cell division is more abundant
on the dorsal side of the blastopore is certainly incorrect, since

1 Lwoff, p. 7.

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No. 3.] THE GASTRULATION OF AMPHIOXUS. 591

our preparations show that cell division is no more frequent
here than elsewhere. Lwoff attempts to explain the gastrula-
tion as a result of the more rapid division of the ectoderm at
the dorsal lip of the blastopore: “ Die Einstiilpung beginnt an
der Grenze zwischen den Ektoderm- und Entodermzellen, wo
der Unterschied zwischen Wachstumsenergien beider Elemente
am grossten ist. Da aber die Zellenvermehrung nicht iiberall
gleichmassig vor sich geht, sondern sich vorzugsweise an einer
Seite konzentriert, die zur Dorsalseite der Gastrula wird, so
erklart sich dadurch die Ungleichmassigkeit der Einstiilpung
und die Entstehung einer radial-unsymmetrischen Gastrula.
Wahrend namlich an anderen Stellen die Entodermzellen ein-
gestilpt werden, stiilpen sich an dieser Seite die Ektodermzel-
len selbst nach innen ein. Mit anderen Worten, die Zellen, die
vom dorsalen Umschlagsrande aus nach innen wachsen, bilden
die dorsale Wand der Hohle, wahrend die eigentlichen Ento-
dermzellen an die ventrale Wand und an die Seiten der Hohle
zu liegen kommen. Zugleich wachst der dorsale Umschlagsrand
nach hinten, und Hand in Hand damit wird der urspriinglich
weite nach hinten offene Gastrulamund allmahlich geschlossen.
Dadurch kommt eine radial-unsymmetrische, aber zugleich, da
die Riickenseite markirt ist, bilateral-symmetrische Gastrula zu
Stande, die keineswegs als Archigastrula zu bezeichnen ist.’’?

We have shown that Lwoff’s statement in regard to the
presence of certain cells free from yolk in the dorsal wall of
the archenteron is correct. The more recent writers, Klaatsch,
Sobotta, and MacBride, have entirely overlooked this important
point, and have needlessly criticised Lwoff in consequence.
Whether those cells that form the dorsal wall of the archen-
teron are to be called ectoderm or endoderm is entirely, it
seems to us, a matter of choice or definition.

It is the old problem of what we shall define as a germ layer
— whether the presence of yolk in certain cells is, in itself, a
sufficient criterion to distinguish the endoderm, or whether all
the cells that are invaginated are, irrespective of their form, to
be called endoderm. For ourselves, the question seems to be
a trivial one, and simply as a matter of personal preference we

1 Lwoff, p. 9.

592 MORGAN AND HAZEN. | VoL. XVI.

choose to speak of all the cells that turn in during gastrulation
as endoderm.

While we agree with Lwoff that some of the cells that are
at first invaginated contain only a small amount of yolk, and
clearly resemble the ectodermal cells of the dorsal side, we
have found no evidence that the ectoderm continues to turn
in at this point during the later period of the closing of the
blastopore.

Lwoff, while admitting that Hatschek’s figures are true to
nature, yet disagrees with Hatschek in regard to the relation
of the egg axis to that of the embryo: “Ich habe gefunden,
dass der Gastrulamund von allen Seiten geschlossen wird,
indem seine Rander einander entgegenwachsen. Die Schlies-
sung des Gastrulamundes vollzieht sich zwar ungleichmassig,
aber ich habe schon gezeigt, dass die Gleichmassigkeit der Ein-
stiilpung und der Gastrulaschliessung durch die Einstiilpung
der Ektodermzellen am dorsalen Umschlagsrande gestort wird,
indem, wie oben erwahnt, der dorsale Umschlagsrand wahrend
der Gastrulaschliessung nach hinten wachst und mehr als der
ventrale und die seitlichen Rander daran Antheil nimmt. Wenn
ich das Wachsthum des dorsalen Umschlagsrandes nach hinten
beriicksichtige, so kénnte ich auch sagen, dass die Schliessung
des Gastrulamundes vorzugsweise von vorn nach hinten sich
vollzieht, aber nicht in dem Sinne, wie es Hatschek will. Denn
ich habe gefunden, dass die Riickenseite der Gastrula selbst
nach hinten wachst, dadurch allmahlig langer wird und den
Gastrulamund schliesst. Wahrend die iibrigen Rander des
Gastrulamundes gleichzeitig sich zusammenziehen, wird der-
selbe immer kleiner. Ich habe dabei keine Spuren der Ver-
wachsung der seitlichen Rander von vorn nach hinten in der
Medianlinie des Riickens (etwa in der Gestalt einer Nathlinie)
weder an ganzen Larven noch auf den Schnitten sehen konnen.
Indessen wirde die Behauptung, dass der hinterste Theil des
Gastrulamundes zuletzt ubrigbleibe, nur dann reelle Bedeutung
haben, wenn es nachgewiesen ware, dass die seitlichen Rander
des Urmundes in der Medianlinie in einer von vorn nach hinten
fortschreitenden Richtung verwachsen. Diese Behauptung ruht
auch auf der Annahme, dass der hintere (ventrale) Rand des

No. 3.] THE GASTRULATION OF AMPHIOXUS. 593

Gastrulamundes wahrend der Gastrulaschliessung unverandert
bleibt. Dies ist aber auch nicht der Fall, und Hatschek’s
eigene Abbildungen sprechen nicht zu Gunsten dieser An-
nahme.”’!

Our view of the method of closing of the blastopore agrees
essentially with Lwoff’s, that is, that the blastopore closes
equally from all sides, and that the gastrula axis corresponds
more or less exactly with the longitudinal axis of the embryo.
On the other hand, we have tried to show that there is no
necessity for supposing that the cells outside the blastopore
on the dorsal side turn in during the period of closure of the
blastopore. It seems more probable that the advance of the
dorsal lip takes place in the same way as that of the lateral
and ventral lips, and even Lwoff does not suppose the latter to
advance as the result of the inturning of cells.

Wilson made the important observation that “the cleavage
pore, which marks the lower pole of the blastula, sometimes
persists up to a stage as late as the gastrula shown in Hat-
schek’s Figs. 26 and 27. In all such cases I examined, it lay
exactly at the central point of the dome—a fact that shows
that the invagination is primarily symmetrical, as originally
described by Kowalevski.”’

