Journal of morphology

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JOURNAL

OF

MORPHOLOGY

FounpDEp By C. O. WHITMAN

EDITED BY

J2 Se KINGS DBY
University of Illinois
Urbana, Ill.

WITH THE COLLABORATION OF

Gary N. CALkINS Epwin G. CoNxKLIN C. E. McCuune
Columbia University Princeton University University of Pennsylvania
W. M. WHEELER WILLIAM PATTEN
Bussey Institution Harvard University Dartmouth College

VOLUME 33
DECEMBER, 1919-MARCH, 1920

THE WISTAR INSTITUTE OF ANATOMY AND BIOLOGY

PHILADELPHIA

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ATHAIGT ATLAS

CONTENTS

No. 1. DECEMBER, 1919

GrorGceE T. Hareirr. Germ cells of coelenterates. VI. General considerations,
discussion, conclusions. Thirty figures (three plates)....................
Cart L. Husss. A comparative study of the bones forming the opercular
Series Of fiSWeS.... ss: PONY PUR ROT PEL ee) VR. AS es cl aN sete ont, LE PT
GrorGE Ortnay SHtnsI. Embryology of coccids, with especial reference to the
formation of the ovary, origin and differentiation of the germ cells, germ
layers, rudiments of the midgut, and the intracellular symbiotic organism.
One hundred thirty-three figures (twenty plates)...................0....
CHARLES L. PARMENTER. Chromosome number and pairs in the somatic
mitoses of Ambystoma tigrinum. Thirty-seven figures (nine plates)......

No. 2. MARCH, 1920

Hacuiro Yuasa. The anatomy of the head and mouth-parts of Orthoptera and
EKuplexoptera. One hundred and sixty-three figures (nine plates).........
Wm. A. KeEPNER AND FRANK HELVESTINE, JR. Pharynx of Microstoma cauda-
hime One text iimure and three, plates)... « saeeiee «es + ve La ote ee
H. D. Resp. The morphology of the sound-transmitting apparatus in caudate
Amphibia and its phylogenetic significance. Eighteen text figures and six
DISteS erty meee tea, Some A... 7: es Se Rae,
GEORGE W. TANNREUTHER. The development of Asplanchnia ebbesbornii
(Rotifer). Twenty-one text figures and seven plates..................
Tracy, Henry C. The elupeoid cranium in its relation to the Seannicndides
feeeonlaen and the membranous labyrinth. Three figures and four
| SLAPS SS ai co eee erie Re ke 8 a” SRR ty ees
O. W. Hyman. The development of Gelasimus after hatching. Twelve plates
[STP SUN 8 1 es CTT 2h) A ec kA 8 cl
Louise Smita. The hyobranchial apparatus of Spelerpes bislineatus. Fifteen

61

73

169

251
309

325

. 389

439

485

PIAES MEDLEY Se VEN OES). Yam oie: An siert’s |< «de nde Sp 5abS le cite better 527

AUTHOR’S ARSTRACT OF THIS PAPER ISSUED
BY THE BIBLLOGRAPHIC SERVICE, OCTOBER 13

GERM CELLS OF COELENTERATES

VI. GENERAL CONSIDERATIONS, DISCUSSION, CONCLUSIONS

GEORGE T. HARGITT

Zoological Laboratory, Syracuse University

THIRTY FIGURES (THREE PLATES)

CONTENTS
LMNELOOUUGETOTIN ES Seren etree te eae elec aeelas siatereeta ie arena Os efor spenaters sumac de 1
filer Orieineor eerm cells 255 520/520 adele ite oc ope SE lS cle do shtte ee aye ote 2
Pep reeam igor mire. tone ke ackvas aie Cgc ates.) 3S hale chek tye eo endosa Ice terns 2
Ady Wheaten ieee a Fea 1) See nC CRS Ca eRe ee cee stn cree eans feces 3
2 (Clone hinerornes Se et Ge io Sic BRE Me SiGe 12
Mik Phe eerm-—plagm theory... 22. ee. c-fos ois + + ol elete misters 2 ee sm ewialo ne see 12
1. General statement and discussion of the theory.................. 12
DE GaGeMGG ATOM ERY GTOZOR. 02-5 oy So.5 ease - tere ee as lee» = else 14
Mea COTITINCE lie ee ee ce tokcntte >: oo. RRR veins © ateveiey 14
[hs Eek TaN aed ee ORS 2 tae Seo AoE eo! aNd Ae Soe 15
t RE PENERAbION. e422 ds ant eAR A Tite 2s «dee bate = shake id (oso ae ho's 21
ak, IDTSRGvermniGG! Calle co oe Cac oan ob umoneeaebo ++ oOnoeeaoe Ob ber 23
3. Evidence from germ cells, outside of Hydrozoa................... 26
A Bvidence from tissue culitmes..+....i....--.--satsaus-s. eo sane Bo. 27
Seebi vid encemmonmi cancernce lsu rerriy slat. « Sekar sts) ie iets rere 29
6: Summary, and canelusions). 446.04... 5. <sabeeegek ies eine 30
OR Chgoh yan oerorme:/oates Lip) Sees a” A ee es eee Cs oc are eae Sina 32
TL WLGri Hoye) Grier ie iyo3 qi AR deklio icn eeSe cic: Saat ome agin 32
PENCE MOT OWtTis <ccle 2 SEPAR ET eects sw o's Dial eheMMetehe ete se tehac= i tots 33
3) @ytoplaspue INeWIsiOns st eae a. Foe 01-0 + ws oo alee ie ese ne ss 36
Wee (Gn iw arenrafs td 1s Cae ee ga R Ce CIO: oe lo cee eC G8 Ore Cee meaeem notes Te 42
Wake Sumanisiey rd: CONCLUSIONS: «cc ce ain 5650 00 a + 0.0 mM ins ni oleh aisleeve se 45

I. INTRODUCTION

The study of the germ cells of the Coelenterates was under-
taken with the primary aim of securing a series of observations
upon the behavior of these cells in a variety of types within the
phylum. The original plan included the study of representative
forms of Hydrozoa, Scyphozoa, and Actinozoa. The last class

1

2 GEORGE T. HARGITT

has been omitted from consideration because of a failure to secure
adequate material, but considerable numbers of the other two
classes have been carefully investigated.

The earlier studies (G. T. Hargitt, ’09 to ’18) presented the
data obtained from the investigation of particular species, with
some discussion of a general sort in interpreting these observa-
tions. It is now necessary to consider the observations in the
light of the accumulated knowledge of the various species and
to correlate the data obtained from different species. It will also
be well to discuss the results of these studies in connection with
observations upon the germ cells of other phyla. Certain phases
of the problem have been reinvestigated and new data obtained;
the results of this new study will be considered in place under the
appropriate headings.

II. ORIGIN OF THE GERM CELLS
1. Place of origin

The generalization was made many years ago that the germ
cells of Hydrozoa always arose from the ectoderm, while in
Seyphozoa it was the entoderm which gave rise to germ cells.
So far as my observations go, the latter statement is confirmed,
but the former is not correct. The genera and species of Hydrozoa
which have been investigated are sufficient in number to show
that neither the ectoderm nor the entoderm may be considered
as the characteristic place of germ-cell origin; on the contrary,
these cells may arise sometimes from one layer and sometimes
from the other, even in the same species.

A survey of the available literature of recent years on the
germ cells of Hydrozoa gave the following results: All who have
worked upon Hydra agree upon the ectodermal origin of the germ
cells. Thirteen authors record twenty-three other species of
Hydrozoa as producing germ cells in the ectoderm and nine
authors record thirty-one species in which the germ cells arise in
the entoderm. In many cases from two to four authors have
studied the same species, in other cases only a single study has
been made of a species. The summary made above includes

GERM CELLS OF COELENTERATES 3

every record, which means that a few species have been recorded
twice when two authors differ in their results. If these disputed
cases were omitted, the ratio would remain practically unchanged.
Four investigators, working upon six species, agreed that the
germ cells might take their origin either in the ectoderm or in the
entoderm; Goette (’07) found five species in which the male
germ cells were formed in one layer and the female cells in the
other; in fourteen species the two sexes agreed in the place of
germ cell origin. Other authors have recorded for single species
a different place of origin for the two sexes.

Those recent investigators who have studied the Hydrozoa
most carefully and extensively are in agreement upon the lack
of definiteness in the place of germ-cell origin. They agree that
the portion of the polyp or colony where germ cells arise is not
always the same, the layer may differ in the same species and in
the two sexes of the same species, and they also agree in dismissing
the place of origin in germ cells of Hydrozoa as of no significance.
The work of the author is in harmony with this opinion.

2. Time of origin

The investigation of the precise time in ontogeny at which
germ cells arise comes within the scope of cytological study, rather
than in earlier embryological investigation. This change of
attitude has developed largely as a consequence of the interest
in the germ-plasm theory of Weismann; it is of much impor-
tance to the theory to determine the time at which germ cells
are differentiated and especially to discover their relation to the
fertilized ovum. The studies of Weismann (’83) upon the origin
of sex cells of Hydromedusae furnished him with the chief material
upon which to formulate his theory. The actual observations of
Weismann, did not, in fact, warrant the enunciation of this
theory, as has been clearly pointed out by Goette (’07), C. W.
Hargitt (11), the author and others. It is only necessary to
refer to the preceding section to note the extent to which Weis-
mann’s claim of the ectodermal origin of the germ cells in all
Hydrozoa is incorrect; indeed Weismann’s own published papers

4 GEORGE T. HARGITT

demonstrate that he often found the germ cells to be first recog-
nizable in the entoderm. The suggestion of an ectodermal
origin was proposed on theoretical grounds. But while the ecto-
dermal origin of the germ cells is proved not to be characteristic
of Hydrozoa, this does not necessarily discount the germ-plasm
theory. If it could be shown that the germ cells arise very early
in ontogeny and remain distinct and unchanged to the time of
sexual maturity and the formation of the gonads, it would be a
matter of no importance where these cells were located in the
interim, provided they remained passive and took no part in the
functioning of the body.

In certain phyla considerable success has attended the inves-
tigation of germ-cell origin; an early differentiation has been
noted and these cells have been followed to their position in the
gonads. Some of these cases will be discussed later. In Hydro-
zoa, on the contrary, there has been an almost universal failure to
observe the differentiation at any time before sexual maturity
wasreached. Weismann’s studies were made on mature hydroids
and medusae, and only as a theoretical suggestion was an early
differentiation urged. Harm (’02), in young hydranths of Clava
squamata, just developing from planulae, found certain cells
which he believed to be primordial germ cells. These cells,
figured and described by Harm, are ectodermal cells similar in
form, size, and position to the interstitial cells; somewhat later
they form elongated, spindle-shaped cells lying directly against
the supporting membrane, and possessing a slightly more deeply
staining cytoplasm. They were not traced beyond this stage.
Wulfert (02) traced the development of Gonothyraea loveni to
the formation of the polyp. While the planula is still within the
gonophore, interstitial cells are produced in the ectoderm and
entoderm, and these were followed through their differentiation
into ganglion cells and nematocysts. After the planula has
begun to transform into the polyp, Wulfert finds, for the first time,
what he believes to be germ cells. These occur in both ectoderm
and entoderm and, according to his figures, are like those cells of
an earlier period which became ganglion cells. Furthermore, the
cells called germ cells differ from other interstitial cells of all
stages only in their staining reaction.

GERM CELLS OF COELENTERATES 5

Harm does not describe the formation of ganglion or nettling
cells in Clava, but his germ cells follow the same course as the
ganglion cells of Wulfert in Gonothyraea, and Wulfert never refers
to the spindle-shaped cells as germ cells. It seems clear that
both these investigators are dealing with interstitial cells. Wul-
fert’s results would suggest that what Harm called germ cells
were in reality differentiating ganglion cells. Wulfert states that
his primordial germ cells arise from interstitial cells, but the
evidence he presents in favor of considering these as germ cells
isnot convincing. The determining characteristic, to him, is the
more deeply staining cytoplasm, and this, I believe, cannot be
considered a sufficient criterion, as I have pointed out in another
place (G. T. Hargitt, 16).

Stschelkanowzew (’06) describes germ cells as present in late
cleavage stages of Cunina proboscidea. While the embryo is a
solid mass of cells and the ectoderm and entoderm are being sepa-
rated as layers of a single cell in thickness, he finds one or two
cells between the ectoderm and entoderm layers, but neither in
description nor in figures does he specify the characteristics of
these cells. Their size, form, color reaction, and the size of
nucleus seem to be the same for the ectoderm cells, the position
alone is different. In this instance, also, we have to do either
with the formation of interstitial cells or with the completion of
the formation of the cells of the central solid mass. Precisely
the same process will presently be described for the formation
of embryos of Tubularia.

There is no question of the early formation of interstitial cells;
these have been found, described, and their differentiation followed
in Hydrozoa by various authors. For example, Schneider, (’90)
noted the characteristic and early appearance of interstitial cells
in Hydra, and their later transformation into ganglion cells,
nematocysts, and germ cells. Morganstern (’01) traced the
development of Cordylophora through the larval period, and
identified the ganglion and nettling cells produced from inter-
stitial cells, but did not find any evidence of germ cells in larvae
or young polyps. The germ cells arose from ectodermal inter-
stitial cells at the time of sexual maturity. Schneider and Mor-

6 GEORGE T. HARGITT

ganstern find, what is probably more or less universal in hydroids,
that some of the interstitial cells remain undifferentiated for a
long time. But such undifferentiated cells are not germ cells,
since they form nettling cells throughout the life of the polyp
and probably act as replacing cells for any of the epithelial cells
destroyed.

In order to test further this question of the presence of germ
cells in embryos, I have made a careful, extensive, and entirely
new study of the cleavage stages and planulae of Campanularia
flexuosa and Gonothyraea loveni; also a similar study of cleavage,
embryo, and young polyp (actinula) of Tubularia crocea. In this
investigation I have followed the formation of the germ layers,
the differentiation of interstitial cells, and especially have searched
for primordial germ cells.

In Campanularia and Gonothyraea cleavage results in the for-
mation of a solid morula composed of yolk-laden cells whose
boundaries are made out with great difficulty, if at all. The
outer cells of the morula arrange themselves into an indefinite
ectodermal layer, and later the cells of the solid central mass pull
apart to form an enteron, but during this time none of the cells
take on a columnar form and no interstitial cells are present.
Figure 1 shows the appearance of the embryo after the formation
of the enteric cavity; the cells are not sharply outlined, and
the nuclei, surrounded by masses of cytoplasm, are irregularly
scattered through both the outer and inner layers. This rather
indefinite condition is replaced in young planulae by the con-
dition shown in figure 2. The ectoderm cells are now columnar
and a few interstitial cells are present, the cells of the entoderm
are assuming a columnar form, and deep in this layer are groups
of interstitial cells. The boundary between the primitive germ
layers is not a definitely formed supporting lamella, but only the
cell outlines of the ectoderm. One is immediately struck by the
appearance of some of the interstitial cells of the entoderm,
and there is little doubt that some of these are similar to the
primordial germ cells of Wulfert. However, some of these are
spindle-shaped or stellate in form and their nuclei do not differ
from the nuclei of the epithelial cells of the entoderm. As the

GERM CELLS OF COELENTERATES aed

planula develops, these interstitial cells divide to produce such
groups as the one shown in figure 3; at the same time the entoderm
cells assume a definite epithelial form. During the progress of
the development of the planula the entodermal interstitial cells
decrease in number, nematocysts are formed from some of them,
and others become elongated, as shown in figure 4. Some of these
spindle-shaped cells extend, or move, toward the free surface of
the entoderm and have the form and appearance of gland cells.
None of the entodermal interstitial cells remain in the form shown
in figure 2, and none of them, in the older planulae, display the
characteristics of germ cells. During these changes in the
entoderm there are very few ectodermal interstitial cells produced
(none were present in the region of the planula from which the
figure was made), and through the entire history of the planula
there are no germ cells in the ectoderm.

Thus, by the time the planula has been perfected, there are no
cells in the ectoderm or entoderm which have even the remotest
resemblance to germ cells. The almost complete absence of inter-
stitial cells from both germ layers of the completed planulae,
and the formation of nematocysts from most of these, renders it
certain that primordial germ cells are not present at this stage.
Consequently, the cells which resemble those interpreted as germ
cells by Wulfert and Harm are not such, but differentiate into
specialized cells of the body.

There are some differences in the formation of the morula in
Gonothyraea loveni, but once the solid mass of cells is produced
the development is so similar to that of Campanularia it has not
been thought necessary to describe and figure this form. But it
may be said that at no stage could I find even a single cell in Gono-
thyraea which showed the characteristics of a germ cell. I am
forced to believe, therefore, that Wulfert described as germ cells
merely interstitial cells which were undergoing differentiation into
ganglion cells, gland cells, or some other specialized cell element.
Certainly, if primordial germ cells were characteristically present,
one should be able to find them, but this study of similar stages
of the same species on which Wulfert worked gave no evidence
of their presence.

8 GEORGE T. HARGITT

A similar solid morula is produced by the cleavage of the egg
of Tubularia crocea, but the cell outlines are sharply defined and
the formation of the germ layers is easily followed. During the
separation of an outer ectoderm from the superficial cells of the
morula, divisions take place (fig. 5) in such a fashion as to result
in the production of interstitial cells. But interstitial cells are
also formed by divisions from the deeper cells which make up
the entoderm (fig. 6). In this early stage the interstitial cells
may, but not always do, show a more deeply staining cytoplasm.
The ectodermal layer becomes more distinct, its cells become more
columnar, and the interstitials increase in numbers to produce
the appearance represented in figure 7. The groups of inter-
stitial cells are formed by divisions of the earlier cells, but others
are also formed from the cells of the outermost layers. By the time
a cavity is present in the center of the embryo the ectoderm has
become separated by a supporting layer (fig. 8). At this stage
the interstitial cells are numerous, so closely packed as to render
their outlines indistinct, and for the most part there is no dif-
ference in the staining reaction of the ectodermal and interstitial
cells. From this embryonic condition the young polyp or actinula
develops. In the development of the polyp the interstitial cells
shown in figure 8 are easily followed through their differentiation
into nematocysts and other specialized cells. At no time can
cells be found which resemble germ cells. This conclusion is in
harmony with the earlier results of the author (’09) on this
species, for it was then found that germ cells first became dif-
ferentiated in the medusoid buds of the hydroid from the ecto-
dermal cells.

Other hydroids, as Clava, Hybocodon, Eudendrium, were
examined in cleavage and larval stages, but the material was not
sufficient in amount to permit a determination of the details
noted for Tubularia, Campanularia, and Gonothyraea. No
evidences of germ cells were seen in these stages of the forms
mentioned. <A study of Clava and Eudendrium was made by
C. W. Hargitt (04 b, ’06), and the formation of germ layers and
interstitial cells was determined. In neither did a differentiation
of germ cells occur in the planulae or earlier stages. Other inves-

GERM CELLS OF COELENTERATES 9

tigators who have studied the development of Hydrozoa and
Scyphozoa record the formation of germ layers and interstitial
cells and the differentiation of the latter into ganglion cells and
nematocysts, but have not observed the presence of germ cells
in these earlier stages.

We may conclude that Wulfert and Harm made no mistake
in their observations, but the interpretation of certain cells as
germ cells is not justified by their own evidence, nor is it confirmed
by this new study. The cells described as germ cells are inter-
stitial cells which were in the process of differentiation into
specialized cells of the body. In all the forms carefully studied
it is clear that germ cells do not occur in larvae or young polyps,
and in the absence of any evidence of their presence in similar
stages of other forms, there is ample reason for concluding that
an early differentiation of germ cells does not occur.

The germ cells of Hydra have been investigated by a consid-
erable number of investigators, and practically all of these agree
upon the origin from interstitial cells of the ectoderm at the
breeding season. Brauer (’91) has observed the formation of the
interstitial cells before the ectoderm and entoderm are fully sepa-
rated, and has followed the differentiation of these into ganglion
cells and nematocysts, confirming the earlier results of Schneider
(90). Downing (’05, ’09) is the only one who has suggested a

different conclusion for the germ cells of Hydra. He observes
the same origin from interstitial cells, but, in the developing
ovary, finds some interstitials to be larger than others; these he
believes to be primordial germ cells which have been segregated
in early ontogeny to form a ‘self propagating’ germinal tissue.
He has not observed these cells in the embryo, indeed he seems
to have studied only the polyps which are producing reproductive
organs, and therefore his conclusions are largely hypothetical.
The presence of larger interstitial cells in the developing ovary and
their identification as germ cells is confirmed by Tannreuther (’08),
who also finds similar cells forming spermatogonia. But in every
case the formation of ovaries and spermaries is initiated by a
rapid growth of interstitial cells and later a multiplication of
these cells. Tannreuther thus accounts for the presence of larger

10 GEORGE T. HARGITT

interstitial cells, but shows they are not a germinal tissue. He
finds no such cells before or after the formation of the repro-
ductive organs and can trace their growth from ordinary inter-
stitial cells. Later, Tannreuther (’09) followed the behavior of
the interstitial cells and could find all gradations between small
and large interstitials, as well as trace the transformation of an
ordinary interstitial through spermatogonia into spermatozoa.

Wager (’09) finds no evidence of a germinal tissue in Hydra,
nor of any difference between interstitial cells. Furthermore, in
the very groups of interstitial cells which grow to form oogonia,
‘“‘one usually finds nematocysts developing in large numbers. In
the course of development of the ovarian area these nematocysts
either migrate out or are resorbed. Frequently they are found
within the egg itself.”’ This is a very striking demonstration
of the equipotency of the interstitial cells and effectually refutes
the belief of a distinct germinal tissue composed of certain inter-
stitial cells. The characters used by Downing to differentiate
germ cells from other interstitials are found to be applicable to
most interstitial cells; there is great variety in size and appear-
ance, and Wager finds all gradations between these variations
in interstitial cells. He strongly confirms the work of the earlier
authors and agrees perfectly with Tannreuther in the absence of
a distinct germinal tissue in Hydra. The work of these two
authors did not include a complete study of the histogenesis,
but the investigations of Schneider (’90) and Brauer (’91) com-
pletely fill this gap. Hegner (’14), in discussing the germ cells
of Hydra, says he “‘is inclined to accept Downing’s position in
the matter.’’ But Downing’s position is untenable, for his
conclusions are refuted by the work of other investigators. The
strongest evidence of the occurrence of a distinct germinal tissue
presented by Downing, viz., the presence of larger interstitial
cells of a distinct sort, is shown by both Wager and Tannreuther
to be merely an incident in the formation of reproductive organs.

From the facts presented in the above discussion there is but
one conclusion which may fairly be drawn, viz., in Hydra and
other Hydrozoa there is no clear evidence that germ cells are ever
differentiated in larvae, young polyps, or any early stage in
ontogeny.

GERM CELLS OF COELENTERATES 11

In addition to the line of evidence just presented, we have
direct observations upon the entire germ-cell cycle of some
hydroids from their earliest differentiation. Some of the thirty
or more species studied by Goette (’07) give very clear evidence
of the method of origin of germ cells. In Podocoryne germ cells
arise from both ectoderm and entoderm; in Corydendrium para-
siticum the egg cells are formed from ordinary entoderm cells by
division, a basal egg cell and a distal epithelial cell resulting, and
only this method of formation is applicable in this species. A
similar division takes place in Clava, and Goette says, ‘‘no doubt
exists, that the egg cells of Clava multicornis proceed only from
transformed half entoderm cells.”’ He observed epithelial cells
dividing, one half forming the egg cells of Sertularia argentea,
Gonothyraea loveni, Obelia longissima, and the sperm cells of
Eudendrium. In Obelia geniculata the eggs develop only in the
medusae by the transformation of entire entoderm cells. Small-
wood (’09) traced the egg cells of Hydractinia echinata back to
single entoderm cells which underwent no division, but trans-
formed directly into oocytes. Campanularia flexuosa produces
its eggs by a similar transformation of entire entodermal epi-
thelial cells or from the basal half of a divided entoderm cell, the
distal end of which persists as an epithelial cell (G. T. Hargitt,
13). The author (’16) also observed egg cells typically arising
from half entoderm cells in Clava leptostyla, though occasionally
from ectodermal interstitial cells.

Such observations upon a number of species by different
investigators leave no doubt of the entire normality of the
described transformation of tissue cells into germ cells. In such
cases there can have been no differentiation and segregation of
germ cells in the early ontogeny, for they came from functional
tissue cells, a portion of which continued as a tissue cell. Such
a cell is a specialized cell and not a latent germ cell. In certain
Hydrozoa, therefore, the origin of germ cells has been precisely
determined and an early differentiation shown to be impossible;
in the absence of positive evidence to the contrary, it would
probably be fair to believe that none of the Hydrozoa show a
differentiation of germ cells till sexual maturity approaches.

12 GEORGE T. HARGITT

3. Conclusions

In the last twenty years the reports of investigations upon the
origin of germ cells of Hydrozoa show more species in which such
cells proceed from the entoderm than from the ectoderm.
Numerous cases are recorded in which the place of origin differs
in the sexes of a single species and where the same individual
may produce germ cells from different layers. Furthermore, the
germ cells come from different sorts of cells. All of this points
to the conclusion that the place of origin is variable and not a
matter of any significance. A few cases are reported of the origin
of germ cells in the embryos or larvae of Hydrozoa, but new
investigation of these gives no confirmation of this. Interstitial
cells are differentiated in early ontogeny and undergo early
specialization into ganglion, nettling, and other cells, but those
not so specialized are alike in all respects and at most persist as
somewhat inactive cells. During all the life of the polyps these
produce nettling cells, form replacing cells, and, in some species, at
sexual maturity produce germ cells.

In none of the Hydrozoa has the differentiation of germ cells
been demonstrated in early ontogeny. On the other hand,
observations of several species have demonstrated that germ cells
may arise from body eells directly, either by the transformation
of an entire cell or from the transformation of one half of such a
body cell. Obviously in such cases an early differentiation of
germ cells is out of the question, and it is believed to be typical
of Hydrozoa to form their germ cells only at the time of sexual
maturity.

Ill. THE GERM-PLASM THEORY
1. General statement and discussion of the theory

This theory has been much discussed and many weighty
objections have been raised against it; at the same time it has
been strongly defended and important evidence brought forward
to uphold it. Probably the lines of defense, as well as of oppo-
sition, are so well known as not to require further review. There-

~ GERM CELLS OF COELENTERATES 13

fore, the present discussion will be limited to a consideration of
the theory in relation to observed facts in the Hydrozoa. In
order to have clearly in mind the essential features of the theory
and its method of application to the Hydrozoa attention is
directed to the statements of the author of the theory.

In every ontogeny, a part of the specific germ-plasm contained in
the parent egg cell is not used up in the construction of the body of the
offspring, but is reserved unchanged for the formation of the germ cells
of the following generation (Weismann, ’91, vol. 1, p. 170).

This splftting up of the substance of the ovum into a somatic half,
which directs the development of the individual, and a propagative
half, which reaches the germ cells and there remains inactive, and later
gives rise to the succeeding generation, constitutes the theory of the
continuity of the germ plasm, which I first stated in the year 1885
(Weismann, ’04, vol. 1, p. 411).

In hydroids the germ cells do not appear in the ‘person’
which is developed from the ovum at all, and only arise in a much
later generation, which is produced from the first by continued budding.

: In all the last mentioned cases the germ cells are not present
in the first person arising by embryogeny as special cells, but are only
formed in much later cell generations from the offspring of certain cells
of which this first person was composed. These ancestors of the germ-
cells cannot be recognized as such: they are somatic cells—that is to
say, they, like the numerous other somatic cells, take part in the con-
struction of the body, and may be histologically differentiated in different
degrees (Weismann, ’93, p. 185).

Invisible, or at any rate unrecognizable, masses of unalterable germ-
plasm must have been contained in the body cells in all cases in which
such a transformation has apparently occurred (Weismann, ’93, p. 19).

In the hydroids, then, Weismann notes the germ cells as unrec-
ognizable till the period of maturity; their origin at that time is
from body cells which are morphologically differentiated and
physiologically specialized to perform certain functions of the
animal. This is a statement of fact which is confirmed by the
work of the authors referred to in section II of this paper. These
facts do not fall into line sufficiently with the theory as stated
in the first two quotations, and Weismann thereupon assumes
the presence of invisible and unalterable determinants which lie
latent in the body cells till activated in some way not specified.
This point of view is one to which the greatest objection has
been raised. Lloyd Morgan (’91), in a very searching analysis

14 GEORGE T. HARGITT

and criticism of this position, points out its weakness and con-
siders the recourse to invisible units as a hindrance and not an
assistance to an understanding of the facts. In any effort to test
the theory by observed results in hydroids one is met by the
distinct statement that when germ cells arise from body cells the
latter contain invisible and unrecognizable materials. If the
germ plasm be really invisible and unrecognizable, the theory
need not be discussed, since it cannot be proved or disproved..
In the following pages evidence bearing upon the theory is pre-
sented from various lines of investigation, but the point of view
is taken that there must be recognizable differences of some sort,
or else an unbroken line must be traceable from germ cell to germ
cell in the life cycle.

2. Evidence from Hydrozoa

a. Germ cells. The earlier section of this paper upon the origin
of the germ cells is pertinent here, and should be considered in
its entirety as a part of the evidence. It may be repeated that
the facts show an absence of differentiation of germ cells in early
ontogeny; an absence of a definite migration and germ-track; and
the formation of germ cells at the time of sexual maturity from
different layers and cells of the body. It has been possible to
trace the germ cells back to tissue cells and observe the method
by which they are produced; Weismann’s own observations
confirm this perfectly. It is even possible to prove that there
cannot be present in the body cells which form germ cells any
invisible germ-cell determinants. Goette (’07) and the author
(’18, ’16) find cases where division of a tissue cell results in the
formation of two cells, one of which becomes a germ cell while the
other persists as an epithelial body cell. If invisible germ plasm
be present in the chromatin, as Weismann distinctly states, how
is it possible for one of the two cells to become a germ cell and
the other a tissue cell when the chromatin is equally divided
and none of it lost? This is crucial evidence, and it gives the
facts demanded by Weismann himself to prove his contention
incorrect, as Goette and the author have already pointed out.

GERM CELLS OF COELENTERATES L5

Without repeating all the evidence presented in sections I and
II, the facts may be summarized as follows: there is no definite
place of origin of germ cells; there is no definite migration of
germ cells and no germ-track; there is no invisible germ plasm
in the body cells. Not only is there no continuous germ plasm,
so far as can be determined by observation, but the evidence is
such as to show the absence of invisible germ plasm. Hegner
(14) is willing to admit the germ cells in Coelenterates do not
belong to any germ layer, but he maintains that germ cells are
present at all times in a dormant condition. This opinion is
based upon the conclusions of Downing, Wulfert, and Harm.
The error in the interpretation of these authors has been pointed
out and consequently the opinion that germ cells are present in
a latent condition at all times is no longer tenable; all the facts
are inconsistent with this view.

b. Budding. Budding has generally been held to be a process
of growth and cell division, often an evagination taking place.
But Weismann says, ‘‘. . . . J reached the conclusion, that
the budding idioplasm, which must be the starting point of the
budding process according to my view, could not be divided
between both germ layers, but probably was to be found in only
certain cells of the ectoderm.’”’ At Weismann’s suggestion, Lang
(92) undertook to test this hypothesis and studied budding in
So a and some hydroids. Weismann believes Lang’s results
SP . . contain a perfect confirmation of my conjecture that
the same [buds] come from the ectoderm and that actually the
‘Budding-idioplasm’ had its position entirely in the ectoderm
cells.”” These quotations from the preface to Lang’s paper
show the application made by Weismann of the germ-plasm
theory to this form of asexual reproduction. Mang believed his
results showed the proliferation of a few ectoderm cells to form
a mass from which the ectoderm and entoderm of the bud de-
veloped. After the two layers were formed, a cavity was pro-
_ duced in the bud, and this became continuous with the parent
enteron. Braem (’94) repeated the work of Lang on the same
and other forms, but could not confirm his results; on the contrary,
he observed the division of cells in ectoderm, interstitial, and

JOURNAL OF MORPHOLOGY, VOL, 33, NO. 1

16 GEORGE T. HARGITT

entoderm, and the participation of all these layers in the for-
mation of the bud by evagination. He says, “‘. . . . conse-
quently I do not hesitate to proclaim the results of Lang as
erroneous, the conclusions drawn from them as utterly false.”
Downing (’05) believed sexual and asexual reproduction in
Hydra to be mutually exclusive, and implied a relation between
budding and germ cells. Montgomery (’06) supposed sexual
reproduction to be the more primitive, and asexual reproduction
to be a secondarily derived process; for him, regeneration and
asexual reproduction were dependent upon the presence of germ
cells. R. Hertwig (’06) found budding and sexual reproduction
proceeding side by side in Hydra and believed buds were produced
by the activity of the cells in all the layers. Mrdzek (’07) and
Nussbaum (’07) confirm Hertwig on the simultaneous presence
of buds and sex organs in Hydra. The view of Hadzi (’09) was
in partial accord with Weismann and Lang, for he again renewed
the claim of the activity of only a certain layer to form buds in
Hydra. In his opinion the interstitial cells were the active
elements in producing buds, the other layers not participating in
any way. According to this view, the interstitial cells are a
source of all new growth, differentiation, and development in
Hydra, but they do not necessarily form a germinal tissue.
Tannreuther (’09) investigated budding still further, and for two
species of Hydra found, first, an increase in volume, and then a
proliferation of interstitial cells in the budding zone. There was
no migration of interstitial cells into the entoderm as Hadzi had
believed, for the layers remained distinct and unbroken through-
out the process. <A distinct evagination occurs and cells of all
layers divide mitotically and are active in the budding process.
Furthermore, the division of cells of the ectoderm and entoderm
began about as soon as in the interstitial cells. Tannreuther’s
work establishes the fact that budding in Hydra is an evagination
due to cell multiplication and growth, all layers in the budding
zone participating in the process. It seems probable that the.
earlier division of the interstitial cells is merely an expression of
a more prompt response on the part of the indifferent cells than
of the specialized ectoderm and entoderm. I believe the fact is

GERM CELLS OF COELENTERATES £7

established that budding in Hydra and hydroids is a process of
evagination, but the work of Lang, Hadzi, and Tannreuther
suggests an earlier activity of the interstitial cells. Even if the
interstitial cells were entirely responsible for the formation of the
bud, proof would not be thereby constituted for the germinal
nature of these cells, for they are differentiating into nema-
tocysts throughout the life of Hydra. Also these same cells
transform directly into ganglion cells earlier in the life history.

Medusae are sexual individuals and ordinarily reproduce only
by eggs and spermatozoa, but there are a considerable number
which undergo a process of asexual reproduction and form other
generations of medusae by budding. The budded medusae later
become mature and form sex cells just as do the parent medusae.
The author (’17) has given a detailed account of this secondary
budding of medusae and of germ-cell formation in Hybocodon
prolifer; the gonads are produced from the ectoderm of the wall
of the stomach, while the new medusae come from the tissues of
the base of the tentacle at the margin of the bell. In a critical
examination of these medusae no evidence was obtained of the
migration of germ cells from the old to the budding medusae, but
the new buds arose from both layers of cells in the tentacle after
these cells had undergone regressive changes and become embry-
onic. In Hybocodon the asexual budding is not influenced by
the formation of sex organs. Miiller (’08) is in error in believing
the two methods to be mutually exclusive, for C. W. Hargitt
(02), Perkins (’04), and the author (’17) have recorded abundant
cases of the simultaneous presence of buds and gonads.

A. Agassiz (’65), Haeckel (’79), C. W. Hargitt (’04), Mayer
(10), and others have described many cases of asexual budding
in medusae. Such buds may be formed, a few at a time, or many
at a time; a single generation of buds may be produced or many
generations; and many regions of the medusae may be concerned
in their formation. Haeckel describes the buds on the stomach
wall of Sarsia gemmifera (S. siphonophora) (fig. 10), more than
twenty being present at one time and several generations being
produced; in different species of Cytaeis (fig. 14) enormous
numbers of medusae may be budded from the stomach wall at

18 GEORGE T. HARGITT

the same time that gonads are present. Medusae are formed
from a single tentacle base in Hybocodon prolifer, Amphicodon
amphipleurus Haeckel and others; from the bases of all tentacles
in Sarsia codonophora (fig. 13); from radial canals of Probosci-
dactyla ornata (fig. 9); from the margin of the bell in Niobia
dendrotentaculata (fig. 11); and from the gonads of Eucheilota
paradoxica (fig. 12) and other forms. These are merely examples
of the variation in the method of budding as recorded for numer-
ous medusae. In many of these the budding occurs during the
immature period, and only after budding ceases do the gonads
form, but others show no such periodicity and may produce buds
and germ cells simultaneously.

The production of the buds from the gonads has been critically
studied. Mayer (10) describes this process for Eucheilota (fig.
12) as involving the activity of the tissues of the gonad and of
the tissues outside the gonad; both ectoderm and entoderm of the
parent take part in the production of the bud by a process of cell
multiplication and evagination. In Phialidium mccradyi buds are
also produced from the gonads, but only indirectly, since a blasto-
style is first formed and from this the medusae arise by budding.
Sigerfoos (’93), in the formation of the blastostyle and medusae,
discovered no difference from ordinary cases of budding, the
ectoderm and entoderm evaginating to produce the new growth.
The germ cells in the gonad play no part in the process other
than to behave as all other cells of ectoderm and entoderm, which
suggests the probability of the germ cells being merely body cells
capable of acting with other body cells or undergoing a growth
in preparation for sexual reproduction.

Budding in medusae is typically an evagination of the two
body layers, irrespective of the part of the animal which produces
the bud, but a few medusae are known to form their buds only
from the ectoderm. Mayer describes such a case in Bougain-
villia niobe, the ectoderm of the stomach wall differentiating to
form all the tissues and organs of the bud. Mayer believes a
possibility exists of the origin of the bud entoderm from parent
entoderm; but could find no evidence of such a connection, nor
of any union of the enteric cavities of bud and parent at any stage

GERM CELLS OF COELENTERATES 19

of the process. Chun (’95) describes a similar process in Rathkea
octopunctata and Lizzia claparedei. He describes the origin of
the bud by the proliferation of a group of ectoderm cells which
becomes isolated as a definite mass, though still held in place
against the stomach wall, from which all organs of the bud are
developed. In these forms the enteric cavities of buds and
parents later unite. When sexual maturity is reached germ cells
are formed in the stomach wall where the bud was developed
earlier, but Chun does not consider the budding as due to a
geminal process. Rather, he believes the ectoderm and ento-
derm of the medusae to be alike in histological and organo-
genetic structure and potency. Braem (’08) reviews and con-
firms the work of Chun, but finds germ cells are present in the
stomach wall at the same time the bud is forming; he believes the
group of cells which start the bud are oocytes, and looks upon
the budding process as a short and rapid method of producing
a new organism out of cells which are germinal in character.
Most budding, he believes, shows no relation between bud and
sex cells, and in these cases all layers are essential to the forma-
tion of the bud because each tissue has retained only the ability
to produce cells of its own kind. Mayer thinks Braem has
produced strong evidence that this sort of budding is a germinal
process, but does not believe the evidence is conclusive. Child
(15) interprets this case as showing both sex cells and asexual
buds come from the functional and more or less specialized cells
of the parent medusa.

Nekrassoff (?11) studied Eleutheria dichotoma, which produces
buds from the outer wall of the ring-canal. In this form budding
parallels sexual development, but does not interrupt it, nor is
budding interrupted by sexual development. In a single indi-
vidual one may find numerous buds, young and old, young and
old eggs, cleavage stages and young polyps—all at the same time.
The budding takes place in the usual way, involving both ecto-
derm and entoderm, and while Nekrassoff finds conditions which
resemble the observations of Chun and Braem in Rathkea and
Lizzia, he can demonstrate the continuity of bud and parent
tissues at alltimes. He does note that the ectoderm and entoderm

20 GEORGE T. HARGITT

cells show a more embryonic appearance after they have begun
to form the bud than they did before; especially is this true of
the entoderm. Nekrassoff concludes: ‘‘on the ground of the
observations on the budding of Eleutheria we may conclude that
in the Coelenterates already differentiated cells have been given
the possibility of a reversible process—the possibility of taking
on anew an embryonic character.’’ Regarding the suggestion
of the origin of buds from germ cells, he finds in Eleutheria no
relation at all between sex cells and buds.

The process of budding in medusae does not, asarule, involve
any difference in principle from budding in Hydra and hydroids,
since both germ layers, by cell multiplication and evagination,
form the outgrowths which, by later differentiation, become the
tissues of the new individual. ‘There are some buds which arise
from a small group of cells of a single layer, but in no case do buds
come from a single cell. Budding is not, therefore, a germinal
phenomenon, even when the new growth is derived from the
tissues of the gonads. Consequently, not only is there no neces-
sity for thinking of the germ plasm as being essential to the for-
mation of buds, but there is no evidence of the presence of germ
plasm in these buds. The conclusion of Nekrassoff, that differ-
entiated cells may take on again an embryonic character, seems
to explain the facts better than the germ plasm theory. Though
quite unaware of this conclusion of Nekrassoff, the author (717)
worked out the budding of Hybocodon medusae and noted the
embryonic character of the cells involved in the budding process.

There is considerable variation in the degree to which this
‘reversible process’ is exhibited by the tissues of medusae, but
an unbroken series may be arranged which includes all the known
types of budding. At one end of the series we may place the
medusae whose tissues do not have such a capacity; these repro-
duce only from fertilized egg cells. Here are included the
majority of medusae. If we accept the conclusions of Braem,
we may next place forms, like Lizzia and Rathkea, in which a
group of unfertilized oocytes may develop into a new organism.
This is a very unusual method and is applicable, so far as known,
only to the two forms named. Here the tissues either have no

GERM CELLS OF COELENTERATES 21

power to change or the stimulus to such change would be lacking.
- Following this would come Bougainvillia niobe; the ability to
form buds is limited to a definite tissue, the ectoderm. Next are
those forms like Hybocodon in which all layers cooperate to form
buds, but this capacity for asexual reproduction is limited to a
definite locality in the parent. In this category one would place
most of the medusae which form buds, and all hydroids and Hydra.
Niobia dendrotentaculata represents a type in which the bud is
partly new growth and partly the already formed organs of the
parent; presumably all the regions of the body in such forms
would have the ability to undergo some transformation. This
type of budding would really be intermediate between regular
budding and fission. A final group would comprise medusae in
which a real fission occurs, and such a method of asexual repro-
duction is recorded by Mayer (’10, vol. 2, p. 280) for Gastroblasta
raffaelei Lang. A gradation such as this would correlate the
various kinds and degrees of asexual reproduction in Coelen-
terates with reproduction in protozoa, with regeneration, and
with sexual reproduction. It may even mark a possible evolution
of reproductive processes in Coelenterates, but would appear to
have no meaning according to the germ-plasm theory.

c. Regeneration. Weismann (’93) takes the position that regen-
eration is due to the presence of germ plasm, since the latter is
the only substance capable of giving rise to all parts of the body.
As applied to plants, this involves the presence of germ plasm in
the cambium tissue wherever it is found. There is postulated
in plants an accessory germ plasm, concerned with the vegetative
development, and a primary germ plasm which is retained un-
changed till the germ cells are produced. But vegetatively
produced buds may later form reproductive organs and cells;
this requires the further assumption that accessory germ plasm
also contains primary germ plasm. This same involved and
intricate explanation is required to account for regeneration in
animals, if we believe that regeneration is due to latent germ cells.

Morgan (’01) discusses a considerable number of theories of .
regeneration and rejects the germ-plasm theory completely,
since he finds so many facts of regeneration utterly contradicting

ae, GEORGE T. HARGITT

it. He found, for example, that the regenerating organs in
annelids came partly from the old organs and partly from new
sources; new muscles came, not from old muscle or even from
mesoderm, but from the ectoderm, the pharynx regenerated
from entoderm instead of ectoderm as in the original development.
Other evidence of the same sort was directly contradictory to
the view that regeneration is due to latent germ cells. Morgan
(01, ’07) believes regeneration is a growth process. Schultz (’02)
thinks regeneration is a primary property of life, limited more or
less in consequence of specialization of tissues, but always poten-
tially present. His conclusion is in accord with that of Morgan,
and implies development, budding, and regeneration to be
exhibitions of the capacity for growth inherent in all protoplasm.
Montgomery (’06) and Hegner (14) reject this view and accept
the germ-cell explanation, the latter stating that regeneration in
Coelenterates is always due to widely distributed germ cells.
C. W. Hargitt (11) points out serious objections to this expla-
nation in hydroids, and Hegner admits the impossibility of
accounting for regeneration of sex organs on this view. But sex
organs are readily regenerated in hydroids. Child (715) has
observed that specialized cells of Pennaria may undergo a de-
differentiation and take part in budding, along with the inter-
stitial cells; the same thing occurs during regeneration. Morgan
has also found abundant evidence of the formation of masses of
indifferent cells by regressive changes, and the production of new
structures from such masses in regeneration. Morrill (718),
working upon the regeneration of appendages in salamanders,
observed the formation of masses of cells by simplification of old
specialized cells, and the differentiation of muscle and cartilage
from these cells.

In Hydra and hydroids regeneration may take place at practi-
cally any point where a cut is made, and almost as often as new
growths are excised. Very minute pieces may also regenerate
complete animals, normal in all respects, including reproductive
organs. The minimal size is always a group of cells, and yet,
according to the theory of regeneration from germ cells, there is
no reason why a single cell might not produce a new organism,

GERM CELLS OF COELENTERATES 23

for the theory supposes the germ cells to be scattered over the
whole body in great numbers. Clearly, there is no evidence that
regeneration in Coelenterates, nor in other animals, is a process
dependent upon the presence of germ cells. And there is abun-
dant evidence that the specialized cells undergo regressive
changes, produce masses of cells or syncytia of embryonic charac-
ter, and then, by differentiation and specialization form new parts
to replace those lost. Itis, of course, equally well known that not
all tissues can undergo such changes or even regenerate their own
kind of tissue to any great extent; but this offers no evidence of
a correlation between regeneration and the presence of germ cells.
It only shows that specialization may proceed to such a degree
that further changes, whether progressive or regressive, are
impossible.

So far from regeneration presenting evidence in favor of the
germ-plasm theory, practically all the experiments and obser-
vations show direct contradictions to this explanation. The
germ-plasm theory is not only inadequate to explain regenera-
tion, but it is shown to be incorrect, so far as this process is
concerned.

d. Dissociated cells. The tissues of sponges have been broken
up by teasing and forcing through fine screens, and the behavior
of the isolated cells followed by Wilson (’07). Such cells showed
amoeboid activities and fused into masses which later regenerated
to form normal sponges. The amoebocytes first began to unite
to form syncytia, but collar-cells and other specialized cells also
took part in the formation of the masses, first passing through a
regressive differentiation. Miller (’11) largely confirmed Wilson,
but believes such specialized elements as collar-cells do not assist
in the regeneration. Fresh-water sponges also undergo normal
degeneration phenomena by a de-differentiation of cells to produce
embryonic masses which later produce new organisms. This
latter process is quite distinct from gemmation.

Later, Wilson (711) extended his experiments to hydroids.
Here also the isolated cells fused into syncytial masses which
secreted perisare about themselves, then formed ectoderm and
entoderm layers, and later regenerated hydranths, complete and

24 GEORGE T. HARGITT

normal, with tentacles, mouth, hypostome, and other structures.
In these changes ‘‘we apparently have . . . . a plain case
of despecialization of tissue elements and their union to form
masses of totipotent regenerative tissue.” Wilson discusses the
question as to whether the tissue cells may not merely retain
their specificity and later produce only cells of the same sort.
By following the isolated cells with the micros¢ope it was possible
to observe the change of the tissue cells from their typical
appearance to that of embryonic cells, and their fusion into a
mass. The retention of their original specificity seems highly
improbable. A histological study of sections of the coalesced
cells showed the cells, first, as embryonic in appearance, and, as
regeneration proceeds, they undergo changes similar to those
seen in normal development and specialization. DeMorgan
and Drew (’14), in similar experiments, for the most part con-
firmed Wilson, but did not obtain hydranths from the regenerating
masses. They differ from Wilson in thinking the cells are segre-
gated and rearranged and do not form syncytia by despeciali-
zation. They also state their belief that their cell masses are
abnormal and pathological, but this does not appear to be the
case, as C. W. Hargitt (15) has pointed out in some detail.
This latter author confirmed Wilson’s observations in practically
every respect, and also noted in detail the behavior of cells
immediately after their isolation. The identification of the
different cells was easily made, but the characteristic features
- gradually became less marked and finally disappeared as the
cells merged into a common mass. ‘‘They have become de-
specialized into potentially embryonic cells, and probably from
this change have acquired their regenerative capacities.”

In discussing these experiments, Hegner (’14) claims there are
always germ cells present, which would exlain the regeneration
from the masses of cells, and therefore a continuity of germ
plasm exists in these phyla. He does not attempt to explain
the de-differentiation actually observed to occur, though this is
a very significant fact and one that cannot be ignored. For, if
tissue cells may become embryonic and form other cells and
tissues by later differentiation, there is no reason for assuming

GERM CELLS OF COELENTERATES 25

the presence of germ cells. The later work on dissociated cells
gives clear evidence on this point. DeMorgan and Drew can
recognize and follow the isolated ectoderm and entoderm cells
and “. . . . in addition such structures as nematocysts,
ova and broken down cells, all of which are subsequently ab-
sorbed and played no part in the future development.” C. W.
Hargitt also finds that the presence of germ cells in regenerating
masses does not influence the behavior: ‘‘ Indeed, in those cases
in which egg cells were present they took no part whatever in
later regenerative activity, either degenerating or being absorbed
as yolk material.” So far from the regeneration being con-
ditioned upon the presence of germ cells, the latter serve no
purpose but to act as food; growth and differentiation are the
result of the activity of the tissue cells alone. Since these
observations have been confirmed by a number of workers, it is
manifestly false to consider regeneration to depend upon germ
cells in these plasmodia. There would appear, likewise, to be
no ground for assuming any regeneration to be dependent upon
germ cells.

The claim of DeMorgan and Drew, of the retention of their
distinct structure by the isolated cells, and a later rearrange-
ment to produce the regenerated structures, is not confirmed
by any of the other workers. The latter agree in being able to
follow the isolated cells through a gradually decreasing sharpness
and a final coalescence into a common mass. No doubt occa-
sional cells persist, but the observations clearly show the fusion
of the cells into a multinucleate mass. From such a mass a
development occurs which parallels the normal development from
the egg.

These experiments give such striking and clear-cut results that
one is enabled to draw very definite conclusions. ‘Tissue cells
have actually been followed through the process of despeciali-
zation to an embryonic condition; such embryonic cells behave
as any other group of similar cells, and develop a variety of
structures which become differentiated and specialized in such a
way as to produce a complex, normally organized, and functional
individual. The totipotency of the tissue cells of the hydroid is

26 GEORGE T. HARGITT

thereby definitely established, though this is clearly dependent
upon the proper stimulus for its exhibition. When we take into
consideration, also, the observations upon the origin of germ
cells from tissue cells; the observations of Child upon the de-
differentiation of cells in a great variety of animals and their
later differentiation into a different sort of cell; the observations
upon the formation of embryonic masses from which new
structures develop in regenerating worms and salamanders; it
would seem as though the germ-plasm theory was the very one
of all theories least capable of accounting for the facts.

3. Evidence from other phyla

Such phyla as the round worms and arthropods give the
strongest evidence of early segregation of germ cells and the best
support of the germ plasm theory. This view is not universally
accepted, however, and the opposing opinions are worthy of con-
sideration. For instance, Child (’15) states that it is not known
whether the primordial germ cells of Ascaris produce only germ
cells or the reproductive organs as well. If the latter be the
ease, ‘‘the germ path of early cleavage has not resulted in the
segregation of germ plasm from the soma, but merely in the
segregation of different organs,” since the walls of the repro-
ductive organs are not germ plasm. The same author points to
the fact that in no case is a segregation of germ plasm and soma
known to take place at the first cleavage, as the theory requires.
He believes, even in these phyla, the theory is unproved, and is
not in accord with many facts.

In many animals the germ cells are produced periodically at
the breeding season, and at no other period is it possible to
recognize germ cells, or even reproductive organs. In these
eases the germ cells obviously arise from the tissue cells; it
does not answer to claim an invisible germ plasm in the tissue
cells, since this is not capable of investigation and evades the
question. Other animals are produced asexually and at a later
period develop reproductive organs; the germ cells to all appear-
ances, in such cases, come from the more or less differentiated
cells of the region involved in the formation of these organs.

GERM CELLS OF COELENTERATES ne

In the vertebrates the germ cells appear, as a rule, only after
most of the other organs are laid down, and in most cases an
early segregation of germ cells has not been proved. A review
of the work on vertebrates is given by Hegner (714) and
Kingery (’17), and only a few cases will be mentioned here.
Von Winiwarter and Sainmont (’08), from studies upon the eat,
describe the degeneration of all the germ cells produced during
embryonic development; the definitive eggs arise from the un-
differentiated germinal epithelium after birth. Bachman (’14)
in Teleosts and Witschi (’14) in Rana temporaria find no evidence
of the origin of germ cells from the peritoneum, while v. Beren-
berg-Gossler (714) believes ‘“‘that one may no longer speak of a
germ track in the Sauropsida,” and Gatenby (’16), in Rana tem-
poraria, observes the majority of germ cells arising from the
peritoneum. Kingery (717), working upon the white mouse,
gets results comparable to those of von Winiwarter and Sain-
mont in the cat; viz., all germ cells formed during the foetal
period degenerate and have nothing to do with the development
of the definitive ova. The latter arise from the germinal
epithelium after birth and all transitional stages between this
germinal epithelium and graafian follicles were observed and the
development followed.

In the vertebrates and some other phyla the evidence seems
to be as clearly opposed to a continuity of the germ plasm as it
is in the coelenterates. There is, especially in mammals, an
increasing amount of evidence that the germ cells arise from
more or less differentiated tissue cells at a time approaching the
period of sexual maturity.

4. Evidence from tissue cultures

While most of the experiments dealing with explanted tissues
have to do with growth, movements, and general behavior of
the cells, there is some evidence of a de-differentiation of the
tissues into a more embryonic condition. There is very little
evidence that such cells re-differentiate into cells of a new kind,
but this return to an embryonic condition resembles somewhat
the despecialization of isolated cells of hydroids and sponges.

28 GEORGE T. HARGITT

In cultures of skeletal muscle of chick embryos, Lewis (’17)
observed the growth of the cut ends of the muscle into em-
bryonic tissue without striations. Streeter (717) observed a
de-differentiation of cartilage cells in the normal development
of the ear in human embryos, the cartilage of the membranous
labyrinth undergoing a despecialization and a return to the
condition of embryonic connective tissue. From experiments
with muscle, kidney, eye, thyreoid, and other organs, Champy
(14) observes a characteristic behavior of the cells of the edge
of the culture where they receive abundant air and food. These
cells form such an indifferent mass as to resemble cells of a young
blastoderm; and this is true for all tissues, irrespective of their
source or the culture medium. Such a de-differentiation takes
place from explanted adult tissues as well as from embryonic
tissues.

Danchakoff (’18) mashes adult spleen and grafts it upon the
allantois of embryos. The spleen tissue forms a syncytium of
embryonic character, and the cells forming the mass contain
endothelial cells of blood-vessels as well as reticular tissue of the
spleen. The syncytial mass develops and forms cells of a dif-
ferent sort than those which composed it. Danchakoff inter-
prets this, not as a de-differentiation, but as an expression of an
inherent capacity of the original cells to undergo a further
differentiation. Her point of view is as follows (p. 161):

The changes undergone by the living matter during development
are not always specific. They may lead to a specialization of tissue
without differentiating them specifically. The difference between
these two processes consists in that specialization does not imply a
limitation of potencies in the cell, while specific differentiation is a
process, by which the constitution of a cell is changed irrevocably and
its potencies to development are narrowed. The distinction between
the two processes would make it unnecessary to introduce a new
concept of dedifferentiation in order to understand certain phenomena.

I am not convinced that this view is simpler or more nearly
interprets the phenomena observed than the view of regressive
changes in the tissues and a later differentiation of these. Nor
does this opinion take into consideration the fact that de-
differentiation has actually been observed to take place; that is,

GERM CELLS OF COELENTERATES 29

specialized cells do actually become embryonic. But for the
present discussion the important point is the observation of the
varied potencies of the tissues of a differentiated adult organ
like the spleen.

This brief account of some of the experimental investigations
upon cells and tissues of adult and embryonic animals is enough
to show the degree to which such tissues may change their
structure and function. It clearly demonstrates that body cells
are not so limited in behavior and so predetermined in potency
as to render a change impossible. The difference between body
cells and germ cells is proved by such investigations not to be
so great as is usually held.

5. Evidence from cancer cells

The studies which have been made upon cancers throw some
light upon the potencies of tissue cells. As is well known, it is
possible to transplant cancers from one animal to another
through many generations. Most of the cancers which have
been experimentally studied are tissue growths, not germ-cell
growths, and the ability of these cells to continue their growth
and proliferation for long periods of time is an indication of the
ability of tisssue cells to live and grow indefinitely. It is, of
course, perfectly clear that these cells do not produce other
cells of a widely different character, but they are more nearly
like embryonic cells, physiologically if not morphologically, than
the cells from which they originally came. This would probably
involve a sort of despecialization of the tissue cells with the
resumption of an embryonic potency. The germ-plasm theory
postulates a difference between the germ cells and the body
cells of such a sort that the former are conceived to have the
ability to live and develop indefinitely, while body cells have a
limited life. The behavior of the cells in cancerous growths may
do no more than show the ability of highly differentiated tissue
cells, under unknown or poorly known conditions, to regain this
power of repeated and indefinite growth; but this tends to break
down the distinction between germ cells and tissue cells in this
particular.

30 GEORGE T. HARGITT

‘ Loeb (’15), who has given much attention to the study of
cancer cells, discusses the matter from that point of view. He
concludes that the observations of fourteen years upon cancerous
growths have established certain facts which are contrary to the
view of the radical difference between germ cells and body cells.
In those cases where it has been possible to detect and study the
earliest indications of cancer in mice, he has been able to trace
the transformation of the normal tissue cells into the abnormally
proliferating tumor tissue, and is thus able to demonstrate the
origin of the tumor from the tissue. He believes that germ cells
and somatic cells are not so different, and possess no such
differences in potency as is often claimed.

6. Summary and conclusions

The germ cells of Hydrozoa are differentiated, at a time just
preceding sexual maturity, from different regions of the animal
or colony, there being no one region or layer which characterizes
the place of origin in this group. These germ cells probably
arise in all cases from tissue cells; in some species such an origin
is demonstrated, since an entire cell or half a divided body cell
produces a single egg or sperm cell.

Budding in Hydra and hydroids involves a multiplication and
growth of the cells and an evagination of all the body layers in
the budding zone. The claim that latent germ cells are re-
sponsible for budding is not sustained by observations. Some
medusae reproduce asexually by budding, and as a rule such
buds are produced in a manner similar to that of hydroids, viz., |
by an evagination of both ectoderm and entoderm. In a few
cases asexual buds of medusae arise from the ectoderm alone, —
but in no case does such a development come from a single cell.
Buds may also come from the reproductive organs of medusae,
but all investigators of this manner of budding agree upon the
activity of ectoderm and entoderm cells of that region; such a
process is not a development from germ cells. The different
types of budding in Hydrozoa suggest an evolution of repro-
ductive processes which may still be in progress. The phe-
nomena of budding give evidence of a considerable degree of

GERM CELLS OF COELENTERATES 31

plasticity in the cells of the body, a regressive change to an
embryonic condition preceding the formation of the bud.

The germ-plasm theory invokes the aid of latent germ cells
to account for regeneration, but there is no evidence of this in
Hydrozoa. So many cases are recorded, in many groups of
animals including vertebrates, of the de-differentiation of tissue |
cells and the formation of the regenerated structures from an
indifferent or embryonic mass of cells, that it may be doubted
whether regeneration is ever related to germ cells. When
coelenterate tissues are ground up and the cells isolated, the lat-
ter coalesce to form masses capable of regenerating complete and
normal individuals, but in all such masses the cells have become
despecialized before the regenerative processes begin. The ob-
servations upon dissociated cells of hydroids show that germ
cells, if present, degenerate and play no part in the ensuing
regeneration, while the body cells, under the same stimulus, lose
their specificity, become totipotent, and produce the variously
specialized cells and differentiated structures of the normal
individual.

Many animals of different phyla are known whose gonads are
present at the breeding season and entirely unrecognizable at
other times, in such cases the germ cells arise from the body
cells of the appropriate region. Recent work upon mammals
gives strong evidence of the degeneration of all germ cells. formed
during embryogenesis, the definitive germ cells only differen-
tiating after birth from the germinal epithelium of the gonad.

Explanted tissues, grown in culture media outside the body,
may undergo a de-differentiation and form cells more or less
embryonic in character. Cancerous growths, originating from
tissue cells, display a capacity for long-continued and apparently
indefinite growth and division. Such facts are indicative of a
less definite distinction between germ cells and body cells than
has usually been maintained, and the possession of a considerable
capacity in specialized cells to undergo a further differentiation,
even in a new direction.

The investigations discussed in this section furnish a great
body of facts utterly inconsistent with the theory of the con-

JOURNAL OF MORPHOLOGY, VOL. 33, NO. 1

32 GEORGE T. HARGITT

tinuity of the germ plasm. This seems to apply to many phyla,
even to vertebrates, but is especially marked in the coelenterates.
There are so many facts which contradict this theory that it may
confidently be held not to apply in the coelenterates, at any
rate.

IV. GROWTH OF EGG CELLS
1. Cytoplasmic growth

The growth of egg cells proceeds by several methods in animals;
nourishment is obtained either without assistance from other
cells, or else follicle cells, nurse cells, or other accessory structures
assist In securing or preparing the nourishment for the egg. In
none of the coelenterates is a follicle present nor are there nurse
cells such as occur in insects. But there are two distinct methods
of growth; one in which the food is obtained directly from the
enteric cavity or from the adjoining cells, and the second in
which neighboring cells are actually absorbed or engulfed. Often
both methods may be employed. ‘The cells which are absorbed
have sometimes been called nurse cells, but they do not function
in the way nurse cells do in other groups, since they are con-
sumed instead of preparing food. Hydra, Tubularia, Pennaria,
and Hybocodon are examples of those eggs which absorb neigh-
boring cells for food, and Campanularia, Clava, Hydractinia, and
Aurelia are examples of those which obtain food from the enteric
cavity.

A different origin has been claimed for nurse cells and egg
cells in those animals whose eggs are so nourished, the germ cells
representing real reproductive cells while the nurse cells are
held to be tissue cells. The Hydrozoa show no such distine-
tion, for all the oogonia of any ovary are alike in origin and
capable of becoming ova; the determination of which shall
grow and whichserve as food is largely a’ matter of chance.
Even after growth has started, the surrounding cells are like them
until degeneration phenomena become apparent in the cells
undergoing absorption. One explanation for the initiation of
growth is the presence of certain bodies in the cytoplasm.
Schaxel (710 a, ’11 a) describes the growth of oocytes of Pelagia,

GERM CELLS OF COELENTERATES 33

Aequorea, Forskalia, and Agalma as beginning only when
chromatin passes from the nucleus into the cytoplasm. Jérgen-
sen (710) found similar bodies in the cytoplasm of Sycon sponges
at the beginning of growth, Downing (’09) in oogonia of Hydra,
and the author (713 to 718) has noted an apparent correlation
between the presence of such cytoplasmic granules and the
initiation of the growth processes in the eggs of other Hydrozoa.
None of these authors have expressed any thought of these
cytoplasmic inclusions acting as indicators of germ-cell or tissue-
cell origin, but Hegner (’14), who has collected data from many
sources, explains them as germ-cell determinants.

After growth has once started, it continues rapidly, and re-
serve food is stored away for future use. The eggs of some
Hydrozoa become filled with large yolk spheres, while in others
the yolk is in fine particles so diffused through the cytoplasm as
to be scarcely noticeable. There is a great deal of variation in
the size attained by these eggs, as the figures.and descriptions
of the following section will show.

2. Nuclear growth

The detailed changes in the nucleus during growth have been
described in the papers dealing with particular species; only
certain more general relations are here discussed. As the eggs
grow, their nuclei also increase, but not in the same ratio.
Hertwig’s suggestion of a constant ratio between nuclear and
cytoplasmic volume is no more supported by the growing eggs of
these coelenterates than it has been by other cells investigated
by many workers. J6érgensen (’13) has made the claim of a
definite relation between the relative size of the nucleus and the
mode of nourishment of the egg, basing his claim upon observa-
tions of egg cells of a number of different animals. According
to this author, egos nourished by nurse cells or follicle cells, or
by the absorption of adjoining ova and oocytes, have very small
nuclei; eggs without special nourishing apparatus, but which
absorb their food directly, possess relatively large nuclei. In the
latter case, he believes, the nucleus of the egg is responsible for its

34 GEORGE T. HARGITT

growth; in the former, the nuclei of the accessory cells govern the
growth of the egg, and the nucleus of the egg is inactive till it
enters upon the prophase of maturation mitoses.

‘A brief survey is sufficient to demonstrate a great variation in
the relative size of nuclei in coelenterate eggs, and I have under-
taken to test Jérgensen’s suggestion. Figures 16 to 30 are the
outlines of a number of eggs with their nuclei, accurately drawn
to the same scale, all representing eggs at the end of the growth
period before the prophase of maturation mitoses. Figure 15
is a similar representation of a starfish egg of the same stage,
introduced for the sake of comparison. In the accompanying
table these eggs are arranged in order, the one with the relatively
largest nucleus heading the list. Since the nuclei are not always
perfect spheres, and the eggs depart even more from a true
spherical form, the figures given in the table for the diameters
are averages of the greatest and least diameter of both struc-
tures. The measurements, in millimeters, were made from pro-
jected images; if each average is multiplied by 1000 and divided
by 137 (the magnification of the projected images) the results
will give the average diameters in microns. From_ these

; diameter of e ‘ae
measurements was obtained the ratio = Bs indi-
diameter of nucleus

cated in the third column. The figures of this column, squared,
surface area of egg

give the ratio , and the same figures,

surface area of nucleus
volume of egg

cubed, furnish the ratio (this computation is

volume of nucleus
given in the last column of the table). The actual volumes are
not important, the relative volumes being the thing desired.
Some inaccuracies result from the computations based upon
formulae for surface and volume of true spheres, but it is believed
these are not great enough seriously to disturb the order given
in the table. These figures also represent measurements and
ratios for particular eggs, and are not of the nature of constants;
there is variation in size of eggs of the same species, but this,
again, is not of such magnitude as to modify the table greatly.

GERM CELLS OF COELENTERATES 35

Table of measurements and computations of relative sizes of various coelenterate
eggs, and their nuclei. Diameters, in millimeters, are made from projected
images of the eggs and nuclei; these multiplied by 1000 and divided by 137 will
give the diameters in microns. The diameters represent the average diameter of
the egg and nucleus, since often these are not perfectly spherical

AVERAGE | AVERAGE | . nrAMETER EGG VOLUME EGG
woes ieee Nas pee ak ata DIAMETER NUCLEUS; VOLUME NUCLEUS

5, al) SUAMMB MR ee oi ectn ec ys 6.0 12.0 2.0 8.0

16 | Nausithoé punctata.... 8.0 20.0 225 15.625
17 | Hydractinia echinata...} 9.0 23.0 2.555 16.581
18 | Pelagia noctiluca....... 11.0 30.0 2.1212 20.153
MOONE MAIS? sys mini oe 3 6.0 18.0 3.00 27.0

20 | Aglantha digitalis......}| 5.0 15.0 3.0 27.0

21 | Campanularia flexuosa. 7.0 22.0 3.1428 30.957
22 | Gonothyraea loveni....| 4.0 14.0 3.9 42.875
23 | Aurelia flavidula....... 5.0 18.0 3.6 52.656
24 | Clava leptostyla....... 4.3 18.0 4.186 72.930
25 | Corymorpha pendula...| 6.0 39.5 6.583 284.848
Pie edly Spit) ne. aha atcha: 7.0 47.0 6.714 302.6469
27 | Eudendriumramosum..| 4.0 31-5 7.875 488 .058
28 | Pennaria tiarella....... 3.3 32.5 9.848 955.088
29 | Hybocodon prolifer.....| 4.0 58.5 14.625 3122.794
30 | Tubularia crocea.......| 3.0 54.5 18.166 5994.8435

Very obviously the table is divided into two parts, 16 to 24
represent eggs with relatively large nuclei, and 25 to 30 have
distinctly smaller nuclei. Within each group there is a rather
marked gradation, but between the groups a noticeable gap.
The relation between the volumes of nuclei and cytoplasm may
be expressed in another way. In the first lot the egg volume
exceeds the nuclear volume by from 15 to 73 times, but the eggs
of the second group are from 284 to nearly 6000 times the volumes
of their nuclei. Each egg of the first lot obtains its nourishment
from the enteric cavity, from which it is separated by a single
layer of cells; the eggs of the second lot (except 27) absorb the
surrounding oocytes and ova and appear to depend upon these
almost entirely for their food supply. Eudendrium (27), in size
of nucleus, belongs to the second series, but does not absorb
oocytes; however, its gonophores are adapted to serve as nourish-
ing organs, and the cells of these are later absorbed, so it may
properly be placed in the second series instead of the first.

36 GEORGE T. HARGITT

These fifteen coelenterate eggs support the claim of Jérgensen,
or at any rate are consistent with his suggestion of the relation
between the mode of nourishment of the egg and the size of the
nucleus. Perhaps this agreement is incidental, for there are some
objections to Jérgensen’s views. His suggestion implies a pas-
sivity of the nucleus in eggs whose nourishment comes from
absorbed ova. I believe, in these as in the others, there is an
exchange of material between nucleus and cytoplasm of growing
eggs, for there is evidence of the passage of chromatin into the
cytoplasm of these eggs during growth. Nor does it seem prob-
able that the nuclei of accessory cells could have anything to do
in directing the growth processes, for in coelenterates these cells
are absorbed and their nuclei may undergo a degeneration before
absorption. All the facts sustain the belief that the nuclei of
growing eggs are responsible for the direction of the functional
activities of these cells. To this extent, at any rate, Jorgensen
is probably incorrect in his interpretation. I think it quite
probable that some relation may exist between the method of
nourishment and the relative size of the nucleus, and the figures
of the table may be an expression of this relation.

3. Cytoplasmic inclusions

In the cytoplasm of growing coelenterate eggs certain bodies
occur as characteristic structures. These inclusions, described _
by the author (13 to 718) as of nuclear origin, appear to be
correlated with the growth processes, either furnishing the stimu-
lus to growth or in some way determining the course and extent
of growth. Similar bodies are present in germ cells of other
animals at corresponding periods, but there is disagreement
regarding their origin and function. Without doubt, some of
the difference of opinion is due to the presence of cytoplasmic
inclusions of different sorts, both as to origin and as to function.
This is clearly established by the work of Cowdry (16) and
other recent writers.

In Campanularia the cytoplasmic bodies in the egg are formed
from the dissolving nucleolus and passed through the nuclear
wall into the cytoplasm, where they participate in the formation

GERM CELLS OF COELENTERATES 3o7

of yolk. While growth begins before such bodies occur, the
period of rapid growth is coincident with the passage of nuclear
matter into the cytoplasm. The nucleolus is partly chromatic,
and the bodies in the cytoplasm derived from the nucleolus also
contain chromatin. Clava shows essentially the same phe-
nomena, but the chromatin which passes into the cytoplasm
appears earlier and comes from the nuclear reticulum, the
nucleolus being a true plasmosome. After the chromatin enters
the cytoplasm of Clava, growth begins. Growth begins in
Aglantha shortly before nuclear substances enter the egg, or at
least before definite cytoplasmic bodies can be recognized. In
this egg it is not possible to determine the fate of the chromatin
particles, except for their rapid solution within the cytoplasm,
nor whether they have any close relation to cell metabolism. In
the egg plasm of Hybocodon, chromatin granules appear before
the growth of the oocyte begins; this migration of chromatin is
abundant during early growth, but soon ceases, and the particles
dissolve within the cytoplasm. Eudendrium shows similar in-
clusions in oocytes as growth begins, and they continue to form
abundantly during practically the whole of the growth period.
They are apparently of chromatic nature.

The interpretation of these cytoplasmic inclusions involves,
chiefly, the consideration of their origin. Do such bodies arise,
in the place where they first appear, out of materials of the
cytoplasm, or do they represent nuclear substances in the
cytoplasm? If the latter be the case, are the bodies composed
of chromatin or of achromatic material? Bodies of cytoplasmic
origin have commonly been called mitochondria, those believed
to be chromatic in nature are sometimes referred to as chromidia.
Tests seem to have demonstrated the reality and difference of
these two classes of inclusions, for Cowdry (716) believes, ‘‘we
have ample evidence that the chromidial substance (Nissl sub-
stance) is a nucleoprotein containing iron . . ._., formed at
least in part through the activity of the nucleus, and the
mitochondria is a phospholipin albumin complex.”

The granulations in the egg cells of the described coelenterates
are certainly not mitochondria, though typical mitochondria

38 GEORGE T. HARGITT

have been found in such cells, and no doubt are present in these.
Their size, position, time and place of appearance, staining
reactions, all seem to distinguish them as extruded nuclear
material. They are present in young oocytes at the beginning
of growth, and sometimes in later growth stages. ‘They appear,
in all cases, first, in the region of the nucleus, usually directly
against the nuclear membrane; their appearance is often corre-
lated with signs of activity within the nucleus and indications of
currents in the cytoplasm; they stain like chromatin. Within
the cytoplasm it is practically universal for them to lie within
vacuoles, while other granules are commonly not so situated.
In this latter respect they seem to produce a vacuolation or
liquefaction of the surrounding cytoplasm in the same manner as
Lillie (02) described for chromatin particles which are free in
the cytoplasm.

Jorgensen (’10) found a relation between egg growth and the
presence of chromatin particles in sponge eggs; Schaxel, an
emission of chromatin into the cytoplasm of coelenterates (’10 a,
711 a), Ascidia (10 b), and echinoderms (’11 b); and the acti-
vation of the cytoplasm upon the entrance of the chromatin.
Schaxel (’11 ¢) finds the mitochondria (chondriosomes) present
in practically all cells at all times, while the extra nuclear chromatin
(chromidia) occurs only at certain times, performs certain func-
tions and disappears. He also recounts differences in appear-
ance and staining reactions of the two sorts of bodies. Tsuka-
guchi (’14), using Altmann’s technique upon Aurelia eggs,
believes Schaxel to be in error, and considers all cytoplasmic
granules as mitochondria. But the behavior of the bodies he
investigated, especially their disappearance in later growth, is
not like the usual behavior of the mitochondria.

Beckwith (14) discusses the origin of the plasma structure of
one of the hydroid eggs, and observes basically staining bodies,
which she calls ‘pseudochromatin-granules,’ scattered through
the cytoplasm. She also observed a second plasma granulation,
“large drop-like masses which appear near the nuclear wall and
which are also probably not chromatin;’” these also are stained
with nuclear dyes. Various stains were tried, and it was common

GERM CELLS OF COELENTERATES 39

to find the nucleus and cytoplasmic granules staining alike, but
some vital dyes gave a difference in staining reaction. If young
eggs were digested in pepsin, the nucleus and the cytoplasmic
granules were unaffected. Beckwith clearly points out the lack
of precision in selective staining, but believes her evidence shows
the non-chromatic character of the protoplasmic granules. ‘‘In
all cases which seem to indicate the contrary conclusion (some
staining and digestive tests and tests for proteid) the results
can be interpreted in some other way.” ‘This author believes
the contrary conclusions of Smallwood, Schaxel, and others are
due to faulty technique. Differences in technique may un-
doubtedly account for difference in appearance, but it would
appear rather improbable that these investigators, in addition
to others not mentioned, all working independently and by
different methods and arriving at similar conclusions, should not
have worked out a reasonably satisfactory technique and should
have been unable to distinguish between artifacts and real
structures. It is permissible for Beckwith to differ in her in-
terpretation of observed facts, but not to attack the methods
of those who differ in this interpretation, with no more grounds
than she offers. According to Beckwith herself, the evidence
implies that these other authors were correct in interpretation;
the weight of evidence of her own observations supports their
contention of the chromatic character of the protoplasmic bodies
under discussion, for she says, ‘‘the balance of the evidence

indicates the non-chromatic nature of the granules
in question.” I do not believe the balance of her evidence
outweighs the evidence in the other direction.

Jorgensen (713) discounts his own earlier work on sponges, all
of Schaxel’s work, the work of Goldschmidt, Montgomery, and
others, so far as they relate to questions like the present one.
He believes undue weight has been placed upon staining reactions;
it is necessary, in his opinion, to identify nucleic acid in plasm
granules in order to show their chromatic origin. Pepsin digestion
experiments convinced him of the presence of nucleic acid com-
pounds in the cytoplasm of some eggs, and he admits the occa-
sional migration of chromatin from nuclei, but he thinks this is

40 GEORGE T. HARGITT

of no significance where it occurs. J6rgensen finds chromatin
stains and mitochondrial stains and technique to be very uncer-
tain, and neither of these, or any other staining method, is to
be depended on, since they do not differentiate bodies of diverse
origin and chemical composition.

An even stronger criticism of our staining methods and all
microchemical tests is made by van Herwerden (13). Our
technique, she holds, is so primitive as to be useless in the identi-
fication of chromatin; evidence from stained, fixed preparations
is not valid; action of weak or strong alkalis or acids does not
give satisfactory results; digestion by pepsin and trypsin leads
to no intelligible information; none of the usual tests are of any
great service. This author uses nuclease as an enzyme in
digestion experiments to test for chromatin (nucleic acid content)
in the basic cytoplasmic granules of echinoderm eggs. Using
ripe eggs, very simple experiments demonstrated the basophile
granules of the cytoplasm to ‘‘consist of a nucleic acid com-
pound.” In younger oocytes, where chromidia had been de-
scribed against the nuclear membrane, the nuclease experiments
show the presence of nucleic acid compounds. Van Herwerden
is somewhat doubtful as to the origin of these chromatin particles
and hesitates to interpret it as a migration of chromatin from the
nucleus. However, by observing living oocytes of Sphaerechinus,
she could follow a movement of refractile granules to the nuclear
membrane where they disappeared, and at the same time granules
appeared in the cytoplasm close to the nuclear wall. Van Her-
werden concludes that there is a possibility of the diffusion of
nucleic acid compounds from the nucleus into the cytoplasm,
but no direct proof of this. I suppose, in the very nature of the
process, one could not expect to secure absolute proof of this
passage, but van Herwerden seems to have obtained evidence
which renders such diffusion highly probable. In all experi-
ments with nuclease, the chromatin of the nucleus was affected
in the same way (though to a much less degree) as the basophile
granules of the cytoplasm. From the experiments and observa-
tions of van Herwerden there would appear to be ample warrant
for the belief that nuclear material passes from the nucleus into

GERM CELLS OF COELENTERATES 41

the cytoplasm of growing eggs; in other words the morphological
conclusions appear to be supported by experimental results.
Outside the forms already mentioned, the insects are described
as showing a passage of chromatin into the cytoplasm. Wassi-
lieff (’07) finds the nebenkern of the cockroach spermatid has
come from chromatin of the nucleus by a diffusion through the
membrane. -Hegner (’15), in the honey-bee and carpenter-ant,
thinks the oocyte nuclei give off chromatin, which appears in °
the cytoplasm of fixed eggs as granules. In echinoderms Dancha-
koff (16) finds basic granules, indications of cytoplasmic move-
ments, and other conditions similar to those described by the
author (713) for Campanularia, but believes these mark the pas-
sage of basic material of the cytoplasm into the nucleus, where
it becomes differentiated and helps to form chromosomes.
There are abundant records in the literature of the presence
of basophile granules in the cytoplasm of eggs and other cells of
animals. These have been observed and studied by cytologists,
following their usual technique and have been interpreted in
accordance with the morphological appearance; relatively few
attempts having been made to check these by chemical or
physiological tests. It would appear from some of the recent
work that staining reactions are much less specific and selective
than has been assumed; conclusions drawn from stained material,
therefore, would have little significance and would be misleading,
since morphological structures of a very diverse chemical com-
position and varied functions may stain alike. From this point
of view, all interpretations based upon staining are of little value
until they have been checked by appropriate chemical or physio-
logical tests. I believe there is a large element of truth in these
criticisms, and we have probably gone to an extreme in inter-
pretations based upon purely morphological studies. For present
purposes we are very fortunate to have had such a test of baso-
phile granules of echinoderm eggs, with an application of these
to the chromidial hypothesis of Goldschmidt and Schaxel. This
hypothesis is not entirely substantiated by van Herwerden, and
some of the ‘chromidial apparatus’ described for echinoderms is
believed to be artificial. But the fundamental principle of the

42 GEORGE T. HARGITT

theory is confirmed, viz., that basic granules in the cytoplasm
contain nucleic acid components, which are similar to the
nucleic acid compounds within the nucleus. Moreover, it ap-
pears quite probable that this cytoplasmic nucleic acid has come
from the nucleus, van Herwerden having followed a nuclear
emission in living echinoderm eggs. From this evidence we are
warranted in believing that the passage of chromatic material
' (nucleic acid compounds) into the cytoplasm is a reality. Ac-
cording to the tests on echinoderms, it is the basophile granules
near the nuclear wall in young oocytes which represent this
material; probably the similarly placed granules in the coelen-
terate eggs are the same substance.

The determination of the functions of these bodies is not so
simple, and there is a good deal of difference in interpretation.
Hegner believes the chromatin bodies in egg cells are germ-cell
determinants; Goldschmidt thinks they represent the chromatin
which is responsible for all the vegetative functions of the germ
cells; Schaxel looks upon them as regulating some of the cell
functions, but not governing all vegetative activities; the author
has held the view that they are related to yolk production, and
possibly have an enzyme action in stimulating growth and
synthesis of reserve food in eggs. Others view these bodies as
of no significance in cell metabolism. If they play a single
definite part in the cell metabolism, further work is necessary
for a decision. My own impression would lead me to discard
the view of a total absence of any significance.

V. CHROMOSOMES

The maturation phenomena, characteristic of germ cells, are
_ exhibited by both male and female germ cells of coelenterates.
In the egg cells polar bodies are formed by means of mitosis, and
a reduced number of chromosomes remain in the egg. This
reduction apparently takes place at the beginning of the growth
of the oocyte, and evidence is not lacking of a conjugation of
chromosomes. The coelenterates do not appear to offer material
favorable for the determination of the method by which such
conjugation is accomplished. Differences are noticeable in such

GERM CELLS OF COELENTERATES 43

details as the form of spindle, distinctness of chromosomes, and
the like, but the principles involved are those characteristic of
similar phases in germ cells generally. In some instances con-
ditions are found which have been interpreted as synizesis, in
other cases such phases were not found. The coelenterates do
not, therefore, add anything definite to the evidence concerning
the normality of this process.

While the chromosomes appear to show a characteristic be-
havior, they are lacking in the variety of form and size which
obtains in the chromosomes of some animals. In most coelen-
terates whose chromosomes have been studied, there is a simi-
larity which renders it very difficult even to identify synaptic
mates in maturation mitoses. Of the forms studied by the
author only Aglantha had chromosomes which offered a reason-
able opportunity for a study of details. Oogonial chromosomes
did not, however, readily lend themselves to a grouping into
homologous pairs. Some doubt was expressed as to whether
these chromosomes behaved in quite the fashion believed to be
characteristic and typical of maturation mitoses. The evidence
is not sufficient to warrant any definite conclusions of a differ-
ence in the chromosome behavior of the coelenterates.

The question of the individuality and continuity of the
chromosomes has been in mind during the study of the coelen-
terate germ cells. On one point the evidence is clear. During
interkinesis there is no indication of the persistence of the
chromosomes, the ‘resting nucleus’ is typically a single vesicle
clearly without division into smaller vesicles. In certain forms
chromosomal vesicles are produced after maturation or cleavage
mitoses, but it is very common for two or more chromosomes to
form a single vesicle. In any event, if the period of inter-
kinesis is long, these vesicles unite into a single one. On the
matter of the maintenance of chromosome individuality during
interkinesis Wilson (713) says:

Some of the most careful recent cytological studies in this direction
seem to show that such is not the case. Nevertheless these same
studies, together with recent experimental evidence, give very strong
ground for the conclusion that a definite relation of genetic continuity

exists between the individual chromosomes of successive generations
of cells.

44 GEORGE T. HARGITT

The evidence obtained from coelenterate eggs would not
permit one to dissent from this view. In the absence of contrary
evidence in this group, the evidence from other groups would
lead me to agree that there is no reason to believe the coelen-
terates differ in this regard.

Recently Robertson (’16), McClung (717), and others have
expressed a-more radical view. Robertson believes the chro-
mosomes are ‘individually indentical’ in succeeding genera-
tions and ‘persist as entities’ from one cell division to another.
McClung is likewise convinced that each chromsome persists as
a distinct structure; during interkinesis the chromosome may
extend its boundaries and diffuse its substance, but each body
retains just as precise a limit (though it is usually unrecognizable)
during this period as it does during its stay in the usual form.
This is a return to the older view of a distinct morphological in-
dividuality which Wilson and others have abandoned. McClung
says of the chromosomes, ‘‘either they actually persist as dis-
crete units of extremely variable form, or they are entirely
lost as individual entities and are reconstituted by some ex-
trinsic agency.” It is quite unwarranted to state that extrinsic
agencies are all that can explain a reintegration of chromosomes
under these conditions. McClung gives us a very valuable
critique of chromosome individuality, and, in his chief argu-
ments, makes use of analogies between chromosomes and other
organic behavior. The restitution of the normal form in re-
generation and the production of a typical adult form by -
developing eggs are due to internal organization and not to
‘some extrinsic agency.’ On the same basis, the restitution of
chromosome form is scarcely to be ascribed to external agen-
cies, even if there have been a loss of identity in interkinesis.
McClung contrasts organization with lack of organization in
urging a persistent and continuous individuality, but organiza-
tion does not involve preformation, as his discussion assumes.

In discussing chromosomal relationships Payne (716) says:
“‘TIt seems to me it is time we were realizing that evolution of
chromosomes as morphological units, in chromosome numbers,
and in chromosome behavior has been as diverse as it has been in

~

GERM CELLS OF COELENTERATES 45

external morphological characters.’”?’ Nor have we any reason
to believe this evolution has ceased. It is quite conceivable that
the chromosomes may be tending toward a persistence throughout
the entire life of the cell in all its changes, and in some cases may
now be distinguishable in interkinesis as well as in mitosis. But
the evidence does not warrant a belief in such a continuity as
Robertson and McClung postulate for all chromosomes of all
organisms. The work of Hance (717) furnishes him with no
evidence of a persistence of individuality during interkinesis,
-and he can only subscribe to such a view by broadening the
present concept. That is, he believes the chromatin particles
may persist from generation to generation, but the bodies which
they form do not persist. This view could hardly be tested,
since we are without means of identifying or following particular
chromatin particles at the present state of our technique. If
such a belief could be confirmed, we should have a chromatin
individuality hypothesis which would be without many of the
objections of the present one.

So far as coelenterate chromosomes are concerned, there is
nothing to disprove the view that the chromosomes of one
generation are descended from the chromosomes of a previous
generation. All the evidence obtainable, however, is quite in-
consistent with the view of the persistence of chromosomes as
distinct entities during interkinesis. A genetic continuity is
very probable, a morphological continuity is highly improbable.

VI. SUMMARY AND CONCLUSIONS

In the Scyphozoa and Actinozoa all observations point to the
entodermal origin of germ cells. The former widespread belief
in the ectodermal origin of germ cells in Hydrozoa cannot be
maintained, for literature records show a greater number of
species whose germ cells arise in the entoderm than of those in
which the ectoderm produces them. The germ cells of Hydrozoa
may originate in either or both germ layers; the same individual
may even produce germ cells from both ectoderm and entoderm.
There is no characteristic place of germ-cell differentiation in
this class.

46 GEORGE T. HARGITT

The germ cells of some animals have been observed to form
relatively early in ontogeny, but such is not the case in coelen-
terates. It has been claimed by some writers that in Hydra
and a few hydroids germ cells are differentiated in the larvae.
This claim has been refuted by later studies upon Hydra, and
a new investigation of larval stages of hydroids furnishes no
evidence of an early differentiation of germ cells in those forms.
Furthermore, there are a number of the Hydrozoa whose germ
cells have been observed to arise directly from differentiated
body cells. This happens either by the transformation of an
entire epithelial cell into a germ cell or by the division of a body
cell and the transformation of one of these division products
into a germ cell. In the latter case the sister cell persists as a
functional tissue cell. In at least ten species of eight different
genera the germ cells have been observed to form in this way.
This positive evidence, together with the refutation of all con-
trary claims, points to a single conclusion, viz., in the Hydrozoa
(probably also in all coelenterates) germ cells are not dif-
ferentiated in early ontogeny, but only much later as the time
of sexual maturity is at hand.

The theory of the continuity of the germ plasm postulates
the formation of a somatic blastomere and a germinal blasto-
mere at the first cleavage of the egg; in no animal is such a result
known. As applied to hydroids, the theory originally admitted
the origin of germ cells from histologically differentiated somatic
cells, but invoked the aid of invisible and unrecognizable germ
substance lying latent in such body cells. The production of a
germ cell by the transformation of half a tissue cell, and the
persistence of the other half as a tissue cell, is sufficient to
disprove the claim of the presence of an invisible germ plasm in
such tissue cells. This fact, together with the origin of germ
cells only as sexual maturity approaches, indicates a lack of
continuity of the germ plasm in the coelenterates.

As explained by the germ-plasm theory, budding is always due
to the presence of latent germ cells. But budding in Hydra
and hydroids involves the activity of all the layers of the
budding zone; in Hydra it is possible that interstitial cells first

GERM CELLS OF COELENTERATES AZ

become active, but there is no evidence that germ cells are
present. The budding phenomena of medusae resemble the
same processes in hydroids, since, in most cases, both body
layers evaginate to form the bud. Ina few forms buds are pro-
duced from the gonads of the parent medusa, but even here this is
not a germinal process, for the buds are formed from all layers of
the animal; the germ cells of the gonad may participate in the
process, but only by behaving as tissue cells. A few medusae form
their buds from the ectoderm alone, and one investigator claims
that the bud originates from a group of oocytes, though he admits
this is a very unusual method, not applicable to most buds in
coelenterates. In no case are buds known to arise from a single
cell. While it may be possible, therefore, that budding is occa-
sionally a germinal process in medusae, this is rare; as an alter-
native explanation, other investigators believe both germ cells
and tissue cells are able to undergo regressive changes and
become embryonic. The embryonic cells have the ability to
form a new organism. The latter explanation would correlate
various types of reproduction, both sexual and asexual, in
coelenterates; would correlate fission and budding in coelenterates
and other groups of animals, and would outline a possible evo-
lution of reproductive processes in coelenterates. The germ-
plasm theory, therefore, may be held not to apply to budding in
coelenterates, for it is contradictory to most of the facts of this
phenomenon.

Regeneration is also held to be dependent upon the presence
of latent germ cells. There seems to be no direct evidence in
favor of this view, and the great body of facts concerning re-
generation in many phyla of animals contradict such an inter-
pretation. Especially do the observations upon regeneration
from isolated cells of hydroids disprove the germ-plasm theory.
When hydroid tissues are broken up into isolated cells the latter
undergo a despecialization and fuse to form syncytia. From
these masses complete and normal hydranths are regenerated.
When germ cells are present they are absorbed as food, and
take no part in the regenerative processes. The behavior of
the isolated cells has been followed with the microscope ‘and

JOURNAL OF MORPHOLOGY, VOL. 33, NO. 1

48 GEORGE T. HARGITT

sections made of the regenerative plasmodia. All the facts
point to the totipotency of the tissue cells under such stimulus.

In some animals of different phyla reproductive organs are
present only during the breeding season, and at other periods no
germ cells can be recognized. In such cases the germ cells
must be differentiated from the tissues of the region which gives
rise to the reproductive organs. ‘There is no evidence of a con-
tinuity of the germ plasm in these animals. In the vertebrates,
especially in mammals, recent observations point to the degen-
eration of all germ cells which are formed during foetal life;
the definitive germ cells are differentiated from the germinal
epithelium after birth.

Pieces of tissue removed from the body will grow in culture
fluids, under certain conditions. In some cases the new growths
from this explanted tissue are embryonic in character, due to a
despecialization of the old differentiated tissues. Cancers, de-
veloped from tissues, are composed of cells more embryonic in
character than those from which they arose. These cells may
continue to live, grow, and divide indefinitely. Such observa-
tions indicate a less marked difference between body cells and
germ cells, and a greater plasticity and a more varied potency
in differentiated tissue cells, than has commonly been believed.
Such a weakening of the line of demarkation between these
two categories of cells tends also to weaken the germ-plasm
theory.

So far as the coelenterates are concerned, the observations
upon the time and method of germ-cell origin; upon budding of
all types; upon regeneration of the usual sort, and regeneration
from plasmodia formed by coalescence of isolated cells, all point
in one direction, viz., that there is no germ plasm in the sense
of Weismann. Furthermore, the origin of germ cells In some
phyla other than Coelenterata, the despecialization of differ-
entiated cells, and their behavior in tissue cultures and in normal
development, and the continued growth and division of body
cells in cancers, also present evidence contradicting the germ-
plasm theory. There are so many facts, from such different
~ sources and from so many phyla, which are inconsistent with

GERM CELLS OF COELENTERATES 49

the theory, that it may be questioned whether the theory applies
at all extensively to animals of any phylum.

As a rule, those coelenterate eggs which secure nourishment
from the adjoining enteric cavity have large nuclei; and the
ones which absorb oocytes or other cells possess relatively small
nuclei. Whether this correlation be incidental, or whether it
have a deeper significance, is not known.

Cytoplasmic granules which stain in nuclear dyes are a char-
acteristic feature of coelenterate eggs. Typically, these appear
in young oocytes about the time growth begins, and they may
also form at other times during growth. From their initial posi-
tion, close to the nuclear wall; from their staining reactions;
from the behavior of other cytoplasmic and nuclear substances;
the author has interpreted these granules as chromatin. Ob-
servations by other investigators, upon the eggs of other
animals, have led them to conclude that chromatin does migrate
into the cytoplasm. The criticism that the usual tests for
chromatin are not specific is justified in large measure, but
digestive experiments have demonstrated the presence of nucleic
acid compounds in cytoplasmic granules, similar to those de-
scribed for coelenterate eggs. Also van Herwerden has observed
a migration of nuclear material into the cytoplasm of living
oocytes of Echinoderms. Using these experiments to check the
other observations, it seems probable that the cytoplasmic
bodies described as chromatin do, in fact, represent this sub-
stance. There is considerable diversity of opinion as to the
functions of these inclusions, and further work is necessary to
determine this with certainty.

The chromosomes of most coelenterates do not lend themselves
to a study of details of behavior to the degree possible in some
animals. This is due, chiefly, to the lack of variety in form and
size. It is not possible, therefore, to determine whether the
chromosomes reappear in each generation in precisely the same
form and size they had in earlier generations. During inter-
kinesis the nucleus is a single vesicle with no subdivisions into
smaller vesicles, and the chromatin is in the form of a continuous
reticulum.

50 GEORGE T. HARGITT

Since there is no evidence to disprove the view that chromo-
somes are genetically related, this may be accepted for the
coelenterates. But all the evidence from this phylum is opposed
to the view of a persistent morphological continuity and an
individuality of the chromosomes retained during interkinesis.

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Witson, E. B. 1913 Heredity and microscopical research. Science, N. S.,
vol. 37.

Witson, H. V. 1907 On some phenomena of coalescence and regeneration in
sponges. Jour. Exp. Zodl., vol. 5.
1911 On the behavior of the dissociated cells in hydroids, Aleyonaria
and Asterias. Jour. Exp. Zodél., vol. 11.

von WINIWARTER, H., AnD Sarnmont, G. 1908 Uber die ausschliesslich post-
fetale Bildung der definitiven Hier bei der Katze. Anat. Anz., Bd. 32.

Witscut, Emin 1914 Experimentelle Untersuchungen iiber die Entwicklungs-
geschichte der Keimdriisen von Rana temporaria. Arch. f. mikr.
Anat., Bd. 85, Abth. 2.

Woutrert, J. 1902 Die Embryonalentwicklung von Gonothyraea loveni Allm.
Zeitschr. f. wiss. Zool., Bd. 71.

EXPLANATION OF PLATES

All figures of plates 1 and 3 were made by the aid of a camera lucida. These
plates have been reduced in reproduction to three-quarters the original size;
the magnifications given for each figure are the actual magnifications as repro-
duced. The figures of plate 2 are copied from the sources indicated.

PLATE 1
EXPLANATION OF FIGURES

1 to 4, Campanularia flexuosa, approximately X 1150; 5 to 8, Tubularia crocea,
X 620.

1 Young planula with coelenteron present. Primitive ectoderm and ento-
derm present, but cells walls are to be detected in only a few places. Both
ectoderm and entoderm are filled with yolk spheres.

2 Older planula with walls becoming more plainly marked. There are few
interstitial cells in the ectoderm, but a number are present in the entoderm. The
cell with the large nucleus may be like Wulfert’s germ cell in Gonothyraea,
but the nucleus is similar to that of other entodermcells. Some of the interstitial
cells are differentiating into gland cells, muscle cells, and the like. No germ
cells are present.

3 Same planula as figure 2, showing only a portion of the entoderm. A group
of typical interstitial cells is represented.

4 A still older planula with definitive ectoderm and entoderm. There are -
fewer entodermal interstitial cells than in earlier stages. The interstitial cells
are undergoing differentiation, but no germ cells are present.

5 Section of egg about the end of cleavage. The germ layers have not been
separated. The cell in division may be forming an ectodermal and an interstitial
cell.

6 Embryo with a definite outer layer of cells and a solid central mass of cells.
Two interstitial cells have been produced, one of which was formed by the
division of a cell of the central mass.

7 A later embryo with cubical ectodermal cells, and groups of intersitial
cells. One of the ectoderm cells is dividing to form an interstitial cell. None
of these interstitial cells form germ cells at this time.

8 An embryo with coelenteron, about the period of the formation of tentacles
and the production of an actinula. The ectoderm and entoderm cells are dis-
tinectly separated by a supporting lamella. Groups of interstitial cells are
present and others are forming from the ectoderm. These interstitials form
nematocysts and other structures, but not germ cells.

PLATE 1

GERM CELLS OF COELENTERATES

GEORGE T. HARGITT

Proboseidactyla orn

PLATE 2
EXPLANATION OF FIGURES

ata. From Mayer, vol. 1, plate 21, fig. 5.

Sarsia gemmifera. After Chun, from Mayer, vol. 1, p. 63.
Niobia dendrotentaculata. From Mayer, vol. 1, p. 187, plate 19, fig. 2.
Eucheilota paradoxica. From Mayer, plate 37, fig. 3.

Sarsia codonophora.
Cytaeis atlantica.

After Haeckel, from Mayer, vol. 1, p. 61.
After Haeckel, from Mayer, vol. 1, p. 134.

56

PLATE 2

GERM CELLS OF COELENTERATES
GEORGE T

GE T. HARGITT

PLATE 3
EXPLANATION OF FIGURES

Drawing of eggs of coelenterates showing form and size of egg and nucleus at
the end of the growth period. The eggs are arranged in the order of the relative
volume of nucleus and egg. All drawn to the same scale, X 103.

15 Starfish egg, introduced for comparison of relative size and volume of
egg and nucleus, with coelenterate eggs.

16 Nausithoé punctata.

17 Hydractinia echinata.

18 Pelagia noctiluea.

19 Obelia sp?

20 Aglantha digitalis.

21 Campanularia flexuosa.

22 Gonothyraea loveni.

Aurelia flavidula.
Clava leptostyla.
Corymorpha pendula.

bo bd po
OU HS OO

26 Hydra sp?

27 Eudendrium ramosum.
28 Pennaria tiarella.

29 Hybocodon prolifer.
30

Tubularia crocea.

GERM CELLS OF COELENTERATES PLATE 3
GEORGE T, HARGITT

’: See

15 16

20 21 22 23 24
25
26
28
ae

30

59

Resumen por el autor, Carl L. Hubbs,
Universidad de Illinois.

Estudio comparado de los huesos que forman la serie opercular
en los peces.

Aunque la estructura y disposicién de los huesos que forman
la serie opercular de los peces han sido descritas en ciertos grupos
por muchos anatémicos, ninguno parece haber consolidado
todavia las pruebas obtenidas, mediante un estudia comparado.
Después de examinar la estructura de estos huesos en una extensa
serie de peces, el autor ha llegado a la conclusién de que son de
tipo diferente en los Malacopterigios mds primitivos, por una
parte, y en los Acantopterigios mas especializados y sus parientes
mds préximos, porotra. Los Isospondyli, el grupo mas primitivo
de peces teleédsteos (tal como se definen generalmente), tienen
places operculares y radios branquiéstegos bastante semejantes
a los de Amiatus, que bajo este aspecto como en tantos otros,
constituye la forma de transicién que llena el hueco existente
entre los Ganoideos y Teleésteos. En los otros grupos’ princi-
pales de peces con radios blandos (Ostariophysi, Stomiatoidea,
Apodes, Heteromi, Lyopomi, Synentognathi, Haplomi, Iniomi)
los opérculos y los radios branquidéstegos son de tipos derivables
aparentemente de los presentes en los Isospondyli. Hay sin
embargo una extensa variacién en la forma y disposicién de
estos huesos, en armonia con su posicién generalizada. .Los
grupos mas especializados de los teleésteos, por otra parte,
(los Microcyprini, Labyrinthici, Hemibranchii, Symbranchii,
Opisthomi, Salmoperecae y el vasto conjunto comprendido en los
Percoidea o relacionados con este grupo) retienen constantemente
una disposicién peculiar fija en los radios branquiéstegos.

Translation by José F. Nonidez
Carnegie Institution of Washington

AUTHOR'S ARSTRACT OF THIS PAPER ISSUED
BY THE BIBLIOGRAPHIC SERVICE, DECEMBER 15

A COMPARATIVE STUDY OF THE BONES FORMING
THE OPERCULAR SERIES OF FISHES

CARL L. HUBBS

Although the structure and arrangement of the bones com-
prising the opercular series of certain species or groups of fishes
have been described by many anatomists, no one seems to have
consolidated the evidence in a comparative study. After ex-
amining their structure in a wide range of fishes, the writer has
concluded that they are of a different type in the more primitive
malacopterygians, on the one hand, and the specialized acanthop-
terygians and their relatives, on the other hand. The Isos-
pondyli, the most primitive of the teleosts (as usually defined),
have opercular plates and branchiostegal rays similar to those of
Amia, which, in this respect as in many others, bridges the gap
between ganoids and teleosts. In the other chief groups of soft-
rayed fishes—Ostariophysi, Stomiatoidea, Apodes, Heteromi,
Lyopomi, Synentognathi, Haplomi, Iniomi—the opercula and
branchiostegals are of types apparently derivable from that of
the Isospondyli. There is a wide variation in the form and
arrangement of these bones in these groups, however, as one
might expect from their generalized nature. In the higher
groups of teleosts, on the other hand—the Microcyprini, Laby-
rinthici, Hemibranchii, Symbranchii, Opisthomi, Salmopercae,
and in that vast assemblage of modern types comprising in the
Percoidea, or clustering about that group—there is maintained a
peculiarly constant arrangement of the branchiostegalrays. From
a taxonomic standpoint, the results of this study are most sig-
nificant in confirming many of the recent refinements, partic-
ularly those suggested by Mr. C. Tate Regan, in the classifi-
cation of the Teleostel.

The primitive structure of the membrane bones which support
the outer wall of the branchial cavity, and thus protect the

61

62 CARL L. HUBBS

branchiae, is retained in certain extinct groups of ganoids, such
as the Palaeoniscidae. ‘Their structure in this family has been
described by Traquair and others. On each side is a curved series
of homologous, suturally conjoined plates, of which the upper
two or three are dilated to form the opercular bones, while the
lower anterior ones, narrower and less modified, comprise the
branchiostegal rays (or branchiostegals); the lowest or most
anterior pair, enlarged to form the plates which may be termed
the branchigulars (or branchigular plates), are suturally united
posteriorly with one another, and anteriorly with the median
gular plate (or intergular), which extends forward between the
mandibular rami to their symphysis.

In Amia calva the bones of the opercular series are modified
in several respects. Each of the upper five plates is imbricate
over the one next below. The gular plate largely covers the
most anterior branchiostegal rays of the left side, which in turn
overlap those of the right side—a feature due to the definite
asymmetric folding of the branchial membranes, an asymmetry
which is more or less definitely retained throughout the entire
teleost series. The uppermost and largest bone of the opercular
series in Amia is the operculum, a large subquadrate plate, in-
curved and thickened along its dorsal and anterior edges, and
provided inside the anterior edge with an oval socket, into which
a peg-like condyle of the hyomandibular fits, allowing consider-
able lateral movement of the operculum. The suboperculum, the
next lower element of the series, is broadly incised by a downward
extending arm of the operculum; its dorsal edge slips just under
the lower margin of the operculum, while its lower edge is closely
joined by membrane with the triangular interoperculum. The
anterior edges of these three plates fit into a posterior groove of
the preoperculum, which is not regarded as a member of the
opercular series. A free dermal fold connecting the opercular
membrane with the mandible, and extending along the lower
edge of the sub- and interoperculum, may be termed the sub-
opercular fold. The fourth bone of the series, fitting between
the sub- and interoperculum, but widely exposed, being nearly
one-third as wide as long, extends downward and forward, lying
free in a conspicuous fold, continuous with the lower edge of the

BONES FORMING OPERCULAR SERIES OF FISHES 63

mandible. This plate, bearing as much resemblance to the
opercula as to the branchiostegals, may be named the branchio-
perculum; its fold, the branchiopercular fold. The remaining
ten plates of the series are attached to the ceratohyal element of
the hyoid arch, near the lower edge of its outer face. Except for
its pointed, rather than truncate anterior edge, the uppermost of
the ten is similar in form to the branchioperculum, which overlies
its dorsal edge. Both this plate and the next, which is similar,
though only about half as wide, are imbricate on the one next
below, having free folds along their lower edges. The lower
anterior seven branchiostegals form a continuous even surface,
each, except the uppermost, fitting tightly into a groove along
the lower outer face of the one next above; they increase in width
anteroventrally. The most anterior branchiostegal, which is
wider on the left or outer than on the right side, may be homolo-
gous with the branchigular plate of the Palaeoniscidae, or may
represent two or three fused branchiostegals. The current alloca-
tion of Amia in a position intermediate between the typical
ganoids and the teleosts is confirmed by the study of its opercular
and branchiostegal plates.

The Isospondyli, comprising the oldest aa most primitive! of
the teleosts, retain certain generalized features of the opercular
series. Thus, in Elops an intergular plate is developed, and in
Albula, although the plate itself is lacking, the intergular fold
remains. The branchiostegals of the typical Isospondyli (at
least the upper ones), persist as thin wide plates. The uppermost
and widest ray (which may be termed the branchioperculum, as
it seems to be homologous with the plate in Amia to which that
name is here applied) is attached closely to the inner margin of
the sub- and interoperculum; not having become concealed under
these bones, it remains visible from the side. The whole series,
in fact, remaining scarcely at all folded together after the fashion
of a fan, is visible from below,? though the branchial membranes

- 1 Excepting of course Lepidosteus and Amia, if these be included in the
Teleostei.