Wilson also states, in contradiction to Lwoff’s statement, that
the entoblastic cells (macromeres) ‘show numerous conspicu-
ous mitoses, and in every part of the entoblastic plate.” 2

Klaatsch has given a few figures, mainly optical sections of
preserved embryos of Amphioxus. He looked for concrescence
but failed to find any evidence of it. In regard to the orienta-
tion of the embryo he found the closure of the blastopore as
described by Kowalevski, “von vorn herein fast genauer aboral
und ganz geringe Neigung zur dorsalen Seite hin.’ ?

Sobotta’s account follows Hatschek’s very closely, and adds
little that is new. He states that no distinction exists between
the dorsal and ventral wall of the archenteron, during the early
gastrula stages, as Lwoff maintained. The explanation of this
lies, no doubt, in the preserving fluids that were used. So-
botta states that in the eggs from Naples the blastopore

1 Lwoff, p. 14. 2 Wilson, p. 586. 8 Klaatsch, p. 229.

594 MORGAN AND HAZEN. [VoL. XVI.

closes later than in the Messina form, as described by Hat-
schek. We have had material from both localities and can
state that the difference was the result of less normal devel-
opment in the Neapolitan form. Sobotta has been unable to
decide how the axis of the gastrula is related to the axis of
the embryo. He believes that the blastopore closes equally
from all points and that no concrescence takes place.

MacBride? and Sobotta have entirely overlooked the pres-
ence of smaller and lighter cells over the dorsal wall of the
archenteron, and in consequence MacBride says that ‘it is dif-
ficult to find words to adequately characterize the artificiality
and arbitrariness of such a view.”’

MacBride continues: “If we examine a transverse section
of a completed gastrula, ... we find no difference in character
between the cells forming the dorsal wall of the alimentary
canal and those forming the ventral wall, such as we should
have the right to expect did Lwoff’s hypothesis in any way
correspond with the facts.” This statement is unquestionably
wrong, as our figures show.

MacBride gives an inadequate account of the process of gas-
trulation: “Thus I regard the gastrulation as a fairly uni-
form pushing in of the under or flattened surface of the
blastula, accompanied by division and multiplication of the
cells, such multiplication being at first most active in the dor-
sal (future anterior) lip of the blastopore. The blastopore,
which is still wide, becomes rapidly narrowed by the upgrowth
of the ventral lip; in contradistinction to what Hatschek
asserts, the dorsal lip remains relatively stationary.” ?

No evidence is offered in support of this opinion, which is,
as we have tried to show, incorrect.?

It is not our intention to enter into a comparison of the gas-
trulation of Amphioxus and of the other Chordata. The resem-
blance of the early larva of the Ascidians to that of Amphioxus
is, however, so close, that a few words seem justified on this
topic. The recent paper by Castle on “ The Early Development

1 MacBride, p. 597. 2 Tbid., p. 591.
8 MacBride’s Fig. 10 shows the endoderm of the dorsal lip of the blastopore
continuing into the ectoderm on the ower side of the nerve tube.

No. 3.] THE GASTRULATION OF AMPHIOXUS. 595

of Ciona intestinalis’ is much more detailed than the work of
previous writers and we shall confine ourselves entirely to
Castle’s account.

In Ciona, at the time when the endodermal plate is bending
in to form the archenteron (Castle, Fig. 78), a few cells at the
dorsal lip of the blastopore also sink in and, in later stages,
form the dorsal wall of the archenteron (Fig. 98). From these
cells the notochord subsequently develops. They would seem,
therefore, to correspond to the cells of the dorsal wall of
Amphioxus. Outside of the semicircle of notochordal cells
(Fig. 62), at the dorsal lip of the early gastrula, lie the ecto-
dermal cells that subsequently form the nerve plate. As the
blastopore closes, these cells are carried backward with the
advance of the dorsal lip, but none of the cells turn in with
the notochordal cells. During the backward growth of the
ectodermal and notochordal cells, these cells increase in num-
ber. The nervous system of Ciona is derived from cells that
lie at first in front of the dorsal lip of the blastopore. The
nervous system of Amphioxus is also derived from cells in
front of the dorsal lip of the blastopore, but in Ciona the
blastopore closes from before backward, while in Amphioxus,
if our view be correct, the closure is not from before back-
ward, but equally from all points of the periphery. It is im-
portant to note that in Ciona, in which the advance of the
dorsal lip is definitely shown to exist, the ectodermal cells that
lie at the free edge of the dorsal lip do not turn in during the
period of closure.

Castle derives the mesoderm of the tail from a number of
cells around the posterior half of the blastopore. These cells
are turned into the archenteric space during the period of gas-
trulation. Castle has followed the lineage of these cells and
has shown that they correspond in origin with the ectodermal
cells of the anterior region that form the nervous system. He
therefore considers them ectodermal in origin, and consequently
derives a large part of the mesoderm from the ectoderm. The
more anterior mesoderm of the trunk comes from cells lying
just within this semicircle of mes-ectodermal cells. This inner
circle is described as endodermal in origin. Hence, in Ciona,

596 MORGAN AND HAZEN. [VoL. XVI.

the mesoderm has a double origin — the duality, however, being
a matter of definition. In Amphioxus there is nothing indi-
cating that the mesoderm is derived from more than a single
source.

In Ciona the blastopore closes on the dorsal side, and the
longitudinal axis of the embryo seems to be at right angles to
the primary or gastrula axis. This would be the case if the
dorsal lip grew posteriorly, without the embryo changing
shape. On the other hand, there are facts in the development
that make it possible to interpret this backward growth of the
dorsal lip as the result of a change in the shape of the entire
embryo. The gastrula axis, in such a case, would shift during
the closure of the blastopore. The shifting would take place
in such a way that the dorsal side of the axis is carried back-
ward and the ventral forward. The result would be that the
anterior end of Ciona would agree with the anterior end of
Amphioxus. We offer this only as a suggestion, for by this
means the orientation of Amphioxus and of Ciona would be
made to agree.

BryN MAwR COLLEGE, BRYN Mawr, Pa.,
May 29, 1898.

96

‘81

S)7/

67

94

’98

96

aa

'93

bs cl THE GASTRULATION OF AMPHIOXUS. 597

REFERENCES.

CASTLE, W. E. The Early Embryology of Ciona intestinalis. Bzdd.
Mus. Comp. Zool. Vol. xxvii, No. 1. January, 1896.

HATSCHEK, B. Studien tiber Entwickelung des Amphioxus. 4A7¢.
Zool. Inst. Wien. Ba. iv.

Kuaatscu, H. Bemerkungen tiber die Gastrula des Amphioxus.
Morph. Jahrb. Bd. xxv, Heft 2.