2 In the clupeoid fishes the expanded preoperculum covers the larger portion
of the middle rays, and all of the rays are mostly concealed in Chirocentrus
dorab.

JOURNAL OF MORPHOLOGY, VOL, 33, NO. 1

64 CARL L. HUBBS

are separate (as they usually are). The plates of the opercular
series in the isospondylous fishes differ from those of Amia in the
following respects: the reduction of the suboperculum, so that the |
interoperculum and operculum are in contact anteriorly;? the
proximal (or anterior) attachment of branchioperculm and branchi-
opercular fold to the hyoid arch; the more complete imbrication
of all the rays; the attachment of branchiostegals to the epihyal
as well as to the ceratohyal; the frequent reduction of the rays
below the main hyoid suture to rather slender rods, and the occa-
sional attachment of these reduced rays to the edge of the cera-
tohyal, rather than to its outer face. These last two features are
apparently caused by the strong development of the musculus
geniohyoideus of the lower jaw, which is attached to the hyoid
arch near the suture separating the ceratohyal from the epihyal.
The number of the larger and flatter rays attached to the outer
surface of the epihyal (the lowermost sometimes on the suture)
varies widely in the Isospondyli and related orders; the writer has
counted one in Bathylagus pacificus; two in Pterothrissus gissu,
Hiodon tergisus, Osmerus thaleichthys, Osmerus attenuatus, Arius
gagora, and Amiurus nebulosus; three in Amphiodon alveoides
Ethmidium maculatum,‘ Alepocephalus agassizii, Coilia ectenes,
and Hypomesus olidus; either three or four in Albula vulpes; four
in Chirocentrus dorab, Salvelinus fontinalis, Osmerus mordax,
Mallotus villosus, Plagyodus ferox, Lestidiops sphyraenopsis,
Bathysaurus ferox, Chlorophthalmus chalybeius, and Neosco-
pelus macrolepidotus; five in Etrumeus micropus, Felichthys
felis, and Dallia pectoralis; six in Oncorhynchus nerka, Saurida
gracilis, and Bathypterois pectoralis; either six or seven in Trachi-
nocephalus myops; seven in Esox lucius, Aulopus japonicus, and
Synodus intermedius; eight in Esox americanus and Synodus

3 According to Woodward’s restoration of the primitive extinct genera Lep-
tolepis and Holcolepis, these genera bridge the gap between Amia and Elops in
the character of the opercula. These bones apparently show no significant
variation among living teleosts, though certain ones are reduced or increased in
size in certain genera; the interoperculum is especially liable to variation, being
occasionally absent.

4 All but the lowermost of the six on the ceratohyal are of similar shape to
those of the epihyal in Ethmidium maculatum.

BONES FORMING OPERCULAR SERIES OF FISHES 65

lucioceps; nine in Elops affinis and Harpodon microchir; ten in
Megalops atlanticus. The total number of branchiostegals is
three in the Cyprinidae and others, twenty-four to thirty-six in
the several species of Elops. Many other figures might be added,
but these are enough to illustrate clearly the inconstancy of the
number of branchiostegal rays in the generalized malacopterygian
fishes.

In the groups of soft-rayed fishes other than the Isospondyl,
the branchiostegals are variable in form and attachment, but
they show many points of similarity to those of the Isospondyli.
In the Ostariophysi the number of rays varies widely, but the
uppermost, at least, remains like that of the isospondylous fishes.
To take several examples from the Nematognathi, there are six
branchiostegals in Arius gagora, seven in Pseudeutropius garna
and Saccobranchus fossilis, nine in Ictalurus punctatus, Amiurus
nebulosus and Shilbe mystus, eleven in Macrones aor. The
characins have only three to five branchiostegals, the cyprinids,.
constantly three. This low number of branchiostegals in certain
malacopterygian fishes is usually correlated with the broad union
of the branchial membranes and with a fresh-water habitat.
Similarly, there are only three branchiostegals in Haplochiton,
Phractolaemus, Kneria, and Cromeria, and but four in the
Gonorhynchidae, Chanidae, and Salangidae (in all of these,
excepting Gonorhynchus,’® the uppermost ray remains visible
below the margins of the opercles). In the Mormyridae and
Notopteridae the branchiostegals are modified in various ways,
as Doctor Ridewood (’04, pp. 191-195, 199, 205) has demonstrated;
in Notopterus there are six to nine branchiostegals, in Xenomystus
but three, according to Boulenger.

The stomiatoid fishes, formerly confused with the Iniomi, have
the branchiostegals short, slender, little curved, evenly spaced,
not folded together, attached to the external surface of the hyoid
arch near its ventral edge (each opposite a photophore), and
largely covered by the opercula. In the Apodes (eels), Heteromi

5 In Gonorhynchus there are four branchiostegals, attached beneath the
opercles on the outer face of the club-shaped end of the hyoid arch, all above the
suture between the ceratohyal and epihyal.

66 CARL L. HUBBS

and Lyopomi, the rays are also all slender, usually numerous and
long, and frequently curved upward posteriorly about the free
margin of the opercular bones. The branchiostegals of the
Synentognathi (Belonidae, Scombresocidae, Hemirhamphidae,
Exocoetidae) are wholly similar to those of the typical Isospon-
dyli; they are rather numerous (ten in Euleptorhamphus), but
not constant in number, flat, imbricate plates; the uppermost
skirting the lower margins of the opercula, and all with their
lower edges exposed. The characters of the branchiostegal rays
of the Synentognathi strongly confirm Regan’s view that the
resemblance between these fishes and the Percesoces is purely
fictitious: the group should be placed among the typical soft-rayed
fishes. In the Haplomi (Esox, Umbra, and Dallia), but not in
the poecilioid fishes which have been confused with them, the
branchiostegals are like those of the Isospondyli. In the Iniomi
(the Synodont fishes and their allies) the branchiostegals vary
greatly in number (from six to twenty, four to eight attached to
the suture between ceratohyal and epihyal, two to twelve below
the suture) ; in Plagyodus the uppermost ray, asin the Isospondyli,
is not wholly concealed, but in most of the genera several of the
upper rays are covered by the opercula; when the rays are numer-
ous several of the upper ones are closely approximated basally.
The group of the ribbon fishes (Taeniosomi) has been accorded
very different positions among fishes, the current tendency being
to place it much lower in the series than formerly, a disposition
of the group which is doubtfully confirmed by the arrangement
of the branchiostegal rays. In Regalecus, according to Parker’s
figure (’86), there are six slender, saber-shaped branchiostegals,
all attached to the outer face of the hyoid arch near its lower
margin; the uppermost, the only one attached to the epihyal,
curving around the lower margin of the interoperculum. In the
still more extremely aberrant genus Stylephorus, as described by
Starks (08), the five rays are inclined upward from their origin
near the upper edge of the ceratohyal, as in no other known fish.
In Trachypterus arcticus, as described by Meek (’90), the branchi-
ostegal rays differ to no considerable degree from those of Rega-
lecus. As in that genus, they are six in number; the uppermost

BONES FORMING OPERCULAR SERIES OF FISHES 67

borders the lower margin of the interoperculum; all seem to arise
from the outer face of the hyoid arch, but the anterior two are
somewhat separated from the upper posterior four, which, unlike
those of Regalecus, are largely covered by the expanded preoper-
culum. In Trachypterus rex-salmonorum the branchiostegals are
concealed by the interoperculum as well, and the lower two rays,
considerably separated from the upper four, are attached to the
outer side of a ligament which extends as a chord across the
concave anteroventral margin of the hyoid arch.

The Ammodytoidea are another group which has been placed
by some ichthyologists among the higher teleosts, by others
among the lower. The branchiostegals in Ammodytes personatus
resemble those of the Acanthopteri in most of their characters:
they are six in number, and are folded up behind the opercula;
the upper four arise from both the cerato- and epihyal behind a
prominent angle of the arch. The lower two rays, however, arise
from the outer surface of the arch, and are closely approximated
to the upper four.

The Microcyprini (Poeciliidae and Amblyopsidae) were long
confused with the Haplomi, but have recently been shown to have
a more advanced organization. The structure of the branchi-
ostegal rays in the two groups confirms this view: those of the
Haplomi are quite like those of the Isospondyli, whereas those of
the Microcyprini are similar to those of the Acanthopteri. In
‘ the Poeciliidae there are six, or fewer, branchiostegals, which are
folded up behind the operculum and above its lower margin. The
upper four saber-shaped rays are attached to the outer surface
of both the ceratohyal and epihyal, postero-superior to the
prominent angle of the hyoid arch; the lower rays arise from the
inner face of the ceratohyal. In examples of the Ophicephalidae
and Anabantidae at hand (representing the order Labyrinthici),
there are four plus two branchiostegal rays, arranged as in the
Microcyprini and Acanthopteri.

Many of the aberrant fishes referred to the order Hemibranchii
have the branchiostegals reduced in number, but in Fistularia
there are four plus one rays, arranged as in typical Acanthopteri.
“Most Lophobranchs have two branchiostegals, but Nerophis has

68 CARL. L. HUBBS

only one which distally bifurcates” (Jungersen, 710). In other
respects also the hyoid apparatus of the Lophobranchii is reduced,
probably from a condition like that of Fistularia.

The Symbranchia were long considered a group of true eels,
but lately have been accorded a distinctly higher position. The
character of the branchiostegals are in harmony with the latter
view. In Monopterus javanensis, the rather narrow hyoid arch
bears two groups of slender branchiostegals: an upper cluster of
four and a lower inner pair, widely separated from the others. An
essentially similar condition is developed in Symbranchus mar-
moratus, but in this species the two groups of branchiostegals are
less widely separated.

The curious Opisthomi (Mastacembelidae) of southern Asia and
Africa have been variously located in the teleost series; lately
Boulenger and Regan agree in placing them among the higher
teleosts, considering them as bearing a relation toward the spiny-
rayed fishes analogous to that which the Apodes bear toward the
soft-rayed group. ‘This view is sustained by the branchiostegals
in Mastacembelus pancelas. From the outer surface near the
lower edge of the ceratohyal and epihyal, along the upper widened
portion of the hyoid arch, four rays arise in close proximity; they
are curved upward posteriorly, as in some of the Apodes, between
the operculum and the branchial aperture; on the inner surface
of the arch, near the concave anterior ventral margin, the two
lower anterior rays are inserted. ;

The Salmopercae, long considered as intermediate between the
soft-rayed and spiny-rayed fishes, have six branchiostegals,
arranged exactly as in the Acanthopteri. Both of the species
usually referred to this group, Percopsis omisco-maycus and
Columbia transmontana, have been examined. Aphredoderus
sayanus, referred by Regan to the same group, has branchiostegals
in all essential respects similar to those of Percopsis and the
following groups.

A definite fixed type of branchiostegal structure has been
retained, almost without deviation, throughout the great groups
of spiny-rayed fishes which flourish so abundantly in the modern
seas, and with peculiar constancy in the numerous highly special-

BONES FORMING OPERCULAR SERIES OF FISHES 69

ized offshoots of the typical Acanthopteri. In fact, it seems safe
to assert that none other of the known characters which separate
this series from the lower teleosts has been more conservatively
maintained throughout the entire group. This statement may
be emphasized by the naming of a few of the more aberrant types
which differ in some notable way—primitive, specialized, or
degenerate—from the group as a whole, yet which agree with
one another and with the more typical members of the series in
the essential characters of their branchiostegal apparatus:
Atherina, Stephanoberyx, Plectrypops, Cepola, Psettus, Toxotes,
Monacanthus, Lactophrys, Tetraodon, Diodon, Agonus, Cyclop-
terus, Cephalacanthus, Echeneis, Solea, Callionymus, Xiphidion,
Seytalina, Gobiesox, Coryphaenoides, Antennarius, Ogcocephalus,
ete. Broad union of the branchial membranes or their complete
separation, membranous or fleshy character of the branchiostegal
membranes, narrow lateral restriction or wide development of
the branchial aperture, and countless other modifications of these
higher teleosts occur—modifications affecting almost every part
and structure of the body, as well as the branchial membranes—
nevertheless, the essential characters of the branchiostegals
remain unaltered.®

The characteristically stout hyoid arch is strongly angulated?
at some distance below and before the (typically) dentate suture
between the ceratohyal and the epihyal, the angle forming the
hinder border of a concavity in which the musculus geniohyoideus
is attached. The strong development of this muscle not only
modifies the form of the hyoid arch, but also modifies the structure
and attachment of the branchiostegal rays, as it also does, usually

6 Certain of the individual rays may become reduced or specialized: for
example, in Tetraodon the uppermost ray basally is an unossified ligament,
while the lowest ray (as in Diodon) is greatly expanded; in Holotrachys the third
to the seventh branchiostegals are strongly armed externally by rows of spinules;
in Polymixia the lower three rays are modified, according to Starks, into a
skeletal support for the barbel.

7 The hyoid arch is also angulated, but in not quite the same way, in a few
of the soft-rayed types, notably in Brevoortia, Dorosoma, Notopterus, and Gono-
rhynchus. In most of the lower teleosts the hyoid arch is a thin plate, and the
suture between the epihyal and the ceratohyal is straight and often margined
with cartilage.

70 CARL L. HUBBS

to a lesser degree and without constancy, in the lower teleosts.
The upper four saber-shaped branchiostegals are always attached
to the outer surface of both epihyal and ceratohyal, at and above
the angle of the arch, and are folded together like a fan above
and behind the opercular margins (except in those cases in which
the branchiostegal membranes are drawn taut by their broad
union ventrally). Below (and before) the angle of the arch, to
its edge or inner surface, usually two or three shorter and slenderer
rays are attached; these may be reduced to one, or, very rarely, ~
to none, and are increased, in certain berycoids and blennioids to
four, but never to a higher number. Thus, the branchiostegals
of the Acanthopteri and related groups are usually four plus two
or four plus three in number, rarely four plus one or four plus
four, and very rarely four plus nought or even three plus nought.®

In formulating the generalizations outlined in the preceding
paragraph, one to many species of each of the families of higher
teleosts, named in the following list, were examined. The
variations in the characters of the branchiostegals rays were found
to be so slight that for present purposes detailed descriptions are
unnecessary.

BIBLIOGRAPHY

BouLEenGcER, G. A. 1909 Catalogue of the fresh-water fishes of Africa in the
British Museum. London.

JUNGERSEN, H. F. E. 1909 On the osteology of the Lophobranchii. Report
Brit. Assoc. Adv. Sci.

Meex, ALEXANDER 1890 On the structure of Trachypterus arcticus. Studies
Dundee College Museum, vol. 1.

Parker, T. J. 1886 Studies in New Zealand ichthyology. I. On the skeleton
of Regalecus argenteus. Trans. Zool. Socy. London, vol. 12, figs. 6
and 15.

RipEwoop, W.G. 1904 On the cranial osteology of the fishes of the families
Mormyridae, Notopteridae, and Hyodontidae. Jour. Linn. Socy.
London (Zool.), vol. 29.

Starks, E.C. 1908 The characters of Atelaxia, a new suborder of fishes. Bull.
Mus. Comp. Zool., vol. 52.

8 A number recorded only for certain cirrhitiform percoids, so far as the writer
has determined.

BONES FORMING OPERCULAR SERIES OF FISHES ra’

A list of the families of the spiny-rayed fishes (Acanthopteri) and their derivatives

examined?

Atherinidae Cepolidae Gobiidae™
Mugilidae Cirrhitidae Echeneidae
Sphyraenidae Embiotocidae Bothidae
Stephanoberycidae Cichlidae Pleuronectidae
Polynemiidae Pomacentridae Soleidae™
Zeidae Labridae Trachinidae
Berycidae Searidae Nototheniidae
Holocentridae Scorpididae Pteropsaridae
Polymixiidae Toxotidae Bathymasteridae
Scombridae Ephippidae Uranoscopidae
Carangidae Ilarchidae Callionymidae
Coryphaenidae Acanthuridae Clinidae
Leiognathidae Siganidae Blenniidae
Centrarchidae Balistidae Stichaeidae
Percidae Monacanthidae Xiphidiidae

_ Apogonidae Ostraciidae Lumpenidae
Centropomidae Tetraodontidae Pholididae™
Serranidae Diodontidae Anarhichadidae
Lobotidae Scorpaenidae Scytalinidae
Priacanthidae Anaplopomatidae Zoarcidae
Lutianidae Hexagrammidae Ophidiidae
Haemulidae Platycephalidae Brotulidae
Sparidae Cottidae Batrachoididae
Gerridae Agonidae Gobiesocidae
Kyphosidae Cyclopteridae Gadidae
Mullidae” Cyglogasteridae Coryphaenoididae
Sciaenidae Triglidae Lophiidae
Champsodontidae Cephalacanthidae Antennarlidae
Malacanthidae Eleotridae™ Ogcocephalidae

° The sequence of families adopted by Doctor Jordan in his Guide to the Study
of Fishes (’05) is here followed.

10 Branchiostegals four plus naught in the species examined.

1 Branchiostegals four plus two or four plus three in all the genera examined,
Eviota excepted.

12 Branchiostegals four plus one in the numerous genera studied.

13 The branchiostegals of all the flat fishes examined are of a very similar
type.

14 The writer follows Regan in the classification of the blennioid fishes.

Resumen por J. 8. Kingsley, por el autor, Georgo Orihay Shinji.

Embriologia de los Céccidos, con especial mencién de la for-
macion del ovario, origen y diferenciacién de las células ger-
minales, capas germinales, rudimentos del intestino medio y
los organismos simbi6dticos intracelulares.

Los 6vulos, células nutridoras y las células del epitelio folicular
se originan a expensas de las células germinales primordiales.
Cuando el huevo entra en el oviducto posee las cubiertas ordi-
narias y una colonia de simbiotos. E] blastodermo se forma por
la emigracién de una parte de las células de segmentaci6n hacia
la superficie, mientras que el resto de ellas forma las llamadas
células vitelinas. La formaci6n de la placa ventral es de tipo
invaginado, solamente la pared dorsal del tubo asi formado es
embrionaria, constituyendo el resto la capa amnidtica. Los
apéndices comienzan a esbozarse en el siguiente 6rden: labio,
maxilas, patas tordcicas, mandibulas, antenas y labro. Existe
una verdadera gdstrula. Los neuroblastos son teloblasticos y
dan lugar, por mitosis, a ganglioblastos. Existe una revolucién
del embrién. Un poco antes de entrar en el oviducto, el huevo
es invadido por una colonia de organismos globulares o en
forma de bast6n, que se rodean de células germinales cuando se
diferencian estas ultimas.

Translation by José F. Nonidez
Carnegie Institution of Washington

AUTHOR’S ABSTRACT OF THIS PAPER ISSUED BY
THE BIRLIOGRAPHIC SERVICE, DECEMBER 15

EMBRYOLOGY OF COCCIDS, WITH ESPECIAL REFER-
ENCE TO THE FORMATION OF THE OVARY, ORIGIN
AND DIFFERENTIATION OF THE GERM CELLS,
GERM LAYERS, RUDIMENTS OF THE MIDGUT, AND
THE INTRACELLULAR SYMBIOTIC ORGANISMS"

GEORGO ORIHAY SHINJI

TWENTY PLATES (ONE HUNDRED THIRTY-THREE FIGURES)

CONTENTS

Pe am TRTEUIOTIA SOLUS) our, tite shes «Ao 25S. -Daeiae aeMie Se cies ete eS 73
ER Cricnl ee te ries serene’ be 3 Serpe ees tae 73

RA herial ane WIebhOGs. sec) eis = les Hi hoon «cies emirate oi mee ein cs Fes 2: 77

2 The origin and differentiation of the ovarian elements.........-----++-> 80
BM te rae. Pah). et tae oes setae geet. one eee mas ce Baer 84
PEG erage ta. si sa lashes fable es acces 9 ot SSA Iepe rea eee Pe 87
5 Establishment of the external form of the body seer. «ee isl 90
6 The formation of the germ layers and embryonic envelopes....-...----- 97
7 The formation of the nervous system.......------+-+srrrrrererettte 101
Br Intracellular symbiosis. {25.0 ¥e-% 222 Wen = + se + oh oeeiease meri e a * Aine c's 105
9 The origin of the germ cells........-..-----s+esseree erste tert 112
10 The formation of the digestive tract........-.---.+seerrseretretsr tees 118
iy Coppedge) Sok a See ees OA ices neo a te at ately 121
Bbliaprapliye, teeta. ake eee heed oe « iS eald yelp ig IS 127

1. INTRODUCTION
Historical

On account of their economic importance, scale insects have
been the object of extensive observations by several inves-
tigators. The literature pertaining to these insects is, however,
limited in its scope, being mostly concerned with the external
morphology, general accounts of the life history, habits, and
methods of control. The accounts of the formation of the egg
and subsequent history of the development of coccids are mostly

1A thesis submitted to the faculty of the Graduate School of the University
of Missouri for the degree of Doctor of Philosophy.

73

Vt: ae GEORGO ORIHAY SHINJI

fragmentary. One of the earliest works of this kind was by
Leydig (’54), who described and figured the general appearance
of the ovaries of Lecanium (Coccus) hesperidum and its eggs
with three nurse cells and an egg cell. Although he did not
actually describe the process of differentiation of the ovarian
elements, he claimed that nurse cells, egg cell, and the epithelial
cells must have arisen from undifferentiated germ glands. He
also described the formation of the embryo from the egg by
multiplication of the single egg cell. The statement that this
form is really viviparous was made in this article. He also pointed
out the presence of numerous pseudonavicellae in this insect.
These organisms, according to him, migrate into the egg at
its posterior end and multiply rapidly by budding.

Leuckart (’58) has also studied the ovarian structure of
Lecanium hesperidum and found that three nurse cells and
an egg cell developed from epithelial cells. Therefore he main-
tained, as his predecessor did, that the nurse cells and the egg
cell are the modifications of the epithelial cells.

Lubbock (’59) also came to the same conclusion, namely, the
nurse cells, the egg cell, and the epithelial cells are all originally
undifferentiated cells of the germ rudiment.

The most complete account of the development of coccids
was, however, presented by Mecznikow (’66). His work on
coccids is not so complete as was that for Aphids, Corixa, and
Cecidomia. Nevertheless, it covers the development of the
Aspidiotus nerii from a single egg-cell stage to the time of hatch-
ing. He described and figured a differentiated egg with its
germinal vesicle. The appearance of the ‘Wulst’ prior to the
formation of the blastoderm and the invagination of the blasto-
derm near the posterior pole of the egg were also mentioned.
One layer of the invaginated ‘Keimhiigel’ degenerated to form
the amnion while the other developed into the embryo proper.
However, he failed to observe the phenomenon of revolution of
the embryo. The entire alimentary canal, he thought might be
formed by further elongation of both stomodeum and proc-
todeum. He described and figured an early appearance of the
germ cells and of the pseudovitellus.

EMBRYOLOGY OF COCCIDS Tas

Brandt (’89), who likewise studied Lecanium hesperidum and
Aspidiotus nerii, with special reference to the embryonic cover-
ings, stated that the embryo of Aspidiotus nerii was bent, as
Mecznikow had already described, with its caudal part over the
oral portion. He further observed the process of the revolution
of the embryo following the rupture of the amnion. In Aspidi-
otus, he observed, as did also Mecznikow, that the ventral plate
of the embryo was found lying closely on the amniotic covering,
even before the revolution, so that the yolk was rapidly removed
from this region.

Then followed the work of Putnam (’78) on the cottony maple
scale, Pulvinaria innumerabilis. This writer evidently thought
the ovarian eggs were In some manner, unknown to him, attached
to the body cavity by their free or anterior end, where the dif-
ferentiation first takes place. Besides this mistake, his figures
are too vague to show anything very definite. However, this
much was sure, that the eggs of the cottony maple scale developed
within the body of the female. He detected the presence of the
pseudonavicellae in the female before mating and also in the
older eggs. The method of infection or the possible migration
of these bodies into the eggs was not studied. However, he
suggested that these bodies, having higher specifie gravity than
water, may represent the metamorphosed state of spermatozoa,
and that their presence in the egg may be comparable to the
phenomenon of fertilization.

Witaczil’s (86) work on the anatomy of the coccids contains
an account of the differentiation of the nurse cells and of the egg
cell from undifferentiated epithelial cells in the ovaries of Leucaspis
pini. Although not figured, there is an account of the presence
of the so-called nutritive string between the nurse chamber and
the egg chamber. He did not find the pseudovitellus in the eggs
of several species, but expressed the view that the pseudovitellus
of Mecznikow may represent a mass of yolk granules.

The investigations above mentioned were entirely made upon
fresh material or at least upon material prepared in toto. The
eggs were usually studied in water to which acetic acid and sugar
were added, or in somewhat similar solutions. The only paper,

76 GEORGO ORIHAY SHINJI

the result of the study of the sectioned material, fixed and stained in
accordance with modern microscopic technique is the contribution
by Emeis (’15). The sole purpose of his article was to present
the history of the three ovarian elements, namely, the nurse cells,
the egg cell, and the epithelial cells. He did not, however, show
whether the epithelial cells, from which the egg and the nurse
cells develop, come from the primordial germ cells or from the
original mesoderm. He was also not sure whether or not a quan-
titative or qualitative cell division takes place among the early
oogonial and oocytal cells. His cytological accounts of the ovarian
cells do not include the phenomenon of the polar body formation.
- Nevertheless, the most interesting feature of the article is the
discovery of the symbiotic organisms in the egg as well as in the
epithelial cells.

As the foregoing brief survey of literature indicates, two phases
only of the development have been confirmed. The rest of the
accounts still remain to be confirmed or rejected, while the origin
and subsequent history of the pseudovitellus and the Pseudo-
navicellae demand a new and careful investigation. Again the
development of several organs (respiratory, circulatory, sensory,
and secretory) remains entirely undescribed.

The purpose, then, of this work is to contribute as much as
possible toward the embryology of certain scale insects, with,
however, especial reference to the history of the pseudovitellus,
the germ cells, germ layers, alimentary canal, and nervous system.

Before going further, I take this opportunity to acknowledge
my indebtedness to Professor Haseman, of the University of
Missouri, with whom the work was carried on. My hearty
thanks are due to Mr. Hollinger, who not only helped me in the
collection and identification of the material, but also gave valu-
able information, and, above all, daily encouragement; and to
Mr. Severance, of the library, through whose effort many valuable
journals in the library of Congress and of other institutions were
made available to me. Last, but not least, obligation is due to
Professor Woodworth, of the University of California, with whom
the study of the cottony cushion scale was originally begun.

EMBRYOLOGY OF COCCIDS CE

Material and methods

Three species of Coccidae belonging to three different genera
were chosen for the present investigation. These are: the
mealy bug, Pseudococcus medanieli Hollinger (ms.); Hunter’s
Lecaniodiaspis, Lecaniodiaspis pruinosa (Hunter); the cottony
cushion scale, Icerya purchasi Mask.

The material of the mealy bug was obtained from its most
favorite host plant, the ragweed (Ambrosia trifida Linn.), on two
trips in the latter part of September and another in early October,
while that of Lecaniodiaspis was collected from time to time on
two elm trees on the campus of the University of Missouri, during
the season of 1917 to 1918. The cottony cushion scale was col-
lected from Acacia and Pitosporum found in the vicinity of San
Francisco Bay, California, during the seasons of January, 1915,
to May, 1917.

Of the three species each had its own advantages. The eggs
of the mealy bug were very easily fixed, sectioned, and stained,
but they were so small that it was difficult to dissect away the
chorions. The eggs of both the cottony cushion scale and Lecan-
iodiaspis are large, and the chorion can be removed easily. The
eggs of the Lecaniodiaspis, however, stain with considerable
difficulty. In fact, the egg of the cottony cushion scale was the
most favorable material, having none of the disadvantages above
mentioned. Yet it must be said here that it was with the study
of Lecaniodiaspis and to a considerable extent with that of the
Pseudococcus that the writer was able to see the true significance
of several organs and inclusions.

The experimental method of determining the age of the embryo
was tried to a considerable extent with the cottony cushion scale.
For this purpose, about thirty adult females with the egg sacs
were collected, together with the infested twigs. After removing
the egg sac with a sharpened bamboo stick, the females were
placed in small paper boxes. Every five or ten minutes the
specimens were observed, and if an egg was seen protruding from
the vaginal orifice, it was transferred into a numbered gelatin
capsule. In the capsule, the egg was able to develop even to the

78 GEORGO ORIHAY SHINJI

time of hatching. Thus the experiment went along with promise
of success until several of the eggs supposed to be of the same age
were fixed and mounted in toto. The examination of these
prepared specimens, however, showed that no two of them were
in the same stage of development. One of them contained an
embryo nearly ready to hatch, another had an embryo with its
appendages well recognizable, while the remainder were mostly
in much earlier stages.

Sectioned material of several adult females of both the mealy
bug and cottony cushion scale brought to light the fact that
the eggs of these two species of coccids undergo a partial devel-
opment in the uterus of the female. The early deposition of
the egg was usually noticed in specimens in which the growth of
the ovarian eggs was in rapid progress. The eggs were not
deposited until the completion of the blastoderm. The eggs of
the Lecaniodiaspis, on the contrary, were deposited at the first
cleavage stage, and should serve as the most desirable material
for this purpose. Unfortunately, I have failed to work with this
species during the past year.

Thus the determination of the age of the eggs by the experi-
mental method alone is not reliable. Therefore the relative ages
of the embryos in this study were mostly determined by the
number of cells, the position of the polar granules, the length of
the embryo and of the appendages, and other morphological
features.

Most of the material for embryological study was obtained
from egg-sacs of the fully matured female scales in the following
manner: Several females with their egg-sacs were collected at
various times of the day, and the egg-sacs were separated from
the body with a sharpened bamboo stick or needle. In some
cases the eggs were lightly shaken out of the egg-sac into a watch- .
glass containing the fixing fluid, but in many cases the entire egg-
sacs were dropped directly into the watch-glass containing the
fixing fluid, and the cottony substance was removed afterward
with a pair of sharpened bamboo sticks. The use of sharpened
bamboo sticks proved to be advantageous, for they can be made
of any desired sharpness and they are not acted upon by such
corrosive mixtures as Gilson’s.

EMBRYOLOGY OF COCCIDS 79

One of the fixing reagents most extensively used was Carnoy’s
aceto-aleohol-chloroform mixture, prepared by mixing thoroughly
equal parts of absolute alcohol, glacial acetic, and chloroform
saturated with corrosive sublimate. Eggs fixed in this mixture
for from one to two hours lost their red pigment and became
transparent. They were then washed in 30 per cent alcohol for
two hours, and passed through 50 per cent to 70 per cent alcohol
with an intermission of one hour. They were then either left in
70 per cent alcohol until needed or dehydrated by passing them
up through 90 per cent, 100 per cent alcohol to xylol and
imbedded in 52° to 58° paraffin.

Sections were cut from 5y to 7u in thickness and stained mostly
with iron alum haematoxylin followed by eosin, orange G, acid
fuchsin, or a mixture of these. The triple stain, saffranin-gentian-
violet, and orange G, was also frequently used with very good
results.

For whole mounts, the eggs were passed from 70 per cent to 30
per cent alcohol, in which the chorion was dissected away under ,
a binocular microscope. Embryos thus freed of their chorions
were stained with a diluted solution of gentian violet for from one
to six hours and then decolorized with 70 per cent alcohol. Dela-
field’s haematoxylin, borax carmine, and alum cochineal have also
been used with fairly good results. The most beautiful specimens,
however, were those that were treated with gentian violet.

For the study of the history of the germ cells not only the
genital organs of the embryo, but also those of several stages of
larvae, pupae, and adult scales were necessary. Ovaries were
mostly dissected out and fixed in either Fleming’s or Zenker’s
solution. In many cases, however, whole larvae, pupae, and
adult were put directly into Gilson’s or Carnoy’s aceto-alcohol-
chloroform solution, sectioned and stained in the same manner as
in the case of embryos.

Perenyi’s solution was also tried, but all except the aceto-
alcohol-chloroform mixtures were useless unless heated to 70°C.,
because the eggs as well as larvae are covered with a waxy sub-
stance which prevents penetration of fluids. When heated, these

JOURNAL OF MORPHOLOGY, VOL. 33, NO. 1

SO GEORGO ORIHAY SHINJI

fixing fluids as well as ordinary water will kill and fix the specimen,
but in such a case the finer details of nuclear structure are often
destroyed or distorted.

2. THE ORIGIN AND DIFFERENTIATION OF THE OVARIAN
ELEMENTS

The germ cells in the ovaries of the larvae at the time of hatch-
ing are similar in size and appearance (fig. 1). By mitosis, these
germ cells multiply during the first, second, and third larval stages
and form a mass of so-called oogonia (fig. 6.) At an early period
in the fourth larval stage, however, all oogonia cease to multiply.
Consequently, all appear alike on account of their being in
the so-called resting stage. This condition is soon followed by
a peculiar phenomenon. A few oogonia situated along the
periphery of the ovary suddenly undergo another, the last,
oogonial division and begin to grow, not only in size, but also in
nuclear complexity. The number of these oocytes of the first
order forming a group varies with the species. In Pseudococcus
there are four oocytes in a group, but in Icerya the number is
five. At first the nuclei of the oocytes in each group appear
exactly the same, all being in the so-called synizesis stage (fig. 4).
From the contracted nuclear contents fine thread-like chromo-
somes emerge (fig. 6). At first these chromosome threads are
distinctly doubled, but later appear as single.

Meantime a sort of protoplasmic substance begins to be
secreted around each of the oocytes except the one situated
toward the proximal end. As their later history shows, these
secretory oocytes nourish the single oocytes located below or
toward the distal end; and thus become the so-called nurse cells.
The cytoplasm areas of the fast-growing nurse cells soon come
into contact with one another. Being colloidal in nature, the
nutritive substances secreted by the nurse cells elongate in the
direction of the least resistance, which in this case is toward the
egg nucleus, for the expanding force of the nurse cells is much
greater than that of a single egg cell situated near the distal end.
Consequently, the nutritive substance, which is elaborated by
the nurse cells, now literally pours over the egg, causing a rapid
increase in its size (fig. 11).

EMBRYOLOGY OF COCCIDS 81

Epithelial cells which surround the nurse chamber above never
multiply, but those around the egg multiply rapidly and help to
accommodate the protoplasmic substance which pours from the
nurse chamber above (fig. 15). Soon a constriction becomes
evident at the junction of the two chambers (fig. 16), due partly
to the ingrowth of the epithelial cells at the base of the nurse
chambers and partly to the rapid expansion of the egg and nurse
cells. The epithelial cells surrounding the egg chamber are
cubical or elongate ovoid in shape and actively divide, while
those around the constriction are smaller and spindle shaped.
No mitosis was observed among the latter.

As the constriction progresses, the space through which the
two chambers communicate becomes smaller. Consequently,
the protoplasmic substance, which has been flowing homogeneously
toward the egg nucleus or the germinal vesicle, now flows out of
this small passage in minute streamlets as molasses does when
poured through a funnel into a flask. The nuclei of the nurse
cells still increase in size, and change from spherical to egg-shape,
the narrow end being directed toward the egg chamber. The
chromatin threads become broken into numerous chromatin
bodies of different sizes. The nucleus of the egg proper moves
toward the center of the egg chamber, but the chromatin threads
are still in the paired condition as last described. Their affinity
for iron-alum haematoxylin is changed. They now stain so
faintly with this dye as to seem almost achromatic in nature.
From this time on, the germinal vesicle begins to migrate from
the center toward the periphery of the egg. During this migra-
tion the chromosomes lose their paired appearance and form
several small, spherical bodies which become scattered along the
nuclear membrane (fig. 17). There is, however, no indication of
their being passed out through the nuclear membrane into the
surrounding protoplasm, as several investigators of other insects
have stated.

The place where the germinal vesicle reaches the peripheral
layer is on the ventral surface, midway between the equator and
the posterior pole of the egg. As soon as the clear transparent
nucleus reaches the periphery, the surrounding protoplasmic

82 GEORGO ORIHAY SHINJI

substance becomes compact, thereby causing an indentation of
the surface.

In the next stage, the nuclear membrane of the germinal
vesicle disappears and the vesicle itself loses its clear appearance.
This change is due to the appearance of the spindle fibers about
the chromosomes (fig. 26). The oocyte is now in the metaphase
of the maturation division. Following this stage the chromo-
somes divide into two groups and move to the opposite poles.
Soon one of the daughter cells gradually protrudes from the egg
proper (fig. 31). In this manner the first polar body is formed. °
The process of the second polar body formation has not been
studied, but that the egg undergoes the second maturation
division may be established by the presence of three polar bodies.
Meanwhile the epithelial cells not only cease to multiply, but
they also become reduced to a membranous structure. The
nurse cells also cease to grow. Their size becomes very much
reduced. The contents of the nurse chamber is withdrawn into
the egg and its epithelial layer shrinks to a small mass. This,
together with the remains of the epithelial cells of the egg, closes
over the opening left by the egg entering the oviduct.

The earliest egg found in the oviduct (or the uterus, as it is
often called) shows a large nucleus at the center of the egg. Since
this large nucleus divides, it must be the first cleavage nucleus.
It follows, therefore, that the union of the male and the female
pronuclei must have occurred during the passage of the egg pro-
nucleus to the center of the egg after the formation of the last
polar body.

Thus my observations on the three species of coccids are in
accord with those of Leuckart (’53) and Emeis (716). It may
also be added that the ovary of Icerya purchasi is the most favor-
able material for the study of this problem, since the cells are
large and numerous.

The origin of the three ovarian elements in aphids has been
differently described by different writers. Lubbock claims that
the egg and nurse cells are modified epithelial follicular cells of
the end chamber. Recent observations of Tannreuther (’07) are
to the same effect, for he declares that the egg cells do not arise

EMBRYOLOGY OF COCCIDS 83

from the inner mass in common with the nurse cells or ovarian
glands, but grow out of the follicular epithelial cells at the base
of the end chamber.

Balbiani (’82) states that the germ rudiments of partheno-
genetic aphids undergo a process of budding previous to their
differentiation into the nurse cells and oocytes.

Mecznikow (’66) derived the end chamber from a mass of cells,
‘“Die am untersten Pole des endfaches legenden Zell sich
bedeutend vergréssert, wobei sie in ein, aus dem Endfachepithel
entstandes Follikel eingeschlossen wird und hier ihre weitere
Entwicklung vollzieht.”’

Stevens (’05) insists that the contents of the end chamber are
of two kinds, possibly corresponding to the summer and winter
eggs, and that those situated on the lower portion of the end
chamber degenerate in the ovaries which produce the agamic
eggs.

In insects (other than Hemiptera) the results of several inves-
tigators differ with the species with which they have worked.
The prevailing idea, up to 1905, was that the three ovarian
elements, nurse cells, epithelial cells, and oocytes, are all derived
from the germ cells. Paulcke (’00) who studied the development
of the honey-bee, however, discovered for the first time that the
nurse cells and oocytes are originally the same, but later become
differentiated by a certain irregular cell division. Similar dis-
coveries of the existence of a quantitative difference between the
nurse ceils and oocytes have since been reported in several
Coleoptera. In Dytiscus, for example, Gunthert (710) found
that the chromatin eliminated from the nucleus passes, in each
successive mitosis, into the pole of a single daughter cell, and
that the cell having this extra chromatin substance becomes the
oocyte and those lacking this the nurse cells. Somewhat similar
observations were made by Giardia (’01) and Debaisieux (’09) in
Dytiscus, and Govert (’13) in Carabus, Cicindela and Trichiosoma.

The oogonial origin of the oocytes, nurse cells and follicular
epithelial cells was clearly established in Polistes and Platy-
phylax by Marshall (07); in Podura by de Winter (713), and in
Leptinotarsa by Hegner (714). In these cases, the nurse cells

84 GEORGO ORIHAY SHINJI

and follicular epithelial cells are regarded as abortive cells. No
differential mitosis was observed.

Hegner (’12) states that the germ cells in Miaster give rise to
nothing but the true oocytes, and that the nurse cells and
epithelial cells are both derived from somatic cells. He deduces
this from the fact that in Miaster americana altogether sixty-
four oogonial cells are formed by six successive divisions of a
single primordial germ cell and that the number of the young
larvae produced is also about sixty-four.

The foregoing survey of the more important literature per-
taining to this subject, brief as it is, indicates that, even among
the same order of insects, there is no definite law governing the
differentiation of the three ovarian elements. The accounts of
the lineage of the ovarian elements in Leptinotarsa (Wieman, ’14;
Hegner, 712), and Hydrophilus (Korschelt, ’89) are good ex-
amples. However, it should be mentioned that in all insects
the oocytes and also the nurse cells, when present, are all
derived from primordial germ cells. As yet no case has been
found in which the primordial germ cells of the insects entirely
degenerate and the secondary or functional germ cells are formed
de novo at a much later period of development.

3. THE EGG

The eggs of all species of coccids studied, at the stage last
mentioned, consist of the following substances:

1. Chorion—the outermost covering or membrane.

2. Protoplasm—the ground substance.

3. Corticular layer—a thick protoplasmic layer next to the
chorion.

4. Fat globules—oily droplets suspended in the protoplasmic
network.

5. Yolk granules—protoplasmic suspension.

6. Pigment oil-fluid filling interspace between fat globules.

7. Germinal vesicle with its nuclear membrane.

8. Yolk membrane—membrane next to, and, in fact, almost
apposed to the chorion.

EMBRYOLOGY OF COCCIDS 85

The chorion is a very thin membranous structure which en-
closes the substances above mentioned. It is formed shortly
before the passage of the egg from the egg chamber into the
uterus, and is secreted by the follicular epithelial cells.

The protoplasm or cytoplasm fills, so to speak, most of the
space between the other inclusions of the egg, with the exception
of the space occupied by the nucleus or the germinal vesicle.
As already stated, this ground substance of the egg is elaborated
by the nurse cells and is literally poured on the egg. At first
the protoplasm is a homogeneous mass uniformly surrounding
the central clear region, the nucleus. Later, however, it becomes
mesh-like, owing perhaps to the more rapid expansion of the egg
than the flow of the nutritive or protoplasmic substance from
above, to the intrusion and consequent suspension of other sub-
stances, and also to the physiological change due to the metabolic
activity of the germinal vesicle.

What seems to me a sort of yolk substance is found in the egg
of the mealy bug of the giant ragweed. ‘This substance may be
spherical, but it is more often irregular in shape. It first appears
at about the time when the germinal vesicle reaches the periph-
ery. The exact origin of this substance remains to be studied
further. The fact that similar granules are abundant in the
body cavity surrounding the ovarioles, and also in spaces between
the chorion and the epithelial cells, strongly suggests that these
particles may actually migrate from the body of the mother
through the epithelial layer into the egg. The presence of
similar substances in the body cavity of the mother is another
evidence in favor of the view just stated. Several investigators
of other insect eggs state that they have observed the migration
of chromatin matter from the germinal vesicle of the egg. No
indication of such migration was observed in the case of the
scale insects studied. .

In the ovarian eggs of these species of coccids, the nucleus or
the germinal vesicle was always found.

The pigment-oil or coloring matter appears in the mature, but
not in the ovarian egg. I have had occasion to observe this
fluid-like matter flowing into the egg at the posterior or pointed

86 _ GEORGO ORIHAY SHINJI

end. This substance was found literally filling the oviducts of
the adult during her egg-laying period.

The presence of the yolk membrane cannot better be illus-
trated than by figure 28. On account of the migration of the
symbiotic organisms after the formation of the yolk membrane,
the latter is pushed in and remains separated from the chorionic
membrane (which is the last to envelop the egg).

A fully matured egg with all its components is elongate oval.
The pointed end corresponds to the cephalic and the blunt end
to the caudal end of the insect. The ventral surface near the
pointed end is slightly indented. Thus not only the antero-
posteriority, but also the dorsoventrality are marked in the eggs
of the coccids, but not so clearly as in the eggs of the Orthoptera
and Coleoptera reported by Heymons (’89), Wheeler (’93), and
others.

Besides such a difference in shape, the anteroposteriority is
well marked by the presence of a dark-staining substance, the
position of which varies with the species. In cottony cushion
scale, it is, at first, visible near the posterior pole, but later
becomes pushed gradually toward the anterior pole by the
invaginating germ-band; while in the case of Pseudococcus and
Lecanodiaspis, it is found always near the anterior end of the
egg. The presence of these polar granules or symbiotic organisms
is a great service in the determination of the position of the
sectioned material. Later on, I shall treat of the history and
significance of this substance under a separate heading.

No micropyle was found.

The longest and shortest diameters of the eggs of the three
species of coccids are respectively as follows:

LONG SHORT
2S) (O29) (SEL NaS DIAMETER DIAMETER
mm mm.
ER UEE AEE OR GSB, Wioc.u.0 5 5s oben nee ae Rete ae eT 1 8.5-9.0 4.5-5.0
PSCC OCOCCUSRINACUUNTEL cc hele intr ei ae eee 4.0 220
Lecemodtas pus PrUInOst... .. . sere seus cs wan egos vee 6.0-4.0 235

EMBRYOLOGY OF COCCIDS 87

4. CLEAVAGE

The type of cleavage in the coccids studied is the pure super-
ficial type which is so common among the Arthropoda. The
first cleavage spindle lies at right angles to the shorter axis of
the egg, so that one of the two daughter cells arising from the
first division wanders toward the posterior pole while the other
cell remains near the position formerly occupied by the mother
nucleus (fig. 43). This behavior of the first two cleavage cells
in coccids is exactly like that of the termite studied by Knower
(00). At first, all the cleaving cells were in the same mitotic
state, but gradually some lag behind others in division so that
in a later stage of cleavage, e.g., at the thirty-two cell stage,
more than one cleavage figure is noticeable among them (fig. 78).
Up to about the eight-cell stage in the eggs of Lecaniodiaspis,
and to a still later stage in Pseudococcus and Icerya, these
cleavage cells are all at some distance from the cortical layers.
Although in each cell the nuclear membrane is distinct, the
cytoplasm presents numerous pseudopodial processes which con-
nect with those of neighboring cells. On account of their
somewhat isolated appearance, they are usually known as pro-
toplasmic islands. In eggs containing a large amount of yolk,
as, for example, those of Chrysomelid beetles studied by Hegner
(14), the winter eggs of plant-lice investigated by Tannreuther
(07) and Webster and Phillips (12), these protoplasmic islands
literally cut up the yolk into blocks. I have noticed this block-
like appearance of the egg contents in the living eggs of Lecanio-
diaspis, but upon sectioning them, I become convinced they
were not comparable to the yolk-blocks found, for example, in
the ova of aphids, because the eggs of Lecaniodiaspis contain no
yolk granules. The eggs of the mealy bug contain a darkly
staining substance resembling the yolk of the ova of aphids, but
they are never cut up into blocks by the cleaving cells (fig. 42).

Weismann (’82) stated that in Rhodites and Biorhiza aptera
(eynipids), the first two cleavage nuclei move apart in the direction
of the longitudinal axis of the egg. One of them, upon reaching
the posterior pole of the egg, remain inactive and _ probably
degenerates, while the other, upon arriving at the anterior pole,

88 GEORGO ORIHAY SHINJI

produces, by rapid multiplication, all of the embryonic cells.
As stated above, the first cleavage products of our scale insects
do not behave in this way, but both cleavage cells continue
multiplying, and some of their products later form the blasto-
derm, while the others remain in the interior of the egg and
constitute the so-called yolk-cells. In this respect, the develop-
ment of the eggs of coccids resembles that of the silkworm and of
Neophylex and Gryllotalpa, studied, respectively, by Toyama
(02), Patten (’84), and Korotneff (’84). Silvestri (11) recently
discovered that in a parasitic Hymenopteran, Copidosoma, one
of the two nucleoli escapes from the nucleus at the end of the
growth period of the oocyte. Later, this escaped nucleolus
passes into one of the two cleavage cells. During a series of
cleavage processes, only one cell remains in possession of this
nucleolar substance and becomes the germ cell. In another
parasitic Hymenopteran the escaped nucleolar bodies become
localized at the posterior end of the egg until one of the first
cleavage cells reaches out and takes them up into its protoplasm.
In both cases the cell which becomes possessed of this escaped
nucleolar substance, differentiates into the germ cells.

In the scale insects I have studied no escape of the nucleolar
substance into the egg was observed and the germ cells do not
appear during the cleavage period. —

All cleavage cells divide mitotically. No case of amitotis, as
described for Blatta by Wheeler (’93), has been observed. The
fact that very many cells are in the process of division during
the early stages indicates the rapidity with which cells divide.
Nelson (’15) states that no case of a single spireme stage was
found in the cleavage cells of the honey-bee. On this point my
specimens agree strictly with his observation. An abundance of
spireme figures are, however, found among the blastoderm cells.
As the number of cleavage cells increase, they migrate, one by
one, toward the periphery and become imbedded in a thick
cortical layer of the protoplasm. In figure 44 the condition of a
loose blastoderm is shown. Although the cells are arranged in a
peripheral layer, they are very far apart from one another. The
spaces between these blastoderm cells are gradually filled by the

EMBRYOLOGY OF COCCIDS 89

division of the blastoderm cells as well as by a further migration
of cleavage cells from within.

The point at which cleavage nuclei, or cells as they are often
called, reach the surface of the egg varies in different groups of
insects. In Muscidae Graber (’79) found the first arrival of
cells at the posterior end of the egg, while in Pieris Bobretzky
(78) observed the appearance of the first blastoderm at the
anterior end. Wheeler (’93) described the first blastoderm cells
on the ventral side, while Heider (’88) stated that the blastoderm
in Hydrophilus was first formed around the middle of the egg
as a transverse girdle, somewhat nearer the posterior pole and
that the development occurred last at the poles. Again, accord-
ing to Nelson, the cleavage cell first reaches the cortical layer
on the ventral side near the cephalic pole in the egg of the honey-
bee. In the winter egg of the aphid, Melanoxanthus (Ptero-
comma, salices), according to Tannreuther (’07), all of the
blastodermic cells spread uniformly over the entire surface
except at the posterior pole of the egg. Therefore, I agree with
Nelson ('15) that the point at which the first cleavage cells
reach the surface has little significance so far as the formation
of the blastoderm is concerned.

The condition of the egg at the time the geet process has
ceased among the cells within the egg and the blastoderm forma-
tion is completed, is shown in figure 80. At the poles and sides
the blastoderm is similar in appearance. A short distance
within the blastoderm is another loose layer of cells. This is
irregular in shape, and the nuclei are much clearer and coarser
than those of the blastodermic cells. These are the so-called
yolk cells of Will (’84), and are no other than the cleavage cells
that failed to migrate to help form the blastoderm. At about
the time invagination occurs at the posterior end of the egg,
these cells move toward the periphery and become closely apposed
to the blastoderm cells.

90 GEORGO ORIHAY SHINJI

5. ESTABLISHMENT OF THE EXTERNAL FORM OF THE EMBRYO

The development of the embryo was traced up to the com-
pletion of the blastoderm as shown in figure 80. I first describe
the development of the embryo as seen mostly from surface
views.

The first change externally visible after the completion of the
blastoderm is a depression or an invagination near the posterior
end of the egg. It is, at first, very shallow, but gradually
deepens forming a V- or U-shaped structure (fig. 45). This
condition is much more pronounced in the case of the cottony
cushion scale than in the other two species studied. The portion
of the blastodermic layer constituting the bottom of the blasto-
pore and its near-by area increases greatly in thickness, while
toward the anterior pole and in the area surrounding the blasto-
pore it becomes thin. In the cottony cushion scale, the colony
of parasitic organisms, originally found at the posterior pole of
the egg, is later pushed, so to speak, toward the anterior pole
by the elongation of the invaginating germ band.? During the
same period of development, the mass of the parasitic organisms
in the eggs of Pseudococcus and of Lecaniodiaspis has also
migrated a short distance from its point of entrance, the anterior
pole, towards the posterior pole. <A side view of a similar embryo
(fig. 52) shows that the invagination occurs, not exactly at the
posterior pole, but a short distance lateral to it. The same
figure also brings out the fact that the layer forming this de-
pression and also the ventral germ band is essentially a con-
tinuation of the outer blastoderm, a portion of which forms the
serosa. Only the lower, or the ventral portion of these, later
becomes the embryo, while the upper or the dorsal wall trans-
forms into the amnion. A later condition of the egg is repre-
sented in figures 59 and 62. The invaginating germ band is
now about two-thirds as long as the egg. In the side view of a
slightly older embryo the germ band appears as though it con-

2 In this paper the term germ band is used to designate the ventral wall of the
invaginated embryonal rudiment in its early stage. The term ventral plate
denotes the same structure after the amnionic layer becomes clearly distinguish-
able as the dorsal wall of the invagination.

EMBRYOLOGY OF COCCIDS 91

sists of two bands separated by the median groove. In reality,
however, the furrow represents the amniotic cavity, the blasto-
pore, within the invaginating germ band. ‘The colony of para-
sitic organisms was not, at first, in contact with the germ band,
but it now appears as though it were in the same relative position
as in the case of the cottony cushion scale.

As the invagination proceeds further, the caudal portion of the
invaginating germ band curls up ventrally, assuming the shape
of the letter ‘S.’ Each turn of the invagination represents a
particular region or division of the body of the embryo as should
have been brought out earlier. The germ band at the stage
last described consists of four main divisions, representing the
cephalic, the oral, the thoracic, and the abdominal regions. (figs.
46, 50, and 64). These regions are shown in figure 6, separated
by three dark areas. The two remaining regions cannot be
shown very well in the picture, but their presence is represented.
Each dark area indicates the junction of two adjacent regions.

It should also be mentioned that the preoral and abdominal
regions are much less extensive in length as compared with the
oral or thoracic region. The colony of parasitic organisms,
which had been located near the growing tip of the germ band
of the cottony cushion scale, now becomes fixed, so to speak,
at the region of the second and third abdominal segments and
does not accompany the growth of the abdominal region beyond
these segments. The condition described for the cottony cush-
ion scale is accomplished in an altogether different manner
in the case of the mealy bug and Lecaniodiaspis. Instead of
pushing the colony of parasites forward, the germ bands of these
two species sends out a few germ cells at first, then follows a
mesodermal extension to the colony, which establishes a relation
with the latter, Just as in the case of Icerya. During this stage
a pair of somewhat curved, dark elevations appear, one on each
side of the germ band near the blastopore. These, as will be
seen later, are the rudiments of the brain. In the next stage
(fig. 66), the first rudiments of appendages, the second maxillae,
become apparent. The abdominal region is very much elon-
gated beyond the colony of parasitic organisms. The division of

92 ' GEORGO ORIHAY SHINJI

the body into four regions becomes plainly marked. A lateral
view of asomewhat older embryo is shown in figure 61. Here the
abdominal region is so much elongated as to be actually folded
over beyond the thoracic part, and reaching almost to the base
of the second maxillae. The brain becomes very conspicuous,
owing to its enormous dorsal growth. Below the rudiment of
the brain another pair of elevations appears (fig. 65). This is
the rudiment of the first maxillae. Segmentation is clearly
visible throughout the oral, cephalic, thoracic, and the abdominal
regions. In a still older embryo (fig. 67) three pairs of appen-
dages become visible. These are the rudiments of the thoracic
legs. Thus the appendages of scale insects, like those of most
other insects, develop first from the maxillae backward to the
last thoracic limb and finally to the antennae and mandibles,
the labrum being the last to appear. In other words, the rudi-
ments of the second maxillae are the first to appear. The first
maxillae and the thoracic legs make an almost simultaneous
appearance, followed by the antennae, mandibles, and the
labrum. In this respect, the scale insects differ from the aphids,
in which, according to Mecznikow and Witlaczil, the antennal
appendages are the first to appear. The order of the appear-
ance of the appendages in Pseudococcus and other scale insects
differs also from that observed in the Orthoptera in which, as
described by Wheeler (’89), Riley (’98), and others, the antennae
are the first to appear, followed by oral appendages, the thoracic
limbs being the last to be formed. My observations also differ
from those made by Brandt on Libellulids, when the rudiment
of the thoracic limbs appears first, then those of the maxillae;
the antennae being the last to develop. In Coleoptera, 1.e.,
Hydrophilus, Melolontha, etc., according to Heymons and
others, the antennal rudiment seems to appear first, while the
mandibular and maxillary rudiments and those of the thoracic
legs make a simultaneous appearance.

In Mantis (Hagen, 717) the antennal rudiments appear first,
followed by those of the thoracic appendages; then appear,
practically simultaneously, the rudiments of the maxillae and
labrum, the last being distinctly an unpaired organ.

EMBRYOLOGY OF COCCIDS 93

The further growth of the appendages is shown in figure 47,
which represents the surface view of a somewhat older embryo.
Mandibles, maxillae, and three pairs of thoracic legs are now
tubular instead of conical elevations. The second maxillae have
migrated somewhat dorsally and therefore are no longer in line
with the rest of the appendages, as was also noticed by Meczni-
kow (’66) for Lecanitum. The three pairs of thoracic legs appear
somewhat constricted at the middle, indicating that they are, in
a sense, two segments.

The first rudiment of the mouth is found in figure 49. This
is essentially a circular depression of the ventral plate in the
medial line between the rudiments of the brain elevations and is
cephalad of the oral or antennal appendages.

Following this stage, the brain undergoes a conspicuous de-
velopment. Heretofore the rudiments of the brain were only °
represented by two crescentic elevations, one on either side of
the median line. It now becomes a large conical structure, due
to its dorsal turning and rapid neurogenesis. It, however, lies
entirely within the serosa sac and not beyond the original
‘blastopore’ of Will, as he maintained was the case with the
development of the pseudovum of aphids. The brain region is
also surrounded, at least ventrally, by the amniotic layer. That
the rudiments of the antennae arise, not from the posterior end
of the brain, but from below it, is also clearly demonstrable
here. The segmentation of the abdominal region into its future
body segments is also clearly shown.

In the next older embryo (fig. 95) several notable changes
are evident. The first maxillary rudiments have migrated still
further dorsally, leaving their alignment with the rest of the
appendages. The three pairs of thoracic limbs now present a
slight constriction approximately at the middle of the appen-
dages. ‘The segmented abdominal region is still folded over the
thoracic as well as the oral regions.

Following this stage, the embryo gradually shortens until it
begins to rotate around ihe transverse axis of the egg. As a
result of this rotation, the poles of the egg coincide with those
of the embryo (figs. 53, 72). Heretofore, the cephalic or anterior

94 GEORGO ORIHAY SHINJI

end of the embryo has been situated at the posterior pole of the
egg because of its being the part first formed from the invaginated
portion of the germ band which occurred at or near the posterior
pole of the egg, but now it has accomplished its exchange of poles
by the process of revolution just mentioned.

A side view of an embryo after its revolution has been com-
pleted is shown in figure 53. The thoracic limbs are now dis-
tinctly four jointed, showing the coxa, trochanter, femur, and
tibia. The segments of the limbs are almost equal in length,
but differ in shape, the second being the largest and the last
the most slender. The antennae also have four segments. The
most conspicuous feature at this stage is the appearance of the
deep invagination of the proctodeal opening on the dorsally
curled end of the abdominal segment. The proctodaeum invagi-
‘ nates on the dorsal side of the ninth abdominal segment as will
become clear when its formation is studied in sections. Another
interesting feature of this stage is the sudden appearance of a
mass of cells on the dorsal side of the brain segment. In reality,
however, this mass of cells is formed during the revolution of
the embryo by the contraction of serosal and amnionic cells.
Another prominent change noticeable at this stage is that which
occurs in the formation of the mouth parts.

The mandibles and the first maxillae in the stage last de-
scribed have migrated laterally and are no more in line with the
second maxillae. A large invagination between the second ~
maxillae and the first thoracic legs appears during the revolution
of the embryo around the shorter axis of the egg, and there
arises, from the pointed distal end of each of the mandibles
and the first maxillae, a slender chitinous bristle-like structure,
which is from the beginning non-cellular. The wall of the
invagination, on the contrary, is cellular at first, but later
becomes chitinous. The cavity thus formed and lined with a
chitinous layer, it must be mentioned, serves to accommodate
the bristles when they are not in use, as when the larvae changes
its feeding place.

The upper lip, with a well-defined suture, does not elongate
or grow very much in size, but a layer of chitinous substance is

EMBRYOLOGY OF COCCIDS 95

secreted all over the surface forming a semiconical structure
above the base of the mouth parts. The second maxillae, which
also remain relatively small, are also invested with the chitin,
and finally form an inverted crescentic underlip. The suture
formed along the midventral line by the fusion of the second
maxillae remains open externally and also opens internally into
the ventral cavity. It is through this external opening that the
bristle-like piercing organs pass out at the time of feeding.

The formation of the mouth parts of the coccids is essentially
the same as that given for Corixa by Mecznikow (’66), who
says:

Die Mandibeln und die ersten Maxillen liegen jederseits dicht
beisammen, die Form kleiner Zapfen zeigend. Die zweiten Maxillen
verwachsen mit ihren unteren Randern, wodurch eine michtige Unter-
lippe zu Stande kommt. Die oberen unverwachsenen Rinder der

beiden Maxillen bleiben und reprasentiren die bekannte Rinne der
Unterlippe.

The same investigator, however, gives a different account of
the formation of the mouth parts in aphids in the following
words:

Sie (the beak) werden von besondern K6rper secernirt, welche jeder-
seits neben den Mandibeln und Maxillen im Laufe der dritten Entwick-
lungsperiode entstehen. Bei ihrem Wachsthum nehmen diese Korper
bald eine retortenformige Gestalt an, wie es auf der Fig. 53 abgebildet
ist; es schniirt sich dann von ihnen eine diinne peripherische Schicht ab,
welche das Licht stirker bricht und, sich verlangerned, einem schmalen
Faden den Ursprung giebt. Es entstehen somit jederseits zwei solche
Faden, welche nunmehr in die fraglichen Stillette tibergehen und dabei
die Fahigkeit erhalten, nach aussen in die Riisselscheide ausgestiilpt
werden zu kénnen. Die Retortenform der bescriebenen, aus einer
Menge kleiner gleichgestalteten Zellen bestehenden Kérper verursacht
es, dass im ruhigen Zustande das fadenférmige Stillette spiralig auf-
gerollt liegt.

Bei weiteren Wachsthum der Miindanhinge nehmen diese an Linge
zu, wobel man an der ersten Maxille die erste Bildung eines Tasters
wahrnimmt.

Mecznikow must have had the idea that the mouth parts of
the coccids are formed in the same manner as in the case of the
aphids, for he states at length the difference between the forma-
tion of the mouth parts in the Heteroptera and Homoptera. My

JOURNAL OF MORPHOLOGY, VOL. 33, NO. 1

96 GEORGO ORITHAY SHINJI

observation on several coccids shows, it should be repeated,
that the mouth parts of these coccids (Homoptera), are formed
essentially in the same manner as in Corixa, a Heteropteran.

The side view of the last embryonic stage is shown in figure 56.
In each of the thoracic limbs, the first joint or coxa becomes
conical. Contraction has also considerably reduced the length
of the trochanter. The embryo is now shortened to the length
of the egg-shell and is much flattened. The alimentary canal is
already completed, and the ventral nerve cord is contracted a
great deal. A great change has also occurred in the oral append-
ages. The tips of the mandibles and first maxillae become
elongated, extending beyond the rest of the oral appendages to
form slender lancelet-like processes, while the rest of the oral
appendages unite and form a sort of case at the base of the
piercing processes. A careful examination of a mounted speci-
men of this or a slightly older embryo shows that the antennae
are covered with a long slender hair-like process at each joint.
There are also tail filaments, three on either side of the posterior
extremity of the tenth abdominal segment. The embryo is now
ready to hatch.

In the case of the cottony cushion scale, I have observed the
process of hatching. The first indication of this process is the
appearance of a transverse slit, usually around the mesothorax,
often, however, on the metathorax. Through the rent thus formed,
the antennae, which have been folded against the ventral surface
of the body, gradually stretch out. By the force thus exerted
by the antennae the cephalic portion of the insect becomes first
lifted and then carried forward from the egg shell by the stretch-
ing antennae, meanwhile the embryo juggles its abdominal
portion by exertion on the part of the long caudal filaments.
Then the femur of the first thoracic leg and a part of the first
tibia appear. With the next exertion the most of the body and
legs become free from the egg shell. The rest of the process is
the kicking off, so to speak, of the shell from the filaments by
means of legs, and the hatching is completed.

The embryo, eight hours after hatching, is represented by
figures 57 and 75, of Pseudococcus and Icerya, respectively.

EMBRYOLOGY OF COCCIDS 97

6. THE FORMATION OF THE GERM LAYERS AND THE [EMBRYONIC
ENVELOPES

At the time the invagination occurs at the posterior end, the
blastoderm is one cell thick, except at the posterior end where
cell proliferation is evident. The yolk cells (cleavage cells that
failed to take part in the formation of the blastoderm) are here
and there closely attached to the blastoderm. Now and through-
out the embryonic period no evidence of cell migration from the
embryo proper into the yolk is noticed.

The first change occurring after the completion of the blasto-
derm is its thinning out in the immediate neighborhood of the
posterior pole and also toward the anterior end of the egg, and
the simultaneous thickening around the rim of the thinned area
at the posterior end of the egg. The thickening near the
posterior end is due to the proliferation of cells, as shown in
figure 81. The proliferating cells at first are all similar, as in
figure 83. The larger and clearer cells found in chains, from
the region of the invagination toward the opposite end of the
egg, are the germ c¢ells, the history of which will be considered
separately. At this stage, then, there are only two kinds of
cells within the blastoderm: the germ cells and the cells that
form the invaginating germ band.

In figure 85, which represents a longitudinal section of a some-
what older embryo through the region of the blastopore, cells of
another or third kind occur between the germ cells and the
cells which form the wall of the blastopore. These are the
so-called entodermal cells. The cells forming the wall of the
invagination and situated next to the entodermal cell mass are
cubical or almost columnar and are in direct continuity with
the blastoderm layer beyond the invagination or the blastopore.
These are the rudiments of the future ventral plate and amnion.

A longitudinal section through the blastopore of a similar
embryo (fig. 86) shows another, a fourth sort of cells besides the
three already mentioned. Although these latter cells may have
been present in sections of the egg illustrated in the preceding
figure, they have perhaps escaped detection on account of the
syncytial nature of the cells at that period. It is at about the

98 GEORGO ORIHAY SHINJI

stage of development here illustrated that the mesodermal cells
become easily distinguishable from the other two cell layers,
namely, the columnar ectodermal cells forming the floor of the
invagination above and the layer of much smaller and feebly
staining syncytial cells below. The mesodermal cells at this
time can be distinguished from the ectodermal cells by their
large size and coarsely granular nuclear contents. The staining
reaction of the two is also different: the mesoderm stains heavily,
the entoderm only very feebly.

Up to this stage, invagination, in the case of Pseudococcus,
is indicated by a slight depression at a short distance ventrad
of the posterior pole of the egg. As the invagination progresses
further, the depression becomes deeper and its walls more closely
compressed (fig. 85). Mention should be made of the fact that
the depression in Icerya and perhaps in case of the other species
is, at first, in the form of a cup with broadly rounded bottom.
The entodermal cells are always found opposite the base of this
cup-like depression. With the further growth of the embryo,
the dorsal wall of the depression, now a compressed tube, be-
comes thinner and, at the time when the embryo reaches the
anterior mass of the parasitic organisms, it becomes reduced to
a layer one cell thick. The ectoderm cells also undergo a marked
change. They are arranged in a layer beneath or ventrad to the
amnion, and, at the same time, become much more elongated,
with their nuclei arranged alternately, giving the appearance of
two layers. The mesoderm, again, has spread out to its full
length below the ectoderm, which is now the ventral plate. The
cells of the ectoderm, mesoderm, and entoderm are still con-
tinuous at the extreme bottom of the invagination where the
primitive condition still exists.

From this time on, the caudal or abdominal portion of the
embryo elongates, extending over and above the ventral surface
of the ventral plate. Consequently, any transverse section
passing through the thoracic region also passes through an
abdominal segment. Figure 103 shows such a section through
the second maxillary segment and one of the abdominal segments.
Here, in the ventral or thoracic section, the amnion appears as a

EMBRYOLOGY OF COCCIDS 99

thin membrane composed of a few flattened cells. The mesoderm
cells, which, on both sides, meet the amnion and ectoderm, form
a layer one cell in thickness. Although the lateral extensions
of the mesoderm have followed the evaginations of the append-
ages, the layer is still one cell in thickness. The dorsal or
caudal section presents nothing new. It resembles a section
through the thoracic region of a younger embryo. Both the
amnion and the mesoderm consist of spherical cells, while the
ectoderm cells are spindle-shaped. All three germ layers are
continuous at the sides.

A longitudinal section of a somewhat younger embryo of
Pseudococcus (fig. 99) and one of Lecaniodiaspis (fig. 89) show
that the amnion actually terminates at both the caudal and
cepahlic ends and that it incloses the parts of the embryo. The
layer of mesoderm, which is much thicker at the caudal region,
gradually thins out toward the oral region, the cephalic region
being entirely devoid of it.

A transverse section of an older embryo (fig. 104) shows,
among other things, a remarkable change in the mesoderm.
On account of the neurogenic swellings, which occur on both
sides of the median line, the mesodermal cells have been pushed
aside, so to speak, from the area immediately dorsal to the
neurogenic area and have been shifted to the sides where the
evagination of the appendages has taken place.

A transverse section through the second maxillae of a much
older embryo is shown in (fig. 107). The dorsal or caudal section
shows, among other things, that the amnion is represented by a
single layer of cells, and that the mesoderm has expanded, not
only into the evagination of the appendages, but also in the
dorsal extension of the ectoderm.

A comparison of figures 103 and 107 will show that the dif-
ferentiation of germ layers takes piace from in front backward,
as in other insects, except the agamic aphids, in which, according
to Will (88), the cephalic portion develops earlier than the
brain.

As the appendages further elongate, the mesoderm follows
the ectodermal evagination, forming a double-layered structure.

100 GEORGO ORIHAY SHINJI

Similarly, the mesoderm keeps pace with the dorsal extension
of the ectoderm in the closure of the dorsal wall, as in the case of
other insects.

As the brain gradually grows in size, the amnion, which is
situated above, is pushed out until it comes into contact with
the serosa, and a rupture occurs at the point of union. Through
the rent thus formed, the embryo emerges from the amniotic
cavity and begins to rotate around its transverse axis until the
poles of the embryo coincide with those of the egg. During this
rotation the serosa and the united portion of the amnion shortens.
As a consequence, the cells that formed the serosa and amnion
aggregate at the dorsal end of the brain as a dense mass of
ellipsoidal cells—the dorsal organ. This dorsal organ lasts but
a short time, for it soon comes into contact with the invaginating
tip of the stomodaeum, where its elongate cells first become
disarranged and finally disappear, probably being taken up by
the cells of the stomodaeum.

The exact origin of the mesoderm cells is very difficult to
determine in the case of Icerya and Pseudococcus, because of
the crowded condition of the cells about the invagination pore.
In figure 83 the germ cells and a few entoderm cells are seen,
but no mesoderm cells. But in figure 86 there are four kinds of
cells, namely, the germ cells, ectoderm, mesoderm, and entoderm
cells. Therefore, mesoderm cells must have appeared during
the period between these two stages. The way in which the
mesoderm forms can, however, be well ascertained in the case of
Lecaniodiaspis, where there are comparatively few cells in the
early period of invagination. In figure 91 both the amniotic and
the ectodermal cells, as well as the mesoderm cells, are all con-
nected with the blastopore. This fact, I believe, indicates that
not only the mesoderm, but also the entoderm cells are both
developed simultaneously with the invagination at the same
point, namely, at the tip of the blastopore of Will. In this
regard, then, my observation differs from that of Witlaczil for
aphids, where the mesoderm layers are supposed to arise from
the ectoderm layer by delamination. Moreover, my interpre-
tation of the mesoderm formation is in accord with that of Will

EMBRYOLOGY OF COCCIDS 101

for aphids. With Will, I therefore conclude that, in these
families of insects, namely, Aphididae and Coccidae, and perhaps
others, there is a process of true gastrulation as in other higher
animals, and that the invagination pore of our insects represents
the blastopore of other animals, because it is here that the
three germ layers arise.

Mention should also be made that, in the case of coccids
studied, the mouth and the anus arise, respectively, at the
anterior and posterior ends of the invagination. Accordingly, I
agree with Will, Wheeler, Kowalevsky, and others that the
blastopore of insects is homologous to that of other animals,
e.g., of amphibia, and that it is much elongate, even to the
extent of forming a loop within the egg.

As previously stated, the entodermal cells in Pseudococcus
macdanieli become localized at the posterior end of the germ
band only. My interpretation of this unique phenomenon is
this: On account of scarcity of yolk, the entodermal cells remain
in a more or less inactive state as compared with the rapidly
proliferating mesoderm cells beneath. Consequently, the former
become entirely cut off from the point of their origin, the blasto-
poric rim. If this interpretation be correct, it follows that the
scale insects are much more specialized than the Orthoptera, in
which, according to Wheeler (’93) and others, the entoderm is
found at the two extreme ends of the germ band.

7. THE FORMATION OF THE NERVOUS SYSTEM

The nervous system of the coccids studied is formed exactly
in the same manner as in the case of other insects, for example,
in Blatta germanica, so well described by Wheeler (’93). No
account, however, of neurogenesis in Hemoptera,—aphids,
coccids, and others—has ever been presented, so some details
of the history of the nervous system of the coccids may not be
out of place.

Although the brain of the scale insects is but a continuation
of the ventral cord, and the ventral cord, in its genesis, is but a
part of the whole nervous system, the formation of these two
parts will be considered separately, as has been customary
among the embryologists.

102 GEORGO ORIHAY SHINJI

A. The ventral nerve cord. The rudiments of the ventral
nerve cord arise from the ectoderm layer of the ventral plate,
the origin of which has already been described. At first the
ectoderm cells forming the ventral wall of the amniotic cavity
are all similar in appearance, size, staining reaction, etc. They
are all cylindrical, almost spindle-like in shape (fig. 103).

Later, however, on each side of the long axis of the embryo,
a few large spherical cells are formed within the ectoderm layer
by mitotic division. Since, as will be shown later, all of the
nerve cells arise from these large spherical cells, the name,
neuroblasts, is applied to them. The remaining cells in the
ventral ectoderm do not contribute to the nervous system, but
go to form the body wall, and consequently they are called the
dermatoblasts. In like manner, the term neurogenic area and
dermal layer are, respectively, used to designate the portion of
the area containing the neuroblasts and that without them.

The number of the neuroblasts increases by a further mitotic
division of ectodermal cells. In figure 104 four such neuro-
blasts are shown, two on either side of the middle line, and in
addition to these, a division figure of an ectoderm cell is given.

In figure 107, which represents a transverse section through
the first thoracic appendages, the separation of the nerve rudi-
ments from the dermal layer is pronounced. ‘The ectoderm layer
now becomes so thin that it is everywhere but one cell in thick-
ness. On the other hand, the nerve rudiments are several cells
thick. <A layer of cells next to the dermal ectoderm in each of
the body segments consists of six large cells. Each of these
neuroblasts is usually seen in a different phase of mitosis. Dorsad
to the neuroblasts, there are about five horizontal rows of smaller
cells, which stain much darker than the surrounding neuroblasts,
the dermatoblasts, or the mesoderm cells. Nevertheless, these
darkly staining cells are the true daughter cells of the neuro-
blasts and are called ganglioblasts, for they form the ganglia.
All ganglioblasts are of the same size and are in the resting
stage. These facts indicate that they do not divide after they
become once separated from the neuroblasts.

EMBRYOLOGY OF COCCIDS 103

Comparisons and measurements of the neuroblasts in different
stages of development show that they are true teloblastic cells.
They do not diminish in size, but resume their original size after
each of a series of cell divisions. Thus, in fact, there is no cell
intermediate in size between the neuroblasts and the ganglio-
blasts. A comparison of figures 103 and 107 indicates that the
development of the ventral nerve cord takes place from in front
backward, as was the casein the development of the appendages
already described.

The abdominal ganglia, the number of which corresponds with
that of the segments, are noticeable in the embryo, a longitudinal
section of which is shown in figure 118. The nerve cord in the
figure is completely separated from the ectoderm of the ventral
body wall in the cephalic as well as in the thoracic segments, but
in a few of the last abdominal segments they are still connected.
In fact, the separation of the ventral nerve cord, like all phases
of differentiation, takes place from before backward. Each mass
of ganglionic cells which now constitutes a ganglion, contains
a mass of fiber-like ‘Punkt Substance’ of the Germans, the
nerve fibers. The nerve fibers are of two kinds, the longi-
tudinal amd the transverse (fig. 120), the former running parallel
to the long axis of the body, connecting with those of adjoining
ganglia, while the latter run at right angles to the longitudinal
fibers.

The formation of nerve fibers takes place much the same as
was described for Xiphidium by Wheeler (’93) and for the honey-
bee by Nelson (’15), namely, by the elongation and subsequent
transformation of the cytoplasm and the disappearance of the
nucleus of the ganglioblasts (fig. 116).

The neurogenesis described above agrees, in general, with the
results obtained by Wheeler in Doryphora and Blatta (’89) and
for Xiphidium (’93), Heymons for Forficula (’59), Graber for
Melolontha (’90), Lecaillon for Chrysomelidae (’90), Nelson for
Aphis mellifera (’15), and several other investigators in the case
of many other insects.

As to the number of neuroblasts which produce the ganglio-
blasts in each half of a body segment, no two investigators

104 GEORGO ORIHAY SHINJI

exactly agree. Wheeler, for example, found four in Xiphidium,
while Nelson stated that in the honey-bee the number of these
cells usually varied from three to six. In coccids there are six
on each side of the median groove.

Again, opinions are at variance as to whether the ganglioblasts
become directly converted into the nerve fiber or into their
daughter cells which constitute the nerve cells of the larva.
Wheeler (’89) for Doryphora and Nelson (15) for the honey-bee
claim that all the ganglioblasts undergo at least one division
before they become nerve-cells, while Wheeler for Forficula
(95), Lecaillon (’98) and Hirscher for Chysomelidae, and Essche-
risch (’02) for Musca maintain that the ganglioblasts do not
divide. In the coccids, as stated, the ganglioblasts do not
undergo any division, but, later, some of them are transformed
into nerve fibers.

The neurogenesis in the case of the brain is exactly the same
as in the ventral nerve cord. The differentiation of the ammion,
mesoderm, and ectoderm is shown in figure 105. The ectoderm
cells soon give rise to the neuroblasts and thus are differentiated
into the dermal and neural layers. The segmentation of the
brain into three regions, corresponding in number to: the future
brain segments, is also clearly noticeable in this figure.

Figure 110 is a corresponding longitudinal section of a some-
what older embryo. There are about five large spherical cells
or neuroblasts along the periphery of each of the brain segments,
as was the case with the formation of the ventral nerve cord.

In Coceids, as in other insects, there is originally one ganglion
in each of the body segments, making three in the brain, three
in the oral region, three in the thorax, and ten in the abdomen,
or nineteen in all. This number of ganglia is best seen in the
specimens shortly after the completion of the revolution (figs.
53 and 118). Shortly after the union of the stomadeum invagi-
nation with the entoderm or the midgut, all of the abdominal
ganglia, except the first, disappear, leaving only the longitudinal
nerve fibers behind. These slender longitudinal nerve fibers, it
may be added, run out from the posterior margin of the only
surviving abdominal ganglia and innervate the abdominal organs,
such, for example, as the ovaries, midgut, etc. (fig. 132).

EMBRYOLOGY OF COCCIDS 105

A special effort was made to locate the presence of the second
antennal appendages and their ganglia, which were found and
described by Riley and others in Blatta germanica. My figure
114, which corresponds approximately to Riley’s figure 5, shows
neither appendages nor ganglia. Throughout the embryonic
period there was no indication of the formation of the second
antennal appendages or the ganglia corresponding to them.

The apparently later increase in size of the brain has already
been mentioned in connection with the formation of the external
form of the body. The statement, however, does not mean that
neurogenesis in this region lags behind that in the ventral nerve
cord. In fact, neurogenesis in the former occurs and progresses
just as early as in the second maxillary segment, and, conse-
quently, is ahead of that in the thoracic segment. In this regard,
then, coccids seem to differ from aphids, in which, according to
Will (’80), the brain arises independently from the ventral nerve
cord, as in the case of the annelids.

Again, as my figures 62 and 67 show, the brain in coccids is
formed within and not beyond the rim of the blastopore, as Will
found in agamic aphids.

8. THE INTRACELLULAR SYMBIOSIS

Intracellular organisms in the eggs of scale insects have been
described under several different names, such as: ‘Pseudonavi-
cellae’ (Leuckart), ‘secundaren Yolk’ (Mecznikow), ‘‘ Highly re-
fractory bodies with specific gravity higher than water and may
represent spermatozoon”’ (Putnam), and ‘Mycetomia’ (Emeis).
The only investigator who denied the presence of such organisms
is Witlaczil, while Johnston, Child, and other students of the
internal anatomy of coccids failed to mention either their presence
or absence.

Mention has already been made of the presence of an organism
in the mealy bug, in the cottony cushion scale, and in Lecanio-
diaspis prunosa. These bodies present an appearance of true cells
at certain times in their history, but they more often stain heavily
and hence look like granules. Whenever they assume a true
cellular form, a cell membrane, chromatic granules, and cytoplasm

106 GEORGO ORIHAY SHINJI

can be made out. That they are really a kind of parasitic organ-
ism may be proved by the fact, according to Bliichner, that they
were successfully raised in culture media by an Italian investi-
gator. As Iam not familiar with the systematic position of the
different kinds of organisms found in the eggs of the several
species of coccids, I describe them under the somewhat indefinite
yet suggestive termsof ‘symbiotic organisms,’ ‘parasitic organisms’
and often as ‘polar colony of organisms,’ or simply as ‘parasites.’

The manner in which the eggs become infected by the organisms
may be observed by examination of older ovarian eggs, such as
is shown in figure 25. Here a few dark granular bodies occur in
the follicular epithelial cells situated above the constriction
between the nurse cells andegg chamber and also in the egg proper.
These dark staining bodies lie at first in the cytoplasm and not
in the nucleus of the follicular cells. Since, as illustrated, these
parasites are found, not only in the epithelial cells, but also in the
lumen between the epithelial layer and nurse cells as well as
within the egg, it is clear that they are carried into the egg proto-
plasm mostly by the flow of the nurse stream. Soon, however,
the chorion is formed around the cytoplasm of the egg, preventing
a further immigration of the symbiotic organisms into the egg.
In many cases I have noticed the organisms between the ovarian
epithelial layer and the chorionic membrane, long after the com-
pletion of the chorion around the egg. But no case has been
observed where these bodies actually penetrated through the
chorion into the egg.

The condition of the nurse chamber and also that of a portion
of a colony of the symbiotic organisms that almost envelops the
ovary at this period is shown in figure 40. A careful examination
of the specimen brings out the fact that these organisms, so
abundant in the cysts adjoining the ovaries, become literally
squeezed out of the walls of the cyst into the space between the
ovary and the symbiotic organisms. In short, all series of tran-
sitional stages between the liberation of the parasites and their
entrance into the epithelial cells, and final location at the posterior
end of the egg can be found in a few sections of a single female.

EMBRYOLOGY OF COCCIDS 107

The parasites, thus finally collected into a single mass, remain
at this position throughout the embryonic as well as the larval
and adult life. This place, it should be mentioned, corresponds
to the third to fourth abdominal segments of the future embryo
and adult.

After the completion of the blastoderm and the subsequent
invagination of the germ band (which occurs at about the pos-
terior end of the egg), the germ cells, the first differentiated cells,
migrate toward, and some of them actually become imbedded in
this colony of symbiotic organisms. Of the migratory cells,
those which become imbedded in the mass of parasitic organisms
are transformed into the secondary yolk cells of Will (’88). The
nuclei of the secondary yolk cells remain almost unaffected for a
long time, even after hatching. Again, some of the cells surround-
ing the colony of organisms transform into epithelial cells, while
a majority of them divide, multiply and finally become the defin-
itive germ cells.

As previously stated, the invaginating germ band gradually
increases in length until the caudal region becomes actually
curled over the thoracic region, but the colony of symbiotic
organisms remains almost stationary at the place where it was
first located soon after the entrance into the egg. This fixed place,
as previously stated, corresponds approximately with the third
and fourth abdominal segments of the future embryo and also
of the larva and adult.

From this time on, the spherical colony of organisms, consisting
of about eight compartments of spores, becomes spread out over
the rudiments of the ovaries. The organisms gradually increase
in number after hatching until they fill the greater portion of the
coelomic area of the adult females.

These organisms are also found in enormous numbers, simi-
larly located, in the larval as well as the adult males. From the
fact that these organisms remain throughout the life history of
the scale insects, I strongly believe that they are not sperm cells
as Putnam (’88) surmises. It also clearly shows that they do
not function as ‘Keimbahn determinants’ as Mecznikow doubted
in the case of Psylla.

108 GEORGO ORIHAY SHINJI

In the case of Icerya, the symbiotic organisms appear in a
somewhat different manner, especially as regards the place where
they first enter. A brief account of the mode and place of
entrance of the Iceryian form, therefore, may not be out of place.

In the cottony cushion scale, the symbiotic organisms first
appear, not around the anterior, but at the posterior end of the
egg. The method of entrance is, however, essentially the same
as in the case of Pseudococcus and Lecaniodiaspis already men-
tioned. After their liberation from the cytes, the rod-like
organisms migrate through the epithelial layer surrounding the
posterior end of the egg. In figure 35 several stages in the migra-
tion of these rod-like organisms into the egg are shown. ‘These,
like those of Pseudococcus, stain exceedingly darkly with iron
alum haematoxylin or any other nuclear stain, and consequently
their finer structure is difficult to make out. Nevertheless, more
favorable specimens show at least three regions in each of them.
These are the outer somewhat lightly staining portion, probably
representing cytoplasm; the inner more densely staining region,
resembling the nuclear region; and a central denser region, proba-
bly representing chromatic matter. Since, as stated, the symbi-
otic organisms, in the case of the cottony cushion scale, become
localized near the posterior end of the egg, where the invagination
of the germ band occurs soon after the final settlement of the
organisms, the germ cells come into contact with the parasites
without migrating far. But, since now the germ cells and the
symbiotic organisms are situated at the point of invagination,
they both become actually pushed into the egg further and fur-
ther, as the invaginating germ band elongates (figs. 62 to 67),
until they become similarly located as in the mealy bug and
Lecaniodiaspis. Assoonas they reach this point, a short distance
from the anterior pole of the egg, they become stationary and do
not accompany a further extension of the caudal portion of the
embryo. This fixed point corresponds, as in the case of other
scale insects, with the third and fourth abdominal segments of
the embryo, larva, and adult. The history of these organisms
from this time on is an exact repetition of what has been
described for the other two species.

EMBRYOLOGY OF COCCIDS 109

The symbiotic organisms in Homoptera were first discovered
in the pathogenetic embryo of aphids by Huxley (’58). Meezni-
kow (’66) detected a similar substance not only in the embryos
of aphids, but also in those of coccids and Psylla. This writer
thought his ‘secundaren Dotter’ to be characteristic of three
families of Homoptera, namely, Aphididae, Coccidae, and Psylli-
dae. Witlaczil (’84) and Will (’88) confirmed the presence of
‘pseudovitelli’ so far as the parthenogenetically developing
embryos of aphids are concerned. The occurrence of similar
granules in the winter or sexual eggs of Aphids was first reported
by Balbiani (’74) and was later confirmed by the researches of
Tannreuther (’07) and Webster and Phillips (’12). All of these
writers, however, did not consider these as living organisms.
Huxley (’58) was also the first who described the origin of the
‘pseudovitellus.’ He states that.the pseudova (parthenogenetic
eggs) of Aphids are eventually converted into cellular germs,
apparently by the same process as that by which an ovum is con-
verted into an embryo. ‘‘In these germs,’ he claims, ‘‘the
central part becomes a granular pseudovitellus, the peripheral a
blastoderm; the rudiments of the different organs next appear,
and the germ becomes surrounded by a pseudovitelline membrane.
Eventually,’”? he supposed, ‘‘the pseudovitellus becomes the
corpus adiposum.” _

Mecznikow (’66), however, observed the origin of the pseudo-
vitellus in aphids. He stated that the so-called secondary yolk
comes from the follicular cells situated at the posterior end of the
egg. This discovery was later confirmed by Witlaczil (’84) and
Will (’88).

Emeis (715) recently found a case of symbiosis in the eggs of
Coccids. These symbiotic Mycetomia were first found in a
certain epithelial cell near the nurse cells. Later, according to
him, they migrated into the protoplasmic portion of the egg.
Their subsequent history has, however, not been studied.

Inclusions other than the secondary yolk, but somewhat related
to this substance, were recorded for insects belonging to several
different orders. Blochman (’87) noticed a group of bacteria-
like organisms which he called bacterial ‘Stabchen’ in the eggs

110 GEORGO ORIHAY SHINJI

of certain Orthoptera. He did not state how the eggs were
infected, but thought it was a case of symbiosis. The occurrence
of non-living substances has also been recorded in the eggs of
several Diptera and Coleoptera. ‘These so-called Keimbahn-
determinants are located at the posterior end of the egg, as is the
case with the secondary yolk or parasites in Hemiptera, but are
known by several names. Thus Ritter (’11) gave the name of
‘Keimwulst’ for the Keimbahn determinants of Chironomus,
while Hasper (’11) termed them ‘Keimbahnplasma.’ | A similar
substance is described as ‘Dotterplatte’ in Calliphora (Kahle,
08), but is called ‘polares plasma’ in Miaster (Hegner, 712).
Recently Hegner (’09) applied the term ‘pole disc’ to a related
substance in the eggs of chrysomelid beetles. ‘The same nomen-
clature was adopted by Wieman (710) two years later for an
allied species.

The origin of the Keimbahn determinants in these insects is
still unknown. Hegner (’08, 710) states that he has attempted
to trace their origin, but failed to arrive at an exact source in the
case of the eggs of Chrysomelids (Hegner, 715). It was thought,
however, that as in the Hymenoptera chromatin granules might
be cast out of the nuclei of the oocytes, and that these granules
might gather at the posterior end to form the pole disc. It was
also suggested that chromatin granules from the nurse cell nuclei
might make their way into the oocyte and later become the
granules of the pole disc. It should not be forgotten, moreover,
that these granules stain like chromatin. In fact, they are non-
living substance and therefore cannot be homologous with the
simbiotic organisms found in Homoptera. There is, however, an
analogy in the relative position of these two kinds of polar in-
clusions, namely, the symbiotic organisms found in Homoptera
and the polar granules of eggs of Diptera and Coleoptera. The
germ cells of these insects become lodged in the polar granules
or symbiotic organisms early in the embryonic period.

As to the function of the symbiotic organisms found in aphids
and coccids, very little can be said. As already stated, the germ
cells of coccids, before their migration into the colony of the
symbiotic organisms, are already differentiated. This excludes

EMBRYOLOGY OF COCCIDS 111

the possibility of the function of the organism as the ‘Keimbahm
determinant,’ as Mecznikow surmised. The fact that these
organisms are present throughout the life history of scale insects
is in favor of the view just mentioned.

However, I cannot think that the presence of these symbiotic
organisms is altogether without significance. The species known
to harbor these symbiotic organisms, so far as present researches
go, belong without exception to the suborder Homoptera, which
are characterized by a long sucking proboscis, very delicate
membranous wings, and thin dorsal body wall. Coccids may
remain more or less stationary, feeding in the same place, for a
long period of time. It is clear, therefore that such Homoptera
as the scale insects and the aphids are apt to be exposed to changes
in the external conditions because they cannot change places as
easily as other insects.

It may be said that the part most susceptible to the environ-
mental change is the sex cells. As a rule, the sex cells of the
Homoptera, unlike those of the Coleoptera, in which the hard
elytra cover the dorsal surface of the body, lie beneath a thin,
almost unprotected dorsal body wall. Unless some special
means be provided for their protection, the sex cells would be in
danger of injury. In free moving Homoptera, such as Jassids,
Membracids, Flugorids, Cercopids, the means of avoiding injury
to the germ cells are found in quick movements by which these
insects are able to take refuge by either dropping to a more
favorable place or running behind the trunk or leaves of the
host plant. In the Coccids, Aphids, and Psyllids this change of
place cannot be effectively made. The presence of a mass of
highly refractory organisms that always surrounds the germ cells
in these three otherwise helpless insects strongly suggests a sig-
nificance—the protection of germ cells against injury and sudden
change in environmental conditions, such as rain, snow, extreme
heat, cold, ete.

JOURNAL OF MORPHOLOGY, VOL. 33, NO. 1

112 GEORGO ORIHAY SHINJI

9. THE ORIGIN OF THE GERM CELLS

In coccids the germ cells first become noticeable simultaneously
with the invagination of the germ band at the posterior end of
the egg. They are characterized by their large size, oval shape,
and the clearer nuclear appearance as compared with the other
cells, and, above all, by the feeble staining reaction. Increasing
in numbers by mitotic division, they remain for a time at the
point where the invagination of the germ band occurs. The
earliest stage in the history of these cells which has been observed
is shown in figure 81.

At the time the invagination becomes clearly visible, the germ
cells begin to migrate, one after another, toward the anterior
end of the egg where the colony of symbiotic organisms is located.
As soon, however, as they reach the mass of organisms, some of
them become actually imbedded between the spores of the
organisms, others elongate to form a sheath around it, while the
remaining cells aggregate superficially around the colony of
organisms (figs. 90, 92, and 133). Those cells that become
imbedded in or encircle the organisms remain in that condition
throughout the rest of the embryonic and larval life and con-
stitute the so-called secondary yolk cells of Will (’88) (fig. 90).

As already mentioned, the elongation of the embryo continues
beyond the anterior end of the egg so that the posterior portion
gradually curls over the thoracic region, but the germ cells and
the colony of the symbiotic organisms remain stationary. The
region where these cells, for the second time, become localized,
corresponds with the third and the fourth abdominal segments
of the larvae as wellas of adult female. With the segmentation
of the abdominal region of the embryo, the germ cells become
divided into the left and right halves.

With the revolution and subsequent shortening of the embryo
an invagination appears at the ventral surface of the ninth
abdominalsegment. The germ cells, which have for a long time
remained stationary around the symbiotic organisms, then
migrate toward and finally settle at the apex of the vaginal invag-
ination (fig. 119).

EMBRYOLOGY OF COCCIDS 113

Following the closure of the dorsal wall of the embryo along
the dorsal midline by the lateral growth of the ventral plate,
and with the further ingrowth of the vaginal invagination, the
germ cells are carried further cephalad and thus again come into
contact with the colony of the symbiotic organisms. But this
time they collect beneath, instead of encircling the organisms,
as was the case at first. Throughout the larval and adult stages
the germ cells and the colony of symbiotic organisms retain their
relative positions. The former is always found beneath or
ventrad of the latter as though they were under the protection
of the latter.

The foregoing account of the history of the germ cells in the
mealy bug is essentially applicable to that of Lecaniodiaspis.
One of the striking differences is that in the case of Lecanio-
diaspis the germ cells begin to migrate at a much later period
than in Pseudococcus macdanieli. The number of germ cells that
migrate toward the symbiotic organisms in the case of Lecanio-
diaspis is very much smaller than in the case of Pseudococcus
(fig. 89).

Inasmuch as the origin and subsequent history of the germ
cells in the cottony cushion scale is somewhat different from
what has already been given for the mealy bug and Lecaniodiaspis,
a short account for this species may be added here.

In the cottony cushion scale the germ cells become noticeable
at the posterior pole of the egg just as in the mealy bug. They
are larger, clearer, and their nuclei stain much more feebly than
those of the other cells around them. Although there is no doubt
that they, like those of the mealy bug and Lecaniodiaspis, are
able to migrate, the movement is not well marked on account of
the comparatively short distance that separates the blastodermic
layer from which the germ cells are derived and the colony of
‘symbiotic organisms. The germ cells of the cottony cushion
scale come into contact with the organisms earlier than in the
other two species, since the migration is a short one. ‘This fact
of the early association of the germ cells with the symbiotic
organisms, and also the posterior position of the latter, led
Mecznikow (’66) to a statement that the symbiotic organisms

114 GEORGO ORIHAY SHINJI

which he called the pseudovitellus may act as the Keimbahn
determinant in coccids and especially in Psylla.

Following this stage, the germ cells become pushed anteriorly
by the invagination of the ventral plate. When, however, the
colony of symbiotic organism becomes stationary near the pos-
terior end of the egg, the germ cells become also fixed, so to speak,
at the place which corresponds approximately to the third and
fourth abdominal segments. When the abdomen begins to show
segmentation, the mass of germ cells becomes detached from the
colony of parasitic organisms and aggregates to form left and right
syncytia, the miniature yet definitive ovaries.

Thus the chief difference between the germ cells of the mealy
bug and of Icerya is that, in the former, the germ cells early
migrate to the anterior colony of symbiotic organisms on their
own accord, whereas in the latter they move anteriorly, not by
their own effort, but by the ingrowth of the germ band.

The migration of the germ cells, singly or as a group, as I have
observed in coccids, is by no means peculiar to this family of
insects. Such an instance has already been described for the.
embryo of the potato beetle, Leptinotarsa decemlineata, by Heg-
ner (714), who found that the germ cells which apparently rest in
the amniotic cavity migrated later on into the embryo through a
sort of canal at the bottom of a groove in the germ band.

In the two families of Homoptera, Aphididae and Psyllidae,
which are closely related to Coccidae in form, life history, and
habits, the primitive germ cells were observed, just as in the case
of the coccids above mentioned, early in the embryonic devel-
opment. Mecznikow (’66) described the primitive germ cells of
parthenogenetically developing embryos of Aphis (Macrosiphum)
rosae before the appearance of mesoderm cells. Eighteen years
later, Witlaczil (’84) not only confirmed Mecznikow’s observation,
but was also able to see a single large clear cell, the primitive
germ cell, near the blastopore.

The primitive germ glands of chrysomelid beetles have also
been found to appear at about the same stage of development
as in the case of aphids. In Clytra laeviuscula, Gastrophysa
raphani, Chrysomela menthantri, Lina populi, and Lina tremulae,

EMBRYOLOGY OF COCCIDS iS

studied by Lecaillon (’98), no differentiation has been observed
among the cleavage cells until their migration to the periphery,
when they become the blastoderm cells. Although the point at
which the first cleavage cell reaches the periphery differs with the
species, yet in all species which have been investigated so far a
complete layer of blastodermic cells forms around the periphery
of the egg. Those cleavage cells that eventually come to lie
around the posterior pole of the egg where the polar granules are
located, become larger and clearer than their neighboring cells.
They then become disconnected.

Wheeler (’89) was not so fortunate as to observe such an early
segregation of the primitive germ glands in Leptinotarsa (Dory-
phora) decemlineata. According to him, these cells arise as two
elongated thickenings. of splanchnic mesoderm. Saling’s obser-
vation (’07) on the development of genital cells in Tenebrio
molitor is in accord with those of Heider and Wheeler in Hydro-
philus and Leptinotarsa. The germ sells of Hydrophilus piceus
also arise at a much later period of development. According to
Heider (’89), the germ glands of this beetle are derived from the
inner wall of the primitive abdominal segments on either side of
the body. At first they are indistinguishable from the neighbor-
ing mesodermal cells from which they originate, but soon they
grow in size, and their nuclei become clearer. In the following
Orthoptera the germ cells have been observed to develop in
such a manner and at about the same period of embryonic
development as in Hydrophilus:

Oecanthus niveus, Ayers (’83).

Blatta germanica, Heymons (’90-’91); Wheeler (99).

Periplaneta orientalis, Nusbaum (’66).

_Xiphidium, Wheeler (93).

The sex cells of the Hymenoptera have been derived from
mesoderm cells in much the same fashion as in the Orthoptera.
This statement agrees with the researches of Grassi (’84) for
Apis, of Carriere and Burger (’97) for the mason-bee (Chalico-
doma), of Petrunkewitsh and of Nelson (’15) for the female
honey-bee.

116 GEORGO ORIHAY SHINJI

Several cases of germ-cell segregation during an early cleavage
stage have been recorded in Diptera. As early as 1862, Robin
described what seemed to be the primordial germ glands of
Tipulides culiciformis. According to this investigator, four to
eight buds were seen at the posterior end of the egg. A similar
observation was made by Weismann a year later in the egg of
Chironomus. On account of their position Weismann called
tllem ‘die Polzellen.’ Both Robin (’62) and Weismann (’63)
failed to trace these cells up to the formation of definite organs.
Metznikow (’65, 66) and Leuckart (’65), however, found that the
‘Polzellen’ of Weismann migrate into the body cavity of the
embryo and become the sex cells in Simula, Chironomus, Culex,
and Miastor. In the following Diptera, studied by several
investigators since then, germ cells first become differentiated
before the completion of the blastoderm:

Chironomus, Grimm (’70).

Chironomus, Weismann (’82).

Chironomus, Balbiani (’’82-85).

Chironomus, Jaworowski (82).

Musca, Kowalevsky (’86).

Miastor, Kahle (’88).

Miastor, Voeltzkow (’89).

Calliphora, Lucilia, Graber (’89).

Chironomus, Ritter (’90).

Musca, Escherich (’00).

Calliphora, Noack (’01).

Chironomus, Hasper (’11).

Miastor, Hegner (’13-14).

Meyer (’49) could not find the genital organs of Liparia auriflua,
one of the Lepidoptera, until the caterpillars were over three
weeks old, while Balnini (’69—’72) found them in the embryo of
Tinea crinella at about the time the segmented appendages make
their appearance.

Woodworth (’89) and Schwangart (’05), however, found a
comparatively early differentiation of germ cells in certain butter-
flies. They found a thickening of the blastoderm near the
posterior end of the egg, the inner cells of which differentiate

EMBRYOLOGY OF COCCIDS 117

into germ cells. Later these migrate singly into the fourth to
the eighth abdominal segment and there become the forerunner
of the ovarian cells.

The primitive germ cells in Forficula auricularis were also
found by Heymons (’95) at about the same stage as in the case
of the butterflies mentioned above.

Heymons (’96), who could not find the genital organs in the
embryos of certain dragonflies and May-flies, is of the opinion
that in the Ephemeridae and Odonata the first genital rudiments
seem to appear during the larval life. This is, indeed, a case of
the latest segregation of the germ cells so far recorded among the
insects.

From the foregoing brief survey of the germ-cell formation in
insects, it becomes clear that a considerable variation is evident
in the manner and also in the time of their differentiation. This
variation is not a continuous one, but is such that the insects,
whose germ cell formation so far has been studied, can be grouped
in the following three categories:

1. Cleavage differentiation: Diptera, possibly Hymenoptera.

2. Blastodermic differentiation: Aphids, Chrysomelids, Lepi-
doptera and Coccids.

3. Mesodermic differentiation: Orthoptera, Aptera, Neurop-
tera, Hymenoptera, parasitic forms, Ephemeridae, Odonata,
Dermaptera and probably Heteroptera.

It is interesting to note that in all insects, the germ cells of
which have not been observed prior to the formation of mesoderm,
no special posterior inclusions have been found. Conversely,
in all the eggs having a certain kind of polar inclusion, whether
it be of nurse cell, epithelial cell or of nucleolar origin, the earliest
segregation of the germ cell has been recorded. Nevertheless, it
must be stated that the differentiation of the germ cells as such
cannot solely be accredited to the so-called ‘Keimbahn deter-
minants’ of Hegner. The germ cells of the Chrysomelid beetles
that possess the polar granules, appear at about the same time
as those of aphids and scale insects where there are no such
granules, but parasitic organisms. Such a striking case is that
of Euvanessa, where there is no possible inclusion of any sort

118 GEORGO ORIHAY SHINJI

comparable to those of Coleoptera and Diptera, yet, even in this
case, the germ cells arise just about the same time as those of
Coleoptera. The facts just mentioned, that the germ cells of
coccids, especially of Pseudococcus, become distinguishable as
such long before their approach to the colony of parasites, in-
dicate that the germ cells are by some chemotaxic action attracted
to the inclusion and do not become germ cells because they come
in contact with the latter.

10. THE FORMATION OF THE DIGESTIVE TRACT

The digestive tract of the embryo of the scale insects consists,
as in other insects, of the fore-, mid-, and hind-gut. The procto-
dzum and stomodeum in the scale insects are formed in exactly
the same manner as in other insects. The stomodeum grows
from its ventral opening, the mouth, dorsally, traversing the
future nervous rudiment almost perpendicularly, until it meets
the long axis of the egg, and then it turns posteriorly and meets —
with the midgut rudiment. The posterior (proctodzum) invag-
ination, on the other hand, takes place on the tenth abdominal
segment, the segment which, at the time when the anus is exter-
nally visible, still lies curled over the thoracic segment (fig. 50).
From this time on until the embryo completes its revolution,
the posterior invagination proceeds very slowly, as a comparative
study of the figures 126 and 127 will show. However, the rate
of invagination after the revolution is exceedingly rapid.

The midgut of Pseudococcus macdanieli Hollinger (MS) arises
from the entodermal cells, the origin of which ‘has already been
described.

The condition of the entodermal cells, at the time the thoracic
appendages begin to have two segments, is shown in figures 93
to 96. These represent consecutive longitudinal sections of the
same embryo. ‘The purpose of giving these serial sections is to
show that the cells forming the entoderm are not in any way
connected with the cells surrounding the colony of symbiotic
organisms; that they are an altogether different kind of cells;
that they are clearly separated from the mesodermal layer, and
that they are also distinct from the ectodermal cells. The

EMBRYOLOGY OF COCCIDS 119

entodermal cells, as illustrated in these figures, constitute two
parallel layers extending from the caudal extremity, where the
three germ layers arise, cephalad as far as the sixth or seventh
abdominal segment. The cells of the ectodermal layer at this
stage are ellipsoidal, and their nuclei stain exceedingly dark with
iron-alum haematoxylin, so they can be distinguished easily
from the spherical cells lying below them. The spherical cells,
which form a layer closely opposed to the ectoderm, are larger,
with richer nuclear contents than the cells of the ectoderm.
Thus it is clear that there is no possibility of confusing the
entoderm cells with the cells of the two other germ layers.

The germ cells situated around the colony of symbiotic organ-

isms, however, resemble the entoderm cells in many respects.
The common origin of the germ cells and entoderm cells, however,
can be disregarded by the facts that they originate at the two
different periods in the development of. the embryo and that,
at any time during their respective histories, the former never
- migrate caudad beyond the third and the latter cephalad beyond
the seventh abdominal segments.

Figures 126 and 128 represent two longitudinal sections of
different embryos, similar to those in figures 48 and 51. In both
cases the entodermal cells are much compacted near the posterior
end of the embryo. A crescentic groove is shown in figure 126.
This compacted condition of the entoderm and the appearance
of a clear groove within the entoderm cells are both due, not to
the actual massing or proliferation of cells as may be surmised,
but to the proctodaeal invagination which causes the elevation

of the posterior portion of the entodermal layer.