KOWALEVSKI, A. Entwickelungsgeschichte des Amphioxus lanceo-
latus. Mém. Acad. Imp. de St. Pétersbourg. Tome xi.

Lworr, B. Die Bildung der primaren Keimblatter und die Entstehung
der Chorda und des Mesoderms bei den Wirbelthieren. Szdl. de la
Soc. Imp. des Nat. de Moscou.

MacBripgE, E. W. The Early Development of Amphioxus. Qvart.
Journ. Micr. Scz.\) Vol. xi.

MorGan, T. H. The Number of Cells in Larvae from Isolated
Blastomeres of Amphioxus. Arch. f. Entwickelungsmechantk d.
Organismen. Bad. iii, Heft 2.

SospoTta, J. Beobachtungen iiber den Gastrulationsvorgang beim Am-
phioxus. Verh. d. Physikal-Medic. Ges. zu Wiirzburg. Vol. xxxi.

Witson, E. B. Amphioxus and the Mosaic Theory of Development.
Journ. of Morph. Vol. xiii.

REFERENCE LETTERS.

a. _ anterior. n. notochord.

b.c. body cavity. op. ect. opening in ectoderm.
6p. blastopore. p: posterior.

@. _ dorsal. S.C. segmentation cavity.
ect. ectoderm. v. ventral.

end. endoderm. Up. vegetative pore.

598 MORGAN AND HAZEN.

EXPLANATION OF PLATE XXXIII.

Figs. I-10, except Figs. 4.4 and 7 A, were drawn with Zeiss 4, oil immersion
2mm. Figs. 4.4 and 7A were drawn with Zeiss 4 D. The figures in the plate
were reduced one-third.

The material from which the figures (except Fig. 6) were drawn was killed in
Flemming’s solution, the stronger formula. Fig.6 was drawn from material killed
in Hermann’s fluid. The section from which Figs. 7 4, B, C, D, and Figs. 9
and 10 were drawn was stained in iron haematoxylin. The remaining figures
were drawn from unstained material.

Fic. 1. Sagittal section of an embryo at midnight. There is an artificial
rupture in the ectoderm; d., dorsal; v., ventral.

Fic. 2. Sagittal section of an embryo at 2 A.M.

Fic. 3. Sagittal section of an embryo at 4 A.M.

Fic. 4A. Oblique section of an embryo at 5 A.M.

Fic. 48. Enlarged drawing of area marked ~ in 4 4.

Fic. 5. Sagittal section of an embryo at 6 A.M.

Fic. 6. Part of a cross-section through the posterior third of an embryo at

Fic. 7 A. Sagittal section through an embryo at II A.M.

Fic. 7 B. Enlarged drawing of area marked x in 7 A.

Fic. 7 C. Enlarged drawing of area marked y in 7 4.

Fic. 7 D. Enlarged drawing of area marked z in 7 4.

Fic. 8. Drawn from the anterior region of a longitudinal section of an
embryo with eight body cavities; ect., ectoderm ; ezd., endoderm; 4.c., body cavity.

Fic. 9. From the anterior wall of an embryo, showing a portion of a cell
after division.

Fic. 10. Dividing cell from the endoderm of Fig. 16.

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Journal of Morphology Vol. XVI

Pl. XXXII

LT ea
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600 MORGAN AND HAZEN.

EXPLANATION OF PLATE XXXIV.

Figs. 11-16, and 19, 20, were drawn with Zeiss 4, oil immersion 2 mm. Figs. 17
and 18 were drawn with Zeiss 4 D. The figures on the plate were reduced one-
third.

The embryo:from which Fig. 11 was drawn was killed in Flemming’s solution
and was unstained. Figs. 16-18 were drawn from material killed in Flemming’s
solution, the stronger formula, and stained in iron haematoxylin. The remaining
figures were drawn from material killed in corrosive acetic and stained with
lithium carmine.

Fic. 11. Blastopore drawn from a total mount at about 2 A.M.

Fic. 12. Blastopore from a total mount at about 3 A.M., showing also endo-
derm cells through the blastopore opening.

Fic. 13. Blastopore from total mount at about 5 A.M.

Fic. 14. Blastopore from total mount at about 6 A.M.

Fic. 15. Optical section through an embryo at 2 A.M.

Fic. 16. Embryo with endoderm cells turned outward around the vegetative
pore.

Fic. 17. Embryo with vegetative pore.

Fic. 18. Embryo with opening at animal pole.

Fic. 19. Embryo, showing vegetative pore at 6 A.M.

Fic. 20. Oblique section of an embryo at 4.30 A.M., showing cells during and
after division (at x).

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PHOTOGRAPHS OF THE EGG OF ALLOLO-
BOPHORA FOETIDA.

KATHARINE FOOT anp ELLA CHURCH STROBELL.

i:

Tus paper is the first of a series in which we hope to
make a careful comparative study — illustrated with photo-
graphs — of the effects of various fixatives on the cytoplasm
of the egg of Allolobophora foetida.!

In addition a number of preparations have been photographed
to illustrate the following points :

The morphological resemblance of the fertilization cone to
the male aster.

The position of the middle-piece in the male aster.

The origin of the sperm granules.

The early stages of the development of the pronuclei.

The presence of osmophile granules in the nucleoli of the
germinal vesicles.

The photographs have been taken at only two magnifications
(660 and 950). No photographic feats have been attempted,
photography being used merely to register the points we wish
to illustrate, and the higher magnification has not been used
where our aim could be achieved by the lower.

Cytoplasmic Reaction to Fixatives.

Our aim is to make a comparative study of the effect pro-
duced not only by the compound fixatives, but by the compo-
nent parts of each. If with a given fixative we find a definite
cytoplasmic configuration, can this be materially changed by
omitting one of the constituents of the fixative? If this

1 A few photographic illustrations of this comparative work were published in
1898. Foot and Strobell, “ Further Notes on the Egg of Allolobophora foetida,”

Zoblogical Bulletin, vol. ii, No. 3.
601

602 FOOT AND STROBELL. [VoL. XVI.

proves to be the case, will this constituent produce a like effect
in combination with another fixative? We hope by a careful
comparative study to be able to determine how much of the
structure seen in fixed cytoplasm is due to the fixation.

The cytoplasm reacts very differently to different fixatives ;
for example, the spaces occupied by the hyalin globules! are
in some cases distinctly defined (photo. 17), while in others the
globules have apparently fused in all directions, producing a
scattering of the intermediary substance and consequent forma-
tion of rays.