Following these stages, the entoderm cells multiply rapidly
and form a coiled tube (fig. 129), so that a transverse section
through the fourth abdominal segment passes through the ento-
derm three times as shown in figure 113. The midgut: tube is
pushed still further in toward the cephalic end of the embryo
by further ingrowth of the proctodaeum until its anterior end
meets and unites with the posterior elongation of the stomodaeum,
the history of which has already been described. The condition
of the alimentary canal shortly after the union of the stomodaeum
with the midgut is shown in figure 14. During the stages

120 GEORGO ORIHAY SHINJI

that follow the wall of the proctodaeum gradually becomes thin
and membranous, while the midgut increases in size (fig. 130).
After hatching, however, the cephalic portion of the midgut
becomes enlarged and finally assumes a shape very much re-
sembling the human stomach, while the stomodaeum remains a
slender tube throughout (figs. 56 and 132). A greater portion
of the midgut of certain coccids, especially that of Aspidiotus
neriil, was thought by Mecznikow to be derived from the elonga-
tion of the proctodaeal invagination, because, as he stated, the
alimentary canal of coccids is very short. In the case of Icerya
it was a very difficult matter to distinguish the rudiments of the
midgut in the early stages, and consequently I might have also
arrived at the same conclusion as Mecznikow did, had I not also
studied its development in the mealy bug.

In all insects both the fore- and hindgut are derived from
ectoderm, according to different investigators, with the exception
of Diptera, in which, according to Voeltzkow (’88) and Graber,
they are of mesodermal origin.

In the aphids, which are closely related to the coccids, the
alimentary system is, however, derived from a different source,
according to investigators. The fore and hindguts are here also
formed from the ectodermal invaginations. The midgut is
claimed by Will (’88) to come from yolk cells, but according to
Witlaczil (’84) the same organ is formed from the proctodaeal
and stomodaeal invaginations and therefore is strictly ectodermal
in origin.

In the majority of insects, however, the midgut is formed from
the anterior and posterior rudiments. These rudiments were
frequently spoken of as entoderm cells, but more often claimed
to be ectodermal in nature. The supposed entoderm cells in the
case of the honey-bee are, according to Nelson’s research, derived
from anterior and posterior portions of the blastodermic tube
which is in direct continuation with the side plate or ectoderm.

Thus, the point of interest in the formation of the midgut in
coccids is this: the midgut is entirely derived from its rudiments,
the entoderm cells, situated at the posterior end of the embryo.
This is, so far as my knowledge goes, a new type of the midgut
formation, never recorded in the case of the class Insecta.

EMBRYOLOGY OF COCCIDS 121

11. SUMMARY

1. The three ovarian elements, namely, the nurse cells, the
egg cell, and the follicular epithelial cells, are derived from the
primordial germ cells. All germ cells are exactly alike during
the oogonial period. The first differentiation begins after the
next to the last oogonial division. Few peripheral cells undergo
the last oogonial mitosis and enter into the so-called growth
period of the oocyte of the first order. Their nuclei are, at first,
condensed near one pole, but later become thread-like. At this
point another change occurs: three or four out of a group of four
(Icerya), or ‘five (Pseudococcus and Lecaniodiaspis) oocytes
situated peripherally overgrow the single cell situated at the
proximal end and secrete a sort of nutritive substance. At the
same time their nuclear contents become granular. These are
the nurse cells, while the single cell situated at the posterior end
becomes the true egg cell. The follicular epithelial cells, which
invest the nurse and the egg cells are oogonial cells that happened
to be at the proximal part of the group. These epithelial cells
multiply, like the somatic cells, by ordinary mitosis and their
nuclear contents do not assume the appearance of either a con-
densed or paired condition.

2. The ovarian egg, at the time of its passage into the oviduct
consists of the following parts: a chorion, yolk membrane, cyto-
plasm, germinal vesicle with its nuclear membrane and contents,
and a colony of symbiotic organisms. In addition to these, the
eggs of Icerya and Pseudococcus contain fat granules and yolk-
like substance.

3. All cleavage cells divide mitotically. No case of amitosis
occurs. Some of the cleavage cells later migrate to the peripheral
-cortical layer and become the so-called blastoderm, while the
rest of them remain within the egg and become the so-called yolk
cells. The first cleavage spindle lies at right angles to the shorter
axis of the egg and the place where the cleavage cell first reaches
the cortical layer is at the posterior pole of the egg.

4. The process of ventral plate formation is the so-called in-
vagination type. The invagination occurs at a short distance
ventrad to the posterior end of the egg. It is, at first, a shallow

122 GEORGO ORIHAY SHINJI

depression, but it gradually elongates to form a long folded tube.
Only the dorsal wall of the invagination becomes the embryo
proper, while the ventral wall gradually transforms into the
amniotic layer. .

The second maxillae, first maxillae, thoracic legs, mandibles,
antennae, and labrum appear in the order given. As the brain
grows enormously in size, the amnion in front of the brain is
pushed against the serosa, with which it finally fuses. At this
point of fusion a rupture occurs, through which the embryo
emerges, first the head, then gradually the rest of the body, and
thus it rotates around the transverse axis of the egg, with the
result that the poles of the egg and those of the embryo coincide.

A short time before the revolution, a large ventral middle
invagination occurs between the maxillae. At the time of the
completion of the revolution this invagination forms the ventral
cavity. The mandible and the first maxilla each produces,
from its distal or pointed end, a long chitinous bristle or oral
seta, while the labrum and the second maxilla together form a
box-like framework.

5. A short time before the egg passes into the oviduct, a colony
of globular (Icerya) or rod-shaped (Lecaniodiaspis) organisms mi-
grate into the egg through the follicular epithelial cells situated
at the anterior (Pseudococcus and Lecaniodiaspis) or posterior
end of the egg (Icerya). The germ cells, the first definitive cells,
migrate toward this colony of organisms and surround it. In the
case of Icerya, the germ cells, as well as the colony of the parasitic
organisms, are actually pushed forward by the invagination of
the germ band to a point a short distance from the anterior end
of the egg. Then both the germ cells and colony of the
parasitic organisms become stationary and do not accompany the
further elongation of the germband. Inthe case of Pseudococcus
and Lecaniodiaspis, the germ cells which are formed at the
posterior end of the egg, voluntarily migrate toward the colony of
organisms located near the opposite end of the egg. From the
colony of organisms the germ cells later migrate toward the tip
of the invagination and form two definitive ovaries.

6. In coccids there isa true gastrula. The entoderm, mesoderm,
and ectoderm are all continuous at the tip of the invagination.

EMBRYOLOGY OF COCCIDS 123

7. The rudiments of both the brain and ventral nerve cord
are derived from ectodermal cells of the ventral plate. The -
neuroblasts are formed on each side of the ventral middle line
by mitotic division of ectoderm cells. Each neuroblast produces
a series of ganglioblasts. The neuroblasts are teloblastic. Gan-
glioblasts transform into nerve fibers, either longitudinal or
transverse.

8. The stomodaeum and proctodaeum are respectively derived
from an anterior and a posterior invagination of the ectoderm,
while the midgut arises exclusively from the entodermal cells
massed at the posterior end of the embryo.

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HorscHett, E., AND Heiper, K. 1900 Text-book of the embryology of inverte-
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EMBRYOLOGY OF COCCIDS 125

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126 GEORGO ORIHAY SHINJI

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Euvanessa antiopa; in Scudder’s ‘‘Butterflies of New England,’’
vol. 1

a., anus

abd., abdomen
Abdl., abdominal
ag., anal gland

am., amnion

ant., antenna

bld., blastoderm

ch., chorion

cuc., cleavage cell
D., dc., dorsal organ
é., eye

ect., ectoderm

epd., epidermis

epl., epithelial cell
fb., fat cell

gangb., ganglioblast
gc., germ cell

gv., germinal vesicle
ha., hair

hag., hair gland

lb., labium

lbr., labrum

Ig1., 1st thoracic leg
lg2., 2nd thoracic leg
1lg3., 3rd thoracic leg
m., mouth

md., mandible
meso., Mesoderm

midint., rudiments of midgut

mus., rouscle

PLATES

ABBREVIATIONS

Mel., first maxilla
mx2., second maxilla
nu., nucleus

nc., nurse cell

nb., neuroblast

ng., neurogenic area
nf., nerve fiber

og., oogonium

ocl., ocular nerve
oyt., oocyte

oent., oenocyte
oesp., oesophagus
pb., polar body
proc., proctodaeum

rd., rudiment of abdomen

salg., salivary gland
stm., stomodaeum
sr., serosa

sp., spiracle

ten., tentorilum

tr., trachea

v., vagina

vg., vaginal gland
vp., ventral plate
y., yolk

yc., yolk cell

ys?., yolk substance?
1., brain segment 1
2., brain segment 2
3., brain segment 3

my., symbiotic organisms

JOURNAL OF MORPHOLOGY. VOL. 33, NO. 1

PLATE 1
EXPLANATION OF FIGURES

Pseudococeus

1 Ovary of larva at the time of hatching.

2 <A portion of sagittal section of the ovary of a larva in early fourth instar.

3 As above. Much later stage.

4to15 Stages in differentiation of the three ovarian cellular elements,
namely, the nurse cells, egg cell, and follicular epithelial cells.

EMBRYOLOGY OF COCCIDS PLATE 1
GEORGO ORIHAY SHINJI

PLATE 2

EXPLANATION OF FIGURES

Pseudococecus —

16 and 17 Stages in differentiation of the three ovarian cellular elements,
namely, the nurse cells, egg cell, and follicular epithelial cells.

18 A portion of transverse section through the egg chamber.

19 Transverse section through the nurse chamber like the one represented
in figure 15.

20 Longitudinal section of an ovariole somewhat older than the one repre -
sented in figure 17.

21 A portion of transverse section of an ovarian egg.

22 Longitudinal section of an early egg.

23 A portion of longitudinal section through germinal vesicle.

24 Longitudinal section of an egg when the germinal vesicle has reached the
periphery of the egg.

130

EMBRYOLOGY OF COCCIDS PLATE 2
GEORGO ORIHAY SHINJI

18

131

PLATE 3
EXPLANATION OF FIGURES

Pseudococcus

25 Longitudinal section of an egg somewhat older than the one represented
in figure 20.

26 Germinal vesicle in the metaphase of the first maturation division,
lateral view.

27 to 28 Longitudinal sections of egg of Lecaniodiaspis a short time before
their passage into the oviduct.

29 An oblique longitudinal section of the ovary of an adult Icerya, one hour
after copulating.

30 A portion of the ovary of an adult Icerya two days after mating.

132

EMBRYOLOGY OF COCCIDS PEATE
GEORGO ORIHAY SHINJI

wee

resto mpl

133

PLATE 4
EXPLANATION OF FIGURES

31, 33, and 34 Stages in growth of the eggs of Icerya.
32 Aportion of longitudinal section of an egg of Icerya fixed in boiling
Flemming’s solution.

134

PLATE 4

EMBRYOLOGY OF COCCIDS

GEORGO ORIHAY SHINJI

135

PLATE 5

EXPLANATION OF FIGURES

35 A portion of transverse section of the ovary of egg-laying female. Icerya-

36 A portion of transverse section of a Pseudococcus larva in the third instar.

37 Longitudinal section of the egg of Pseudococcus after the polar body
formation.

38 The egg of Pseudococcus after the protrusion of the first polar body.

136

PLATE 5

EMBRYOLOGY OF COCCIDS

GEORGO ORIHAY SHINJI

137

PLATE 6

EXPLANATION OF FIGURES

39 Longitudinal section of an egg of Pseudococcus, showing only one blasto-
derm cell and several cleavage cells.

40 Longitudinal section of an egg of Pseudococcus.

41 A portion of figure 43, more enlarged.

42 Transverse section of the egg of Pseudococcus at the time of the comple-
tion of the blastoderm.

138

PLATE 6

EMBRYOLOGY OF COCCIDS
GEORGO ORIHAY SHINJI

139

PLATE 7

EXPLANATION OF FIGURES

43 The first cleavage cell of Lecaniodiaspis.

44 Longitudinal section of an egg of Pseudococcus in an early blastoderm
stage.

45 to 47 Surface views of embryos.

48 and 49 Surface views of embryos, somewhat older embryos.

50 Lateral view of embryo.

51 Surface view of a somewhat older embryo than the one represented in
figure 50.

140

EMBRYOLOGY OF COCCIDS PLATE 7
GEORGO ORIHAY SHINJI

50

my miding
.
ly3
[g2
Igl

m idint

mx2

Mx!
md

14]

ys)
56

PLATE 8

EXPLANATION OF FIGURES

Pseudococcus. ‘Toto preparations.

Lateral view of an embryo during the process of revolution.

Lateral view of an embryo after the completion of revolution.

Surface view of an embryo like the one represented in figure 53.

Surface view of a much older embryo.

Lateral view of still older embryo at the time of the completion of the

alimentary canal.

57
58
59
60

A Pseudococcus larva at the time of hatching. Toto preparation.

An early cleavage stage; toto preparation of an egg of Lecaniodiaspis.
Surface view of a Pseudococcus embryo in an early invagination stage.
Surface view of an Lecaniodiaspis embryo at the time of the appearance

of the mouth rudiment.

142

EMBRYOLOGY OF COCCIDS PLATE 8
GEORGO ORIHAY SHINJI

| rege

ix

~~~ mtdint

--193

midint-+--

145

JOURNAL OF MORPHOLOGY, VOL. 33, NO. 1

PLATE 9
EXPLANATION OF FIGURES

61 Lateral view of an Lecaniodiaspis embryo like the one represented in
figure 60.

62 to 67 Stages in the growth of the embryo of Icerya.

68 and 69 Stages in the development of Iceryan embryo.

144

EMBRYOLOGY OF COCCIDS PLATE 9
GEORGO ORIHAY SHINJI

---——am

145

PLATE 10

EXPLANATION OF FIGURES

70 to 75 Stages in the development of Iceryan embryo.

72 Lateral view of an embryo shortly after the completion of revolution.

73 Lateral view of older embryo almost ready to hatch.

74 Embryo at the time of the union of the midgut rudiments with the pos-
terior prolongation of the stomodaeum.

75 Ventral view of a larva eight hours after hatching.

76 Longitudinal section of an Iceryan egg in early cleavage stage.

146

EMBRYOLOGY OF COCCIDS PLATE 10
GEORGO ORIHAY SHINJI

Ae
midint

147

PLATE Or

EXPLANATION OF FIGURES

77 Polar view of the metaphase figure, early cleavage cell of Icerya.

78 <A portion of figure 76, much magnified.

79 ‘Transverse section of an egg of Lecaniodiaspis in early cleavage stage.

81 Posterior half of longitudinal section of the egg a short time after the
completion of the blastodermic sac. Pseudococcus.

148

“

EMBRYOLOGY OF COCCIDS
GEORGO ORIHAY SHINJI

149

PLATE 11

Te

PLATE 12
EXPLANATION OF FIGURES

Pseudococeus

82 and 83 Posterior half of longitudinal sections of the eggs a short time
after the completion of the blastodermic sac.

83 Longitudinal (oblique) section of a somewhat older embryo than the one
represeented in figure 84.

85 to 87 Oblique longitudinal sections of more older embryos.

150

EMBRYOLOGY OF COCCIDS PLATE 12
GEORGO ORIHAY SHINJI

x 28,
> goe

20,00 0500s Sia 2; <

eg 959.8059 95 —_-mup

Rp, ww a 3 38 ra

89
90
91
92

PLATE 13

EXPLANATION OF FIGURES

Longitudinal section of an egg of Pseudococcus.

A magnified view of figure 99 between vp. and my.

Posterior half of a longitudinal section of an early Lecaniodiaspis embryo.
A portion of figure 96, much magnified.

152

EMBRYOLOGY OF COCCIDS PLATE 13
GEORGO ORIHAY SHINJI

” |
" midint

153

PLATE 14

EXPLANATION OF FIGURES

93 t096 Four consecutive longitudinal sections of anembryo. Pseudococcus.

97 Transverse section of an embryo, Lecaniodiaspis, approximately in the
stage represented in figure 89.

98 Oblique longitudinal section of an egg of Pseudococcus.

99 Longitudinal section of a Pseudococcus embryo.

190 A portion of transverse section through the blastopore of Pseudococcus.

154

PLATE 14

EMBRYOLOGY OF COCCIDS

GEORGO ORIHAY SHINJI

- prot

155

PLATE 15

EXPLANATION OF FIGURES

101 Oblique transverse section through the posterior end of an egg of Icerya.

102 A portion of longitudinal section of an egg like the one represented in
figure 66.

103 Transverse section through the second maxillae of an Pseudococcus
embryo like the one represented in figure 50.

104 Transverse section through the second maxilla region of an embryo like
that represented in figure 50.

156

EMBRYOLOGY OF COCCIDS PLATE 15
GEORGO ORIHAY SHINJI

meso ect
'
: i
\
!

4, PFO A )

PLATE 16

EXPLANATION OF FIGURES

106 Oblique frontal section of the embryo like the one represented in figure 55.

107 Transverse section of a Pseudococcus embryo somewhat older than the
one represented in figure 55.

108 Transverse section through thoracic region of the embryo represented in
figure 105.

109 Transverse section through the last thoracic region of an embryo some-
what older than the one represented in figure 55.

158

PLATE 16

EMBRYOLOGY OF COCCIDS

GEORGO ORIHAY SHINJI

106

-gangb

108

159

JOURNAL OF MORPHOLOGY, VOL. 33, NO. 1

PLATE 17

EXPLANATION OF FIGURES

110 Longitudinal section through the brain of an embryo like the one repre-
sented in figure 51.

111 Transverse section through the thoracic region of a Pseudococcus embryo
like the one represented in figure 72.

112 Transverse section through the salivary glands of Pseudococcus.

113 Transverse section through the third abdominal segment of the embryo
almost ready to hatch.

114 Transverse section of a Pseudococcus embryo like the one represented
in figure 51.

115 Another section of the same embryo as figure 114.

116 A portion of figure 117 magnified.

117 Oblique longitudinal section of an embryo.

160

EMBRYOLOGY OF COCCIDS PLATE 17
GEORGO ORIHAY SHINJI

“~-meso

gangbh

161

PLATE 18

EXPLANATION OF FIGURES

118 Median longitudinal section of a Pseudococcus embryo a short time before
the completion of the alimentary canal.

119 Longitudinal section of the embryo of Pseudococcus at the time of the
completion of the alimentary canal.

120 Longitudinal section of an Iceryan embryo.

121 Transverse section of an Iceryan embryo like the one represented in
figure 119.

PLATE 18

EMBRYOLOGY OF COCCIDS

SHINJI

GEORGO ORIHAY

N,
3
>

maces ae
foes me ra Ors & i
SUE aa el

dint

ate,

163

PLATE 19

EXPLANATION OF FIGURES

122 Transverse section of Iceryan embryo like the one represented in
figure 119.

123 and 124 Hair glands of a newly hatched Pseudococcus larva.

125 Transverse section through the eyes of an embryo like the one represented
in figure 119.

126 Caudal portion of an embryo Pseudococcus like the one represented in
figure 70.

127 and 128 Stages of the growth of the rudiments of the midgut of
Pseudococcus.

164

PLATE 19

EMBRYOLOGY OF COCCIDS

GEORGO ORIHAY SHINJI

124

123

at

2 Do0G GL:
< Denke

Be
‘So 089

+)

eo!
e
°

— meso

+3)
=———— meso
midint

t

165

PLATE 20

EXPLANATION OF FIGURES

129 and 130 Stages of the growth of the rudiments of the midgut of
Pseudococcus.

131 Longitudinal section of an Iceryan embryo a short time after the com-
pletion of revolutions.

132 Longitudinal section of an Iceryan embryo like the one represented in
figure 119.

133. The colony of symbiotic parasites at the time of the hatching of larva.

166

EMBRYOLOGY OF COCCIDS PLATE 20
GEORGO ORIHAY SHINJI

22 te
Soe
26

@o

e®,

icy
set
e

midint®
Gna.

Resumen por el autor, Charles L. Parmenter,
Universidad de Pennsylvania.

Numero de cromosomas y parejas de cromosomas en las mitosis
somiaticas de Amblystoma tigrinum.

El autor dd a conocer en el presente trabajo un estudio del
numero de cromosomas y sus relaciones de longitud en las células
de varios tejidos somdticos de Amblystoma tigrinum, cuyos
resultados suministran pruebas en favor de la teorfa de la indi-
vidualidad de los cromosomas En sesenta células pertenecientes
a veinte y tres individuos diferentes el ntimero de cromosomas
es constantemente veinte y ocho. Las medidas lineales de los
cromosomas de un ntimero limitado de complejos seleccionados
cuidadosamente indican que los cromosomas de una célula forman
una serie duplicada en tamafio y forma, lo que presta apoyo a la
suposicion de que estan formados por pares de cromosomas
homdlogos maternos y paternos. También existe una constancia
aproximada en la relacién de tamafo entre los pares de cromo-
somas de los complejos de diferentes individuos. Los datos
mencionados favorecen la teorfa de la individualidad de los
cromosomas y no confirman el aserto de Della Valle, que supone
que la variacién del nimero de cromosomas es la regla y que las
longitudes de dichos cromosomas en una célula se deben meramente
a una casualidad.

Translation by José F. Nonidez
Carnegie Institution of Washington

AUTHOR’S ABSTRACT OF THIS PAPER ISSUED
BY THE BIBLIOGRAPHIC SERVICE, OCTOBER 13

CHROMOSOME NUMBER AND PAIRS IN THE
SOMATIC MITOSES OF AMBYSTOMA!
TIGRINUM

CHARLES L. PARMENTER

Zoological Laboratory, University of Pennsylvania

THIRTY-SEVEN FIGURES (NINE PLATES)

CONTENTS

TURTON 9. dts 2s Ho SUS Ae Boba toe otto oid Cloke toe neo ero mace ORCC aca Reena
so 7A
56 ZS
5 AlzAG}
meal vi7
so Mer
Sis
b. Possible variation in number in uncounted romp lene: foe Bae
. 182

Technique..
(nee amons.. of Pees
A. The eves a Pinemocauese : Saint
a. Method of determining ine. “aerial ere
1. Procedure. , Anis
2. Clearness and apecii cota oF ae spl cee.

c. Abnormal complexes.........

Bees OMA tie, CHLOMOSOME PAIS! serps bo. 2:44 eles ss oe oo Ae A Dae ee ee
CmlMtrOdUGtonyastatementinwins cece sein ao cela ae ee eee ee
. 185

b. Mensuration.. aS:
1. Types of cells Be ea ey nee RR AA tM un ate
D. ie ek DE a Ae Oi | an NO a aah

Debvidence fon theyexissencerol PAlnsee.... sce acca seer ae
Ore OUMTT Any ee ear ee T eeers yeee hr tty ttk foe Gone oe oe een
Discussion. . week
A. imeeeductany Premiere weer
B. Constancy of Tinta en univ uae wo 8 RY OOM ROMs OMY
Gry arigbions mother Wrodeless..-..tsse ss cnc. 0% «2 so) Davie he nie ek choevaaiees
1D), WENO SOI IM ClaienioMnis > ae te poem ene Ga Deebod be odocoo uaHe ona ees
E. Fragmentation. .
F. Existence of pairs..

a. Pairs in the germ ncelise.

b. Pairs in the somatic SU Le oe Ee ake eee ae

1. Meves’ measurements............
PewWella Vallessamedsurements. 2 os cc cacao « tek acct
Si Jeesiuillush tm VNamongsiorane) inlsenMOwON, | aa ooono ne ooo bn gosuooKe ene
emConstanienelapiversizeure avionss s-sneeetenere meee seine oe eee nee
GuSuniun a Gy Olemiensuneme nda)..." d4 ao «Facto ckeoharslerg reise Ge eiec
SuanN Ty gO tGOMCIUSLOMS sf, Y8t) a: Gio sells wun o% oelcgre oeeoreteatait aletns: daueie se-se ee

169

181

184
184

186

. 186
SE SOURCESEOMELROL ya rere t Tie Aon ee aes EEL ihan coe Toe
Guhesulliszot measurements cacent meat cok tc ose ciees, cuclouysl > dimers
Jee Cribkenia ford eherminimoe paisa eee). scl a te satan eee

186
191
191
194
199

. 200

200

200

206
206

. 207

208

. 208

210
211
215
216
220
220
221

1 Also known as Amblystoma.

170 CHARLES L. PARMENTER

INTRODUCTION

It is believed (McClung, 717, pp. 536-38) that the chromatin
of an organism is, for the most part at least, the idioplasm, and
consists of a definite linearly arranged series of differentiated
materials which is perpetuated from generation to generation.
The chromosomes which are essentially constant in number in
an individual are thought to constitute the visible mechanism
for this perpetuation. This conception is known as the theory
of the individuality of the chromosomes, which is quite generally
accepted by all who have an intimate acquaintance with chro-
mosome behavior. However, there are a few not so acquainted
who strenuously oppose the theory.

Among these is Della Valle (’09, 711, ’12), who presents some
data and a large amount of discussion in an effort to disprove
this theory upon the claim that the chromosome number in an
individual is not constant, but is simply the quotient of the
quantity of chromatin divided by the average size of the chro-
mosomes. ‘This removes from them any constancy of organiza-
tion and contradicts the above theory. These observations have
been cited by other opponents of the theory as cytological
evidence in favor of their contentions. Della Valle’s conclusions
are based upon observations made upon dividing cells of the
peritoneum and blood-cells of Salamandra maculosa, together
with a large amount of data taken from the observations of .
others.

Meves (11) and Della Valle (12) further oppose the theory
upon the basis of linear measurements made upon the spermat-
ogonial and somatic chromosomes of Salamandra maculosa in
denying Montgomery’s (’01) and Sutton’s (’02) claim that the
chromosomes occur in pairs whose homologues are of equal
length, and that approximately constant size relations among
chromosomes are maintained from one cell generation to
another.

In the spring of 1916 I was fortunate in obtaining peritoneal
and other somatic tissues of Ambystoma tigrinum. This made
it possible to repeat Della Valle’s observations upon the somatic

CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA HEL

cells of the same and other tissues of this closely related species
and thus to determine whether such a variation as he claims is
present in the somatic tissues of other Amphibians. Also the
chromosomes of some cells in my material are sufficiently
favorable for measurements to permit a reconsideration of their
length relationhips.

Since this paper is regrettably controversial, it is necessary to
give careful attention to all the methods and conditions under
which the preparations and the observations were made. Della
Valle also lays much emphasis upon this point, and therefore
considerable space is devoted to this problem.

For facilities in collecting and preparing this material I am
indebted to the courtesy of the Department of Zoology of the
University of Minnesota, and to Prof. C. P. Sigerfoos I owe the
loan of several very excellent preparations. The work was done
under the direction of Prof. C. E. McClung, of the University
of Pennsylvania, toward whom I feel especially grateful for
constant encouragement and valuable criticism, and for his
characteristically generous and kindly interest at all times. I
am also greatly indebted to other members of the department,
especially to Dr. Eleanor Carothers and Dr. D. H. Wenrich, for
helpful suggestions and very painstaking criticisms.

TECHNIQUE

The material used was obtained during the spring of 1916
from larvae of Ambystoma tigrinum, which were abundant in
the ponds and lagoons near the College of Agriculture of the
University of Minnesota. Mitotic figures in epithelial cells of
the tail, gill plates, and lung, and of the endothelium from
peritoneum and mesentery were studied.

Tail epithelium

Very excellent preparations of this tissue were kindly loaned
me by Prof. Charles P. Sigerfoos, of the University of Minnesota.
These were made from the tails of larvae 2 to 14 inches in length
obtained during the last of May and the first of June during
several years. |

172 CHARLES L. PARMENTER

The living larvae were thrown into Flemming’s stronger solu-
tion. After about four hours of fixation, the tails were split
dorsoventrally? into two thin plates of cells. These two plates
of cells were then fixed twenty hours longer. After washing
in running tap-water for twelve hours or more, the pieces were
stained in toto in Heidenhain’s haematoxylin, carefully dehy-
drated, and cleared in xylol and mounted in damar.

Gill-plate epithelium

The most successful gill-plate preparations were also obtained
from larvae ? to 13 inches long. In each larva there are eight
gill plates, one subtended from each gill arch and another behind
each posterior gill cleft. These gill plates contain very numer-
ous mitotic figures. They are composed of two epithelial
lamellae with connective-tissue cells and capillaries lying between
them. The two layers, unseparated, are so thin that they give
very excellent preparations.

The material was fixed in situ by dropping the living larvae
into the Flemming’s stronger solution as soon as they were taken
from the net. They were fixed in situ twenty-four hours and

2 Haecker (’99) describesa very successful method of separating these two plates
of cells. The posterior end of the larva is cut off after fixation just in front of
the cloaca. With a sharp scalpel the thick cephalic end of the tail is split dorso-
ventrally through the middle of the vertebra to a depth of an eighth of an inch
or more. By grasping with the forceps the ends thus made free, the two layers
of epithelium can be pulled apart in a manner similar to separating two sheets
of fly-paper with adhesive surfaces sticking together. Professor Sigerfoos advises
separating the two layers after about four hours of fixation and then allowing to
fix about twenty hours longer.

The numerous large mitotic figures in various stages with clear cell walls
which can be studied without an immersion lens makes this material excellent
for the class-room. The gill-plate preparations are equal or superior to those
of the tail epithelium. Those of larger larvae are too thick when mounted in
toto, but give very satisfactory preparations when separated. Peritoneal prepa-
rations of larger larvae contain fewer mitoses and the cell walls are indistinct.
However, preparations can be made from the more rapidly growing shorter
larvae from which the gill-plates were taken and would probably contain more
divisions. Preparations of Ambystoma punctatum are less favorable than those
of A. tigrinum because there are fewer figures, more pigment cells in the tail
epithelium and the gill-plates are small and thicker.

CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA Ls

washed in running tap-water. The larvae from which the gill-
plates were taken were fixed for other purposes, and no special
effort was made to insure good fixation of the plates. They
were preserved_in position in 5 per cent formalin, which was
later gradually replaced with 80 per cent alcohol.

The fixed gill-plates were carefully removed from the larvae in
80 per cent alcohol and were left attached to the gill arches,
which, in subsequent handling, were grasped by forceps to prevent
injury of the plates. Hydrogen peroxide was added to the 80
per cent alcohol drop by drop through a fine glass capillary
siphon until the solution amounted to equal parts of each.
In this the plates were bleached for four to twelve hours and then
transferred to the mordant by the above-mentioned drop-process
and stained in iron haematoxylin. They were dehydrated by
this drop method, cleared in cedar-wood oil followed by xylol,
cut from gill arches, after being transferred to the slide, and
then covered with damar and a thin cover-glass. While the
damar was hardening they were kept for twenty-four hours or
more under slight pressure to insure flat preparations.

The peritoneum, mesentery, and lungs

These preparations were made from larvae 3 to 4 inches long.
All the tissues of a given individual were not only fixed together
in the same fixative for the same length of time, but also received
the same treatment in all subsequent processes. They were put
into the fixatives within an estimated maximum of two minutes
after the first incision. Two methods of procedure were used in
preparing these tissues for fixation:

1. In order to avoid any possible unfavorable effect of cap-
tivity, the tissues were fixed in situ in the field as soon as the
larvae were taken from the net. The animals were prepared for
fixation as follows: With sharp scissors the body wall was cut
open along the midventral line and also lateral incisions were
made on each side at right angles to the first incision behind the
pectoral girdle and in front of the pelvic girdle, so that the two
halves of the body wall fell away from the viscera and opened

174 CHARLES L. PARMENTER

wide the body cavity. The folds of the viscera were pulled
apart and the whole larva was plunged into the fixative. This
secured immediate and uniform fixation. The operation requires
less than a minute and the incisions are apparently painless, for
the larva does not often struggle.

2. The body walls, lungs, and viscera were removed from the
body of the larvae before fixing, either in the field or at the
laboratory. The peritoneum was fixed in situ on the body walls.
Only normally inflated lungs were used, and these were ligated
anteriorly before removal from the body to prevent them from
collapsing. After fixing one or two hours, they were cut into
two or more flat longitudinal strips and returned to the fixative.
The mesentery, attached to the intestine, was spread out flat
on a piece of glass and the whole immersed in the fixative with
the tissue beneath.

Fixatives

The fixatives used were: 1) Flemming’s stronger solution,
thirty hours; 2) Rouin’s solution, forty-three hours; 3) Bouin’s
solution, to which was added 14 grams of chromic acid crystals
per 100 ec., twenty to twenty-four hours; 4) Hermann’s solution
with two parts of osmic acid (Lee, ’13, p. 38) twelve to eighteen
hours; 5) a solution of saturated picrie acid 75 ec., formalin 15
ee., glacial acetic acid 10 cc., urea crystals 2 grams, thirty to
forty-three hours. The urea should be added gradually to the
solution warmed to about 40°C., otherwise a precipitate is
formed.

It is a difficult matter to decide which solution gave the best
fixation. The prettiest cells were fixed in Hermann’s and the
chromic acid modification of Bouin’s fluid. However, the peri-
toneum preparations of the osmic fixatives were a little thicker
and less transparent than the others. If any fixative should be
exclusively chosen, I believe it should be the chromic acid modi-
fication of Bouin’s solution, because of its excellent fixation,
convenience, and economy.

The peritoneum was removed as follows: The two sides of
the body wall were detached by an incision along the back

CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 175

close to the spine. Under the binocular lens, in water, the
peritoneum was carefully loosened from the underlying tissue
by scraping it with a sharp scalpel, first along the edge cut
from the back. Sections of this loosened edge were then grasped
by the forceps and relatively large sheets were easily pulled off
from the underlying tissue. _The peritoneum covering the dorsal
and lateral portion of the body wall is deeply pigmented and to
it adhere considerable muscle and connective tissue when the
peritoneum is removed. This portion was grasped with the
forceps in removing the peritoneum from the body wall, as well
as in all subsequent handling. Consequently the cells in the
ventral transparent region available for study have been undis-
turbed by instruments. However, there still remains a possi-
bility that the strain of pulling the peritoneum loose might
disturb some cells.

Peritoneum fixed in Flemming’s and Hermann’s solutions was
stripped from the body wall after four hours of fixation and then
fixed twenty hours longer. That treated with the various picric
acid mixtures was stripped immediately after fixation. However,
the peritoneum fixed in the chromic acid modification of Bouin’s
solution may be preserved in alcohol for as much as a year before
stripping. That of Ambystoma punctatum, fixed in Flemming’s
stronger solution and preserved in 5 per cent formalin, can be
stripped at least six months after fixation.

Material fixed in osmic acid fluids was washed five to fourteen
hours in frequent changes of tap-water. Picric acid preparations
were gradually transferred to 70 per cent alcohol, beginning with
10 per cent and progressing through successively stronger grades
differmg by 10 per cent. They remained in each grade five to
ten minutes. The tissues remained in 70 per cent alcohol con-
taining a few drops of saturated aqueous lithium carbonate
solution until the picric stain was removed, and before staining
they were returned to water by reversing the above process.
All of the material was stained in Heidenhain’s haematoxylin after
mordanting in a 23 per cent solution of iron alum for four to six
hours. No counterstains were used.

JOURNAL OF MORPHOLOGY, VOL. 33, NO. 1

176 CHARLES L. PARMENTER

Dehydration was accomplished by passing the material through
the above grades. The fluids were removed from, and added to,
the containers without handling the material. Alcohols were _
followed by half xylol and half absolute alcohol, and finally by
xylol.

The pieces of peritoneum were transferred from xylol to a
slide where the above-mentioned pigmented area, with the at-
tached muscle fibers, was removed quickly with a sharp scalpel
just before mounting. After mounting in damar under a cover-
glass, they were put under a light pressure for twenty-four
hours or more while drying to insure as flat a preparation as
possible.

OBSERVATIONS

It should be emphasized that the preparations upon which
these observations were made are unsectioned surface mem-
branes. This makes it possible to study the mitotic figures with
the confidence that all of the chromosomes are present and that
none have been cut and are being counted more than once.
This is an important consideration in determining whether the —
number of chromosomes is constant.

A. The number of chromosomes

There are twenty-eight chromosomes in the somatic complexes
of Ambystoma tigrinum. In forty-five unquestionable enumera-
tions and in eighteen which contained either one or two chro-
mosomes that might possibly be considered subject to interpre-
tation, there are none which vary from twenty-eight. In three
complexes, because of the alternative interpretations possible at
one or more points, the number cannot be definitely determined
and is interpreted to be either twenty-seven or twenty-eight.
The fact that these numbers are so close to twenty-eight is
strong evidence that these cells contain the usual number of
chromosomes.

The counts as indicated in the accompanying table have been
obtained from twenty-three different individuals varying in age

CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 177

approximately from six to ten weeks. The preparations of tail
epithelium loaned by Professor Sigerfoos were taken from a
collection which he has been accumulating for a number of years.
It is probable, therefore, that the counts in these preparations
represent the chromosome number present during a series of
years and that the number is constant from year to year.

Table showing for each tissue studied, the number of different individuals repre-
sented and the number of complexes with their distribution into classes as de-
scribed on page 178. The total number of different individuals represented is
twenty-three

NUMBER CLASSES
TISSUE —9RD EE — EE ————————EE————— es TOTAL
VIDUALS I II III
IReniioneumey ons aah eee wore oen 7 14 6 1 PAY
INTESemibe type tcr te cievaiticstises sists te, ahese 1 1 0 0 1
UIT Meare etnrcte chars aia tccucra Bisgeter aa 7 2 il 10
“LU ey OTH CC) Ws a ee A 5 9 4 0 13
CUB DIRTESHREL State ood oh vtec age ast 8 14 6 1 21
USTED ie SA a aa eee 45 18 3 66

a. Method of determining number. Since one of the chief pur-
poses of this study is to determine accurately whether there is
any variation in the number of chromosomes, considerable care
has been taken to eliminate from the evidence every possible
source of error. An important part of the presentation of this
evidence is, then, a concise description of the exact procedure
employed in obtaining it.

1. Procedure. In order to avoid overlooking any mitotic
figures, the entire surface of every piece of tissue was completely
surveyed systematically before beginning to count any of the
chromosomes in any of the complexes. The survey was accom-
plished with a 4-mm. objective and an 8x ocular supplemented
by a mechanical stage. |

In determining the number of chromosomes in each complex,
a camera lucida sketch of it was first made at a magnification of
2633 diameters. This sketch was carefully compared with the
cell in order to make certain that no errors had been made in

178 CHARLES L. PARMENTER

sketching it. The chromosomes were then numbered consecu-
tively, the number being placed on both ends of each chromo-
some. This method avoided any possibility of overlooking any
chromosome or of counting any chromosome twice.

2. Clearness and classification of the complexes. All the com-
plexes counted were polar views of late prophases and of meta-
phases and have been divided into three classes on the basis of
their clearness. The first class consists of forty-five complexes
in which every chromosome was so Clearly separated from adja-
cent chromosomes that it could be optically traced continuously
over its entire length, without losing sight of it at any point.
Only the counts from complexes of this group are submitted
as data which are unquestionably free from objection and
uncertainty.

In the second class of cells there are eighteen complexes in
which the chromosomes are all exactly as clear as those of the
first class, with the exception that either one or two chromosomes
cannot be clearly traced over their entire length as they could
be in class I and therefore might possibly be hypercritically
considered to necessitate interpretation.

The three cells of the third class differ from those of the
second class in that they each contain places in which the number
of chromosomes cannot be determined with confidence and
consequently are actually subjects for interpretation.

Complexes of the first class. Complexes of this class are
represented by figures 1 to 8 which have been made in carbon
and are attempts to represent the actual appearance of the
chromosomes and their relative positions inthecomplexes. Rep-
resentative cells from each of the tissues studied, except the
mesentery and lung, have been so drawn. Other complexes of
this group have been outlined in ink, figures 9 to 20, to give a
further assurance of the nature of the complexes constituting
this class of conditions.

Since it is impossible to represent chromosomes in a drawing
as clearly as they are seen in a cell, it is necessary to consider
briefly this situation in order to prevent misunderstanding, and
incorrect impressions concerning the clearness of the cells and

CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 179

the faithfulness of the description of the conditions under which
the number of chromosomes was determined. The difficulty
lies in the necessity of representing on a plane surface chromo-
somes which in the cell occupy several levels. The effect can
be produced by shading, but at the same time at points where
chromosomes cross or overlap each other for various distances
they might create the impression in the drawing that they
cannot be ‘‘optically traced continuously over their entire
length.’”’ There are such cases in every drawing. This is
especially true of the late metaphases of the tail epithelial com-
plexes (e.g., figs. 7, 8) where every chromosome in the cell can
be clearly and faithfully traced as described above.

There is also the condition in which parts of the same chromo-
some are so related to one another that their appearance in the
drawings might create a doubt as to their clearness in the cell.
Examples of this are represented in figures 6 and 8, chromosome
‘a,’ in which the two arms of the same chromosome turn abruptly
upon one another and the appearance might be subject to the
criticism that there are two different chromosomes involved—a
portion of one lying exactly upon another with their ends termi-
nating at the same point. Such cases were carefully examined
end the two arms can clearly be seen to follow into each other.

In four of this first class of cells there is another condition that
needs mention. These cells contain one or two chromosomes
which appear to be broken into two parts (e.g., figs. 19 and 20, f).
The parts in each case are separated by very short spaces and
are exactly in line with each other. Della Valle (’09, fig. 11)
shows two cases of this sort as one chromosome, but discusses
them (p. 116) as uncertain. That there is a single chromosome
concerned in each of these cases is further evidenced by the fact
that there are twenty-one similar cases in other cells of this
class (e.g., figs. 5, 7, 14 and 15, f) and thirty-five cases in cells
of class II in which the parts are connected by various amounts
of chromatin. In some instances the connection is seen as
faintly staining chromatin, in others as a single or double darkly
stained thread.

180 CHARLES L. PARMENTER

Complexes of the second class. In fourteen cells of this class
there is one point in one chromosome and in four cells there is
one point in each of two chromosomes which, to persons hyper-
critically inclined, might possibly appear uncertain. To one
acquainted with the material, each of these points is entirely
clear, and even when accepted as subject to interpretation it is
very plain how the interpretation should be made—so plain that
I am certain that the count of twenty-eight chromosomes is
accurate and dependable. But for the sake of unquestionable
fairness I have placed these cells in a separate group. As to
the exact nature of the interpretations in these eighteen com-
plexes, four of them have some small portion of only one chro-
mosome so covered by others that it cannot be traced over its
entire length without losing sight of it as stated above (p. 178).
Two other cells had two chromosomes of this nature. Five
complexes have a single chromosome lying in such a relation to
another chromosome that it might possibly be interpreted as a
part of the other chromosome (e.g., fig. 23, chromosome 7), and
in three more cells there were two such chromosomes. In the
remaining five complexes a single chromosome was so situated
or otherwise involved, that it might be interpreted that there
were two chromosomes present (e.g., fig. 21, 2).

In considering all the interpretation possible in each of these
eighteen cells the minimum number in any one of them would
be twenty-seven and the maximum number thirty. Even grant-
ing this much variation, it is far removed from that expected in
a series of chance variants as Della Valle claims them to be.

The points in question were sketched as described above
before the chromosomes were counted, so that the determination
of the number of chromosomes was not influenced, either con-
sciously or unconsciously, by a knowledge of how many chromo-
somes were present or by how they should be sketched in order
to produce the expected number. This procedure and the fact
that the number counted always agreed with the number present
in the forty-five cells of class I make it practically certain that
the enumeration is correct. It should be emphasized again that
these cases are only subject to question when hypercritically

CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 181

considered and would otherwise constitute a part of class I. In
fact, an experienced cytologist of this laboratory, in examining
these, without a knowledge of the number of chromosomes
present, could see no reason for considering them as subjects
for interpretation, and it seems almost absurd to place them in
a separate class. : ft

Complexes of the third class. There was a very large number
of cells which were beautifully clear everywhere except in regard
to one or two chromosomes. However, only three of these were
sketched, because the number of clear counts was so large that an
increased number of these uncertain counts is of little value.

Each of the three cells drawn contains two points of uncer-
tainty as to whether there are one or two chromosomes present.
The number is interpreted as either twenty-seven or twenty-eight.
The minimum number of chromosomes possible of interpreta-
tion in one cell is twenty-six, the maximum is twenty-eight; in
the other two cells the minimum is twenty-seven, the maximum
is twenty-nine.

These three cases were interpreted while the sketch was being
made and before it was known how many chromosomes were
present. It is not true, therefore, that the interpretations were
prejudiced nor- that any cases which did not agree with the
expected numbers were cast aside and consequently ignored.
On the contrary, they are here included as part of the evidence in
forming the conclusions drawn from this study.

Rationally considered, then, of the cells sketched there are
sixty-three in which the enumeration of chromosomes is accurate
and dependable and three in which there are unavoidable
interpretations necessary. These sixty-six complexes constitute
very strong evidence that the number of chromosomes in Am-
bystoma tigrinum is constant.

b. Possible variation in number in uncounted complexes. As to
whether or not there was any variation in chromosome number
in this species can be judged from the results obtained from the
sixty-six cells which were studied. If as few as 2 per cent of the
total complexes studied varied from: the usual number, at least
one of these should have made its appearance. Furthermore,

182 CHARLES L. PARMENTER

Della Valle (’09, p. 117) claims that variation of chromosome
number is probably a general law and (p. 120) that his counts
strikingly bear out the expectation expressed by Newton’s theo-
retical binomial curve. Were this the condition in Ambystoma
tigrinum, a good proportion of the sixty-six complexes should
have shown variation in number. Since no variation was found,
it is safe to conclude that there is none in the cells that could
not be counted.

c. Abnormal complexes. Seven apparent variations from the
usual number were found. These were groups of chromosomes
in which the number was clearly other than twenty-eight (figs.
22, 24, 25, A.B., 26, A.B.). But when these groups are thoroughly
analyzed it is certain that they are nothing else than cases of a
very unusual behavior of four cells and do not constitute a
variation from the usual number of chromosomes.

Figure 22 shows a peritoneal cell which has lost a part of the
chromosomes. Chromosome a is but part of a chromosome,
showing very unmistakable evidence that a portion of it has been
broken off and there is a conspicuous depression in the tissue
from which it is evident that the remainder of the chromosomes
of this cell have been lost. The cell lies close to a tear in the
peritoneum. It is a bare possibility that the tear and the loss
of the chromosomes is due to the same cause.

The second case isa very early metaphase from the peritoneum.
It consists, as represented in figure 24, of one group of twelve
chromosomes and another group of sixteen immediately adja-
cent to it.’ These two groups and figures 2 and 20 are very
similar to Della Valle’s dicentric cell (’09, fig. 6) and Flemming’s
(91) figures 31 to 39, table 40. <A study of these two groups
makes it practically certain that they are separated parts of one
‘ and the same cell. This is evidenced by the following facts: 1)
these two groups together constitute the normal number twenty-
eight. 2) The chromosomes of both groups of cells are in the
same stage of mitosis. 3) Both groups represent a half circle
and indicate strongly that they are separated parts of one cell
which have rotated a total of 180° to their present positions. 4)
When these chromosomes are arranged side by side linearly they

CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 183

form a series (fig. 31) like that (figs. 27 to 30) made by a similar
arrangement of the chromosomes of normal cells (figures 1 and
3). The length relationships of these chromosomes as shown
graphically in figure 37 are practically identical with those of
normal cells (figs. 33 to 36). Unfortunately, the cell walls are
not visible. 5) Both of the homologues of chromosome pairs,
as determined by measurements and indicated in figure 24 by a
duplicate series of numbers, in some cases are found in the
same separated part of the cell and in other cases one homologue
is found in part a and the other in part b.

The third case is a compact metaphase in the epithelium of
the lung (figs. 26 A and B) and is similar to the second case.
These two groups are somewhat more separated than those of
figure 24. Figure 26 B represents the chromosome number and
characteristics and figure 26 A shows the relative positions of
the two groups omitting some of the chromosomes in a.

That these two groups of chromosomes are parts of the same
cell which have become separated is made highly probable by
the following facts: 1) As in the second case (fig. 24), the two
groups are near together, one containing eight and the other
twenty chromosomes—a total of twenty-eight. 2) The chromo-
somes are in the same stage of mitosis, the chromatids of those
of b, however, being separated a little more than those of the
twenty chromosomes in a which may be due to a less crowded
condition. 3) The two groups are practically of the same
diameter and of the same shape. An outline of a on transparent
paper can be perfectly fitted to b. 4) These chromosomes also
form a linear series of lengths (fig. 32) similar to those of normal
cells. 5) Group a is not a complete cell because the cytoplasm
can be seen only below the chromosomes, while above the
chromosomes are bare. The boundaries of the cytoplasm of 6
cannot be seen. 6) The homologues of the chromosome pairs
are numbered and distributed in the two groups like those of
figure 24.

The fourth case is the peritoneum of another individual. It
evidently is a cell which has been divided into two parts like
those of cases 2 and 38. Figure 25 A is a camera-lucida: drawing

184 CHARLES L. PARMENTER

representing the relative positions of the two groups and figure
25 B shows the chromosomes enlarged and numbered consecu-
tively. As in case 3, the two groups are not immediately
adjacent, but are separated by the longer diameter of a resting
nucleus.

In group a there are eleven chromosomes. In the other group
there are apparently seventeen, but unfortunately in this second
group the chromosomes are so overlapped at one point that
they cannot be counted with confidence. There are, however,
thirteen chromosomes which can be clearly delineated and the
interpretation that there are four chromosomes in the group
(14 to 17) which so badly overlap is likely correct. The total
number of chromosomes in the two groups is then probably
twenty-eight.

The chromosomes are so much foreshortened, and at fhe
above-mentioned point so crowded, that I have not attempted to
measure and arrange them in a series as was done for the chro-
mosomes of figures 24 and 26. However, a glance at figure 25 B
shows that such a series might be arranged.

The shape and size of both groups of chromosomes, and of
the cytoplasm about them, are such that one can be fitted upon
the other. Although these two relations are not positive evidence
they indicate that one of these groups, possibly the smaller, has
been separated from the other.

To summarize, it may be said that in the first case considered
(fig. 22) it is certain that the smaller number of chromosomes is
due to a loss of a part of the chromosome complex from the cell.
Although the facts stated for cases 2, 3, and 4 may not be con-
sidered absolute proof, they do constitute a very strong prob-
ability, which closely approximates a proof, that in each of these
cases a cell has been separated into two parts.

B. Somatic chromosome pairs

a. Introductory statement. Since Van Beneden’s (’83) hypoth-
esis that one-half of the chromosomes of an individual are of
paternal origin and that one-half are of maternal origin there

CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 185

have been many confirmatory observations and some that
oppose it.

Montgomery (’01, p. 220) advanced evidence that for each
of the chromosomes of maternal origin there is a homologous
mate among the chromosomes of paternal origin, and that these
homologues unite during synapsis. He also maintained that
these pairs? can be recognized in the spermatogonia. This
view has been supported by many authors. Among these are
Sutton (02), who compared numerous camera-lucida drawings
of spermatogonial complexes of Brachystola magna; Meek
(12a), who measured the lengths of spermatogonial chromo-
somes of a somewhat wide range of animals, and Hance (’17 b,
"18 a), who made linear measurements on the germinal and
somatic chromosomes of the primrose, Oenothera scintillans and
the pig. On the other hand, Meves (11), on the basis of meas-
urements made upon the spermatogonial and somatic complexes
of Salamandra maculosa, fails to confirm the claim for the
former and denies it (p. 282) for the latter. Della Valle (12),
who measured the chromosomes of peritoneal cells of the same
form, also denies the existence of pairs.

Some of the somatic cells studied in Ambystoma tigrinum are
quite favorable for a linear measurement of chromosomes, and
these complexes have been used to obtain further data upon
the query as to whether the chromosomes of the somatic cells
form a duplicate series (based upon length and form) as is shown
by their progenitors in the germinal line during the maturation
period.

b. Mensuration. Since the possibilities of error In measure-
ments are so great, it is necessary to consider the conditions

3 The two mates constituting a pair are usually of equal length, so that homo-
logues may be recognized by such equality. In some cases, for example, in
the Diptera, Stevens (’08, 711), Metz (’14, ’16 a and b), Holt (’17), Whiting (’17),
Hance (717), the two members lie near each other or even closely approximated
in the spermatogonia and somatic cells, while in many other cases, for example,
in Orthoptera and Amphibia, the homologues may be widely separated in these
cells. In the present paper the term ‘pairs’ will refer to the two chromosomes
which are homologues as determined by length and form regardless of the
relative position in the cell.

186 CHARLES L. PARMENTER

under which these measurements were made in order to judge
their value correctly.

1. Type of cells. Only cells were used in which every chro-
mosome was perfectly clear and, except as noted (p. 189), lay
exactly level in the equatorial plane throughout their entire
length. Only three cells (figs. 1, 3, and 9) of this quality
were available, and these were polar views of early metaphase
stages in cells of the peritoneum and lung. The chromosomes
of one other cell (fig. 10) approximated this condition and were
also measured. The care with which these cells have been
chosen may be judged from the fact that they were the only
suitable cells in material from over one hundred larvae con-
taining large numbers of division figures. In material with chro-
mosomes so long and so numerous it is not surprising that so few
cells were perfect enough for measurement.

2. Method. In addition to choosing cells with chromosomes
of the above character, three different camera-lucida sketches of
each chromosome were made on different days with extreme care
at a magnification of 2633 diameters. Each of these sketches
was measured three or more times along the median line with an
Ott compensating planimeter modified for this purpose, or with
an opisometer. These nine determinations obtained for each
chromosome were averaged to represent its length. This method
is important because the extremes of these nine measurements in
about one-fifth of the cases may differ 1 mm. from the average
(and occasionally more). This demonstrates that one measure-
ment upon a single drawing might give rise to an erroneous
difference in the lengths of the homologues of some pairs rang-
ing from 1 to 2 mm., the actual amount depending upon the
respective errors in each homologue. Averages largely eliminate
this error.

3. Sources of error. The various sources of error may be classi-
fied in three groups: 1) instrumental errors, 2) personal errors, 3)
errors inherent in the condition of the material.

In the first place, it should be emphasized that no attempt
has been made to determine the actual length of any chromo-
some. These measurements have all been made on the drawings

CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 187

described above and have to do only with relative lengths. This
fact eliminates at once a number of errors which would otherwise
be very serious.

The instrumental errors. The possible instrumental errors are
a) failure to maintain a critical illumination, b) failure to maintain
a constant wave length of illumination, and c) errors inherent in
the planimeter and opisometer.

It so happened that the strongest hght was obtained a little
above the point of critical illumination, and might therefore
cause an error in measurement. However, several chromosomes
were drawn a number of times under both conditions and no
perceptible difference was observed. Any slight error overlooked
would be equal in the homologous chromosomes and would not
interfere with a relative measurement.

Farmer and Digby (’14) state that errors can arise from the
use of varying wave lengths of ight. The same optical equip-
ment and illumination were maintained in all of my operations
so that relative values were unaffected.

In making measurements with the planimeter the polar arm
was held rigidly stationary in two grooved blocks so that the
tracing point moved around in a circle having a diameter of 33
em. The sharp tracing point was kept upon the median line of
the chromosome figure by moving the drawing around (without
slipping) into line with the path of this tracing point, which was
used as a pivot for orienting the drawing. A constant, repre-
senting the value of each of the divisions of the vernier, was
determined by measuring a series of known distances on a straight
line.

The accuracy with which this instrument was operated is
indicated by the fact that the average difference between the
extremes of measurements made upon each of several drawings
is 0.3 mm., the standard deviation, computed from the combined
measurements of several drawings, is 0.17 mm. The measure-
ments, obtained more quickly with the opisometer, are slightly
less accurate, the average of the above extremes and the standard
deviations being 0.4 mm. and 0.26 mm., respectively. Finally,
the average of the nine measurements made upon the three
drawings of each chromosome reduces this instrumental error

188 CHARLES L. PARMENTER
to approximately zero. This error is, of course, inherently
included as a part of the personal error discussed below.
Personal errors. Probably the greatest personal error was
due to inaccuracies in making camera-lucida drawings. To
reduce this error to a minimum, each chromosome, as stated
above (p. 186), was drawn three times with extreme care. These
sketches were made at the same point on the drawing-board so
that any error due to different drawing distances and consequent
differences in magnification was eliminated. The estimated
median line of the sketch, upon which the measurement -was
made, was indicated with a lead pencil. The average deviation
from the mean of the nine measurements is 0.6 mm. and the
standard deviation, computed from combined measurements of
several drawings, is 0.37 mm., which indicates that the instru-
mental and personal errors in the average of these measurements
are practically zero for relative purposes.
Errors due to conditions inherent in the material. This class
of errors is much more important than the preceding. The
errors of this kind are an unequal shortening of the whole chro-
mosome and a foreshortening of parts or all of the chromosome.
Measurements made without very careful attention to fore-
shortening are of questionable value, for small amounts can
give rise to large errors, especially inshort chromosomes. Shorten-
ing is caused by a twisting of the chromatids about one another
(figs. 1 to 8, 27 to 30). The amount of shortening in each twist
of the chromatids, as determined by computation,’ at the mag-

4The amount of shortening in each twist of a chromosome was determined
by adding together the separately computed amounts of shortening due to the
lateral deviation of the chromatids and that due to the vertical deviation of
the chromatids. The shortening in each twist due to the lateral deviation was
computed by averaging the lengths of the two chromatids of a chromosome and
substracting the length measured upon the median line of the whole chromo-
some. This total difference divided by the number of twists is 0.2 mm., which
is approximately the amount of shortening due to the lateral deviation in each
twist. In determining the amount of shortening due to the vertical deviation
of the chromatids, the width or thickness of the chromatid, as determined with
an ocular micrometer was assumed to be the amount of vertical sag of the chro-
matid in each twist. This thickness multiplied by the magnification amounted
to 1mm. This was used as the altitude of a right triangle, the base of which
represented half of the measured longitudinal length of the shortened part, and
the hypotenuse of which then represents very closely one-half of the true length

CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 189
nification of the drawings (2633 diameters), amounts very
closely to 0.4 mm. However, the effect of this condition is
either completely or largely neutralized by equal or nearly equal
amounts of twisting in the homologues of each pair. The maxi-
mum amount of error due to this cause may be judged by an
examination of figure 28 which contains the most twisting. In
this cell there are seven pairs in which the amount of twisting
is equal and the error completely neutralized, two pairs con-
taining an error of 0.4 mm., two pairs with 0.8 mm. error, one
with 1.2 mm., and two pairs in which it is uncertain. Since
these errors that occur at critical points will be considered indi-
vidually later, no corrections for them are included in the
measurements.

Foreshortening occurs only in certain chromosomes as indi-
cated in figures 27 to 30 and 33 to 36. This is, however, in the
cells represented by figures 27, 28, 33, and 34 so slight that the
whole chromosome can be seen at one focus, the foreshortened
part appearing only slightly hazy. In the cells represented by
figures 29, 30, 35, and 36 it is a little more. Measurements
with the fine-adjustment graduated wheel, made more accurate
with a sharper pointer made of a pin, indicated this sagging to
be not more than 2.5 (one division of the fine adjustment wheel)
in any case. Corrections’ made for this foreshortening are

of the shortened portion. Double the length of this hypotenuse minus the orignal
measurement of the shortened portion is 0.2 mm., which is the maximum amount
of shortening due to the vertical sag of any twisted portion. This amount added
to that caused by the lateral deviation made the total shortening in one twist
amount to0.4mm. Although this determination cannot be considered entirely
accurate, it is a close approximation.

5 The correction was made as follows. The amount of vertical deviation was
read from the fine-adjustment wheel when the objective was focused as nearly
as could be judged upon the middle of the lowest and highest points of the fore-
shortened portions. All measurements for a given complex were made with the
same part of the fine-adjustment screw, thus avoiding different pitches in the
thread. The reading (2.54 for each division) gave the actual differences of
vertical positions. This multiplied by the magnification and divided by 1000
converted the figure into millimeters, the units made use of in the drawings.
By using this distance as the altitude of a right triangle and the measured longi-
tudinal extent of the foreshortened portion as the base of the triangle, the hypot-
enuse (which represented approximately the correct length of the foreshortened
part) was determined. This was substituted for the original measurement of
the foreshortened portion.

190 CHARLES L. PARMENTER

only approximate, because it is very difficult to determine
accurately the amount of vertical deviation, and its longitudinal
extent, as well as its exact course. In figures 27 to 30 and 33
to 36 corrected figures are used, and the amount included in each
measurement for foreshortening is indicated.

There are two other conditions which do not give rise to actual
errors in measurement, but do interfere with precision of results
and may well be considered here. 1) A possible unequal con-
traction of chromosomes. Wenrich (’16) observes that chromo-
somes A and B condense before the other chromosomes in the
spermatogonia and tetrads of Phrynotettix, and (717) he shows
that one homologue of chromosome 4, cell E, plate 2, contracts
more rapidly than the other. 2) Since so many of the chromo-
somes of Ambystoma are so long and composed of two inter-
twined chromatids, there is considerable possibility of a stretching
due to bending and other stresses still present in the complexes
nearing the metaphase. As Meves (’11, p. 247) points: out,
under these conditions two chromosomes could be of different
length and of equal volume. Even an imperceptible difference
in diameter of parts or all of two chromosomes of equal volume
might cause considerable difference in their lengths. This dif-
ference would of course be proportionally greater in the longer
chromosomes so that measurements of the shorter chromosomes
of a cell might strongly indicate the presence of pairs while the
homologues of the longer pairs would show quite wide differences
in length. A case of very perceptible stretching is to be seen in
chomosomes ‘s,’ figures 9 and 12.

Effects of technique. The differences which may arise in the
chromosomes of different cells of even the same tissue due to
different effects of fixatives, and all other effects of technique,
do not affect relative measurements of chromosomes in the same
cell. For it is extremely improbable that the lengths of chro-
mosomes of the same cell which are so equally close to the
surface of these membranes would not be similarly affected by
the action of these various reagents and processes. It is also
improbable that inherent differences among the homologues
would cause a differential change of length under these
conditions.

CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 19]

Summary. The combined instrumental and personal errors
are reduced to practically zero by averaging several measurements
made upon different drawings. The errors due to twisting of
chromatids about one another are largely neutralized and are
considered individually later. Therefore, except for possible
unequal contraction and stretching of homologues, only the
measurements of those chromosomes which are foreshortened
contain appreciable errors. It is thought that these errors,
after corrections have been made, probably do not in any case
exceed 1mm. The presence and amount of error due to unequal
contraction and stretching in any particular chromosome is an
uncertainty, but if existing would probably be greater in the
longer chromosomes. .

c. Results of measurements. 1. Criteria for determining pairs.
Before considering the results of the measurements, it seems
desirable to state what the criteria are that will demonstrate
whether pairs (p. 185) are present among the chromosomes at
these particular stages of mitosis. In the absence of definite
minute morphological characteristics, such as a repeated occur-
rence of marked granules, constant in position and size, which
Wenrich (’16) describes for certain Orthopteran chromosomes,
the next most exact criterion for determining the presence of
pairs in diploid cells would be a duplicate series of chromosomes
of equal volume. But in these chromosomes trustworthy volu-
metric determinations cannot be obtained, for the above-
mentioned intertwining of the chromatids and the stretching of
the chromosomes would cause variations in diameter which
could not be measured accurately, and these errors would be
cubed in the volume. Consequently linear measurements, sup-
ported by form, have been chosen as giving more trustworthy
data.

Upon this basis, in order to constitute undeniable evidence
that the chromosomes form a duplex series, there are two con-
ditions which should be met. First, when the chromosome
lengths are plotted in a graph (e.g., figs. 33 to 37), they should
definitely associate themselves in twos of equallengths. Second,
the differences in length between successive pairs, as indicated

JOURNAL OF MORPHOLOGY, VOL, 33, No. 1

192 CHARLES L. PARMENTER

by the first condition, should exceed the errors of measurement
by a good margin. Unless the above conditions are met, the
errors of measurement make it possible to contend that the
chromosomes are arranged in a series of successively increasing
lengths which bear no relation to one another and therefore do
not represent pairs.

In addition to the above, the form of the chromosome, which
is probably determined in large part by the position of the
spindle fiber attachment, may be used as an aid in determining
which chromosomes are homologues. McClung (’14, p. 674)
pointed out that, although the spindle fiber attachment may be
different on different chromosomes, nevertheless, for each chro-
mosome “it is most precise and constant’’ in the individual.
Carothers (’17, p. 470) has shown that for certain tetrads (e.g.,
figs. 32 and 63) the point of spindle fiber attachment on one
homologue is different from that on the other. But she also
finds that the point of fiber attachment is constant on a given
homologue for each individual. She shows (figs. 32, 32a and
63, 63a) that the point of spindle fiber attachment on these
homologues in the spermatogonia is preserved in the tetrads.
However, an exception to constancy of fiber attachment in the
individual has been noted by Wenrich (716). He found in a
rod-shaped tetrad of another genus that the fiber attachment
might shift from one end of the chromosome to the other in
certain individuals. Therefore, according to the theory of the
individuality, in the somatic chromosomes the homologues of
certain pairs of chromosomes may be expected to be unlike in
form. However, individuals showing such conditions are very
few and should be considered exceptions rather than the rule.
There is the possibility that these heteromorphic homologues
may not be confined to the Orthoptera, and certain cases in
Ambystoma make this appear to be so.

Finally, it would be remarkable if any material satisfied the
above criterion in all points. The small difference in length
between some chromosomes makes it impossible to demonstrate
beyond doubt the presence of pairs among them. Again, the
possibility that the chromosomes may not condense at equal

CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 193

rates, and that unequal stretching, especially between the
longer chromosomes, may occur during mitosis, increases the
difficulty in obtaining accurate metric comparisons, and may
interfere with perfect certainty in the interpretation of the
results. Furthermore, some variation is characteristic of living
material and hence slight differences in relative length and form
in different cells would be expected rather than absolute uni-
formity. McClung (17, p. 567) finds in certain Orthoptera
that the accessory chromosome, although unmistakably dis-
tinguished from the euchromosomes, is not always-of the same
relative length in different individuals of a given species, for it
sometimes occupies the fourth and sometimes the fifth position
in the series of lengths. However, it must be remembered that
this is the sex chromosome which at the metaphase (the stage
of greatest condensation of the euchromosomes) is already be-
coming diffuse. Differences in the degree of condensation might
therefore be involved in the differences of relative lengths. And
again, since individuals vary in their morphological character-
istics, why should it be expected that the chromosomes of two
different individuals should be of exactly the same relative
lengths at the same stage of mitosis? In view of universal
variability, homologous chromosomes, which are derived from
different individuals and which may be expected to maintain
their individuality, should not invariably be of exactly the
same length. As discussed (p. 217), the observations on different
Orthoptera by several authors indicates this to be so.

On account of these interfering factors it cannot be expected
that homologous chromosomes will always be of exactly the
same length at any particular stage of mitosis. Therefore,
length and form, considered in a limited number of cells, from
different individuals, cannot be regarded as conclusive evidence
for or against the presence of chromosome pairs. Much more
conclusive evidence would be had in a comparison of several
somatic complexes from a single individual and with those of
other individuals, a comparison of these with the diploid and
haploid chromosomes of the germinal line, and a comparison of
the complexes of parents and progeny.

194 CHARLES L. PARMENTER

However, although the measurements may not meet the above
criteria in all chromosomes, there are certain cases which do meet
them definitely, and strongly evidence the existence of pairs.
This fact, together with the above consideration, makes it
possible that all the chromosomes are in pairs. .

Furthermore, it may be mentioned here that conditions which
do not meet the above criteria fall far short of proving that
pairs do not exist. The possibility still remains that two or
more pairs may be of equal or nearly equal length. Such a
condition is known to exist in certain Orthopteran chromosome
pairs (Carothers, ’17, pl. 1, tetrads 7 and 8) where the chromosomes
are unquestionably known to be paired.

2. Evidence for the existence of pairs. On plate 9, figures 33
to 37, are five rows of vertical lines representing the relative
lengths of the chromosomes of as many cells. The differences
in the lengths of these lines and also the space between adjacent
lines represent relative differences in chromosome length. For
convenience the lines are made twice the length of the chromo-
somes as drawn and the width of the spaces are made eight times
the difference in length. The lengths of the chromosomes, the
amounts included for foreshortening, and the form of chromo-
somes for each of these cells are also shown, respectively, in
figures 27 to 31 and 33 to 37. ;

A part of the evidence which these graphs present for the
existence of pairs is three outstanding characteristics common
to all of them. First, there is a graded series of chromosome
lengths from the shortest to the longest; second, there is a
marked sameness in the relative chromosome lengths of these
cells which appears in the approximately constant presence of
groups containing the same chromosome pairs, and, third, a
similarity of form between homologues.

The pairs, in accordance with the above criteria, were de-
termined primarily on the basis of chromosome length, supported
by a comparison of form. The graphs and figures of each com-
plex measured show that certain chromosomes are very probably
homologues. In other cases a number of chromosomes are so
nearly of the same length that, according to the criteria, the

CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 195

homologues of the pairs cannot be determined with certainty,
but they doubtless exist. Since the measurements of figures 33
and 34 are very nearly accurate and present the most reliable
evidence, these will be considered separately from the others.
For convenience the pairs may be considered in two groups, the
first including those which differ greatly in length from their
neighbors (pairs 1, 2, and 8) and the second including the
remainder in which the pairs are not so clearly distinguished.
It is all but certain that the chromosomes represented in each
of pairs 1, 2, and 8 in both of these figures are homologues, for
they almost completely satisfy the criteria outlined above. In
these pairs there is foreshortening in only one chromosome and
in pair 8 the error of 0.8 mm. in both complexes due to twisting
of the chromatids is negligible. The homologues are of approxi-
mately equal length, and the difference between each pair and
the adjacent pairs is so much greater than the error of measure-
ment that it is improbable that the condition represented by
these three pairs in both complexes is merely a matter of chance.
Furthermore, there is a close resemblance of form between these
homologues. A comparison of other cells of the same individual,
if available, would be expected to show that this condition is
constant in all the cells as is shown by comparable cases of
constancy in the germ cells of individuals of certain Orthoptera
(p. 219). It can, therefore, be maintained with considerable
confidence that these particular chromosomes of equal length
and sameness of form actually constitute pairs. The measure-
ments of the chromosomes of these pairs in other cells as dis-
cussed below give similar although less conclusive evidence.
Among the chromosomes of the second group in these two cells
there is strong evidence for the existence of pairs, but the small
difference in length and the errors due to twisting and possible
stretching make it inconclusive. In figure 33 the homologues as
shown in each of pairs 3 to 7 are so nearly of the same length
and form (fig. 27) that one may believe that they constitute
pairs as represented. Pair 3, in addition, is well separated from
those adjacent. Although pairs 4 to 7 appear to be actual
pairs, the chromosomes of this series differ so little in length that

196 CHARLES L. PARMENTER

the criteria adopted are not entirely satisfied. There is a chance
of doubt, therefore, of the validity of the pairs as indicated.

As mentioned above, it will be noted that the groups into
which the chromosomes of this cell are associated are repeated
in the other cells. The similarity of grouping is very marked,
especially in the formation of two large and distinctly separated
groups, one containing pairs 3 to 7 and the other pairs 9 to 14.
In pairs 3 to 7 of figure 34 the condition present in figure 33 is
duplicated, except that pair 3 is not so well separated from pair
4, due to the fact that both homologues of pair 4 are relatively
shorter in the former complex. Concerning the homologues of
pair 6 there is some doubt. I have interpreted the end of
chromosome 30 + (fig. 28) as bending back upon the main
portion of the chromosome, and have estimated the length of
this portion.

Of the remaining six pairs (9 to 14) in both cells several are
quite clear, but on the whole the possibilities of error and the
differences in length between successive pairs is too small to
satisfy the second criterion fully. In figure 33, on account of
the practical absence of twisting in pair 12 and adjacent pairs,
the condition for determining pairs is very closely satisfied.
The chromosomes of pair 9 were considered homologues through
a process of elimination. They differ 12.4 mm. in length, but
agree in form (fig. 27). This condition will be discussed later
(p2i217),.

In figure 34, pairs 9, 11, and 18, although not sufficiently
separated to constitute an unquestionable demonstration of pairs,
are fairly well separated and the homologues of each pair, after
allowance is made for errors due to twisting, are of nearly equal
length. One homologue of pair 10 (fig. 28) is imperfect. Pair
14 is well separated in the graph from pair 13. Approximate
corrections made for the kink in the shorter member and for
the slight foreshortening at that point make its length approxi-
mately the same as that of the longer member. The homologues
of all these pairs agree very well in form (fig. 28), in spite of the
fact that some may not yet have assumed their final shape.

CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 197

The measurements represented in figure 35 are nearly as
reliable as those of the preceding cells. The amounts included
for very slight foreshortenings are indicated in figure 29. The
relative chromosome lengths form approximately the same
groups as those of the other cells, and the evidence for the
existence of pairsis strong. Ascontrasted with figures 33 and 34,
the chromosomes of pairs 1 and 2, when approximately corrected
for foreshortening, do not entirely meet the conditions of the
criteria, but their lengths and form strongly support the prob-
ability that they are homologues. The chromosomes of pairs 7,
8, and 10 differ somewhat in length. They do not appear fore-
shortened, and although possibly present there is no perceptible
stretching in them. ‘These differences may be due to an un-
equalness of homologues as discussed later (p. 217). On the
other hand, the chromosomes of pairs 3 to 6, 9 and 11 to 138 are,
except as noted in figure 29, unforeshortened and of equal length,
they agree in form and present strong evidence for the exist-
ence of pairs. The greater part of the difference in length
between the homologues of pair 14 is due to stretching.

The measurement of the chromosomes of the cell represented
in figures 30 and 36 are somewhat less favorable for measurement
than those of the preceding cells, because in addition to a
moderate amount of twisting there is slight foreshortening in
many of them. Although the lengths have been approximately
corrected for this foreshortening (figs. 30 and 36) the measure-
ments cannot be considered so accurate and reliable as those of
figures 33 and 34. ‘The relative lengths of the pairs closely
parallels that of the preceding figures which results in a similar
distribution in the series. As contrasted with figures 33 and 34,
the chromosomes of pairs 1 and 2 fail to satisfy the criteria,
and this is apparently not due to errors in measurement. The
large difference of 5.6 mm. between the homologues of pair 7
recalls a similar difference in pair 9 of cell 33. Pairs 8 and 9
clearly satisfy the conditions of the criteria and the remainder
of the pairs duplicate the conditions in figures 33 and 34.

The chromosomes of the cell represented by figures 24, 31,
and 37 are foreshortened in nearly every case and were measured

198 CHARLES L. PARMENTER

only in order to learn whether they constitute a series which
would indicate that they belong to one cell. Only one set of
measurements was made. Consequently, the figures are not so
accurate as those of the other cells. Pairs 1 and 2 are readily
recognized because they are well separated from each other and
adjacent pairs. Pair 8 which stood out clearly in figures 33, 34,
and 36, occupies a similar position here, but its homologues
according to these less correct measurements differ about 4 mm.
in length. ‘The relative positions of the pairs practically dupli-
cate those of the other cells. I have not attempted to make
corrections for foreshortenings, but as nearly as I can judge, the
chromosomes of pair 1, if corrected for foreshortening, would
differ in length a little more, homologues of pair 2 would differ
less in length, and the homologues of pair 8 are foreshortened
about equally. Approximately the same condition exists in the
other pairs, so that the matching as indicated would not be
disturbed sufficiently to alter the grouping of the pairs. In
this cell chromosomes of pair 12 differ by approximately 10 mm.,
which recalls a second time the condition in pair 9 of figure 33.

Further consideration of the form of the chromosomes in all
these figures furnishes additional strong evidence that the chro-
matin is definitely organized. As indicated in anaphases, the
general form of the chromosomes in the metaphase of these
somatic mitoses is determined by the point of spindle fiber
attachment. The complexes represented in figures 27 to 32 are
early metaphases, and the final form which the chromosomes will
take is quite apparent, although in some cases (e.g., pair 8,