It is a significant fact that in those preparations where the
spherical spaces occupied by the hyalin globules are destroyed,
we usually find more definite rays, this being clearly shown by
comparing photo. 17 with photos. 15, 19, 21, and 22 (these five
photos. showing nearly the same stage of development of the
egg).

The last four, with many of the chromo-acetic preparations
(é.g., 2, 8 and g), might be called in evidence for the reticular
theory of cytoplasm, and photo. 17 supports with equal force
the alveolar theory.

We are convinced that the indication of rays we have seen
in the /zving eggs is not comparable to the rays seen in prep-
arations where the hyalin globules have fused and scattered.

Twelve fixatives are represented in our three plates, but we
do not wish to assert that any one of these preparations repre-
sents the typical effect of the special fixative, the reaction of
the egg to any one fixative being very inconstant. This com-
plicates the problem and a comparative study can be profitably
carried out only by an exhaustive collection of photographs.
In the present paper attention will be called to a few suggestive
comparisons, awaiting further data before attempting to draw
definite conclusions.

1 At Dr. Whitman’s suggestion we have adopted the term hyalin globules to
designate the substance which in an earlier paper we called sap globules. This
substance, which is in globular form only at certain stages, we interpret as synon-
ymous with the hyaloplasm of some authors. We retain the term globule,
because when this substance is pressed out of the living egg, it keeps its globular
form, not fusing with water for several minutes. Treatment with osmic acid has
failed to demonstrate any fatty constituent.

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No. 3.] EGG OF ALLOLOBOPHORA FOETIDA. 603

Archoplasm in the Male Attraction Sphere

Sometimes the archoplasm is quite uniformly distributed, and
again it is represented by rods or rays or granular masses. As
these varying forms are seen at the same stage of development
of the egg, is it not probable that they represent varying
expressions of the effect of the fixatives?

An examination of the photos. of eggs killed in corrosive-
sublimate (12, 14, 15, 18, 20) reveals a marked similarity in
the distribution of the archoplasm. It forms a granular, flocky
circle around the male attraction sphere, and this arrangement
is relatively constant for corrosive-sublimate preparations.

A comparison of these with photo. 17 shows a striking
difference, and we are forced to conclude that the approxi-
mately even distribution of archoplasm in this section is more
suggestive of the living condition, because the hyalin globules
have not fused, as is probably the case in the corrosive-sublimate
preparations, and the action of the fixative must be thus less
injurious. In the above list of corrosive-sublimate prepara-
tions we did not include photo. 16, for the arrangement of
the archoplasm of that section is exceptional—it is more
like that shown in photo. 17, and it is a significant fact that
the fixation of the rest of the cytoplasm is also more like
that of photo. 17, z.e., there has been less fusing of the hyalin
globules. The egg of photo. 17 was fixed with Hermann’s fluid,
without acetic acid; the same stage fixed with Hermann, in
which the acetic acid had been retained, shows a very different
cytoplasmic configuration.

The archoplasm of photo. 21 (Perenyi’s fluid) and of 19
(Flemming’s fluid strong) is aggregated into decided rays, and
there are no indications of the presence of hyalin globules.

The archoplasm of photo. 7 is more condensed than is the
case in any of the other preparations. The fixative used (osmic

1 We are preparing a paper to demonstrate (with a series of photographs) the
presence of the archoplasm throughout the cytoplasm and its homology to
the so-called yolk-nucleus. We hope to defend this broader use of the term, em-
ployed in an earlier paper, and to support the interpretations there suggested. Foot,
“ Yolk-Nucleus and Polar Rings,” Journ. of Morph., vol. xii, No. 1, 1896.

604 FOOT AND STROBELL. [VoL. XVI.

and acetic) shrunk the egg nearly one-half its diameter, and the
archoplasm is shrunken into compact masses that resemble rods.

The archoplasm of photo. 9 (chromo-acetic) forms a sharp
contrast to that of photo. 7. Part of it is aggregated at the
center of the attraction sphere, where it is stained so deeply
that the middle-piece is completely obliterated.

Fertilization Cone.

Photo. 1 shows the size of the cone in relation to the
entire egg (at this stage the first maturation spindle is in
the metaphase and at the periphery of the egg). Photo. 2
is a section of such an egg, cut longitudinally through the
cone. The thinness of the section enables us to see the head
of the sperm within the cone, on its way to the center of the
egg. The sperm continues its course until its middle-piece is
near the inner aster of the first maturation spindle, slightly
turning and bringing its head again near the periphery, some
of the preparations (for example, photo. 12) producing the
false impression that it has entered from the latter point,
and the aster has formed at the apex of the head. The prog-
ress of the sperm towards the inner aster of the spindle
appears to be dependent upon the stage of development of
the egg. When the spindle has reached the anaphase, the
sperm aster is formed and the progress of the sperm towards
the center of the egg then ceases, the head separates from
the middle-piece and contracts into a short thick rod (photos. 8,
16, 17, and 18). The spiral twist of the sperm shown in
photo. 2 is an uncommon form, but it probably does not indi-
cate an abnormal condition of the egg.

In photo. 5 we have an unmistakably pathological cone, this
enabling us to determine the pathological condition of the
rest of the cytoplasm and giving a standard of comparison
which may prove of service.

Photo. 6 probably represents another example of patho-
logical cytoplasm, as the egg contains four spermatozoa, two of
which are shown in the section photographed. It is a question

1 Over-printing at the apex of the cone has obliterated the sperm at that point.

INO33°] EGG OF ALLOLOBOPHORA FOETIDA. 605

how much of the difference in the cytoplasmic configuration of
photos. 5 and 6 is due to the difference in fixation, the egg
of photo. 5 having been killed in corrosive-sublimate and that
of photo. 6 in picro-formalin. Photo. 6 represents, however, a
little later stage of development of the egg.

Morphological Similarity of the Cone and Male Aster.

It is impossible to avoid drawing conclusions as to the mor-
phological significance of the resemblance between the male
aster and transverse sections through the fertilization cone.