fig. 27) it is not entirely clear.

In all of these figures, as previously mentioned, the form of
the homologues of each pair is practically the same, even in
cases where the final form has not been reached. Only three
pairs (5, fig. 29; 7, fig. 30; 5, fig. 31) are exceptions, and this may
be expected as indicated by a like condition in the heteromorphic
pairs of certain Orthoptera (Carothers, ’17). A comparison -of
several complexes from the same individual would probably
show this condition constant for the individual as in the Or-
thoptera. A further comparison of each pair in any figure with

CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 199

the corresponding pairs of all the other figures also shows a
striking correspondence of form in these chromosomes. The
homologues of the unusual cells represented by figures 31 and 32
may not be correctly determined, as previously mentioned.

Such agreement and constancy of form between homologues
and between corresponding pairs of different individuals cannot
well be considered as due to chance and indicates a definite
organization of the chromatin.

3. Summary. The strongest evidence for the existence a pairs
is the fact that the chromosomes indicated as pairs 1, 2, and 8
in figures 33 and 34 completely satisfy the criteria. Although
these pairs fail to do so in figures 35 and 36, they are recognizable.
Among those chromosomes composing the two large groups in
which the chromosomes differ so little in length (pairs 3 to 7
and 9 to 14) the evidence presented by the lengths of the chro-
mosomes does not conclusively demonstrate nor deny the exist-
ence of pairs because of the various factors inherent in the
nature of the material. However, as represented in the graphs
and figures, the lengths and forms of these chromosomes strongly
indicate such a duplexity. The cases in which the chromosomes
of a pair differ somewhat in length do not constitute contrary
evidence since homologues are not always of equal length as
explained on page 217. Furthermore, the constancy of chromo-
some number, the presence in all the cells of certain groups com-
posed of the same number of chromosomes with approximately
the same relative lengths is further strong evidence of a con-
stancy of chromatin organization and that the lengths of the
chromosomes are not due merely to chance.

It seems to me to. be a very difficult task to demonstrate
conclusively by means of measurements the existence of pairs
in these and similar somatic complexes. To furnish anything
more than strong supporting evidence is almost impossible
because of the various difficulties inherent in the nature of the
material and because of the fact that homologues, as shown in
exceptional cases, are not always of equal length, a fact which
has been actually observed in the germ cells (tetrads) of certain
Orthoptera by several authors. The same conditions make it

200 CHARLES L. PARMENTER

just as difficult to demonstrate the absence of pairs. It seems
to me that the evidence in the Dipteran somatic complexes,
where the members of a pair le parallel and adjacent to one
another, together with the already large and well-supported
evidence of pairs in the various generations of the germ cells
throw the balance greatly in favor of the presence of homologues.

DISCUSSION
A. Introductory statement

The foregoing observations upon the constancy of chromosome
number and the existence of pairs in the somatic chromosome
complexes have their chief importance in their relation to the
Roux-Weismann hypothesis that the chromatin is the idioplasm,
which is differentially organized and linearly arranged, and
that this organization is perpetuated. This hypothesis received
important support from the theory of the individuality of the
chromosomes as set forth by Van Beneden (’83) and strongly
maintained by Rabl (’85), Boveri (’88, ’02), and numerous other
more recent investigators. The morphological evidence ac-
cepted as supporting this proposition is’an essential constancy
of number, size, form, and behavior.

Since McClung (17) has so recently thoroughly considered
the theory of individuality, this discussion is confined to the
particular phases of the supporting evidence which are directly
related to the observations made upon this material. These
phases are essential constancy of number, of size, and of form.

B. Constancy of chromosome number

Della Valle has strongly attacked this theory on the basis of
inconstancy of chromosome number. He arrives at the con-
clusion (’09, p. 120 ff.) that the number of the chromosomes is
the quotient of the quantity of the chromatin divided by the
average size of the chromosomes; that their size is variable
according to the nature of the elements and the conditions in
which they are found, and (’11, p. 188) that the size and number

CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 201

of the chromatic elements are directly comparable to the size
and number of the fluid crystals which are formed in a solution
under different conditions.

These contentions he supports with the claim (’09) of a
variation of nineteen to twenty-seven chromosomes in forty
mitoses of the peritoneum, and (711) by a very large variation
in the blood cells of Salamandra maculosa. These observations
he supplements with a long list of citations of chromosome num-
bers which he interprets as supporting his contention.

But, following an apparently frank and critical discussion of
the accuracy of his observations in the peritoneal cells, he says
(09, p. 116) that he is only sure of his enumeration in twenty-
five of the forty cells discussed. These twenty-five complexes
he describes.as being very clear. The range of variation in these
cells is as follows:

Number of chromosomes.... 19 21 22 23 24 25 26 27
INtimberOlmmuasesseee eee I lee GG M12 oe total4o
Number of mitoses.......... IRL SsilOngs we total 25

An examination of his descriptions and figures may indicate to
some extent the reliability of these counts.

His count of 22 chromosomes was made upon a polar view of
a very late anaphase (fig. 2) in which he states the smaller chro-
mosomes in the center of the complex were beginning to go to
pieces and becoming indistinct, and his only doubt is whether
the chromosome numbered 18 is one or two chromosomes. But,
judging from similar stages in my material, it seems to me that
where the chromosomes are beginning to go to pieces in as
crowded a condition as this must be, such a complex is not a
safe object for an exact chromosome count.

Instances of this kind make it seem possible that conditions
which he considers clear for an exact count might be much less
conclusively clear to others, and that his drawings do not represent
the actual conditions in his complexes.

Since his citations of chromosome number variations found in
the literature have been discussed by Montgomery (710), Wilson
(10), and by McClung (17, p. 548 ff.), it is not necessary to

202 CHARLES L. PARMENTER

review them extensively. But Della Valle’s attitude and his
conception of what constitutes clearness and certainty may be
better understood in the light of some of these citations of
chromosome variations, especially in the Amphibia, which he
presents as valid evidence of variation in chromosome number.
He quotes (’09, p. 35) Flemming (’81, [’82] p. 51) and Rabl
(84, [’85] p. 248 to 250) as reporting variations of from seventeen
to twenty-four in the gill epithelium of Salamandra maculosa.
Flemming explicitly states (pp. 51 and 52) that in the three
cells which admit an exact count there are twenty-four chromo-
somes, that in about twenty other cells he counted from seven-
teen to twenty-four, but was not certain of the number, and
assumed that there were twenty-four. Rabl says (’85, p. 248)
that up to that time only eleven unquestionable counts had been
made and each of them showed twenty-four chromosomes. In
no exact counts in any cell had a different number been found.
Della Valle seems to think that Térék’s (’88) figures of erythro-
cytes of Salamandra maculosa show a variation. This work
was not concerned with chromosome number and the figures
were not intended to show the number of chromosomes in the
cell. His citations of the work of Carnoy and Lebrun (’00) on
Rana temporaria, and of Lebrun (’02) on Diemyctylus and Bom-
binator may be criticised because the authors were primarily
concerned with other considerations and only gave approximate
number determinations. Winiwarter (’00, p. 699), as cited by
Della Valle, reports a variation of chromosome number in the
rabbit; but he states that he is uncertain of his counts. The
variations reported by Barratt (07, p. 376), in proliferating
epithelium of the rabbit are in pathological tissue, and, more- |
over, his counts are uncertain. Montgomery (710) has shown
that many other such citations are misinterpreted.

The above cases represent Della Valle’s inexact and uncritical
attitude in relation to data that seem to serve his purpose, and
this creates the suspicion that his attitude interfered with the
accuracy of his observations when he counted the chromosomes
in his own material. This suspicion approaches a probability
in view of the fact that numerous competent investigators who

CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 203

have made extended and careful studies of the germinal and
somatic mitoses of the same species mention no variation in
chromosome number. Furthermore, Meves (711), who attacks
the theory of individuality, fails to substantiate Della Valle’s
observations, and it is not at all likely that he would have failed
to mention any variation observed. He appears to believe (p.
296) that the number is constant. However, Heidenhain (’07,
p. 176, figs. 80 and 81) shows a polar view of a late prophase
and a lateral view of a metaphase with twenty-six and twenty-
two chromosomes, respectively, and states that such irregu-
larities occasionally occur. An occasional variation is not sur-
prising, but variations as numerous as Della Valle claims to be
present are unusual. Flemming (’90, p. 78) states that he
observes in the lungs of Salamandra maculosa numerous atypical
mitoses with very short chromosomes. He gives no further
discussion and no figures to indicate what kind of cells they are
nor whether they are normal. In the ten cells of the lung of
Ambystoma tigrinum (table, p. 177) there were no variations in
chromosome number, and with the exception shown in figure 26
I observed no abnormalities. Della Valle’s (11) figures of blood
cells in Salamandra maculosa, which he claims show an extreme
variation in chromosome number, appear very much like cells
undergoing disintegration.

To the above evidence of the questionableness of Della Valle’s
results may be added the results of the sixty-six counts in Amby-
stoma tigrinum showing no variation innumber. The important
fact that these counts were made with extreme care (p. 177) in
the somatic cells of the same and other tissues of a closely related
species, and made in uncut membranes (which Della Valle
emphasizes as important for accurate counts), further strengthens
the already strong probability that his number determinations
are incorrect.

There are certain characteristics in his figures that also indicate
that his drawings are none too accurate. He notices that the
chromosomes are twisted, but he does not show what constitutes
the twist. That the peritoneal chromosomes of Salamandra
maculosa are each composed of two separate chromatids twisted

204 CHARLES L. PARMENTER

about one another is plainly evident in Meves’ (11) figures 11
to 15 which show each chromosome to be of variable width.
These figures are exactly comparable to my figures 1 to 8, and 9
to 23 which demonstrate that this variation in width is due to
the twisting of the chromatids. Della Valle represents each
chromosome to be of uniform width excepting an occasional
split in the end of some chromosomes. If he does not see chro-
matids in any of the chromosomes which he has drawn, either
his observations, his technique, or both are faulty. Further-
more, the above evidence together with his attitude make it
uncertain whether his preparations were as clear or the chromo-
somes as distinctly separated from one another as his drawings
indicate.

Finally, Della Valle’s above demonstrated attitude, the ab-
sence of confirmatory evidence for his contentions, his question-
able ability as an observer as indicated by his drawings, and the
results of critical counts in Ambystoma tigrinum, all support
the view that his observations and conclusions are incorrect. —

But upon the assumption that they may be partially correct,
there are some possible explanations for the presence of variation
in the peritoneum of Salamandra maculosa. 1) One or more
chromosomes of a complex easily could have been disturbed, as
is evident from my figures 22 and 24 to 26. This could account
for number deficiencies and perhaps also for excesses. 2)
Champi (713, p. 181) claims that chromosome number can vary
by fragmentation under the influence of certain external stimuli.
Della Valle (09, p. 86) says the number of mitoses can be in-
creased by keeping the larvae covered with a blue glass. If
Della Valle did this, and if such a stimulus could produce frag-
mentation, a bare possibility is offered for a disturbance of
chromosome number. 3) There is also a slight possibility that
the larvae had been kept in captivity and might in consequence
have been sufficiently pathological to produce abnormal mitoses.
4) In an investigation on certain Orthoptera now in progress in
this laboratory, Mr. Carroll observes that in three individuals
some of the few dividing spermatogonial cells contain, in ad-
dition to the normal number of twenty-three, one, and some two

CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 205

extra chromosomes (dyads). This is a small variation in chro-
mosome number in the individual of from twenty-three to twenty-
five. In each of four individuals, including the above three, he
finds that in from one to three of the primary spermatocytes
observed in division, one of the eleven tetrads normally present
is replaced by two separate dyads. Both of these dyads in the
division of the cell may pass undivided to either daughter cell
with or without the accessory chromosome. One of the secondary
spermatocytes resulting from the division of the cell in which
these two dyads accompany the accessory chromosomes receives
twelve dyads plus the accessory and the other receives ten
dyads. The other spermatocyte in which two dyads do not
accompany the accessory chromosome gives rise to one secondary
spermatocyte with ten dyads plus the accessory, and another
with twelve dyads. This would make possible four classes of
spermatozoa containing ten to thirteen chromosomes.

If, similarly, in Salamandra maculosa one of the twelve
tetrads normally present in the spermatocytes and in the oocytes
should be replaced by two dyads, there would be produced
gametes with eleven, twelve, and thirteen chromosomes. ‘These
gametes would produce zygotes (individuals) having twenty-two
and twenty-six chromosomes, respectively, a variation com-
parable to that claimed by Della Valle. Further, if extra chro-
mosomes can thus appear in the germ cells of an occasional
individual, the same might also occur in the somatic cells. But
this variation should be expected in but few of the total indi-
viduals, making the proportion of cells containing the normal
number greatly predominating. In Della Valle’s counts about
one-half contain the normal number which is far too small a
portion unless such variation is much more common than is at
present known.

Although the above is a clear case of a small variation in chro-
mosome number in the individual, it must be clearly understood
that these cases are exceptional and do not represent the normal
condition. ‘The chromosome number may vary in the species,
but it is usually constant for the individual, as has been
especially pointed out by Wilson (’09, 710), Carothers (717),

206 CHARLES L. PARMENTER

McClung (’05, ’07, and 717), and others. But it is very im-
portant to note that these irregular chromosomes arise and
perpetuate themselves in a manner entirely consistent with a
definitely organized chromatin and furnishes no support what-
ever for Della Valle’s contention that the chromosomes are
comparable to crystallizations of a salt solution and that their
number in any cell is dependent upon the law of chance.

C. Variations in other Urodeles

Snook and Long (’14) find in the spermatogonial cells of
Aneides lugubris nine containing clearly the usual number of
twenty-eight chromosomes, and one cell with twenty-three.
There are no other authentic reports of variable chromosome
number in individuals of the Urodeles. The counts of Kélliker
(89), Fick (93), and Jenkinson (’04) in the cleavage stages of
Axolotl (Ambystoma tigrinum) were made for other purposes,
and were not presented as accurate number determinations.
Likewise, the counts of about eighteen to twenty-four, Champi
(713, p. 124) in several other Salamanders are only approximate.

D. Variations vn other forms

Since a somewhat extensive tabulation of comparative germi-
nal and somatic counts has been made and discussed by Hoy
(16), Harvey (16), and briefly reviewed by Hance (17b), a
repetition of this discussion is of little value. However, in the
review of reported cases of variation a few general considerations
have impressed me as worthy of mention. These may seem
commonplace, but are evidently not altogether realized by some
who are none too critical in their discussion of the significance
of these enumerations. 1) As Montgomery (’01) long ago sug-
gested, it is important to distinguish between variation in the
chromosomes in the germinal line and those of differentiating
somatic cells. In the germ cells I believe it can be stated safely
that there are no certainly demonstrated variations in number
which do not conform to a definite organization of chromatin.
From time to time cases of apparent variation have appeared

CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 207

and again disappeared when thoroughly understood (e.g., Meta-
podius Wilson, *10, and the sex group of Ascaris lumbricoides
Edwards, 710, and multiple chromosomes of certain Orthoptera
Woolsey, 715, Robertson, 716, and McClung, ’05, and 17). 2)
In considering the significance of variations, it should be remem-
bered that there are normal and abnormal conditions (p. 202).
3) Metz (16), Hance (17 a, b), and Whiting (’17) have called
attention to the necessity of proper technique. This is not
always an easy matter to judge, especially in absence of material
for comparison. 4) In determining the presence or absence of
variation in any material, a very rigid line should be drawn
between accurate enumerations and those involving varying
degrees of interpretation, e.g., Winiwarter’s (’00) cited varia-
tions in the amnion and omentum of rabbit embryos were uncer-
tain and interpreted. 5) Finally, observers should maintain an
exacting standard in distinguishing between that which is con-
sidered ‘certain’ and that which is interpreted. This is
especially true in counting small chromosomes.

E. Fragmentation

Hance (’17 b, ’18 a) found the spermatogonial number in the
pig, and the diploid pollen-mother cells in Oenothera scintillans
to be constantly forty and fifteen, respectively. But the somatic
chromosomes vary from forty to fifty-seven in the pig and from
fifteen to twenty-one in Oenothera scintillans. He presented
metrical evidence that this variation is due to a fragmentation,
probably of the longer chromosomes. He maintains that these
fragments divide normally with the other chromosomes, and
that therefore this fragmentation does not oppose the theory of
the individuality of the chromosomes.

However, the probability that this variation in Oenothera is
much less, and that most if not all of these fragmentations are
invisibly connected with the main part of the chromosome is
strongly supported by the conditions in Ambystoma material
and by Hance’s (18 b) later observations upon additional
Oenothera material from the same source. Both he (’17 b, p. 90)

JOURNAL OF MORPHOLOGY, VOL. 33, NO. 1

208 CHARLES L. PARMENTER

and Hoy (16, p. 356) review other cases of fragmentation in
Ascaris megalocephala (Boveri, ’99, ’04), Angiostomum (Schleip,
711) and Fragmatobia (Seiler, 713).

In Ambystoma tigrinum (p. 179) and in Salamandra maculosa
(Della Valle, ’09, fig. 11), and as stated above, in Oenothera, the
fragmented portions are directly in line with the main portion
of the chromosome. ‘This may be due to the absence of chro-
matin on the linin or failure of the chromatin to stain at that
point. The fact that in several cases (e.g., f, figs. 5, 6 and 7)
the space between the fragment and the main portion of the
chromosome was uniformly faintly stained lends support to the
suggestion. Other cases exhibited connections consisting of
various amounts of strongly stained chromatin (e.g., chr.f., figs.
5, 14, 15, and 21). All mitoses in the gill plates (the most i
prophases, fig. 5) showed the largest number of instances of this
condition; the peritoneum contained scarcely any. This might
be explained as an effect of inferior fixation (p. 173) were it not
for the fact that a considerable amount of apparent fragmenta-
tion is present, even in the metaphases of the tail epithelium
which are fixed under the most favorable conditions. The
reason for this is not clear.

F. The existence of pairs

The question whether the chromosomes exist in a duplicate
series is significant in two respects: 1) in its relation to the
mechanism ‘of heredity as suggested by Janssen’s chiasmatype
theory and by the brilliant work of Morgan and his co-workers;
2) as a further index of the constancy of the organization of
the chromatin. This constancy is vitally related to the theory
of the individuality of chromosomes.

It will be convenient to consider separately the evidence of
the existence of pairs in the germ cells and in the somatic cells.

a. Pairs in germ cells. Van Beneden’s (’83) hypothesis, that
one-half of the chromosomes of an individual are of maternal
origin and that the other half are of paternal. origin, has been
verified in many cases. That this double set of chromosomes

CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 209

exists in pairs in the germinal line is evidenced by their behavior
during the maturation period.

Montgomery (’01) presented evidence for the recognition of
pairs in the spermatogonia, basing his argument upon the signifi-
eance of the chromosome number in Euschistus. Sutton (02)
showed by means of a comparison of many camera-lucida draw-
ings of spermatogonial complexes of Brachystola magna that
these chromosomes form a duplicate series of lengths, and by
means of measurements with a pair of dividers that the chro-
mosomes of the early primary spermatocyte prophases are graded
into the same series of relative sizes. Meves (’11) interpreted
his measurements upon spermatogonia of Salamandra maculosa
as failing to demonstrate pairs. Meek (’12) has made linear
measurements upon the spermatogonia and secondary spermat-
ocytes of a number of animals, interpreting his results as con-
firming the claim of the existence of pairs. . Robertson (’15, 716)
also supports this view with metrical data in certain Orthoptera,
and Hance (17 b; 718 a) confirmatively interprets his measure-
_ments in the germinal and somatic cells of Oenothera and the
pig. In unmeasured spermatogonial chromosomes of the Dip-
tera, Stevens (708, 710, 711), Metz (14, ’16), and Whiting (’17)
show very convincing evidence of pairs for the homologues are
associated side by side. Wilson’s (’06, p. 11) figures of Anasa
and Hoy’s (716, p. 336; 718) figures of Anasa, Epilachna, and
-Diabrotica also support this conclusion. The majority of other
authors as a result of their general observations have expressed
the belief that the chromosomes exist as a duplicate series.

Furthermore, the existence of pairs in the spermatogonia is
practically proved by parasynapsis where the chromosomes of
the last spermatogonial division unite side by side and remain
so until separated by the reduction division. This statement is
made possible by Wenrich (’16) who, besides confirming the
already numerous and all but conclusive evidences of parasynapsis
by Janssen (05, 709), A. and K. E. Schreiner (’06, a and b;
08, a and b), Wilson (712), and many others, carries the demon-
stration a.step further by actually tracing a well-marked chro-
mosome pair A (p. 76) continuously through every stage of

210 ; CHARLES L. PARMENTER

spermatogenesis from the early spermatogonia to the spermatids.
He thus demonstrates that the conjugating elements are chro-
mosomes and are morphologically identical with the spermat-
ogonial chromosomes. That one of the homologues of each
conjugated pair is maternal and the other paternal is very
probable, as has been shown by the observations of Van Beneden
(83) and numerous later authors, especially Mulsow (’12).

It remains to be seen whether the pairs of maternal and
paternal homologues present in the germ cells during the matura-
tion period maintain their identity in the germinal line between
the time of fertilization and the first observations upon the sper-
matogonia. This has been accomplished in part. Mulsow (712)
has followed the actual chromosomes of the living spermatozoon
of a parasitic trematode, Ancyracanthus, into the egg, and has
found the expected number of chromosomes in the two pro-
nuclei and cleavage stages. He also observes that the chromo-
somes of the cleavage nuclei show in many cases a tendency to
lie parallel to one another, and suggests that this is an approxi-
mation of maternal and paternal chromosomes. Boveri (’87,
’92) traced the chromosomes of the primordial germ cell of Ascaris
univalens through the cleavages from the two-celled stage, .
and Moenkhaus (04), Morris (14), Richards (17) have traced
the persistence of individual chromosomes through several
cleavages of hybrid eggs of Fundulus. If this persistence of the
chromosomes is permanently maintained, the observations of the
above authors make it probable that the maternal and paternal
chromosomes form a duplicate series throughout the germinal
line.

b. Pairs in somatic cells. Since the existence of chromosome
pairs can be considered to be all but proved throughout the
germinal line, it remains to be seen whether or not the chromo-
somes of the somatic cells, which are really descendants of those
of the germinal line, still retain this duplicate series and thus give
evidence of maintaining their individuality.

The earliest observations bearing upon this question were
made on Salamandra maculosa by Flemming (’82) and Rabl
(85), who observed that the chromosome segments were not of

CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 2k

equal lengths in the early spireme and metaphase stages. The
general observations of many later authors, especially those
studying Dipteran somatic cells (Metz, ’14, ’16 a, b; Hance, 717;
Holt, 17; Whiting, ’17) indicate these chromosomes to be paired.
Observations of pairs in a large number of animals and plants
have been extensively reviewed by Metz (716, p. 245).

But no cases are recorded in which an attempt was made to
determine accurately by measurement what relation the lengths
of somatic chromosomes bore to one another until the work of
Meves (711). Asa result of the discussion centering around the
observations of Montgomery (’01) and Sutton (’02), he was led
to attempt measurements in an effort to obtain more definite
data, as suggested by Della Valle (’09, p. 109). He measured
both spermatogonial and somatic chromosomes of various tissues
in Salamandra maculosa. Della Valle (12) made further meas-
urements upon the same form, and agrees with Meves that their
results do not confirm the observations of Montgomery and
Sutton. Hance (717 and ’18a) interprets his measurements
upon the somatic chromosomes of Oenothera scintillans and the
pig as confirmatory.

1. Meves’ results. Since Meves has (p. 282) failed to confirm
the results of other authors, it is desirable to reconsider his data
in comparison with linear measurements upon cells of the same
nature in the same kind of preparations and of the same tissues
of another salamander, Ambystoma tigrinum, in an effort to form
a judgment of the validity of his conclusions. For this purpose
it is necessary to recall what criteria are required (p. 191)
definitely to affirm or to deny the existence of pairs and under
what conditions these criteria were satisfied.

The following conditions under which Meves’ measurements
were made allow the introduction of such a varying amount of
error that the conclusions drawn from his results are of question-
able value.

Instrumental and personal errors. Meves made his measure-
ments evidently upon a single drawing, probably somewhat care-
fully executed, which means, according to a series of tests in my
own attempts to be accurate, that he has a minimum instru-

PN CHARLES L. PARMENTER

mental and personal error of about 0.6 mm. at his magnification.
This, of course, is negligible in comparison with the large errors
arising from foreshortening in numerous chromosomes.

Concerning the favorableness of the spermatogonial chromo-
somes for measurement, Meves says (p. 274) that by no means
do all of the chromosomes lie in the equatorial plane; without
exception the bend lies in the plane while the ends lie outside;
in the drawings such chromosomes seem shortened and therefore
the measurements upon these chromosomes would give only an
approximate value. Judging from these statements and from
the magnitude of error due to the slight foreshortenings in my
material, his measurements very likely contain errors which
amount to as much as 4 or 5 mm. .

For measurements of somatic chromosomes he chose (p. 280)
polar views of the transformation stages between the prophase
and metaphase stages in the epithelium of the gill plates (figs. 16
to 18) and extraordinarily well-flattened polar views of pro-
phases (figs. 11 to 18) and metaphases (figs. 14 to 15) in the
peritoneum. In the three prophases of the peritoneum the chro-
mosomes lay nearly or entirely parallel with the upper surface
of the cell. According to this description, it is evident that these
three prophases are the most favorable cells, and even in these
the chromosomes are not entirely free from foreshortening. The
chromosomes of the other cells probably were more foreshortened.
Therefore, judging from results in Ambystoma, his measurements
contain errors due to foreshortening which probably vary from 2
to 5 mm.

The amount of the errors which are due to the twisting of the
chromatids of these chromosomes is uncertain. Such twisting is
evidently present, as indicated by the irregular contour of his
chromosome drawings which are similar to those of my own.
The errors due to this twisting may largely neutralize each other
as explained above (p. 189). He mentions also the possibility of
different rates of contraction of the chromosomes, especially in
the earlier stages. Having before us the conditions under which
Meves made his measurements, we are in a position to judge
their value more or less correctly.

CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 213

As Meves states (p. 276), the differences in length between the
spermatogonial chromosomes are too small, and the possible
errors too great to affirm that these chromosomes are present in
pairs of equal length. On the other hand, it should be added
that these conditions offer no evidence against this claim.
Furthermore, he shows a practically constant difference in
length of 6 half-millimeters between chromosomes 8 and 9 in
his table of measurements. An examination of these figures
shows unmistakable evidence that there is foreshortening in chro-
mosome 9 in three of these cells, and a probability that chromo-
some 8 is much less, if any, foreshortened than chromosome 9.
There results, therefore, in all of these cells a relatively uniform
distribution of the chromosomes into two constant groups, a
point which supports the claim that the relative lengths of these
chromosomes remain constant.

Concerning the somatic measurements, Meves concludes (p.
282) ‘‘Die Fragen, ob bei gew6hnlichen Gewebszellen des Sala-
mandra Chromosomenpaaren unterschieden werden kénnen, ist
bereits von C. Rabl (06, S. 72) verneint worden, ich muss mich
ihm auf Grund der mitgeteilten Zahlen anschliessen”’ and ‘‘ Die
erhebliche Lingendifferenz zwischen chromosomen VIII und
IX, welche wir bei den Spermatogonien festgestellt haben,
besteht bei den gezeichneten somatische Zellen nur in der Hiilfte
der Faille.” This means, of course, that he believes that the
chromosomes are not distributed into two constant groups in
each cell and that therefore the evidence of the constant organi-
zation of the chromatin is lacking in this respect.

As already stated, the possibilities of error are so great that
nothing is conclusively affirmed or denied by his measurements.
However, an examination of the drawings of the chromosomes in
his figures indicates certain probabilities.

1. In three of the four cells (his figs. 15 to 17) in which the
marked difference between chromosome 8 and 9 is not present,
chromosome 9 is conspicuously foreshortened, and is in reality
longer than his measurements indicate. Furthermore, chromo-
some 8 in these cells may or may not be foreshortened, at any
rate it is probably much less foreshortened than chromosome 9.

214 CHARLES L. PARMENTER

A conservative estimate of foreshortening upon chromosome 9 in
these cells, based upon computation of foreshortening in my
material, amounts to a minimum of 2 mm., which restores in
these cells the difference in length between chromosomes 8 and 9
found in the spermatogonial and the other somatic cells. These
same chromosomes in the remaining cell (fig. 13) which lack this
difference do not appear foreshortened, but owing to the various
sources of error pointed out in the preceding pages, it is entirely
possible that such a difference may also be present in this cell.
These considerations make it probable that the difference be-
tween chromosomes 8 and 9 is present in eleven and possibly in
all of the twelve somatic cells which Meves measured. It is
beyond expectation that this difference should be exactly the
same in every cell whether of the same or of different stages of
mitosis.

2. Meves also observes a difference of 3 to 4 mm. between
other chromosomes which theoretically should be homologues.
In figure 11 between chromosomes 17 and 18 and between
chromosomes 23 and 24; in figure 12, between chromosomes 11
and 12; in figure 13, between chromosomes 7 and 8, 15 and 16,
17 and 18, 23 and 24.

Attempts to apply corrections for the foreshortening evident
in these cells, estimated upon the basis of his drawings as com-
pared with similar cells in Ambystoma (figs. 5, 13, and 14),
leave the situation about as it was. This is not surprising, con-
sidering the difficulty of judging the amount of foreshortening
that is conspicuously present in some chromosomes and the
uncertainty of its presence, or absence, in others. Meves’ state-
ment quoted above concerning the type of cells used for measure-
ments makes it quite possible that a great many of these chro-
mosomes are foreshortened in amounts varying from 2 to 5 mm.
This opinion is strengthened by his generalized and unprecise
statement concerning foreshortening and by comparisons with
similar cells in Ambystoma.

The conspicuous foreshortening in some chromosomes, and
the probability of it in others, seriously weakens the validity of
his measurements. This is especially true of figures 11, 12,

CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 215

and 13 showing late prophases in which there is usually much
foreshortening and considerable inequality of homologues, as
stated above. In five other cells (figs. 14 to 18) Meves’ figures
show but one case of a difference of 3 mm. and only five cases
of a difference of as much as 2.5 mm. between homologues, as
they are indicated by his measurements. But the various
sources of error already mentioned make it uncertain as to what
the actual lengths are.

To summarize the examination of the results of Meves’
measurements, it may be said, 1) that the chromosomes of the
spermatogonial cells fall into two groups, one containing chro-
mosomes | to 8, the other chromosomes 9 to 24. 2) In the
somatic cells, when corrections are made for evident fore-
shortenings, it is probable that in eleven of the twelve cells, and
possibly also in the twelfth, the same grouping is present. This
indicates a constancy of organization of the chromatin. 3) It
is impossible either to demonstrate conclusively or to deny that
these chromosomes are paired because, a) of the various sources
of error present and, 6) the small differences in length in the
majority of cases between adjacent chromosomes.

2. Della Valle’s measurements. Della Valle (12) measured
the lengths of chromosomes in the peritoneal cells of Salamandra
maculosa shown in his (’09) figures 1 to 3, 8 to 9, and 12. The
length of each chromosome was obtained by averaging two
measurements made with a curvimeter upon a single camera-
lucida drawing. He also attempts to determine the degree of
concordance between the measured lengths of each of these
chromosomes and the dimensions which would exist if the
lengths of these chromosomes were determined by the laws of
fluctuating variation. These latter figures he obtains by calcu-
lation from a table of figures compiled by Sheppard and pub-
lished by Galton (07). He interprets his data as demonstrating,
1) that the chromosomes of Salamandra maculosa do not exist in
pairs; 2) that there is no constant grouping of chromosomes,
such as is evident in the measurements of Meves and myself,
and, 3) that the chromosomes are a series of variants subject
to the laws of fluctuating variation as shown by the comparison,

216 CHARLES L. PARMENTER

given in his tables and curves, between the measured lengths of
the chromosomes and the computed lengths that would be
expected if the chromosomes were such a series of variants.

I believe Della Valle’s conclusions are incorrect for the fol-
owing reasons: 1) He fails to demonstrate the presence of
chromosome pairs because, a) as discussed on page 201, his chro-
mosome enumerations are probably incorrect and therefore his
measurements do not represent the actual conditions; 6) his
measurements probably contain numerous errors of varying
magnitude due to foreshortening (as well as to errors arising
from measurements upon single drawings) even though he chose
for measurements strongly flattened cells (p. 126); c) the dif-
ferences in the lengths of these chromosomes are so small and
the errors so great that it is impossible either to demonstrate or
to deny a presence of pairs. 2) Failure to find a constant
grouping among the chromosomes would result from the causes
given in (a) and (b). 3) His interpretation that the chromosome
lengths are controlled by the law of fluctuating variations is
untenable because, even if his measurements were reliable and
whether pairs do or do not exist, the differences in length between
the chromosomes of Salamandra maculosa are so small that the
degree of correspondence between their measured lengths and the
calculated lengths of a series of variants, corresponding respec-
tively to each of these chromosomes, would be fully as close as
those which he presents in his tables and curves containing
numerous and large differences.

3. Resultsin Ambystoma tigrinum. The chromosomes of Am-
bystoma tigrinum, fortunately, are more favorable subjects for
measurements than those of Salamandra maculosa, because the
relative differences in length between many pairs is so large
that certain pairs and certain groups of pairs stand out con-
spicuously. The evidence presented in figures 33 and 34 is free
from all errors of measurement except those due to twisting of
the chromatids and to minute foreshortenings at non-critical
points in the series. These errors have been approximately
eliminated (p. 188) and do not seriously disturb the critical evi-
dence of the chromosome pairs which are much shorter or longer

CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA yAWs

than adjacent pairs. The evidence in the other figures is but
little inferior to that of figures 33 and 34. However, measure-
ments of the chromosomes of so few cells are insufficient to
furnish more than strongly supporting evidence of the existence
of pairs, and of an approximate constancy of size relations
between pairs in different individuals.

It may appear that these measurements support equally well
Della Valle’s claim that there are no chromosome pairs, but that
the chromosomes form a series of variants. However, the con-
sistent evidence of the presence of pairs among the shorter
chromosomes, the possibility of unequal stretching of the longer
homologues together with the known condition in Orthoptera
that homologues of tetrads may be of unequal lengths lends
greater support to the probability of the existence of homologues.

The following points need further consideration.

Large differences in length between homologues. ‘The difference
in length of 12.4 mm. between the homologues of pair 9 in
figure 33 and the similar difference of 9.5 mm. between the
homologues of pair 12 in figure 37, the smaller difference in
pairs 7 and 8, figure 35, pair 7, figure 36, and pair 8, figure 37,
may possibly be explained as follows:

1. Unequal homologues have been reported in Orthoptera by
Baumgartner (711), Hartmann (713), and more thoroughly studied
by Carothers (13), Robertson (’15), and Wenrich (’16). The
latter’s observations are particularly significant. He found dif-
ferent conditions of inequality in two of the small tetrads of
Phrynotettix. He designated these two tetrads as ‘B’ and
‘C’ and traced their history from the pachytene stages through
the first maturation division. The homologues of tetrad ‘B’
were unequal in eleven of the thirteen individuals studied and
were equal in the other two. Tetrad ‘C’ was found in three
forms, designated as ‘C,, Cs, C3.’ ‘Ci is composed of very
unequal elements, the larger of which possesses a relatively large
terminal knob or granule which is not present on the other two.
‘C,’ is a pair with equal members, each of which appears to be
homologous to the smaller member of ‘C;.’? ‘C;’ is a pair of
unequal elements, neither member of which appears to be

218 CHARLES L. PARMENTER

exactly homologous to the components of ‘C;’ and ‘C;,.’ The
smaller member resembles those of ‘C,’ and may be homologous
with them. It is important to note that the three last-mentioned
authors find that each particular condition is constant for the
individual in which it is found.

Although tetrads with unequal homologues among the longer
chromosomes have not been observed in the Orthoptera, they
might possibly exist in other animals. The above observations,
especially those of Wenrich, offer a possible explanation for the
inequalities between homologues observed in Ambystoma as well
as in Salamandra maculosa. Furthermore, the condition found
in tetrad ‘C.’ may offer a parallel explanation for the different
relative lengths shown in some cases between corresponding
pairs in complexes of different individuals (e.g., pr. 4 and pr.
9). Of course much further data from both the somatic and
germinal chromosomes is necessary before the above can amount
to anything more than a suggestion.

2. Certain inequalities might be explained as due to the
presence of a multiple chromosome similar to that which has
been described by McClung (’05, 717), in Orthoptera, by Boveri
(09), Edwards (’10, 711), and Frolowa (’12), for Ascaris megalo-
cephala, Boveri (’11) and Edwards (’11) for Ascaris felis, Stevens
(711) in Anopheles, and by King (’12) for Necturus. In these cases
the sex chromosome has been interpreted as being attached to one
of the euchromosomes, and thus there is present in the male an
unequal pair of chromosomes which may parallel the condition
in pair 9 of figure 33 and pair 12 of figure 37. In certain Or-
thoptera McClung (717) finds that the accessory may be attached
to different chromosomes in different individuals, which lends
support to the possibility that this is the condition in Ambystoma.
The presence: of an X and a Y chromosome would also produce
unequal homologues.

If either of these explanations be valid, such an unequal pair
should appear in all the diploid complexes of approximately
one-half of a somewhat large number of individuals, and similar
conditions should be found in the maturation period. Unfor-
tunately, the difficulty of obtaining a sufficient number of suitable

CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 219

cells prevents extensive investigation of this point in the somatic
cells for the present and makes this explanation only suggestive.

Constancy in the indiwidual. It should be emphasized that the
observations in the Orthoptera concerning the unequal tetrads
(p. 217) and other heteromorphic tetrads (p. 192) strikingly
demonstrate a constancy in the individual for the particular
characteristic of each homologue concerned. In Ambystoma it
has been impossible to obtain sufficient material to verify this
point.

Whether the members of these Orthopteran unequal and other
heteromorphic tetrads maintain their organization from one
generation of animals to the next is yet to be demonstrated by
breeding experiments now in progress in this laboratory. The
expectation is that they do, since Wenrich (716) and Carothers
(17) find every possible combination which would arise from the
segregation and recombination of the members of these various
types of tetrads. :

The presence in the Orthoptera of unequal tetrads does not
indicate a lack of individuality. On the contrary, the per-
sistence of this condition throughout the individual, and perhaps
from generation to generation, is strong evidence to the con-
trary. Of course a change has taken place at some time (if it
be correct to assume that the homologues were all alike at some
earlier period), but this is to be expected if these chromosomes
are to parallel genetic behavior.

Bridges (17, p. 445-6) presents parallel genetical data in
connection with the chromosomes of Drosophila. He finds in
certain cases that the genes for ‘bar’ eye and ‘forked’ bristles,
whose loci are located near one end of the sex-chromosome, have
been lost and that the region between these two loci has also
been affected. He suggests that this deficiency may be due to
a physical loss of this portion of the chromosome. He also
reports (’19, p. 357) a case in which ‘‘a section of the X-chromo-
some, including the loci for vermilion and sable, became
detached from its normal location in the middle of the X-chro-
mosome and became joined on to the ‘zero’ end (spindle fiber)
of its mate.” In other instances the locus for sable alone, as

220 ; CHARLES L. PARMENTER

far as known, has been lost from one homologue and joined to
the end of its mate. Another case is the transposition of a piece
of the II chromosome to the middle of the II] chromosome.
He has exhibited definite cytological evidence (unpublished)
supporting a part of the above. This condition produces
homologues of unequal length which parallels the observations
in the germ cells of the Orthoptera and the apparent similar
condition in certain somatic homologues of Ambystoma.

c. Constant relative size relations. In addition to verifying
Montgomery’s (’01) and Sutton’s (’02) observations of paired
homologous chromosomes of equal length in the germ cells,
Meek (712), Robertson (716), and Hance (717 b, ’18 a) confirm
Sutton’s (02) observation (based upon comparisons of camera-
lucida drawings of many spermatogonial cells and upon measure-
ments of early prophase tetrads) that the proportional difference
in size between any two pairs in one nucleus is practically the
same as that between the corresponding pairs in any other
nucleus. In Ambystoma tigrinum, as is seen in figures 33 to
37 and the table of percentages accompanying them, while
the relative lengths are not exactly the same in every cell, there
is in general a marked constancy of relative lengths. Were
Meves’ and Della Valle’s measurements correct, the same would
probably appear there. :

d. Summary of measurements. The data here presented in
connection with measurements upon the chromosomes of Amby-
stoma tigrinum and Salamandra maculosa cannot well be inter-
preted as a confirmation of Meves’ and Della Valle’s contention
that pairing of the chromosomes and a constant organization of
the chromosomes do not exist because: 1) Their data, on
account of errors inherent in the material, are too unreliable to
command confidence; 2) the differences in the lengths of the
chromosomes of Salamandra maculosa are too small to permit
one to deny or to affirm the existence of pairs, and, 3) it has
been shown that because of obvious foreshortenings, individually
mentioned above, which are unaccounted for in Meves’ measure-
ments, there probably is a somewhat constant difference between
chromosomes & and 9 in eleven out of the twelve cells which he

CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA Zot

measured. This confirms the contention of a constancy of
chromatin organization so far as is possible in material having
chromosomes differing so little in length as those of Salamandra
maculosa. 4) The measurements of chromosomes in the so-
matic cells of Ambystoma tigrinum show duplication of sizes,
especially pairs 1, 2, and 8 in figures 33 and 34, pairs 8, 9, and 10
in figure 36 and indications of the same in the other chromo-
somes of all the cells which differ too little in length to constitute
reliable evidence. Explanations are offered for cases in which
the homologues differ in length. 5) The chromosome lengths
show approximately constant relative sizes in all of the cells
measured.

Based upon the above considerations and upon the unequal
and other heteromorphie tetrads in Orthoptera, my expectations
are that the pairs and their relative lengths in the somatic cells
of Ambystoma are constant for the individual, and although
not exactly the same, they are approximately the same in dif-
ferent individuals. However, as stated above (p. 199), the meas-
urements cannot be considered to demonstrate conclusively the
presence of a duplicate series of chromosomes.

SUMMARY OF CONCLUSIONS

1. No variation is found in the somatic chromosome number
of twenty-eight in Ambystoma tigrinum.

2. Della Valle’s contention that variation’ in chromosome
number is the rule is unconfirmed.

3. The chromosomes form approximately a duplicate series of
sizes and forms, supporting the contention that they consist of
pairs of maternal and paternal homologues.

4, An approximate constancy of size relations between pairs
in the complexes of different individuals is also maintained.

5. Della Valle’s claim that the chromosome lengths are a series
of variants is not substantiated.

6. There is evidence of unequal homologues in these cells.

7. There is also a suggestion that there is a sex chromosome
attached to a euchromosome.

pis CHARLES L. PARMENTER

8. There is apparently but little complete fragmentation of
the chromosomes.

9. The above observations support the theory of the indi-
viduality of the chromosomes.

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PLATES

EXPLANATION OF PLATES

The drawings were made with the aid of a camera lucida, using a Zeiss 2-mm.
apochromatic immersion objective, N. A. 1.30, and a Spencer compensating
ocular 20 X which produced a magnification of 2633. The illumination consisted
of light from a 100-watt frosted-globe Mazda concentrated-filament lamp passed
through a daylight glass or a common cobalt-blue glass filter, and an Abbe con-
denser. The observer was shaded from the light in front of him and from the
sides by a black-cloth screen.

In reproduction plates 1 to 8 have been reduced one-third, and plate 9 three-
fourths, giving a final magnification of 1755 and 1316, respectively.

Figures 1 to 20 represent complexes of class I; figures 21 and 23, complexes of
elass IT.

PLATE 1

EXPLANATION OF FIGURES

1to3 Very late peritoneal prophases.

4 <A peritoneal metaphase.

The numbered pairs of chromosomes correspond to pairs bearing, respectively,
the same numbers in figures 27 and 28, and in the legend of figures 33 and 34.

228

CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA PLATE 1
CHARLES L. PARMENTER

PLATE 2
EXPLANATION OF FIGURES

5and6 A gill-plate prophase and metaphase.
7and8 Metaphases of the tail epithelium.

CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA PLATE 2
CHARLES L. PARMENTER

PLATE 3

EXPLANATION OF FIGURES

9to12 Late prophase complexes of the lung epithelium. a
The pair numbers correspond ,respectively, to those in figures 29 and 30, and
in the legend of figures 35 and 36.

232

PLATE 4

EXPLANATION OF FIGURES

13 to 16 Gill-plate complexes. The chromatids are shown only in figure 13.

a |
>
)
Zs
FP

AW
\\
C

PLATE 5

EXPLANATION OF FIGURES

17 to 19 Two prophases and one early metaphase of gill-plate epithelium.
20 A peritoneal metaphase.

236

©

JAS WE
Ba eecss

CO

——)

Vin
cSSS ‘a
——5

PLATE 6
EXPLANATION OF FIGURES

21 and 23 Gill-plate prophases of class Il. Chromosomes ‘/,’ figure 21, inter-
preted as one; ‘i,’ figure 23, as two (p. 12).

22 A peritoneal complex with part of the chromosomes missing.

24 A peritoneal complex separated into two parts. The pair numbers are
duplicated in figure 31 and_in the legend of figure 37.

238

WM |) .
14 i 5

if
<

PLATE 7
EXPLANATION OF FIGURES

25A Another peritoneal complex separated into two parts which are drawn
in their relative positions. 866.

25B The chromosomes of figure 25A. X 1755.

Chromosomes 14 to 17 are interpreted; note that the chromatids of these four
chromosomes are well separated.

26A A lung epithelial cell separated into two parts which are drawn in their
relative positions. XX 866.

26B Chromosomes of 26A. X 1755. Pair numbers duplicated in figure 32.

240

PLATE 7

CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA
CHARLES L. PARMENTER

PLATE 8

EXPLANATION OF FIGURES

27 to 32. Chromosomes of figures 1, 3, 9, 10, 24, and 26, respectively, arranged
in pairs with pair numbers and figures indicating their lengths in millimeters
at X 2633. The amount included in any figure for foreshortening is indicated
above that figure. The pair numbers are duplicated in the above figures and in

the legend of figures 33 to 37, respectively.

CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA
CHARLES L, PARMENTER

Seu C LS Ub

12.5 13 15.1 16.2 22.5 233 265 26.8 27.2 27.2

9 ON o6 ML

28 14145 178179 25.5 26.9 278 28.6 316 32.1

acl
29 ig a5 139 195

eos
30 145 175 18 20

iv av vb vu LY

Sl 85105 14 165 20 20 22) 22712315 23.5
S2) ia: a I 14 15 7 175 IS 19
| 2 a 4 5

$3.5 355

wy CL ai t¢ hd

404 562.8

44.4 45.3 45.7 45.8

Lo ve SY

33.6 33.6

iv a” JV

36.5 39.5
VA Vv yy
36.5 42 44.8 44.8 4g 49.5
25 26 29 33.5 39 4\
pe) 26 26 26 o7

244

$2.3

55.4 568

55.5 55.5 565 56.5
\ +12
573 58.8 61.3

¥ ey

PLATE 8

48.9 49.3 51 52
3
58.7 58.9 62.2 64
+1 2
585 585
60 62
%

47 47.5 48 49.5
30 30 30 33
13 14

245,

PLATE 9

EXPLANATION OF FIGURES

33 to 37 Representing the lengths of the chromosomes in the cells indicated
in the table below at a magnification of 1316. The differences in the lengths of
the lines and also the spaces between the lines represent relative differences in
chromosome lengths. For convenience the width of the spaces between the
lines are made eight times the differences in lengths. The lengths of the chromo-
somes and their percentage of the average length in the cell is shown in the
table below. The amount of foreshortening in any chromosome is indicated in
plate 8.

FIGURES

PAIRS 33 (1, 27) 34 (3, 28) 35 (9, 29) 36 (10, 30) 37 (24, 31)

Milli- Per Milli- Per Milli- Per Milli- Per Milli- Per
metres} cent | metres| cent | metres|} cent | metres; cent | metres| cent

12.5| 37 | 14.0| 35 | 18.0| 44 | 14.5] 34 8.5 | 27
13.0 | 38 | 14.5 | 36 | 18:35} 45 | 17.5) 41 | 10.5! 338

15.1} 44 | 17.8) 45 | 19.0} 46 | 18.0] 42 | 14.0] 44

Z 16:2) 47 | 47.9) 45 | 1975.) 47 "| 2070 | "47 | AGsoiee2
3 22.5 | 66 | 25.5 | 64 | 26.7] 65 | 24.7) 58 | 20.0] 68
23.3 | 68 | 26.9], 68 | 27.5 | 68 | 25.0] 59 | 20.0] 638
ie ZO | EC | 2H 10" 29090.) el 2Oe| © Gs! eee
26:8 | 78 | 23/6.) 72.) 29-5 | “72—) 29:0) | 68" 4), 22.00 70
5 2h.2 | 80, \-Sl-6 3) <f9" | 31208), 76" [Stn 7S: 235 aes
2t-2 | 80-| 32.1) 80. | 30:6.) 77 .| 33:0 | 78-23. 55/075
6 21-6/) 8b) '80-- | — | 33750) 82 4) 35.4) 84 9) 24 Oca
27.6) 81 | 33.0] 83 | 35.5 | 87 | 35.5) 8&4 | 24.0] 76
= 28.4 | 83 | 33.6] 84 | 36.5] 89 | 36.5] 86 | 25.0| 79
f 28.9 | 84 | 33.6] 8 | 39.5] 97 | 42.0) 99 | 26.0) 81
8 36.0 | 105 | 42.0 | 105 | 40.4} 99 | 44.8] 103 | 29.0] 91
36.0 | 105 | 42.0} 105 | 46.0] 112 | 44.8] 103 | 33.5 | 106
9 40.4} 118 | 49.7 | 125 | 47.5 | 116 | 49.0 |) 116 | 39.0 | 124
52.8 | 154 | 49.7 | 125 | 48.0] 117 | 49.5 | 117 | 41.0} 130
10 43.0 | 126 | 50.7 | 127 | 50.0 | 122 | 54.0 | 127 | 41.0 | 130
44.0 | 129 | 52.3 | 182 | 54.0 | 182 | 55.0] 130 | 41.0 | 1380
i 44.4 | 130 | 53.8 | 1385 | 55.5 | 1386 | 57.3 | 185 | 43.0 | 136
45.3 | 182 | 53.8 | 1385 | 55.5 | 186 | 58.8 | 1388 | 44.5 | 141
12 45.7 | 184 | 55.4 | 139 | 56.5 | 188 | 60.0 | 142 | 44.5 | 141
% 45.8 | 1384 | 56.8 | 143 | 56.5 | 1388 | 61.3 | 145 | 54.0} 171
13 48.9 | 143 | 58.7 | 148 | 58.5 | 143 | 62.2 | 147 | 47.0 | 149
49.3 | 144 | 58.9 | 148 | 58.5.) 143 | 64.7 | 153 | 47.5 | 150
14 51.0 | 149 | 62.2 | 156 | 60.0 | 147 | 66.3 | 156 | 48.0} 152
52.0 | 152 | 64.0 | 161 | 62.0 | 151 | 69.3 | 163 | 49.5 | 157

CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA PLATE 9
CHARLES L. PARMENTER

oe Ul |

33 | 2

wt Mil || | | | |
a all tl

oe NUE | | |

249

AUTHOR’S ABSTRACT OF THIS PAPER ISSUED
BY THE BIBLIOGRAPHIC SERVICE, JANUARY 19

THE ANATOMY OF THE HEAD AND MOUTH-PARTS
OF ORTHOPTERA AND EUPLEXOPTERA!

HACHIRO YUASA

ONE HUNDRED AND SIXTY-THREE FIGURES (NINE PLATES)

CONTENTS
CLS HP CTU GLa yore, Ae BE Ae eee atte Ae ae ee ee a Og ee ee ee 251
DASA TE" DUNT g PEC SuH FY A ee ee ay a a See, ny ee mre NP 253
OTS CTa Al ONS Mere NN Seite eter eres hides inca id Ae sed AE oe Bes 253
AU RIXeOUpPAbLsTOulNeRNeAGrsyderats, ote tk Cee ep eeer eee Te en) Ya! 253
B. Movable parts of the head.............. sepa Ns: Sai ee eer Mert Be oe 264
STOLEN DEA ele. cole SEE Oe Bb CRORE CAT eS a Re Oe eA Naa 284
INTRODUCTION

Huxley (’78), with good reasons, turned to the generalized
Orthoptera for a type for study and description as a representa-
tive of the class Hexapoda. Blatta orientalis was selected for
this purpose, and its anatomy received a careful consideration.
A few years later, Packard (’83) discussed rather briefly the ho-
mology of the head and mouth-parts of orthopterous and other
insects. Miall and Denny (’86), three years later, published a
book on the morphology and biology of the cockroach which is
still considered a classic. The descriptive and theoretical as-
pects of the skeleton of the head of the more generalized insects,
including the tentorium, were thoroughly studied by Comstock
and Kochi (’02). After defining the sclerites and areas of the
head capsule, these writers attempted to ascribe each sclerite and
appendage to some one of the seven primordial segments of which
an insect’s head was supposed to be composed. Riley (’04), who
investigated the embryological development of the skeleton of
the head of Blatta germanica, came to the conclusion that ‘‘so

1 Contributions from the Entomological Laboratories of the University of
Illinois, no. 55.

251

252 HACHIRO YUASA

intimate a relation between the segmentation and the sclerites
cannot be shown.”’ More recently the mouth-parts of the cock-
roach were studied by Mangan (’08) and Bugnion (’13, 716).

In spite of the fact that an immense amount of valuable infor-
mation is found in the more comprehensive works on general
entomology, notably Kolbe (’89), Packard (’98), Henneguy (’04),
Berlese (’06), and Schréder (12), and, contrary to the general
impression that the external anatomy of insects in general and
especially of the more common insects such as Orthoptera has
already received sufficient attention from morphologists, it is sur-
prising, when the subject is scrutinized a little more closely, to
find how few studies of homology have been attempted by the use
of a series of forms. The possible exceptions in America are
the successful attempts to homologize the wing veins of different
orders by Comstock, Needham, MacGillivray, and others and
the careful investigations of Crampton (’09, 714, 717), Snodgrass
(09, 710), and Peterson (’16) on the different regions of the insect
body. The confusion in the terminology used by insect morphol-
ogists and the greater confusion of the anatomical terms now cur-
rent among taxonomists, particularly among specialists interested
exclusively in restricted groups, amply justify attempts to in-
vestigate and in many cases to reinvestigate the more funda-
mental structures of insects.

The following pages present a résumé of a detailed study of the
salient characteristics of the external anatomy of the head and
mouth-parts of the generalized biting insects as represented by
typical species belonging to the orders Orthoptera and Euplex-
optera. Particular attention was given to structures hereto-
fore little studied—the prepharynx and tentorium. ‘This study
was undertaken under the supervision of Prof. Alex. D. Mac-
Gillivray, of the University of Illinois, and to him I extend my
sincere thanks for suggestions and encouragement and for the
permission to use his unpublished terminology? and forthcoming
outline for the study of insect anatomy. This outline proved
most valuable and indispensable as a guide.

2 The new terms used in this paper are defined in the following pages. On
pages 285-286 they are tabulated in their relations to other parts.

HEAD, MOUTH-PARTS, ORTHOPTERA EUPLEXOPTERA 253

MATERIAL AND METHODS

In order to make the study as comprehensive and representa-
tive as possible, the following species have been selected and
examined: Blatta orientalis (Blattidae), Mantis religiosa (Man-
tidae), Diapheromera femorata (Phasmidae), Gryllus pennsyl-
vanicus (Gryllidae), Orchelimum vulgare (Locustidae), Steno-
pelmatus sp. (Locustidae), Melanoplus differentialis (Acrididae),
Tettix arenosus (Acrididae), and Anisolabis maritima (Euplex-
optera).

The specimens were treated, as a rule, with a 5 per cent solu-
tion of potassium hydroxid from five to twenty-four or more
hours (depending upon the degree of chitinization of the struc-
tures) and examined in 70 per cent alcohol under a binocular
microscope. The drawings were made free hand after measuring
the dimensions of the specimens by means of an ocular microm-
eter.

OBSERVATIONS
A. Fixed parts of the head

The conditions in the cockroach have been taken as typical,
and those of other families have been discussed only where they
differ from this type. Detailed descriptions have been omitted;
with the accompanying figures it is believed that no difficulty
will be experienced in identifying on specimens the structures
shown. In order to avoid confusion, the skeleton of the head and
its appendages have been described in every case as if the mouth
were directed cephalad.

The epicranial suture in Blatta (fig. 1) is the inverted Y-shaped
median suture. The stem (es) of the Y begins at the occipital
foramen, extends cephalad on to the dorsal aspect for some dis-
tance, then bifurcates. Each arm (ea) extends obliquely toward
an antacoria and terminates in the whitish area mesocaudad of
the latter. The epicranial suture is well developed in Gryllus
(fig. 5) and Anisolabis (fig. 12); in the former, however, the cau-
dal portion of the stem is obsolete in untreated specimens, and
the arms are short and terminate at the mesal margins of the

Aga HACHIRO YUASA

lateral ocelli, while in the latter, the epicranial arms are very
conspicuous, semicircular, and extend to the middle of the mesal
margins of the compound eyes and apparently continue cephalad
to the lateral margins of the antacoriae. In Mantis (figs. 3, 4),
Stenopelmatus (fig. 6), and Melanoplus (fig. 10), the stem is dis-
tinct, represented by a parademe in the latter. The stem is
represented in Orchelimum (fig. 7) by a longitudinal median fur-
row which becomes obsolete near the dorsal margin of the caudo-
dorsal median prominence of the epicranium. In Diapheromera
(fig. 8) the stem is the long faint line, seen only in treated speci-
mens, which bifurcates between the antacoriae. It is obsolete
in Tettix (fig. 11) and its position is indicated by a short para-
deme on the caudal aspect of the head. In Mantis the epicranial
arms are represented by the parademe caudad of the lateral
ocelli; they are very short and faint in Diapheromera; obsolete
in Orchelimum, although the deep transverse furrow at the
cephalic margin of the epicranial prominence may represent them.
In Stenopelmatus their position is indicated by the parademes
which terminate near the mesal margins of the antacoriae. They
are short in Melanoplus and bend caudad between the caudal
ends of the compound eyes; obsolete in Tettix, their position is
indicated by a short curved parademe between the compound
eyes.

The frontogenal suture (fgs) in Blatta extends laterad for some
distance from the lateral end of each pretentorina, then caudad
toward the cephalomesal angle of the compound eye, where it
merges into a short furrow which originates near the caudo-
lateral margin of the antacoria. It is only slightly curved in
Mantis and Gryllus, becoming obsolete near the cephalic part
of the mesal margin of the compound eye; it is very short in
Diapheromera and terminates near the middle of the cephalic
margin of the eye; is depressed in Melanoplus and short in Tettix
and terminates at the cephalomesal angle of the eye; in Aniso-
labis apparently merges into the cephalic end of each epicranial
arm near the antacoria, and is wanting in Orchelimum and Sten-
opelmatus. The frontogenal sutures are considered as the ce-
phalic portions of the epicranial arms which have been isolated

HEAD, MOUTH-PARTS, ORTHOPTERA EUPLEXOPTERA 255

from the caudal portions by the encroachment of the antacoriae
and lateral ocelli.

The vertex (v) in Blatta (figs. 1 and 13) extends on the caudal
and dorsal aspects from near the occipital foramen to the epi-
cranial arms, and is divided on the meson by the epicranial stem.
On the lateral aspect each half of the vertex extends cephalad
between the compound eye and the occipital suture to the man-
dibularia (mb), then dorsad to the frontogenal suture, and is
connected with the dorsocaudal part by a narrow area between
the compound eye and the antacoria. The vertex, therefore,
occupies nearly one-third of the surface of the head and includes
the compound eyes and lateral ocelli. |

The extent of the vertex depends upon the shape and size of
the head and the position of the epicranial suture. It reaches
its maximum development in Diapheromera and ‘is much re-
stricted on the caudal aspect in Orchelimum and Tettix. The
lateral ocelli are not situated on the vertex in Mantis, Melano-
plus, and Tettix. A furrow with an accompanying parademe ex-
tends cephalodorsad from the occipital foramen on each lateral
half of the vertex in Mantis, Diapheromera, and Anisolabis.
Whether these furrows are homologous with the vertical furrows
found in the larvae of the Tenthredinidae and other Entomet-
abola is not known.

The portion of the vertex cephalad of each compound eye is a
gena (g). It is restricted in Mantis and Diapheromera, ample
in Melanoplus and Tettix, and fused with the front in Orcheli-
mum and Stenopelmatus. In Anisolabis it is completely iso-
lated from the rest of the vertex by the cephalic encroachment
of the occipital suture.

The compound eyes (ce) in Blatta are large, kidney-shaped,
emarginate on the mesal margin, and occupy the dorsal, lateral,
and caudal parts of the head. They are present in all the genera
studied, but vary in size, shape, and position.

The narrow annular sclerite surrounding the periphery of each
compound eye is the oculata (ol). It is produced entad as a
ring-like plate (fig. 45). Oculatae are always present and the
ental rings are more or less well developed, reaching the maxi-

256 HACHIRO YUASA

mum size in the species having large compound eyes, as Mantis
(fig. 46) and Melanoplus (fig. 49).

The lateral ocellus (lo) in Blatta (fig. 1) is in the smooth oblong
area located in the whitish spot in which each epicranial arm
terminates; the median ocellus (mo) is wanting. In other spe-
cies the ocelli vary in size, shape, number, and position (figs.
4,5, and 10). In Diapheromera the median ocellus is wanting,
while in Stenopelmatus and Anisolabis all ocelli are absent.
When the usual number of the ocelli, three, is present, they are
arranged in a triangle asin Mantis. There is a sexual dimorph-
ism in this genus (figs. 3 and 4).

The front (f) in Blatta is well developed and is bounded by the
epicranial arms, antacoriae, and frontogenal sutures on the
caudal and lateral aspects, respectively. The cephalic boundary
is an imaginary line connecting the mesal ends of the frontogenal
sutures or the pretentorinae. A. smooth oblong spot, meso-
cephalad of each antacoria, is a muscle impression (mz). The
front varies in extent and position. Its cephalic boundary is
marked by the frontoclypeal suture (fcs) in all, except Orcheli-
mum and Stenopelmatus where the lateral boundaries are also
indefinite. It includes all or part of the antacoriae, frequently
the lateral ocelli, and the median ocellus when present. Muscle
impressions on the front, which have been mistaken for ocelli,
occur in Gryllus, Melanoplus, Tettix, and Anisolabis (fig. 12).
Tettix has a prominent inverted Y-shaped ridge on the front,
which is not connected with the frontoclypeal suture.

Frontoclypeal sutures (fcs) are present except in Orchelimum
and Stenopelmatus.

The antacoriae (an) are the circular membranous areas in
which the antennae are inserted. They are adjacent to and
mesad of the compound eyes in all the genera studied, except
Anisolabis where they are cephalad of them. They are located
on the front, except in Diapheromera and Stenopelmatus where
the position of the epicranial arms would indicate that they
belong to the vertex.

The antennaria (ar) in Blatta is the chitinized annular sclerite
forming the periphery of each antacoria. Its inner margin is

HEAD, MOUTH-PARTS, ORTHOPTERA EUPLEXOPTERA 207

connected with the flexible membrane which is attached to the
seape of the antenna. There is a narrow pointed projection,
antacoila (aa), extending caudad from the cephalic margin of
the antennaria (fig. 54). This is the ‘chitinized pin’ of Miall
and Denny (89). Another fine slender bar is attached to the
caudal margin of the scape. These two projections, together
with the antatendons (at) control the movements of the antenna.
The minute chitinized spots (ch) in the antacoria mark the at-
tachment of these tendons. The antennariae are always pres-
ent. The articulation of the antennae is either like that of the
cockroach, as in Mantis (fig. 56) and Anisolabis (fig. 61) ; by means
of less definite projections, as in Gryllus (fig. 60), Stenopelmatus
and Melanoplus, or by means of the triangular projection of the
scape which fits into the emargination of the antennaria (fig. 62).
There are two antatendons, and their points of attachement can
be identified in all the genera.

The pretentorina (pn) in Blatta (figs. 1 and 2) is the linear,
transverse furrow along the caudomesal margin of each man-
dibularia. In all the genera studied, with a possible exception of
Anisolabis, the pretentorinae are distinct and definite in their
location and afford excellent dependable landmarks in orienting
other structures. Although they are always associated with the
front, mandibularia, frontogenal and mandogenal sutures, they
differ somewhat in their extent and appearance. The lateral or
caudal portion of each pretentorina terminates either on the fron-
togenal suture, more or less remote from the mandibularia, as
in Blatta, Mantis, and Anisolabis, or on the mandogenal suture
beyond the cephalic end of the frontogenal suture, as in Gryllus,
Melanoplus, and others.

The suture forming the caudal margin of each mandibularia
and separating it from the vertex or gena and front is the mando-
genal suture (mgs). This suture is present, except in Anisolabis,
but varies in length and direction on account of the differences
in size, shape, and position of the mandibularia.

The mandibularia (mb) in Blatta (fig. 2) is the small triangular
area extending from the precoila to the cephalic end of the occip-
ital suture and, on its cephalic margin, which is often submem-

258 HACHIRO YUASA

branous, is connected with the proximal portion of each mandible.
The extensacuta (ec) is located near the ventral part of the ce-
phalic margin of the mandibularia. The mandibulariae, although
they vary in size and shape, are present and more or less well
differentiated in all except in Anisolabis (fig. 17).

The occipital foramen (fig. 23, of) is the large subquadrate
opening located in the caudal part of the ventral aspect of the
head. Jt varies in size, shape, and position. The elongation
and the peculiar position of the foramen in Diapheromera (fig.
29) are due to the natural position of the head which is horizontal
instead of vertical as in the case of the other genera.

In Blatta (fig. 23) the occipital suture (0s) is distinct; it begins-
at the lateral end of each postcoila and extends caudad, becom-
ing obsolete some distance from and slightly beyond the caudal
margin of the occipital foramen. The occipital sutures are pres-
ent in all except Diapheromera. They are practically complete
in Gryllus (fig. 28) and Tettix (fig. 35) where the caudal ends of
the sutures unite with the epicranial stem near its origin. In
other genera the caudal ends of the sutures are either free, as in
Melanoplus (fig. 30), Orchelimum (fig. 27), and Stenopelmatus