Photo. 3 shows a transverse section of a fertilization cone,
near its apex, and a comparison of this with a section through
the male aster of photo. 9 will serve to illustrate this point.
The rays and the central aggregation of archoplasm are as
pronounced in the one as in the other, suggesting that each
end of the head of the sperm — the spine and the middle-piece
(see photo. 37) produces on the cytoplasm of the egg a like
morphological effect. This would indicate that the spine and
the middle-piece are of the same substance, though the iden-
tity cannot be complete, as the cytoplasm does not react to the
two structures at the same stage of development of the egg.
There are a few investigators who claim to have traced a sub-
stance in the spermatid to both spine and middle-piece. The
effect produced by the spine is made, however, by a moving
object (the sperm entering the egg), and we have thus a different-
shaped ‘aster’? —a cone-shaped aster. Is it possible that
this may have any bearing on the opposing interpretations of
various authors, some asserting that the anterior end of the
head of the sperm produces the male aster, and others that
the posterior end of the head (the middle-piece) produces it?

If we accept the interpretation of those authors who claim
to have traced a part of the aster of the spermatid to doth
spine and middle-piece, may we not regard that part of the
spermatozoon (including spine, head, and middle-piece) as an
attenuated spindle,! and expect that each end of this spindle

1 Foot, “‘The Centrosomes of the Fertilized Egg of Allolobophora foetida,”
Biol. Lect., Marine Biological Laboratory. Boston, 1896.

606 FOOT AND STROBELL. [Vo.. XVI.

will produce a like morphological effect upon the cytoplasm
of the egg? We have been unable to differentiate in either
spine or middle-piece any special structure that we feel justi-
fied in interpreting as a centrosome.

Further Observations on the Middle-Piece of the Sperm and
zts Morphological Role in the Male Aster.

In 18971 one of us differentiated in color the centrosome
of the male aster from the middle-piece of the spermatozo6n,
this leading to the interpretation that the centrosome of the
male aster is of purely cytoplasmic origin —the middle-piece
merely producing the cytoplasmic phenomenon known as the
aster, but no definite part of the sphere being formed of the
middle-piece substance.

Further investigation has demonstrated that the middle-piece
can remain for a definite period intact within the aster, and
that the later differentiation in color is probably due to chem-
ical change, for it disintegrates before disappearing.

The photos. of Pl. XXXVI show the middle-piece within
the aster. Although it is by no means always in the center,
we have been able to find no other structure that we feel
justified in interpreting as a centrosome.

The middle-piece finally disintegrates and totally disappears,
and there is no evidence that it takes any part in forming the
cleavage centrosomes. As stated in the paper above referred
to (I), during the formation of the young pronuclei both the egg
and sperm centrosomes totally disappear, and there is no evi-
dence that either takes part in forming the cleavage centrosomes,
this egg supporting the theory that the cleavage centrosomes
arise de novo in the cytoplasm.

In photo. 12 we see a portion of the head of the spermato-
zoon, its middle-piece within the aster, and a part of the tail
(the missing part of the head is in the next section). The
head at this stage shows constrictions at definite intervals
(these are lost in some of the reproductions of photo. 12, but

1 Foot, “ The Origin of the Cleavage Centrosomes,” Journ. of Morph., vol. xii,
No. 3, 1897.

—

No. 3.] EGG OF ALLOLOBOPHORA FOETIDA. 607

two are shown in photo. 14). We are unable at present to
homologize these divisions of the sperm head with the chromo-
somes of the egg, not having seen the requisite number.

If the archoplasm shown at the periphery of this sphere is
comparable to Boveri's archoplasm in ascaris, it certainly is not
brought in by the sperm, as claimed by several authors for the
ascaris egg, for here we see the spermatozoon still intact.

Photo. 13 shows part of the head of the sperm and the
entire middle-piece, at about the same stage as that of photo.
12, though the magnification is somewhat greater (950).
Without more data we hesitate to interpret the two tiny
filaments at the posterior end of the middle-piece. One of
them may be the proximal end of the tail of the spermato-
zon, or they may both represent the splitting of the proximal
part of the tail and its fusing with the cytoplasm of the egg.
This egg was killed in 2 per cent osmic in 70° alcohol, and it
is interesting to compare its cytoplasmic structure with that of
photo. 12, in which case the egg was killed in corrosive-sublimate.

In photo. 14 we have the same stage of development of
both sperm and middle-piece.

In photo. 15 we have transverse sections of two middle-
pieces within the aster, and it is scarcely necessary to call
attention to their resemblance to a dividing centrosome. The
heads of the two spermatozoa are in the adjacent sections.
They are contracted rods such as those shown in photos. 16,
17, and 18.

Photo. 16 shows the head of the spermatozoon separated
from the middle-piece and contracted into a relatively thick
short rod —the middle-piece has (apparently) contracted some-
what, and we interpret the filament to the right of the middle-
piece as a part of the tail of the spermatozoon.

In the preparation represented by photo. 17 there is a middle-
piece distinctly seen within the sphere; but it has been oblit-
erated by too dark printing in the reproduction, over-printing
producing the same results as over-staining.

In photo. 18 the middle-piece is by no means in the center
of the sphere; but the spherical form of the latter remains
intact. If the middle-piece does more than stimulate the egg

608 FOOT AND STROBELL. (Vo. XVI.

to the expression of cell activity known as the male attraction
sphere, if it is the organic center of the sphere, forming with the
rays an organic connected whole, would not the spherical form
of the sphere be disturbed by the middle-piece moving away
from the center? In reality the form is no more disturbed than
is the form of the fertilization cone, when we find the head of
the spermatozoon quite out of the center of the cone.

In photo. 19 the middle-piece is in nearly the center of the
sphere. (The contracted head of the sperm is in the adjacent
sections.)

In photo. 20 we have a transverse cut through the middle-
piece, which bears a marked resemblance to a centrosome,
though its position in the sphere is eccentric ; the spherical
form of the sphere, however, remains undisturbed. (The con-
tracted head of the sperm is in the next section.)

Photo. 21 shows a little later stage of development than
that of 18; the middle-piece is beginning to disintegrate,
and at a still later stage it has entirely disappeared. The
differentiation in color which one of us obtained between the
middle-piece of the spermatozo6n and a distinct spherical body
within the male aster was probably due to the chemical change
which must take place at this time of disintegration.

Photo. 22 shows about the same stage of development as
that of photo. 21; the middle-piece is beginning to disinte-
grate, showing three tiny spherical bodies. The middle-piece
and rays finally disappear at about the same time.

Further Observations on the Origin of the Sperm Granules.

Photos. 10 and 11 indicate an origin of the pathological sperm
granules different from that suggested by one of us in 1897.1

They were interpreted then as being formed at the expense
of the archoplasm, for the reason that in those cases where
they were present in the cone they were surrounded by an
area free from archoplasm, indicating that they arose by a
concentration of the archoplasm substance at that point.

1 Foot, “The Origin of the Cleavage Centrosome,” Journ. of Morph., vol. xii,
No. 3, 1897.

vives. ] EGG OF ALLOLOBOPHORA FOETIDA. 609

We should expect to find them formed at the expense of
some substance in the cell, as they are not constant structures
and appear to be a pathological feature. In photo. 10 we have
an exaggerated expression of the phenomenon (an unusual
number of granules), this aiding us in interpreting their
origin. This photograph ‘indicates that they have arisen at
the expense of the head of the sperm itself. Less than
half of the length of the head is shown in this section; but a
comparison of its diameter with that shown in photo. 2 indi-
cates a great loss of substance.

In photo. 11 we see a part of a contracted head of a sperm
which appears to have been fixed in the act of constricting off
a sperm granule.!

Early Stages of Development of the Pronuclet.

The photographs of Plate XXXVII illustrate the early
stages of development of the male and female pronuclei —
four fixatives being represented.

Photo. 23 shows a transverse view through the second matu-
ration spindle, which has reached the anaphase of development.
These are the chromosomes approaching the inner pole of the
spindle, and which are destined to form the female pronucleus.
There are eleven chromosomes in the first and second spindles,
and in-this photo. each one of the eleven is shown. This
photo. was taken by Dr. Fuller, from a slightly crushed odcyte
second order.

Photo. 24 shows a little later stage of development (telo-
phase). This section contains five or six of the chromosomes,

1In the Journ. of Morph., vol. xvi, No. 1, 1899, Byrnes describes small round
bodies often accompanying the sperm-nucleus in Limax agrestis, and says they
may owe their origin — although she does not illustrate this point — to particles
of chromatin constricted off from the sperm-nucleus before it becomes vesicular,
having seen a “few cases in which a portion of the chromatin seemed to be in
process of constricting from the sperm head.” As she does not suggest they are
a pathological expression, on the contrary implying they have a function, it is a
question whether they are the same structures we show in photos. 11 and 12. We
regret the necessity of mentioning Miss Byrnes’s paper in a footnote. The number

of the journal in which it appeared was issued in June, 1900, and her paper was
not read until just before the receipt of our final proofs.

610 FOOT AND STROBELL. [VoL. XVI.

which have reached the inner pole of the second spindle, the
rays of the aster still persisting. A comparison with photo. 23
indicates that these ring chromosomes have been formed by
the uniting of the free ends of V-shaped chromosomes, such
as those shown in photo. 23.

Photo. 25 shows two (of the eleven) chromosomes at a little
later stage of development. Besides the ring, a tiny spherical
body appears in connection with each chromosome, and we
interpret these as the first appearance of the nucleoli of the
female pronucleus, the periphery of each ring representing
the chromatin of the chromosomes.! If this interpretation is
correct, then the periphery of the vesicles which characterize
the /ater stages must be interpreted as chromatin, and the
spherical body in connection with each, as nucleolar substance.
These later stages are shown in photo. 26 (where three of
the eleven vesicles are represented) and in photos. 27, 28, 20,
30, 31, and 32. In photo. 27 one of the vesicles shows thick
threads of chromatin connecting the nucleolus with the periph-
ery of the vesicle.

Photo. 33 shows part of the head of the spermatozoon
breaking up into similar vesicles — the periphery of the vesicle
upon which we have focussed we interpret as of chromatin,
and the tiny spherical body as nucleolus.

Photo. 34 shows a later stage of the development of the
spermatozoon into the male pronucleus, several of the vesicles
having fused into one, the rest being in adjacent sections.

Osmophile Granules in the Nucleolr.

It is exceptional to find osmophile granules in the nucleoli,
and we are therefore inclined to regard them as abnormal
features. A nucleolus from a germinal vesicle of an unstained
ovarian egg is shown in photo. 35. The ovary was fixed in
chromo-acetic, washed in water and immersed for one hour in
osmic in order to blacken the dentoplasmic granules.

1 In these vesicles, at a little later stage of development, one of us differentiated
in color the nucleolus from the peripheral chromatin ring. Foot, “‘ The Origin
of the Cleavage Centrosomes,” Journ. of Morph., vol. xii, No. 3, 1897.

Cg a

No. 3.] EGG OF ALLOLOBOPHORA FOETIDA. 611

After photographing, this ovary was immersed for fifty-three
hours in turpentine and the same nucleolus again photographed
without staining (photo. 36). The fat granules (osmophile
granules) had neither dissolved out nor faded in any part of
the ovary. This discredits the advice of those authors who
recommend placing sections for twenty-four hours in turpen-
tine or xylol to remove the fat granules before staining. In
some cases, perhaps as a rule, warm xylol or turpentine will
fade the blackening caused by osmic, but it is by no means
infallible, and, moreover, the xylol very often does not dissolve
the fat substance. Photographs have enabled us to discover
its presence after a careful examination under the microscope
had convinced us it had dissolved out completely. Again, after
long immersion in turpentine, and after staining, the granules
are still sharply blackened, and confidence in the certainty of
their removal by turpentine leads one to misinterpret them as
other than fat granules.

Further details regarding the nucleoli and the ovarian egg
will be discussed and illustrated by photographs in a paper
now being prepared for press.

Method.

Further experiments with the mechanical mode of focussing
described in our last paper! have led to the development of a
simpler and more rapid method of overcoming one of the
practical difficulties encountered by the cytologist in photog-
raphy.

After abandoning the effort to focus fine details on the
ground glass of the camera, or through a transparent portion
marked off in the ground glass, we used the method described
in detail in the paper just referred to —ascertaining by experi-
ment with a large object, easily focussed on the ground glass
(a sharply stained nucleolus, for example), just what difference
the pointer on the micrometer screw registered, between the
focus through the microscope and the focus on the ground

1“ Further Notes on the Egg of Allolobophora foetida,” Zodlogical Bulletin,
vol. ii, No. 3, 1899.

612 FOOT AND STROBELL. [VoL. XVI.

glass. This difference we found to be 3, of one of the
twenty-five divisions marked on face of micrometer screw.}

In the practical use of this method, the suggestion for
increasing its accuracy came through observing the variation
in the turn required by the micrometer screw, dependent
upon the operator’s eyeglasses, whether reading or distance
glasses were worn. This suggested that there must be a
lens which, when once adapted to the operator’s eyes, would
give with unfailing accuracy the plane required for the focus
on the ground glass. A series of spherical lenses, from —1.D.
to —5.D., were tested in the following manner:

A number of small microsomes were carefully focussed
through the microscope, with projection ocular IV (diaphragm
at o), the operator wearing ordinary distance spectacles, or in
a case where the sight was normal, no glasses were used.
The micrometer screw was then turned to raise the focus
the number of points found by former tests, to give the cam-
era focus. By this change of screw the microsomes origi-
nally focussed upon were of course completely lost sight
of. Leaving the screw at this point, the spherical (minus)
lenses were tested, beginning with the lowest number, placing
them one at a time on the projection ocular, until one was
found which brought the microsomes in sight and gave the
desired focus, exact in every detail. In making this test, if
the camera focus has not been obtained mechanically (7.e., the
difference in the two foci measured by points on micrometer
screw) the most accurate way of getting at it is to take a
series of photographs, focussing through the minus lenses.
Beginning with the lowest number, develop each plate as it is
taken, until a negative giving the desired focus demonstrates
which lens is needed. The proper lens, when found, can be
mounted in ordinary spectacle frames, or simply laid on top of
the projection ocular, as the operator finds most convenient.

To insure faithful results it is advisable to make a careful
sketch, for reference, of a few of the most minute details,
in the section to be photographed. After focussing for a

1 These figures are given merely to illustrate the method, each microscope
requiring a special test.

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No. 3.] EGG OF ALLOLOBOPHORA FOETIDA. 613

photograph, let the microscope stand undisturbed for at least
ten minutes before pulling down the camera, and never attempt
to take a photograph unless the focus has held absolutely true
during this interval. After the photograph is taken, raise the
camera and again examine the preparation to see that the
focus has held while the plate was being exposed. Atten-
tion should be given to the working condition of the microm-
eter screw; it should be tight enough to preclude any chance
of slipping after the focus is finally adjusted.

A special test must be made for each magnification, but this
need not mean undue experimenting, for two magnifications
should be adequate for practical use, the lower for photo-
graphing entire sections, and the higher for special details,
such as centrosomes. Restricting one’s photographs, as far
as possible, to one magnification, offers a great advantage for
comparison of shrinkages.!

We feel sure that our photographs in this paper are merely
a promise of better results. It has been necessary in the
reproduction to sacrifice many minor details to bring out a few
important points. Our original prints are partly responsible
for this, for in them, to insure a successful reproduction of
the salient points in a section, we sacrificed the general cyto-
plasmic structure. For example, we have experimented with
photo. 17, and have made a much stronger impression of the
cytoplasm, and at the same time have not obliterated the
middle-piece in the aster by over-printing.

1 We have taken a photograph of a stage micrometer at the two magnifica-

tions we use, 660 and g50. A print from this negative gives a scale by which any
detail in the photograph can be readily measured.

EXPLANATION OF PLATES.

All the photographs, except Nos. 1, 8, 9, 23, 35, 36, and 37, were taken in the
winter of 1899, on the Lumiére (France) or Nye (Belgium) plates.

In three or four cases a detail in the original print has been slightly strength-
ened with a lead-pencil, merely enough to overcome in part what is lost by
reproduction.

If any of our readers should wish to compare the reproductions with the solar
prints, one or more may be obtained on request.

614 FOOT AND STROBELL.

EXPLANATION OF PLATE XXXvV.

Puoto. 1. An entire egg (odcyte first order). This photograph was taken
by Dr. Charles G. Fuller, of Chicago, in the fall of 1893. He focussed on the
fertilization cone, and although the magnification is not great, the photograph
appears to us as very satisfactory in showing the size of the cone in relation to
the entire egg. Fixative, chromo-acetic. Stain, alum-cochineal. The reproduc-
tion of the cone in this figure has not been uniformly successful.

PHorTo. 2. Section (3) of odcyte first order. Fertilization cone cut longi-
tudinally. Head of the sperm within the cone, showing a spiraljtwist. Cytoplasm
typical of chromo-acetic preparations at this stage. The osmophile granules are
stained. Fixative, chromo-acetic. Stain, iron-haematoxylin. x 660.

PuHoTo. 3. Section (3 4) of odcyte first order. Fertilization cone cut trans-
versely near its apex. Fixative, corrosive-sublimate. Stain, iron-haematoxylin.
x 660.

A comparison of photos. 2 and 3 illustrates the relative shrinkage produced
by the two fixatives and the difference in the topography of the cytoplasm.

PHorTo. 4. Vignetted section (3) of odcyte first order. Fertilization cone
cut transversely about midway between its base and apex. Fixative, osmic and
acetic acid. Stain, iron-haematoxylin. x 660.

PHoTo. 5. Section (3) of a pathological odcyte first order. Fertilization
cone cut longitudinally. Fixative, corrosive-sublimate. Stain, iron-haematoxylin.
x 660.

Puoto. 6. Vignetted section (3 ~) of a polysperm odcyte first order. The
fertilization cone has almost disappeared, but shows that it contained two sper-
matozoa. Fixative, picro-formalin. Stain, iron-haematoxylin. x 660.

PHoTo. 7. Vignetted section (3 4) of odcyte second order, showing a sperm
aster, with the middle-piece of the sperm near its center. The head of the sperm
appears in the next section as a rod, as:shown in photos. 8, 16, 17, 18. Fixative,
osmic and acetic. Stain, iron-haematoxylin. x 660.

Puorto. 8. Section (7 “) of odcyte second order, showing a part of the sperm
head contracted into’a rod and an indication of the sperm aster, the section being
through the periphery of the aster. This photo. was taken by Dr. Fuller, 1894.
Fixative, chromo-acetic. Stain, iron-haematoxylin. x about 400.

PHOTO. 9. Section (7 «) of odcyte second order, showing male aster, the
center of which is stained so deeply that the middle-piece is completely obliter-
ated. Photographed by Dr. Fuller, 1894. Fixative, chromo-acetic. Stain, iron-
haematoxylin. x about 400.

PHOTO. Io. Vignetted section (3 «) of odcyte second order, showing a part of
the head of the sperm surrounded by sperm granules. Fixative, corrosive-sub-
limate. Stain, iron-haematoxylin. x 950.

PHoTo. 11. Section (3 “) of odcyte second order, showing a part of a con-
tracted head of a spermatozo6n which appears to have been fixed in the act of
constricting off a sperm granule. Fixative, Flemming’s fluid (strong). Stain,
iron-haematoxylin. x 660.

4
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XXXKY.

JOURNAL OF MORPHOLOGY, Vol. XVI.

THE CENTRAL BUREAU OF ENGRAVING, N. Y.

PHOTOGRAPHS BY FOOT & STROBELL.

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616 FOOT AND STROBELL.

EXPLANATION OF PLATE XXXVI.

PHOTO. 12. Section (3) of odcyte second order, showing the head of the
spermatozo6n, its middle-piece within the aster and a part of the tail. (The miss-
ing part of the head is in the next section.) Fixative, corrosive-sublimate.
Stain, iron-haematoxylin. x 660.

PHOTO. 13. Vignetted section (3 u) of odcyte second order, showing part of
the head of the spermatozoon, the middle-piece, and (probably) a tiny piece of the
proximal end of the tail. Stage of development about the same as that of photo.
12. Fixative, 2% osmic in 70° alcohol. Stain, iron-haematoxylin. Xx 950.

PHOTO. 14. Vignetted section (3 “) of odcyte second order. Stage of devel-
opment about the same as that of photo. 13. Fixative, corrosive-sublimate.
Stain, iron-haematoxylin. x 950.

PHOTO. 15. Vignetted section (3 4) of odcyte second order. Stage of devel-
opment about the same as that of photo. 14. The section has cut transversely
two middle-pieces which are within the aster. (The heads of the two sperma-
tozoa are in adjacent sections.) Fixative, corrosive-sublimate. Stain, iron-
haematoxylin. xX 950.

PHOTO. 16. Vignetted section (3 «) of odcyte second order, showing the head
of the spermatozoon separated from the middle-piece and contracted into a rela-
tively thick short rod. Within the aster the middle-piece and probably a piece of
the tail. Fixative, corrosive-sublimate. Stain, iron-haematoxylin. x 950.

PHoTO. 17. Section (3) of odcyte second order, showing the contracted
head of the spermatozodn separated from the middle-piece, which is within the
sphere, but over-printing has obliterated it. The hyalin globules larger than those
seen in the normal living egg at this stage. Fixative, Hermann’s fluid without
acetic acid. Stain, iron-haematoxylin. X 660.

PHoTo. 18. Vignetted section (3 «) of odcyte second order. Stage of devel-
opment about the same as that of photos. 16 and 17. Middle-piece not in the
center of the sphere. Fixative, corrosive-sublimate. Stain, iron-haematoxylin.
X 950.

PHOTO. Ig. Vignetted section (3) of odcyte second order. Middle-piece
nearly in the center of the sphere. The contracted head of the sperm is in the
adjacent section. Fixative, Flemming’s fluid (strong). Stain, iron-haematoxylin.
X 950.

PHOTO. 20. Vignetted section (3 “) of odcyte second order, showing a trans-
verse cut through the middle-piece of the spermatozo6n, its position in the sphere
eccentric. The head of the spermatozo6n is in the next section. Fixative,
corrosive-sublimate. Stain, iron-haematoxylin. x 950.

PHOTO. 21. Vignetted section (34) of odcyte second order, showing a little
later stage of development than that of 16-19. The middle-piece is beginning to
disintegrate. The contracted head of the sperm is seen in the next section.
Fixative, Perenyi’s fluid. Stain, iron-haematoxylin. x 950.

PHOTO, 22. Vignetted section (3 “) of odcyte second order at about the same
stage of development as that of photo. 21, the middle-piece showing like evidences
of disintegration. Fixative, picro-formalin. Stain, iron-haematoxylin. x 950.

JOURNAL OF MORPHOLOGY, Vol. XVI.

sed gee

PHOTOGRAPHS BY FOOT & STROBELL.

THE CENTRAL BUREAU OF ENGRAVING,
157 169 WILLIAM STREET, N. Y.

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618 FOOT AND STROBELL.

EXPLANATION OF PLATE XXXVII.

The photos. of this plate show the early stages of development of the male
and female pronuclei, four fixatives being represented.

PHOTO. 23. Shows a transverse view of the second spindle, which has reached
the anaphase of development. Photo. taken by Dr. Fuller. Fixative, chromo-
acetic. Stain, alum-cochineal. x about 1000.

PHOTO. 24. Vignetted section (3 «) of mature egg, showing five of the eleven
chromosomes which have reached the inner pole of the second spindle, the aster
rays of which are still present. Fixative, 2% acetic. Stain, iron-haematoxylin.
X 950.

PHOTO. 25. Section (3) of mature egg, showing a little later stage of the
chromosomes than that of photo. 24. In this section only two of the eleven
chromosomes are seen. Within each tiny vesicle is a minute spherical body which
we interpret as the first appearance of a nucleolus. Fixative, picro-acetic.
Stain, iron-haematoxylin. x 660. The reproduction of this photograph is not
satisfactory. In the solar print the chromatin rings are nearly as sharp as those
shown in photo. 24, and on the inner side of each ring there is a tiny sharp
granule.

PHOTOS. 26-32 are vignetted sections (3) of mature eggs, showing later
stages of development of the vesicles, from three to five vesicles in each section.

PHOTO. 31 shows the second polar body on the periphery.

Fixative, photos. 26 and 28, 2% acetic. x 660. Fixative, photos. 27, 29, 30,
and 32, chromo-acetic. x 660. Fixative, photo. 31, Flemming’s fluid (strong).
Stain, iron-haematoxylin. x 660.

PHOTO. 33. Section (3) of mature egg, showing part of the head of the
spermatozoon breaking up into vesicles similar to those destined for the female
pronucleus, .e., photos. 24-32. Fixative, picro-acetic. Stain, iron-haematoxylin.
x 660.

PHOTO. 34. Vignetted section (3 ~) of mature egg, showing a later stage of
development of the male pronucleus than that of photo. 33. Only a part of the
pronucleus is present in this section. Fixative, chromo-acetic. Stain, iron-
haematoxylin. x 660.

PHOTO. 35. A nucleolus from a germinal vesicle of an ovarian egg, showing
the presence of osmophile granules in an unstained preparation. Fixative,
chromo-acetic, followed by osmic acid. x 950.

PHoTo. 36. The same nucleolus (at a slightly different focus) after the sec-
tions had been immersed for fifty-three hours in turpentine. Preparation still
unstained. x 950.

PHOTO. 37. Spermatozodn showing spine, head, middle-piece, and tail. This
was taken from a slime tube in which the two cocoons were in process of forma-

tion. Spine and middle-piece only are stained. —5

JOURNAL OF ee Vol. XVI. PI. XXXVI.

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PHOTOGRAPHS BY FOOT & STROBELL.
THE CENTRAL BUREAU OF ENGRAVING, N. Y.

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