(fig. 31), or they merge into the furrows which extend on to the
vertex, as in Mantis (fig. 24) and Anisolabis (fig. 32). In Mantis
(fig. 41), Orchelimum (fig. 44), and Melanoplus (fig. 49) the
sutures are connected with the lateral margins of the occipital
foramen by an ental thickening or parademe (pm). The ceph-
alic portion of each occipital suture is produced as a parademe
in Gryllus and others.

The occiput (oc) in Blatta (fig. 23) is the narrow crescentic
area surrounding the caudal one-third of the occipital foramen.
Since there is no suture (excepting the occipital suture on the
lateral boundaries) either on its caudal margin where it meets.
the vertex or on its cephalic margin where it merges with the
postgenae, the exact boundaries of the occiput cannot be estab-
lished. The imaginary line drawn across the occipital foramen
just cephalad of the odontoidea (od) is here considered as the
cephalic limit of the occiput. In other genera the occiput varies
in size and shape and its caudal and cephalic boundaries are

HEAD, MOUTH-PARTS, ORTHOPTERA EUPLEXOPTERA 259

more or less indefinite. In Melanoplus and others the oblique
parademes mentioned above form its cephalic margin.

The postgenae (pgn) in Blatta (fig. 23) is the flat area cephalad
of the occiput and mesad of each occipital suture. The caudal
half of its mesal margin is bounded by the lateral margin of the
occipital foramen and the cephalic half is roundly emarginate and
continuous with the microcoria (ma). The postcoila (pil) is lo-
cated on the cephalic margin. The relation of each postgena to
the adjacent areas is similar in all the genera studied, but it varies
in size and shape due largely to the difference in size and shape of
the occipital foramen. There is a distinct crassa (cr) adjacent to
and parallel with the cephalic part of the mesal margin of each
postgena which extends from near the middle of the postcoila in
Gryllus (fig. 28) and from the lateral end in Anisolabis (fig. 32).
On the ental surface of the crassa, there is a corresponding para-
deme. This parademe is present in all and extends from each
postcoila to the occipital foramen along the mesal margin of
each postgena.

The postcoila (fig. 23, ptl) is the blackish acetabulum in which
the postartis of the mandible articulates. It is located at the
cephalic margin of each postgena. It is usually distinct and well
developed, being largest in Gryllus, comparatively shallow in
Diapheromera, and located on the mesal angle of the cephalic
margin of the postgena in Melanoplus and Tettix.

The metatentorina (mn) in Blatta (figs. 13 and 23) is the elon-
gated opening located on each side of the cephalic margin of the
occipital foramen between the maxillaria and the postgena and
leading into the corpotentorium. It is always present and dis-
tinct and similar in position, but varies in size and shape.

The paracoila (pl) in Blatta is the condyle-like projection
formed by the cephalolateral angle of the maxillaria protruding
ventrocephalad on each side of the occipital foramen. It is
folded longitudinally upon itself, the crest of the fold forming a
condyle and the concavity an acetabulum. The margin, near
the lateral portion of each paracoila, is emarginate and forms the
other side of the acetabulum. The exparartis of the maxilla is
articulated against this condyle and acetabulum. ‘The para-

260 HACHIRO YUASA

coilae are always present and distinct. ‘They are not well differ-
entiated in Diapheromera, but are prominent mesocaudal pro-
jections in Tettix.

The maxillariae (my) in Blatta (fig. 23) are narrow plates sur-
rounding the lateral and caudal margins of the occipital foramen.
The ectal surface of each maxillaria is closely applied to the
ectal surface of the postgena and the occiput. The lateral mar-
gins are folded, forming a roll. The caudal part of each roll is
produced into a cone-shaped projection, an odontoidea (od).
The microcoria (ma) is attached to the ventral margin of the
maxillariae. The caudal side of each maxillaria is reduced to a
narrow band, the cephalic part of each lateral portion is expanded
and produced to form a distinct paracoila, and the cephalomesal
margin is fused with the corpotentorium. The maxillariae are
simple in Orchelimum, Melanoplus, and Tettix. In other gen-
era they vary in size and shape, are very complicated and have,
besides odontoideae, many projections, some of which bear ten-
dons (figs. 27 and 31).

The odontoideae (od) are not well differentiated in Orcheli-
mum and Melanoplus, but are distinct in the other genera. In
Blatta, Stenopelmatus, and Anisolabis they occur on the caudal
third of the lateral parts of the maxillariae; in other genera near
the cephalic part of the maxillariae and extend ventrad or caudo-
ventrad of the corpotentorium.

The clypeus (c) in Blatta (fig. 1) is the convex sclerite attached
to the cephalic margin of the front. The caudolateral angles are
produced into thickened, transverse lobes, and each bears a dis-
tinct precoila. The lateral margins are rounded and the ce-
phalic boundary is the distinct clypeolabral suture. The clypeus
is transverse, the lateral margins are entire and converge cephalad.
In Gryllus (fig. 5) and Melanoplus (fig. 10) there are oblique
furrows extending from the middle of the lateral margins, which
may represent the incomplete clypeal suture (cs) dividing the
clypeus into preclypeus and postclypeus. In Diapheromera (fig.
8) the mesal part of the clypeus is dilated and produced, forming
a prominent elevation.

HEAD, MOUTH-PARTS, ORTHOPTERA EUPLEXOPTERA 261

The clypeolabral suture (cls) is distinct and complete in all,
except Orchelimum, where it is represented by a transverse
fold; in Stenopelmatus the mesal portion and in Diapheromera
the lateral portions are obsolete. The suture in Melanoplus is
irregular, the clypeus is emarginate on the meson.

The laterocaudal angles of the clypeus are produced into small
lobes which are emarginate on the cephalic and ventral aspects.
These emarginations form the precoilae in which the preartes of
the mandibles articulate. The precoilae (pr) are distinct and
practically similar in all. They are conspicuous in Mantis,
Diapheromera, and Stenopelmatus (fig. 6).

The small chitinized structures at the lateral ends of the cly-
peolabral suture are the dorsal parts of the tormae (tm). They
extend on to the ventral aspect and are the landmarks indicating
the boundary between the clypeus and labrum. 'Tormae are
present in all the genera studied.

The labrum (l) in Blatta is flexible, its lateral margins are
rounded, and its cephalic margin is emarginate. The emargi-
nation is marked on each side by an oblique blackish thickening
and bears spinulae. The labrum, although varying in size and
shape, is distinct and well developed in all.

The tentorium is the endoskeleton of the head and is always
well developed. Its form is closely related with and to a large
extent influenced by the development and direction of the mouth-
parts. It is expanded and comparatively thin in Blatta (fig. 36);
thick and heavily chitinized in Mantis (fig. 41), Gryllus (fig. 37),
Stenopelmatus (fig. 43), Melanoplus (fig. 39), and Anisolabis
(figs. 32 and 42). The cephalic portion is reduced in Diaphero-
mera (fig. 38) and elongated and enlarged in Anisolabis. The
location of the external markings or invaginations of the arms of
the tentorium have been described elsewhere. The tentorium in
Blatta (figs. 36 and 45) is composed of the typical parts, namely,
the metatentoria (mt), corpotentorium (ct), pretentoria (pt), lam-
inatentorium (/t), and supratentoria (st). The corpotentorium
is the cuticular plate connecting the postgenae within the head;
it is formed by the fusion of the mesal portions of the meta-
tentoria. The ental plate surrounding the lateral and caudal

262 HACHIRO YUASA

margins of the occipital foramen are considered as belonging to
the metatentoria. The fan-shaped plate connected with each
pretentorina is a pretentorium. Its cephalic margin is thickened,
and its dorsomesal margin is thickened and turned mesad. The
expanded caudal parts of the pretentoria converge, fuse on the
meson, separate bounding a small opening, and then fuse with
each other and the cephalic portion of the corpotentorium. The
quadrate plate, the cephalic part of which is arched, is the lam-
inatentorium. The two tendinous projections which extend
cephalad from the opening in the tentorium are the oesotendons
(ot). The circular opening is the foramen through which the
nerve cord passes. The line of fusion of the pretentoria on the
meson is indicated by a ventral ridge located cephalad of the
opening. The thin, cuticular, triangular plate continuous with
each caudolateral margin of the laminatentorium is a supra-
tentorium. The apex of the triangle is produced into a thin, ©
delicate, slender extension which is directed laterodorsad toward
an antacoria. The point of attachment could not be determined,
but it has been stated that it is attached to the caudolateral
margin of the antennaria. )

The metatentoria are always distinct and afford excellent land-
marks for beginning the study of the tentorium. The corpo-
tentorium connects the postgenae and forms the cephalic margin
of the occipital foramen. It is usually vertical in position, but
in Anisolabis it is a narrow horizontal plate. Each metaten-
torium fuses with a postgena on the dorsal side and with a max-
illaria on the ventral, and extends as a more or less flaring band
along the lateral and caudal margins of the occipital foramen.
The flaring inner margins are produced as tendons (figs. 23, 28,
and 32).

The pretentorium is generally fan-shaped or expanded where
it 1s attached to the pretentorina on the ental surface of the fron-
togenal suture. Each is directed caudomesad for a short dis-
tance, then is twisted, again expanded, extends to the meson,
and fuses with the other pretentorium, and forms a more or less
distinct laminatentorium. The pretentorium in Mantis is turned
caudad and follows the frontogenal suture to its caudalend. The

HEAD, MOUTH-PARTS, ORTHOPTERA EUPLEXOPTERA 263

fan-shaped portion is divided into a large, concave mesal part
and a small, triangular, cephalolateral part. A somewhat simi-
lar condition occurs in Gryllus, where the median carina is small
and affects the contour of the pretentorium only slightly. In
Diapheromera and Orchelimum a lateral furrow extends longi-
tudinally and tends to fold dorsad forming the lateroventral mar-
gin. In Stenopelmatus, Orchelimum, Melanoplus (fig. 39), Tet-
tix (fig. 40), and Anisolabis (fig. 42) the dorsomesal margin is
thickened and the lateral expansion concave. This concavity
reaches its maximum development in Anisolabis, where it forms
a cup-shaped pocket along the frontogenal suture and the
antennaria. .

The laminatentorium is usually well developed and its caudal
margin is often constricted and fused with the corpotentorium.
It is small in Diapheromera and almost wanting in Orchelimum
and Stenopelmatus. ‘The line of fusion is distinctly indicated in
Mantis, Diapheromera, Gryllus, and Tettix. In Anisolabis the
line of fusion extends to the caudal margin of the corpotentorium.
There is a circular opening in the laminatentorium of Mantis, as
in Blatta, but the oesotendons are wanting. The laminaten-
‘torium is triangular and concave in Gryllus and Tettix and emar-
ginate on the dorsal aspect in Melanoplus. The plate-lke pro-
‘jection on each mesal side of the pretentorium in Orchelimum
(fig. 43) and Stenopelmatus (fig. 44) may represent the vestige
of the laminatentorium. The dorsomesal thickenings of the
pretentoria are frequently continuous with the dorsolateral thick-
enings of the metatentoria, forming the X-shaped dorsal ridges
across the laminatentorium and corpotentorium. In Anisolabis,
however, this connection is obsolete and the caudal part of the
laminatentorium is elongated, an inverted trough-shaped struc-
ture, with a longitudinal depression on each side instead of a
dorsal thickening.

The supratentoria arise from the cephalic part of the dorso-
lateral margins of the laminatentorium in all, except in Diaph-
eromera (fig. 51) where they issue from the pretentoria. They
are always small, linear, and expanded, adjacent to the ‘amina-
fentorium; and in Gryllus (fig. 47) they are attached to the caudo-

264 HACHIRO YUASA

lateral angles of the antennariae; in Tettix (fig. 52) they are
attached on the front cephalolaterad of the antacoriae; and in
Anisolabis (fig. 53) near the caudomesal margins of the com-
pound eyes. In others they are directed laterodorsad or latero-
dorso-caudad toward the antacoriae or points near them and are
apparently unattached.

B. Movable parts of the head

The antennae (a) of Blatta’ (figs. 54 and 55) are long, slender,
setaceous, and multisegmented. The scape (sc), pedicel (p), and
the segments of the flagellum (fl) are cylindrical and setigerous.
The proximal margin of the scape is emarginate, forming an
antartis (ad), and articulates with the antacoila (aa). Two
antatendons (at) are attached to the scape near the emargina-
tion. The first segment of the flagellum is longer than the pedi-
cel in the female and shorter in the male. The antennae of
other species are either filiform or setaceous and articulate with
the antennaria. The scape is quadrate and flattened in Gryllus
(figs. 59 and 60) and elongated in Anisolabis (fig. 61).

The mandibles (md) in Blatta (figs. 70, 72, and 73) are convex
on the dorsal aspect and concave on the ventral. The preartis
(py) is a combination of condyle and acetabulum, and is located
on the dorsal aspect, while the postartis (ptc) is globular, prom-
inent, and situated near the lateral margin of the ventral aspect.
The small spatulate extensotendon (et) is attached near the post-
artis and the large branched rectotendon (rt) to the proximal
part of the mesal margin on the ventral aspect. The acia (ac)
is well differentiated and the mola (ml) is the triangular tooth
cephalad of the acia. The dentes (d) are sharp and three in
number in the dextral mandible and five in the sinistral. The
mandibles are always strongly chitinized and fitted for cutting
and grinding. They are so constructed that they interlock when
at rest. Asymmetry of various degrees exists between the dentes —
and concavities. Aside from such differences, the mandibles are
similar in form on both sides and more or less similar in both
sexes. ‘They usually differ, in different species, in size, shape,
and manner of interlocking, but most of the structures in the

HEAD, MOUTH-PARTS, ORTHOPTERA EUPLEXOPTERA 265

cockroach can be identified. The acia is generally absent, al-
though the digit-like cuticular projection (ac) in Mantis (figs.
74, 75, and 76) may represent it. The base of the rectotendon is
more or less distinctly chitinized and forms, in many cases, @
sclerite-like area, the rectacuta (rc) of the ambipharynx. The
dentes are always present, but are not differentiable into proxa-
dentes (pd) and distadentes (dd) except in Anisolabis (figs. 86
and 87). The dentes and mola cannot be distinguished in Diaph-
eromera (figs. 80 and 81).

The maxillae (mz) in Blatta (figs. 97 and 100) are typically
orthopterous in structure and each consists of a cardo, stipes,
galea, lacinea, palpifer, and maxillary palpus. The cardo (ca)
consists of two segments (fig. 98), the subeardo (sa) and ala-
eardo (al). The parartis is dorsal in position and bifurcate, the
exparartis (ey) is a combination of a condyle and acetabulum.
The entoparartis (en) is triangular, prominent, and bears the
small premaxatendon (pmt) near its distal end. The alacardo
is much smaller than the subcardo and attached obliquely to the
lateroventral margin of the latter. The suture between these
segments is distinct on the ventral aspect and forms a para-
deme. The exparartis articulates with the paracoila and the
entoparartis is inserted near the paracoila dorso entad of the
mesal margin of the head. The stipes (s) is large, distinctly
chitinized on the ventral aspect, and, excepting a triangular area,
is largely membranous on the dorsal aspect. It articulates with
the alacardo at the caudolateral angle of the latter and is separated
from it by a distinct suture. The caudomesal angle of the stipes
on the ventral aspect articulates with the laterocephalic angle of
the subcardo. Thus the stipes has two points of articulation
with the cardo, one on the subeardo and the other on the alacardo.
The narrow longitudinal sclerite attached to the mesal margin of
the stipes on the ventral aspect is the subgalea (sg). The suture
between them is modified into a distinct parademe. On the ven-
tral aspect the cephalic margin of the subecardo and the mesal mar-
gins of subgalea, and of the stipes, cephalad of the subgalea, are
continuous with the labacoria (lc), while the cephalic and mesal
margins of the cardo and stipes, respectively, are continuous on

JOURNAL OF MGRPEOLOG?Y, VOL. 33, NO, 2

266 HACHIRO YUASA

the dorsal aspect with the maxacoria (mc). The palpifer (pf) is
the small sclerite on the dorsal aspect, near the cephalolateral
part of the stipes and is separated from it by a subchitinized
area. The galea (gl) is two-segmented. It is attached to the
laterodistal margin of the stipes without any indication of a
suture. The proxagalea (pg) is short, subcylindrical, and its
distal margin on the dorsal aspect is marked by a transverse
chitinous band and on the ventral by a distinct fold. The
distagalea (dg) is much longer and slightly narrower than the
proxagalea. It is hood-shaped and overlaps the distal portion
of the lacinia. The distom¢sal margin is flaring and spinulate.
The lacinia (la) is the flattened, chitinous, distal appendage
mesad of the galea which fits into the mesal concavity of the
latter. The lacinia is separated from the stipes by the distinct
suture already mentioned, but its caudolateral portion is ex-
panded into a subquadrate area which encroaches upon the
stipes, and the suture is obliterated for a short distance. The
suture is obsolete on the dorsal aspect. The maxadentes (ms)
are sharp, curved, strongly chitinized, and two in number. The
hamadens (h) is located near the maxadentes and is minutely
tridentate. The mesal margin of the lacinia is convex, sharply
flattened, and bears prominent lacinarastra (rs). The maxil-
lary palpus (mp) is five-segmented and the mesocephalic margin
of the distal segment is membranous and covered with setae
and spinules.

The cardo is two-segmented in all species studied. The sub-
cardo is irregular in outline, its entoparartis is always promi-
nent and bears the premaxatendon, and its exparartis is often
bifureate and combines the function of a condyle and an ace-
tabulum. The alacardo is convex, triangular or semitriangular,
and obliquely attached to the subcardo. The ental surface of
the suture between the two segments bears a parademe. The
alacardo articulates with the stipes at its laterodistal angle, and
the subcardo with the caudomesal angle of the stipes on the
ventral aspect. In Mantis (figs. 96 and 104) and Anisolabis
(figs. 93, 94, and 95), the alacardo is triangular, smaller than
the subcardo. In Mantis, Diapheromera (figs. 110, 115, and

HEAD, MOUTH-PARTS, ORTHOPTERA EUPLEXOPTERA 267

116), Stenopelmatus (figs. 101, 109, and 111), and Anisolabis the
subeardo extends along the caudal and dorsal margins of the
alacardo. In Gryllus (figs. 99, 102, and 103), Orchelimum (figs.
105, 107, and 108), Melanoplus (figs. 112, 118, and 114), and
Tettix (figs. 117, 118, and 119) the subcardo is deeply emargin-
ate and surrounds the alacardo on the ventral, caudal, and
dorsal aspects. In these genera the suture between the two
segments is obliterated, probably due to the irregularity pro-
duced by the complicated parademes. In Diapheromera and
Melanoplus the exparartis is entire and in other genera it is
shallowly or broadly bifurcate.

The stipes, except in Anisolabis, is subquadrate and chitinized
on the ventral aspect; on the dorsal aspect the chitinous area is
small and triangular, and the remainder of the surface is mem-
branous or submembranous. In Anisolabis the stipes is divided
into two parts, the proximal of which is small and triangular
and located on the ventrolateral aspect, the distal is triangular
and located on the ventral aspect. They are separated on the
ventral aspect by the encroachment of the greatly enlarged sub-
galea (fig. 94, sg) and on the dorsal aspect by the elongated
palpifer (fig. 93, pf); the two parts are connected by a narrow
membranous area.

The subgalea is always present, is narrow, and extends longi-
tudinally, except in Anisolabis where it is very large, flat, sub-
pentagonal, and occupies the greater part of the ventral sur-
face. It extends along the entire mesal margin of the stipes in
Mantis, Gryllus, Stenopelmatus, and Anisolabis. In the others
it does not quite reach the cephalic margin of the stipes. The
suture between the stipes and subgalea is distinct except in
Mantis, where a furrow marks the line of fusion of the two
sclerites and its ental surface is thickened.

The palpifer is uniformly present and in Mantis and Dia-
pheromera it is very indistinctly separated from the stipes. It
is distinct, although on the dorsal aspect it is submembranous
and its boundary is more or less obliterated, except in Anisolabis,
as already noted. In Melanoplus it is confined to the ventral
aspect.

268 HACHIRO YUASA

The lacinia is hook-shaped, depressed, chitinized, and dentate
in all; its surfaces and the mesal margin are convex. In Mantis,
Gryllus, Orchelimum, and Stenopelmatus, the lateral half of the
proximal part of the lacinia is concave on the dorsal aspect.
In Melanoplus and Tettix, the mesoproximal angle is pro-
duced into a cone-shaped elevation. The suture between the
lacinia and the stipes is obsolete on the dorsal aspect, but dis-
tinct and complete on the ventral aspect in all except Mantis,
where it is interrupted, and Melanoplus, where it is represented
by a furrow. An oblique suture on the ventral aspect of the
stipes extends caudolaterad from the mesodistal angle in Gryllus,
Orchelimum, and Anisolabis, and a mere indication of it occurs
in Stenopelmatus and Tettix. Directed caudolaterad from the
lateroproximal corner of the lacinia, there is another suture in
Mantis, Gryllus, Orchelimum, and Melanoplus and slightly indi-'
cated in Diapheromera. The maxadentes are two in number,
except in Melanoplus, which has three, and Tettix (fig. 99")
which has four, arranged in a transverse row. In Diapheromera,
the single dens may be a product of fusion of two or three
dentes. There is a non-dentate hamadens (h) on the mesal
margin near the maxadentes in Gryllus, Orchelimum, and
Stenopelmatus. The lacinarastrae are distinctly developed in all
except Melanoplus and Tettix. In these genera, however, there
are a number of long distinct setae (7s) on the mesal and ventral
aspects. They are small and few in number in Diapheromera.

The galea is always two-segmented, and the suture between
the segments is distinct on the ventral aspect. This suture, in
Diapheromera and Orchelimum, is cbsolete on the dorsal aspect.
The proxagalea is short, transverse, and subcylindrical in Gryllus,
Orchelimum, Stenopelmatus, and Anisolabis; is short and deeply
concave on the mesal aspect in Mantis, and flattened, mem-
branous, and slightly concave on the mesal half of the dorsal
aspect in Melanoplus and Tettix, with the distal part dilated in
the former and obtusely pointed in the latter. The distal end
of the galea, except in Melanoplus, is membranous and pro-
vided with minute setae. The suture between the stipes and
the proxagalea is obsolete on the ventral aspect in Mantis, Dia-

HEAD, MOUTH-PARTS, ORTHOPTERA EUPLEXOPTERA 269

pheromera, Stenopelmatus, Melanoplus, and Tettix, and dis-
tinct on the dorsal aspect in Gryllus, Melanoplus, and Tettix.
Crampton (’16) has named a small secondary lobe on the lateral
margin of the distagalea of Diapheromera, the ‘galealobulus.’

The maxillary palpus contains five segments. The proximal
segment is small and cylindrical, with the distal portion fre-
quently thickened; the second is larger than the first, except in
Diapheromera, Tettix, and Anisolabis; the third is long and
cylindrical—in Mantis the longest segment of the five; the third
and fourth are usually subequal in length; and the fifth is clav-
ate, subequal to the fourth in length in Gryllus, Orchelimum,
Stenopelmatus, Melanoplus, Tettix, and Anisolabis. The distal
segment in Mantis is small, cone-shaped, and subequal in length
to the second; the largest and flattened in Diapheromera; and
the tip provided with a small distinct papilla in Anisolabis. The
two distal segments in Tettix are somewhat flattened. Except-
ing Mantis and Diapheromera, the distal end of the fifth seg-
ment of the palpus is covered with numerous setae.

The maxillae articulate with the head at the precoilae, dor-
sad of which the entoparartes always extend. On the dorsal
aspect the margins of the cardo and stipes are continuous with
the maxacoria and on the ventral with the labacoria.

The labium (lb) in Blatta (figs. 120 and 128) consists of the
submentum (sm), mentum (m), and ligula (li) which includes
- the stipulae (sp), glossae (go), paraglossae (pgo), palpigers (pp),
and labial palpi (lp). The submentum is a large subquadrate
basal sclerite. Its caudal margin is transversely emarginate and
is continuous with the microcoria (ma). The cephalic margin
has a deep round emargination into which the semicircular
mentum fits. The lateral margins of the submentum are folded
over on to the dorsal aspect and form the lateral lobes (Il), the
margins of which are continuous with the labicoria. The men-
tum is much smaller and narrower than the submentum, and
the distal portion is only slightly chitinized. The suture be-
tween submentum and mentum is sometimes obsolete at the
middle. The distal margin of the mentum is membranous and
is folded dorsad and then caudad so that the ligula is placed at a

270 HACHIRO YUASA

higher level and overlaps this portion of the mentum. The
stipulae are distinct, quadrate, and bear a palpiger on each lateral
aspect. The suture between each stipula and palpiger is dis-
tinct for a short distance on the ventral aspect and obsolete
on the dorsal. The stipulae, except the fundarima, are mem-
branous on the dorsal aspect. The fundarima (fr) is chitinized
and distinct for some distance on the ventral aspect. The small,
elongated, triangular appendage which is obliquely attached to
the mesocephalic margin of each stipula is a glossa. Its distal
end is covered with fine setae. The cephalic lobe, laterad of
and separated by a distinct latarima (lr) from each glossa, is a
paraglossa. The suture between the stipula and paraglossa is
distinct. On the dorsal aspect the glossa and paraglossa are
fused to the cephalic margin of the stipula without the indication
of a suture. The proximal part of the mesal margin of each
paraglossa is concave and the mesodistal portion is dilated and
covered with minute setae. The lateroproximal portions of the
glossae fit into the concavities of the paraglossae. The labial
palpi are three-segmented and geniculate. The distal portion of
the third segment is hemispherical and covered with minute
setae. According to the interpretation of Crampton (’17), the
labium articulates with the maxillariae. If so, this articula-
tion is only slightly or not at all differentiated. The caudal
angles of the submentum ordinarily are continuous with the
microcoria.

The submentum in Mantis (figs. 121 and 129) is elongated, and
narrowed toward the cephalic margin. In Diapheromera (figs.
127 and 1384) it is considerably elongated and is deeply emargin-
ate on the caudal margin. The condition in Gryllus (figs. 122
and 130) is similar to that in Blatta. It is wider than long in
Orchelimum (figs. 125 and 133) and Stenopelmatus (figs. 123
and 131) and its caudal margin in the former is roundly emargin-
ate. In Melanoplus (figs. 124 and 135) and Tettix (figs. 126
and 132) it is crescentic, and its caudal margin is deeply emar-
ginate. The submentum of Anisolabis (figs. 136 and 1387) is
large, strongly chitinized, and subquadrate, with the cephalic
and caudal margins only slightly emarginate. There is a nar-

HEAD, MOUTH-PARTS, ORTHOPTERA EUPLEXOPTERA 271

row transverse sclerite (ch) caudad of the submentum. This
piece, which is separated from the submentum by a dist net
suture, has been designated as the ‘submentum’ by Packard
(83) and by practically all other writers, who have considered
the large sclerite cephalad of it as the mentum and which is
here designated as the submentum. This so-called ‘submentum’
is one of the sclerites of the microthorax. It is interesting to
note that Hansen (’94) found a similar piece in Hemimeius,
and the labium, according to his figures, is very similar to that
of Anisolabis. In Diapheromera the caudal two-thirds of the
submentum is membranous except for a narrow area along each
lateral margin and a variable ovate area on the meson. The
suture between submentum and mentum is distinct and com-
plete, except in Melanoplus where the mesal portion is cbsolete.
The lateral lobes are distinct, except in Melanoplus and Tettix;
they are best developed in Anisolabis.

The mentum is narrow, transverse, and distinctly smaller
than the submentum in Diapheromera, Gryllus, Stenopelmatus,
and Anisolabis; is more or less distinct in Melanoplus and
Tettix; and is indistinct in Mantis and Orchelimum, where it
is fused with the stipulae without indication of sutures. The
caudal margin of the mentum in Orchelimum, Stenopelmatus,
Melanoplus, and Tettix slightly overlaps the cephalic margin of
the submentum and is at a higher level than the latter.

The stipulae are subquadrate and distinct in all except those
where the mentum has fused with the stipulae. They are
elongated in all except Gryllus and Melanoplus, where they are
transverse. The mesarima is very deep in Mantis; moderately
deep in Orchelimum and Melanoplus, and reaches the cephalic
margin of the mentum in Anisolabis. The fundarima extends
caudad as a chitinized thickening in Diapheromera, Gryllus,
Orchelimum, Stenopelmatus, and Tettix. In the last four
genera, the caudal end is connected with thickenings which
extend laterad. The suture which separates each stipula from
a glossa and paraglossa is complete in Gryllus, Melanoplus, Tet-
tix, Anisolabis, and incomplete in the others. It is entirely
wanting on the dorsal surface.

272 HACHIRO YUASA

The palpiger is small and lateral in position in all except
Melanoplus, where it is located on the ventral aspect. The
suture between the palpiger and stipula of each side is obsolete
or indistinct in Mantis, Diapheromera, Orchelimum, Tettix, and
Anisolabis.

The glossae are small and e'ongate. In Gryllus, Orchelimum,
Stenopelmatus, and Tettix, they are pointed, in the remaining
genera more or less rounded. In Melanoplus the dextral glossa
is distinctly larger than the sinistral, which is rudmmentary.

The paraglossa are very mugh larger than the glossae, except
in Mantis, where they are slightly smaller. In Melanoplus
they are enormously expanded and decidedly larger than the
glossae; are more or less flat in Mantis, Diapheromera, Melano-
plus, and Tettix; thicker and folded mesad in Gryllus, Orcheli-
mum, and Stenopelmatus. In all of the latter genera the mesal,
cephalomesal, or dorsal portion of each paraglossa is concave
and overlaps the glossa to a greater or less extent. In Gryllus
and Stenopelmatus a furrow, some distance caudad of the
suture, separates each paraglossa and stipula. This furrow may
be an indication of the suture which separates the paraglossa into
two segments—a condition comparable to the two-segmented
galea of the maxilla. Anisolabis has only a single appendage at-
tached to the distal end of each stipula. As to the interpretation
of this, the literature is confusing. Fabricius (1776) characterized
the labium of Forficula as ‘trifidum,’ but Olivier (1791) correctly
spoke of the two equal lobes of the labium. Later, writers seem
to have noticed the bifurcated condition of the labium, but, with
the exception of Borman (’00), have not recorded their opinion
as to the homology of these lobes. This author states that the
glossa and paraglossa have fused and formed the single distal
lobe attached to each stipula, but I have been unable to find his
‘deutliche Trenungslinie,’ and there is no indication of the real
nature of this appendage. It may represent the glossa, the
paraglossa, or the product of the fusion of the two.

The labial palpi are invariably composed of three segments.
The basal segment of each palpus is the smallest of the three
except in Mantis, where all are subequal. The middle segment

HEAD, MOUTH-PARTS, ORTHOPTERA EUPLEXOPTERA 273

is smaller than the distal one, except in Mantis and Anisolabis.
The distal segment is long, cylindrical, clavate, and its hemi-
spherical tip is covered with minute conical setae in Gryllus,
-Orchelimum, Stenopelmatus, and Melanoplus, and is more or
less depressed in Diapheromera and Tettix. In Anisolabis the
last segment is clavate and smaller than the second and its tip
bears a small, cuticular papilla (fig. 137). The labial palpi are
nearly straight in Mantis, but are more or less geniculate in
other genera. :

The prepharynx of generalized biting insects consists of a well-
differentiated propharynx (pra), parapharynx (ppx), and ambi-
pharynx (ax). The propharynx is dorsal in position and in-
cludes the epipharynx (ex), a pair of tormae (tm), and the
epigusta (eg). The parapharynx is ventral in position, is lingui-
form, and consists of two parts, the proximal. basipharynx (bz)
and the distal hypopharynx (hx). The former includes the
pharyngeae (prg), the paralinguae (pln) with the linguacutae
(lg) which bears the linguatendons (lg), the lingulae (/n), and
the subgusta (sw); while the latter includes a pair of saliviae
(sl), which support the salivos (so), and the ventral membranous
oscula (ox). That part of the pharynx caudad of the pre-
pharynx is the postpharynx (pox). It is well to remember
that the parapharynx corresponds approximately to the ‘hypo-
pharynx’ of most writers.

__. The prepharynx of Blatta (figs. 151, 152, 153, and 154) in-

cludes all the parts enumerated above. The epipharynx is the
same in size and shape as the labrum and the surface is concave
near the cephalic margin. The tormae are the distinct, brown-
ish, X-shaped sclerites. One arm of the X extends. to the
laterocaudal angle of the labrum, forming on the dorsal aspect, a
distinct landmark for the lateral end of the clypeolabral suture
and another arm extends caudad for a short distance, where it
disappears from the surface, becoming an ental bar to which
the principal retractor muscles of the labrum are attached. The
other two arms of each torma extend mesad and enclose a clear
membranous circular area which is provided with minute sen-
-sory pits. A brownish claw-shaped chitinized area, extending

274. HACHIRO YUASA

cephalad from the cephalic margin of each circular area, bears
about fifteen, short, conspicuous, subdecumbent, spine-like
setae. The surface of the epipharynx between the two brown-
ish areas and the area cephalad of them is densely and uni-
formly covered with short fine spinulae, the majority of which
are directed more or less mesocaudad. The cephalic emargina-
tion of the epipharynx is beset with stiff, brownish spinulae.
The epigusta is flat or slightly concave, and is uniformly covered
with short fine spinulae or solid ‘hairs.’ The ambipharnyx is
rather broad, smooth, and thinner than the adjoining coriae.
The parapharynx is linguiform and its various parts are well
differentiated. The pharyngea is the long, slender sclerite along
each side of the subgusta. Its caudal portion is expanded, less
chitinized, and extends dorsocaudad, supporting the lateral
wall of the postpharynx. The entrance to the latter is located,
therefore, near the middle of the pharyngeae. The paralingua is
the convex subquadrate sclerite fused to the cephalic end of
each pharyngea and extends caudoyentrad. A long, narrow,
crescentric sclerite, the linguacuta, lies near the caudal angle of
each paralingua. It extends for a short distance dorso-caudo-
laterad and merges into a slender ental tendon, the linguatendon,
which, after passing over the dorsal side of the rectotendon of
the mandible, extends into and toward the lateral side of this
structure. Mangan (’08) figured, for the first time, this tendon
in Periplaneta australasiae. The lingula is the subcrescentic
brownish sclerite fused to the cephalic end of and dorsad to each
paralingua., It is sparsely covered with short, distinct, spine-
like setae. The mesal end of the lingula is produced meso-entad
and approaches the one on the other side, but remains separate
onthemeson. This part of the lingulae produces the constriction
of the propharynx between the basipharynx and hypopharynx.
The membranous area caudad of the mesal arms of the lingulae
is convex and slightly elevated, forming a ridge which is pro-
vided on each side with a band of brownish spinulae. Bugnion
(16) erroneously designated and figured this ridge in Blatta
americana as ‘l’entrée du pharynx.’ There is a group of small
distinct sensory pits on each side and slightly cephalad of the

HEAD, MOUTH-PARTS, ORTHOPTERA EUPLEXOPTERA 275

median ridge. The subgusta is broad, membranous, and pro-
vided with a shallow, median, longitudinal furrow. The hypo-
pharynx is the subquadrate structure cephalad of the constric-
tion of the parapharynx. Its dorsal surface is convex, slightly
elevated on the meson, and is densely covered with spinulae.
The distal end of hypopharynx is also spinulate. There is an
elongated, triangular, slightly chitinized area cephalad of and
indefinitely separated from each lingula. The salivia is the large
lanceolate sclerite, ventrad of the cephalic part of the lingula
and the triangular area cephalad of it. It extends cephalo-
dorsad to the caudal margin of the distal spinulate area. Its
caudal third is strongly chitinized, its caudal third moderately,
and its middle third only slightly. This sclerite bears several
spine-like setae. The ventrocaudal angle of each salivia is
rounded and produced into a chitinized, tapering bar which
extends ventro-caudo-mesad and meets the one from the other
side, forming a deep semicircular structure. The salivos is lo-
cated cephalad of this median structure. Each ventrolateral
angle of the hypopharynx, ventrad of the salivia and cephalad
of the bars supporting the salivos, is obtusely rounded and mod-
erately chitinized. The ventromeson of the hypopharynx is
concave, the concavity widens caudad, and is provided with a
shallow median furrow. On each side and cephalad of the
salivos is a pit leading into a short blind pouch which Mangan
(08) has suggested to be a ‘salivary receptacle,’ but its function
has not been determined. The oscula is the membranous area lo-
cated ventrad of the hypopharynx. It extends laterad and then
dorsocaudad and is continuous with the labacoria and lingula.
The salivos is protected by a portion of the oscula which extends
ventrocephalad of the salivial bars. The salivary duct is large
and extends caudad for some distance before bifurcating.

The general plan of the organization of the prepharynx in all
the genera studied manifests a striking similarity, and the homol-
logy of each component part is demonstrable with a fair degree of
certainty. The differences in size, shape, and position in these
structures, though great in some cases, are reducible in general
to a gradual series of modification.

276 HACHIRO YUASA

The epipharynx is setiferous, membranous, and usually con-
cave and is similar in size and shape to the labrum. The sur-
face bears setae and spinulae which are local in their distribu-
tion in Gryllus and others. There are often chitinized structures
which are usually more distinct on the ental than on the ectal
surface and which may be Y-shaped as in Melanoplus or wedge-
shaped as in Gryllus.

The tormae are always present, distinct, well chitinized, and
twisted, and bear definite relation to the clypeolabral suture.
The dorsal arm of the twisted body is attached to the lateral
angle of the suture and the ventral arm to the caudolateral angle
of the epipharynx, while the body of each torma connects the
two arms. ‘There is a variation in size, shape, and complexity
from the simple type found in Stenopelmatus to the many-
branched type of Gryllus. The mesal ends of the tormae are
sometimes connected by a thickening (fig. 159).

The epigusta is membranous and often includes a few tendinous
thickenings. It is frequently spinulate and bears thickenings
and sensory pits. It gradually merges with the postpharynx
and ambipharynx; there is never a sharp line of demarcation
between them. The epigusta is never extensive.

The ambipharynx is membranous and not well differentiated.
It is usually restricted by the encroachment of the mandibles.
In Gryllus it is spinulate near the entrance to the postpharynx
‘and bears a small chitinized area cephalad of each mandible.

The parapharynx is well developed and, since it is complex in
organization.and since there is no adequate description published,
it will be described with more or less detail. The distribution
and localization of the setae and spinulae on the chitinized and
membranous portions of the parapharynx differ considerably.
The asymmetry of the surface structures observed in many
genera (Gryllus, Tettix, Anisolabis, and others) is due to the
adjustment necessary to secure the close fitting of - different
elements of the mouth-parts, both when in repose and in use.

In Mantis (fig. 161) the parapharynx resembles that of the
cockroach. The pharyngea is distinct, slightly chitinized, and
extends caudad along the ventrolateral margin of the subgusta

HEAD, MOUTH-PARTS, ORTHOPTERA EUPLEXOPTERA 277

into the lateral wall of the postpharynx. The caudal end is
on the dorsolateral margin of the postpharynx and supports its
dorsal and lateral walls. The paralingua is an inverted Y-shaped
selerite on the lateral aspect cephalad of and fused with each
pharyngea. To the strongly chitinized dorsal stem of the Y a
prominent linguatendon is attached. It extends laterad, and,
after passing over the rectotendon of the mandible, enters this
structure. The caudal portion of the subgusta is broad and the
cephalic is constricted between the two paralinguae. The lin-
gula is the distinct setiferous sclerite fused by a narrow neck
to the cephalic end of each paralingua. The dorsal surface of
the parapharynx is constricted between the two lingulae. The
salivia is a large chitinized area occupying the ventral half of
each lateral aspect of the hypopharynx. Its caudal angle is
produced into an arm which supports the salivos on the ventro-
meson. A slightly chitinized area lies cephalad of each lingula
and dorsad of each salivia. The ventral aspect of the hypo-
pharynx is concave and furrowed on the meson and the chitinized
extensions of the saliviae occupy the caudolateral portions. The
salivos is protected on the ventral side by a thick triangular
membrane. The salivary duct is very small and extends caudad
for some distance before bifurcating. The oscula is narrow and
indefinite and the salivos is located dorsad of the cephalic part
of the mentum.

- The parapharynx in Diapheromera (fig. 163) is compressed and
boot-shaped. The pharyngea is a large irregular sclerite extend-
ing into the lateral and ventral walls of the postpharynx. Its
cephalic end terminates indefinitely in the membrane laterad of
the subgusta. The subgusta is deeply furrowed on the meson,
and each caudolateral half is strongly chitinized and fused to the
pharyngea without indication of a suture. The paralingua is the
ax-shaped sclerite on each lateral aspect of the subgusta. Its
dorsocaudal angle is pointed, the caudoventral bifurcated, and
the linguatendon is attached to its laterodorsal arm. The
cephalodorsal angle is more chitinized than the other parts and
curves cephalomesad, while the opposite angle extends cephalo-
ventrad and meets the dorsal extension from the caudal part

278 HACHIRO YUASA

of the salivia. The lingula is the indefinite, but more chiti-
nized, area cephalad of each paralingua. ‘The two lingulae fuse
on the meson without indication of a suture, and their dorso-
caudal margin is thickened and constricts the parapharynx.
The greater part of each lingula lies cephalad of this constric-
tion instead of caudad of it as in the other genera. The para-
pharynx caudad of this constriction is decidedly elongated on
account of the increase in the size of the mandibles. The salivia
is uniformly chitinized and occupies the entire ventral half of
each caudo-lateral aspect and the greater part of the ventral
aspect of the hypopharynx. Its caudoventral angle is sharply
produced and the mesal portion extends caudad as a broad tube
and supports the salivos. The ventral surface cephalad of the
salivos is submembranous and convex. The salivary duct is
small and bifureates immediately caudad of the salivos. The
oscula is narrow on the sides and membranous. The modifi-
cations of the mouth-parts due to the cephalization of the mouth
subsequent to the elongation of the head have produced the
peculiarities of the parapharynx noted above.

In Gryllus (fig. 157) the parapharynx is large and linguiform.
The pharyngea is a distinct L-shaped sclerite on each caudo-
lateral aspect of the parapharynx. One arm of the L is vertical
and is produced into a thinner extension which extends caudad
along the lateral margin of the subgusta and, on reaching the
postpharynx, is bent dorsad and extends along its lateral wall:
A small, but distinct, chitinized pocket, immediately ventrad
of the horizontal arm of the L, may be the homologue of the
cuticular pouch near the same position in Blatta. The para-
lingua is a brownish, subquadrate, non-setiferous sclerite cepha-
lad of each pharyngea. Its cephaloventral margin is thickened
and reflected slightly entad. Each lingula consists of two parts,
an ectal and an ental. The ental part is thick, short, strongly
chitinized and curved toward the meson. The sinistral arm is
slightly caudad of the dextral. The ectal part of each lingula is
convex, setiferous, triangular, and chitinized, and is subdivided
by a short suture-like furrow which defines the less chitinized
convex area on the dorsal side from the distinct larger area on

HEAD, MOUTH-PARTS, ORTHOPTERA EUPLEXOPTERA 279

the ventral. The caudal portion of the subgusta is flattened,
the cephalic portion is convex, and there is a constriction near
the middle. Laterocephalad of each lingula is a less chitinized
triangular area similar to the one in Blatta. The tendency to
asymmetry occurs in the ental arms of the lingula, in the ellip-
tical area cephalad of them, and in the patches of brownish
spinulae. Those on the dextral side are more advanced in posi-
tion and more conspicuous than the sinistral. The salivia is a
large, distinct, triangular sclerite cephaloventrad of each lingula
and ventrad of the triangular area mentioned above. The apex
of the triangular salivia is produced ventromesad into a long
pointed extension which, on reaching the meson, supports the
salivos. The ventral surface of the hypopharynx is mem-
branous and broadly and deeply folded. The middle portion of
the membrane is folded over itself and forms a deep pocket into
which the distal portion is retracted. This retractile portion is
covered with pseudotrachea-like thickenings which extend from
the main mesal trunk to the sides in an oblique parallel manner.
These smaller thickenings unite on the lateral margin into a
large lateral trunk which converges cephalad and extends over
the distal margin onto the dorsal surface of the hypopharynx.
This pseudotracheated portion may be evaginated and protruded
much further cephalad than the distal margin of the labium.
Figure 157 represents this portion slightly out of place. The
oscula is narrow cephalad of the salivos, deeply folded around
the ventral margin of the salivia, and is produced on each lateral
aspect where it merges into the labicoria.

In Orchelimum (fig. 158) the parapharynx is well developed
and linguiform. The pharyngea is long, slender, slightly chit-
inized, and extends along each side of the subgusta and then
obliquely dorsad into the lateral wall of the postpharynx. The
paralingua is the ax-shaped sclerite fused to the cephalic end of
each pharyngea. Its ventrocaudal arm is directed mesad and
then laterad and is connected with the mandible by a poorly
differentiated linguacuta. The lingula is distinct and fused to
the dorsocephalic angle of each paralingua; a mesal extension
from its dorsal margin constricts the parapharynx at this point.

280 HACHIRO YUASA

The subgusta is slightly concave and is continuous with the
broad ventral wall of the postpharynx. The cephalic ends of
the paralinguae constrict the subgusta. There is a narrow, dis-
tinctly setiferous, moderately chitinized area cephalad of each
lingula corresponding to that in Blatta. The salivia is large and
chitinized. A nearly vertical arm extends dorsad from it into
the setiferous area above. This may correspond to the similar
arm in Gryllus. The cephalic part of each salivia is longitu-
dinally convex and forms a shelf along the lateroventral margin
of the hypopharynx. The ventral aspect of the hypopharynx is
membranous and longitudinally furrowed on the meson. The
caudolateral angles are strengthened by the chitinized part of
the saliviae. The oscula is narrow and the salivos is dorsad
and slightly cephalad of the middle of the mentum.

The parapharynx in Stenopelmatus (fig. 159) is large, quad-
rate, and linguiform. The pharyngea is a long slender sclerite
along each side of the subgusta. Its expanded caudal part sup-
ports the lateroventral margin of the postpharynx. The cephalic
half is uniformly narrow and extends cephaloventrad. The dis-
tinct brownish sclerite located on each lateral aspect, cephalad
of the pharyngea, is the paralingua. A membranous linguacuta
is attached to the caudoventral angle of each paralingua. It
extends laterad toward the rectotendon of the mandible. The
lingula is the large triangular sclerite cephalad of each para-
lingua; its dorsocephalic angle is produced into a curved ental
arm which approaches the arm on the other side. The caudal
part of the subgusta is wide and flat, and the cephalic part
converges narrowly. The salivia is located along the ventro-
lateral margin of the hypopharynx. Its caudal part, ventrad
of each lingula, is a distinct bar which extends nearly hori-
zontally to the ventro-caudolateral angle of the hypopharynx
and then bends mesocaudad to the wide hooded salivos. The
distal part of the salivia is longitudinally convex so as to form a
shallow shelf on each ventrolateral margin of the hypopharynx.
The dorsal aspect of the hypopharynx is distinctly bent near its
middle. The oscula cephalad of the salivos is very much nar-
rowed by the chitinized area caudad of the mesarima of the

HEAD, MOUTH-PARTS, ORTHOPTERA EUPLEXOPTERA 281

labium. The apex of the triangular area is applied against
the laterocaudal angle of the salivia. The oscula on the lateral
aspect forms a small membranous fold at the proximal end of
the hypopharynx.

In Melanoplus (figs. 156 and 162) the parapharynx is sub-
globular and obtusely pointed at the distal end. The pharyngea
is long and slender, its caudal part extending along the ventro-
lateral margin of the postpharynx; the cephalic part is strongly
chitinized. The subgusta is deeply furrowed on the meson, and
constricted between the cephalic ends of the pharyngeae. The
paralingua is the small V-shaped sclerite ventrad of and fused
with the cephalic part of each pharyngea. The ventral arm of
the V extends ventrad and then caudolaterad into the poorly
differentiated linguacuta which is attached to the mesocephalic
end of the rectotendon of each mandible. The lingula is the
brownish subquadrate sclerite located slightly ventrad and
cephalad of the cephalic end of each paralingua. Its cephalic
margin is expanded and its mesocephalic end bent mesad. The
hypopharynx is narrowest between the distal ends of the lingulae
and is produced laterad and strongly convexly cephalad of this
constriction. There is a constriction near the distal third of the
hypopharynx where the lateral surface is produced like a shoulder.
This prominence caudad of the distal constriction was inter-
preted by Folsom (’00) as a rudimentary ‘superlingua.’ The
salivia consists of two subdivisions. The dorsal is a long strongly
chitinized sclerite extending obliquely from the dorsal constric-
tion along the ventral margin of the proximal part of the hypo-
pharynx. The other is a large chitinized plate on the lateral
and ventral aspects. The ventral part is irregular and is con-
tinuous with the dorsal part, with a faint suture-like furrow be-
tween them. It extends mesad to form a support for the salivos.
The ventral aspect of the hypopharynx is distinctly convex and
is prcduced into a small pocket under the salivos. The salivary
duct is slightly dilated and depressed immediately caudad of the
salivos. It extends caudad for a short distance, then bifurcates.
The oscula is flat under the free part of the hypopharynx, pro-
tects the salivos, and extends laterad, then caudad, to the
lingulae and paralinguae where it is continuous with the lebicoria.

JOURNAL OF MORPHOLOGY, VOL. 33, NO. 2

282 HACHIRO YUASA

The parapharynx in Tettix (fig. 160), somewhat similar to
that of Melanoplus, is well developed and linguiform. The
pharyngea is the short distinct sclerite located at each caudo-
lateral angle of the subgusta. Its main part is on the ventral
side of the lateral wall of the postpharynx. Fused to the end of
each pharynx is the long curved paralingual sclerite. The con-
nection between these is weaker and less distinct on the right
side than on the left. The ventral arm of the paralingua curves
ventrad and merges into the linguacuta. The strongly chitinized
crescentic sclerite fused to each paralingua is the lingula. The
greater part of it is on the ental surface and extends mesad.
The subgusta is longitudinally coneave and is constricted be-
tween the paralinguae. The position of the well-defined sub-
triangular chitinized area cephalad of each lingula suggests that
it is homologous with the long oblique part of the salivia of
Melanoplus. The larger chitinized sclerite on the ventrolateral
aspect of the hypopharynx is the salivia and is separated from
the triangular area on the dorsal side by an oblique suture.
An oblique thickening near its caudodorsal part has its ventral
end produced into a prominent blunt extension, probably homol-
ogous with that in Stenopelmatus. The mesal extension of the
salivia is heavily chitinized and nearly transverse. The oscula is
more or less flat cephalad of the salivos. The salivary duct
bifurcates immediately caudad of the salivos, the latter being
dorsad «f the caudal margin of the mentum. There is an asym-
metry in that the right mesal arm of the lingula is slightly
caudad of the left one and the adjacent large patch of spinulae is
much more extensive on the right side than on the left.

The parapharynx in Anisolabis (fig. 138) has its own charac-
teristics, but in the main conforms to the orthopteran type. Itis
well developed, broad, and trilobed at the distal end. A long
pharyngea lies each side of the subgusta. Its caudal half is
expanded and extends obliquely dorsad into the lateral wall .of
the postpharynx. Its cephalic half is narrow and _ strongly
chitinized. The oblique L-shaped sclerite fused to the cephalic
end of each pharyngea is the paralingua. The linguatendon is
fused to the ventral, wider, less chitinized arm of the paralingua

HEAD, MOUTH-PARTS, ORTHOPTERA EUPLEXOPTERA 283

and extends toward the rectotendon of the mandible. The
prominent X-shaped sclerite cephalad of each paralingua is the
lingula, the lateral aspect of which is concave and is produced
into a strongly chitinized caudal arm and a narrow arm which
is reflected ventrocephalad. The transverse arm of each lingula
apparently fuses on the meson with the one on the other side.
A narrow semicircular projection, extending caudad from the
mesal end of each transverse arm, touches the cephalic end of
the paralingua and a prominent mesal spinulate ridge caudad of
the lingulae. The subgusta is concave and carinate as noted
above. The distal end of the hypopharynx is trilobed. The
median lobe is large, symmetrically triangular, and its apex is
pointed. Each lateral lobe is concave on its mesal aspect and
overlaps the lateral margin of the proximal part of the median
lobe. On the ventral two-thirds of each lateral aspect of the
hypopharynx is a large, moderately chitinized, convex sclerite,
the salivia. Its caudal angle is produced into a mesal arm sup-
porting the salivos on the ventromeson. The ventral aspect of
the hypopharynx is deeply concave near the proximal portion,
but the median third is notably convex, while the distal third is
membranous. A narrow chitinized area extends cephalad along
the lateral margin of the hypopharynx and supports the ventro-
mesal part of the lateral lobe. The mesal arms of the saliviae
form on the ventromeson a cone-shaped structure whose apex is
directed caudad and whose cephalic margin is arched over by a
narrow transverse bar which is provided with a small median
notch. The cone is fused solidly to the strongly chitinized
V-shaped vertical frame which arises from the keel-like ental
ridge at the base of the mesarima of the labium. The salivary
duct is moderately large and attached to the caudal end of the
cone and its opening is at the median notch of the transverse
bar. There is a distinct asymmetry of parts on the dorsal
aspect of the hypopharynx. The dextral ligula is straighter
and narrower than the sinistral, but its cephalic process on the
meson is very much larger than the sinistral; the right paralingua
is slightly in advance of the left, and the sinistral lateral lobe of
the hypopharynx is smaller and overlaps the median lobe to a

284 HACHIRO YUASA

less extent than the dextral. There is also a difference in the
number of extensions on the epigusta. All these peculiarities
seem to be of secondary importance, since their presence can be
explained by the asymmetrical conditions of the mandibles
which, when brought together, leave an irregular space for the
parapharynx to fill. The nature of the lateral lobes is more
difficult to explain. They are the ‘paraglossae’ of the older
writers, and correspond to the ‘maxillulae’ of Hansen (’94) and
‘superlingue’ of Folsom (’00). Berlese (06) considers the
entire parapharynx, including the lateral lobes, as derived from
the labium, and thus disagrees, like many others, with Folsom’s
view concerning the origin and nature of these lobes of the
hypopharynx. Carpenter (’16) figures the labium of Isolepisma
bisetosa with a ‘maxillula’ which simulates the conditions found
in Orthoptera and Euplexoptera.

SUMMARY

1. This study on the comparative anatomy of the head and
mouth-parts of orthopteran insects has resulted in establishing
the homology of all of the structures present in generalized and
specialized Orthoptera, and in elucidating the hitherto but
little studied structures, the propharynx and tentorium. Ex-
cepting variations in size, shape, and other superficial differ-
ences, not of primary importance, all of the structures examined
reveal a remarkable uniformity in their general organization and
at the same time indicate the trend of evolutionary processes
which are responsible for the apparent gradation in specialization
manifested by these structures.

2. Since morphology offers fundamental evidence for the deter-
mination of phylogeny, a word may be added, with due appre-
ciation of the limitation of the data at hand, as to the probable
genealogical relations of the insects under consideration. Briefly
stated, the anatomical evidences presented in this paper show
that the Blattidae is the most generalized; that the Mantidae is
closely related to the Blattidae, that the Gryllidae follows the
Mantidae, although it is not likely to have developed directly

HEAD, MOUTH-PARTS, ORTHOPTERA EUPLEXOPTERA 285

through the latter; that the Locustidae and Acridiidae come
next in the order named, and that the Phasmidae appears to be
most remote from the Blattidae, although its exact phylogenetic
position cannot be determined. The Forficulidae possess evi-
dences of orthopteran affinity in their general morphological
organization, but differ from the Orthoptera in the character of
the maxillae, parapharynx, and labium.

The structures dealt with in this study are summarized as
follows:

3. The exoskeleton of the head consists of vertex, front,
clypeus, labrum (preclypeus and postclypeus), mandibulariae,
occiput, postgenae, and mazxillariae. The vertex includes the
genae and bears the compound eyes, ocelli, antacoriae, and
antennariae. The occipital foramen is bounded by the occiput,
postgenae, and maxillariae.

4. The endoskeleton of the head:

BODY ARMS EXTERNAL MARKS ASSOCIATED WITH
Corpotentorium Metatentorium Metatentorina Postgena
Pretentorium Pretentorina Front, clypeus, and
Laminatentorium mandibularia
Supratentorium | Supratentorina | Antennaria

5. The movable parts of the head:

STRUCTURE BORNE ON SCLERITE INCLUDING

Antennae | Antennariae Scape, pedicel, flagellum, antatendons

and antacoriae

Mandibles | Mandibulariae | Dentes, mola, acia (Blatta), brustia, artes,

tendons

Maxillae Maxillariae Cardo (alacardo and subeardo), parartes, stipes,
subgalea, palpifer, palpi, lacinia, galea (proxi-
galea and distagalea), maxacoria

Labium Microcoria Submentum (lateral lobes sometimes), mentum,
ligula, labicoria. Ligula includes stipula, pal-
piger, palpus, glossa, paraglossa (dista- and
proxa-)

286 HACHIRO YUASA

6. The parts of the prepharynx:

Postpharynx
Shanken ee Tormae
Pharynx Epieuete /
Subgusta
Ambipharynx Pharyngeae
| Prepharynx pace Paralinguae (lingua-
cutae, linguaten-
| | dons)
oo Lingulae
eaenar as Saliviae
| Hypopharynx Salivos (salivary
duct)
(Oscula
7. The articulation of the appendages:
APPENDAGE pada ARTES CcOILA TENDON
Antennae | Scape Antartes or | Antennaria Antatendons
base of scape
Mandibles | Proximal part | Preartis Precoila on | Extensotendon
of mandible clypeus
Postartis Posteoila on | Rectotendon
postgena
Maxillae Subeardo Parartis
Exparartis | Paracoila on | Postmaxatendon
) Entoparar- maxillaria Premaxatendon
{ tis
Labium Submentum
(associated
with micro-
coria)

8. The seers of the head:

SUTURE LOCATION BOUNDARY BETWEEN

Epicranial

Stem Epicranium Two halves of vertex

Arms Epicranium Vertex and front

Frontogenal Front Front and gena
Frontoclypeal Front Front and clypeus
Clypeal Clypeus Preclypeus and postclypeus
Clypeolabral Clypeus Clypeus and labrum
Occipital Postgena Vertex and postgena
Mandogenal Mandibularia Mandibularia, front, and gena

HEAD, MOUTH-PARTS, ORTHOPTERA EUPLEXOPTERA 287

BIBLIOGRAPHY

There has been no attempt to make this bibliography complete; only such

papers as are actually cited in this paper are listed.

BeERxLESE, A. 1909 Gli Insetti. Milan.

Bormans, A. pE 1900 Forficulidae und Hemimeridae. Schulze’s ‘Das Tier-
reich.’ Berlin. 11 Lief.

Buenton, E. 1913 Hexapoda. Lang’s Handbuch der Morphologie der wir-
bellosen Tiere. Jena.
1916 Les pieces buccales de la Blatte. Mitt. d. Schweizer Ent.
Gesel., Bd. 12.

CaRPENTER, G. H. 1916 The Apterygota of the Seychelles. Proc. Roy. Irish
IACAG EV Ole dass no. Le

Comstock, J. H., anp Kocur, C. 1902 Theskeleton of the head of insects. Am.
Nat., vol. 36.

Crampton, G. C. 1909 A contribution to the comparative morphology of the
thoracic sclerites of insects. Proc. Acad. Nat. Sei., Phil., vol. 61.
1916 A comparative study of the maxillae of the Acrididae (Oedi-
ponae and Tettiginae), Phasmidae and Phylliidae. Psyche, vol. 23.
1917 The nature of the veracervix or neck region in insects. Ann.
Ent. Soc. Am., vol. 10.

Fasricius, J. C. 1775 Systema Entomologiae. Flensburg and Leipzig.

Foutsom, J. W. 1900 The development of the mouth-parts of Anurida maritima
Guer. Bull. Mus. Comp. Zool., vol. 36.

Hansen, H. J. 1893 Zur Morphologie der Gliedmassen und Mundtheile bei
Crustaceen und Insecten. Zool. Anz., Bd. 16. Translated in Ann.
and Mag. Nat. Hist., vol. 12.
1894 On the structure and habits of Hemimerus talpoides Walk.
Ent. Tidsskrift.

Hennecuy, L. F. 1904 Les Insectes. Paris.

Huxtey, T. H. 1878 A manual of the anatomy of invertebrate animals.
London.

Kose, H. J. 1889 Einfiihrung in die Kenntnis der Insekten. Berlin.

Mancan, E. 1908 On the mouth-parts of some Blattidae. Proc. Roy. Irish
Acad., vol. 27, B, no. 1.

Mratu, F. L. C., anp Denny, A. 1884 The natural history of the cockroach.
Hardwicke’s Science-Gossip, vol. 20.
1886 The structure and life-history of the cockroach. London.

Outvier, M. 1791 Encyclopédie Méthodique. Histoire naturelle des Insectes.
Paris.

PackarpD, A. 8S. 1883 The systematic position of the Orthoptera in relation to
other orders of insects. Third Rept. U. 8. Ent. Comm. Washington.
1909 A text-book of entomology. New York.

PETERSON, ALVAH 1916 The head-capsule and mouth-parts of Diptera. III.
Biol. Monographs, vol. 3.

Ritey, W. A. 1904 The embryological development of the skeleton of the
head of Blatta. Am. Nat., vol. 38.

Scuroper, C. 1912 Handbuch der Entomologie. Jena.

ade.

a, antenna

aa, antacoila

ac, acia

ad, antartis

al, alacardo

an, antacoria

ar, antennaria

at, antatendon
ax, ambipharynx
b, brustia

bx, basipharynx

c, elypeus

ca, cardo

ce, compound eye
ch, chitinized area
cls, elypeolabral suture
cr, crassa

cs, clypeal suture
ct, corpotentorium
d, dentes

dd, distadentes
dg, distagalea

ea, epicranial arm
ec, extensacuta
eg, epigusta

en, entoparartis
es, epicranial stem
et, extensotendon
ex, epipharynx

ey, exparartis

f, front

fes, frontoclypeal suture
tgs, frontogenal suture
fl, flagellum

fr, fundarima

g, gena

gl, galea

go, glossa

h, hamadens

hx, hypopharynx
1, labrum

la, lacinia

Ic, labicoria

Ig, linguatendon or linguacuta
ll, lateral lobe of submentum

In, lingula
lo, lateral ocellus
lp, labial palpus

LIST OF ABBREVIATIONS

lr, latarima

lt, laminatentorium
Ix, lateral lobe of ligula
m, mentum

ma, microcoria

mb, mandibularia
mc, Maxacoria

md, mandible

mi, muscle impression
ml, mola

mgs, mandogenal suture
mn, metatentorina
mo, median ocellus
mp, maxillary palpus
mr, mesarima

ms, maxadentes

mt, metatentorium
mx, maxilla

my, maxillaria

oc, occiput

od, odontoidea

of, occipital foramen
ol, oculata

os, occipital suture
ot, oesotendon

ox, oscula

p, pedicel

pd, proxadentes

pf, palpifer

pg, proxagalea

pgo, paraglossa

pgn, postgena

pl, paracoila

pln, paralingua

pm, parademe

pmt, premaxatendon
pn, pretentorina
pot, postmaxatendon
pox, postpharynx
pp, palpiger

pr, precoila

prg, pharyngea

pt, pretentorium
ptc, postartis

ptl, postcoila

py, preartis

re, rectacuta

rs, lacinarastra

289

ABBREVIATIONS—Continued

rt, rectotendon sn, supratentorina
8, stipes so, salivos
sa, subeardo sp, stipula
sb, scrobe st, supratentorium
sc, scape su, subgusta
sg, subgalea td, tendon
sl, salivia tm, torma
sm, submentum v. vertex
PLATE 1

EXPLANATION OF FIGURES

Blatta orientalis, dorsal aspect of head.

Blatta orientalis, articulation of mandible.
Mantis religiosa, male, dorsal aspect of head.
Mantis religiosa, female, dorsal aspect of head.
Gryllus pennsylvanicus, dorsal aspect of head.
Stenopelmatus sp., dorsal aspect of head.
Orchelimum vulgare, dorsal aspect of head.
Diapheromera femorata, dorsal aspect of head.
Mantis religiosa, female, lateral aspect of head.
10 Melanoplus differentialis, dorsal aspect of head.
11 Tettix arenosus, dorsal aspect of head.

12 Anisolabis maritima, dorsal aspect of head.

13 Blatta orientalis, lateral aspect of head.

14 Gryllus pennsylvanicus, lateral aspect of head.
15 Diapheromera femorata, lateral aspect of head.
16 Diapheromera femorata, articulation of mandible.
17. Anisolabis maritima, lateral aspect of head.

18 Stenopelmatus sp., lateral aspect of head.

19 Melanoplus differentialis, lateral aspect of head.
20 Tettix arenosus, lateral aspect of head.

21 Anisolabis maritima, lateral aspect of head, appendages removed.
22 Orchelimum vulgare, lateral aspect of head.

IO or Wh FH

c OO

- HEAD, MOUTH-PARTS, ORTHOPTERA EUPLEXOPTERA PLATE 1
HACHIRO YUASA

PLATE 2

EXPLANATION OF FIGURES

23 and 24, 27 to 32, 35 Ventral aspect of the head, appendages removed.
36 to 41 Endosekleton of the head, dorsal wall of the head capsule, append-
ages, and internal organs removed.

23
24

Blatta orientalis.

Mantis religiosa.

Diapheromera femorata, caudal aspect of the head, appendages removed.
Mantis religiosa, postcoila enlarged.

Orchelimum vulgare.

Gryllus pennsylvanicus.

Diapheromera femorata.

Melanoplus differentialis.

Stenopelmatus sp.

Anisolabis maritima.
Diapheromera femorata, cephalic aspect of the head, appendages in situ.
Melanoplus differentialis, postcoila enlarged.

Tettix arenosus.

Blatta orientalis.

Gryllus pennsylvanicus.

Diapheromera femorata.

Melanoplus differentialis.

Tettix arenosus.

Mantis religiosa.

292

HEAD, MOUTH-PARTS, ORTHOPTERA EUPLEXOPTERA PLATE 2
HACHTRO YUASA

zi

NY

oO

Lear)

PLATE 3

EXPLANATION OF FIGURES

42 to 44. Endoskeleton of the head, dorsal wall of the head capsule, append-
ages, and soft internal tissues removed.

45 to53. Endoskeleton of the head in median longitudinal section, soft internal
tissues removed. ;

54 to 69 Antacoria, antennaria, and proximal segments of the antennae.

42 Anisolabis maritima.

43 Stenopelmatus sp.

44 Orchelimum vulgare.

45 Blatta orientalis.

46 Mantis religiosa.

47 Gryllus pennsylvanicus.

48 Stenopelmatus sp.

49 Melanoplus differentialis.

50 Orchelimum vulgare.

51 Diapheromera femorata.

52 Tettix arenosus.

53 Anisolabis maritima.

54 Blatta orientalis, female, cephalic aspect.

55 Blatta orientalis, female, caudal aspect.

56 Blatta orientalis, male, cephalic aspect.

57 Mantis religiosa, cephalic aspect.

58 Mantis religiosa, caudal aspect.

59 Gryllus pennsylvanicus, cephalic aspect.

60 Gryllus pennsylvanicus, caudal aspect.

61 Anisolabis maritima, cephalic aspect.

62 Orchelimum vulgare, cephalic aspect.

63 Stenopelmatus sp., cephalic aspect.

64 Stenopelmatus sp., antennaria, antenna removed.
65 Orchelimum vulgare, antartis and antatendon.
66 Diapheromera femorata, portion of head capsule with proximal segments
antenna.

67 Melanoplus differentialis, ental aspect of antacoria.
68 Melanoplus differentialis, cephalic aspect.

69 Tettix arenosus, cephalic aspect.

294

PLATE 3

HEAD, MOUTH-PARTS, ORTHOPTERA EUPLEXOPTERA

HACHIRO YUASA

PLATE 4

EXPLANATION OF FIGURES

70 to 92. Mandibles with tendons.

70, 80, 85, 86, 90, 91 Dorsal aspect of sinistral mandible.
71, 81, 82, 87, 92 Ventral aspect of sinistral mandible.

73, 76, 78, 79, 84, 89 Dorsal aspect of dextral mandible.

72, 75, 77, 83, 88 Ventral aspect of dextral mandible.

70 to 73 ~Blatta orientalis.

74 to 76 Mantis religiosa. 74, mesal aspect of dextral mandible.
77 and 78 =Gryllus pennsylvanicus.

79 and 82. Orchelimum vulgare.

80 and 81 Diapheromera femorata.

83 to 85 Stenopelmatus sp.

86 and 87 Anisolabis maritima.

88 to 90 Tettix arenosus.

91 and 92 Melanoplus differentialis.

93 Anisolabis maritima, dorsal aspect of sinistral maxilla.
94 Anisolabis maritima, ventral! aspect of sinistral maxilla.
95 Anisolabis maritima, caudal aspect of cardo.

HEAD, MOUTH-PARTS, ORTHOPTERA EUPLEXOPTERA PLATE 4
HACHIRO YUASA.

JOURNAL OF MORPHOLOGY, VOL. 33, NO. 2

PLATE 5

EXPLANATION OF FIGURES

96 to 119 Mazxillae.

98, 99, 101, 106, 108, 113, 115, 117 Caudal aspect of cardo.

96, 97, 103, 107, 109, 114, 116, 118 Ventral aspect.

100, 102, 104, 105, 110, 111, 112, 119 Dorsal aspect.

96, 104, 106 Mantis religiosa.

97, 98, 100 Blatta orientalis.

99, 102, 103 Gryllus pennsylvanicus.

101, 109, 111 Stenopelmatus sp.

105, 107, 108 Orechelimum vulgare.

110, 115, 116 Diapheromera femorata.

112 to 114 Melanplus differentialis.

117 to 119 Tettix arenosus.

99! Tettix arenosus, mesal aspect of the distal portion of lacinia, showing
the arrangement of maxadentes.

298

HEAD, MOUTH-PARTS, ORTHOPTERA EUPLEXOPTERA PLATE 5
HACHIRO YUASA

3 Pmtzy

PLATE 6

EXPLANATION OF FIGURES

120 to 135 Labium.

120 to 127 Ventral aspect.

128 to 135 Dorsal aspect with hypopharynx turned over to show its ventral
aspect.

120, 128 Blatta orientalis.

121, 129 Mantis religiosa.

122, 130 Gryllus pennsylvanicus.

123, 131 Stenopelmatus sp.

124,135 Melanoplus differentialis.

125, 133 Orchelimum vulgare.

126, 132 Tettix arenosus.

127, 134 Diapheromera femorata.

300

HEAD, MOUTH-PARTS, ORTHOPTERA EUPLEXOPTERA PLATE 6
HACHIRO YUASA

PLATE 7

EXPLANATION OF FIGURES

136 and 137 Anisolabis maritima, labium. 136, ventral aspect; 137, dorsal
aspect with the hypopharynx turned over to show its ventral aspect.

138 Anisolabis maritima, mouth-parts dissected out and prepharynx flat-
tened out to show its parts.

139 Gryllus pennsylvanicus, sagittal section of head showing prepharynx.

140 Orchelimum vulgare, sagittal section of head showing prepharynx.

141 Mantis religiosa, mouth-parts dissected out showing lateral aspect of
prepharynx.

142 Diapheromera femorata, sagittal section of head showing prepharynx.

143 Anisolabis maritima, sagittal section of head showing prepharynx.

144 Stenopelmatus sp., sagittal section of head showing prepharynx.

145 Tettix arenosus, mouth parts dissected out showing lateral aspect of
prepharynx.

146 Anisolabis maritima, part of salivia near salivos showing the fusion to
mesarima of labium.

147 Anisolabis maritima, lateral aspect of hypopharynx.

148 and149 Gryllus pennsylvanicus, dorsal and lateral aspect of parapharynx.

150 Stenopelmatus sp., lateral aspect of parapharynx.

f
HEAD, MOUTH-PARTS, ORTHOPTERA EUPLEXOPTERA PLATE 7
HACHIRO YUASA
i
;

———

ea,

303

a a eV

re Saiaal

PLATE 8

EXPLANATION OF FIGURES

151 Blatta orientalis, showing different structures of mouth-parts. This
view is obtained by dissecting out the mouth-parts intact and arranging the
prepharynx so that the observer looks straight down into the mouth cavity.
In this way practically all the structures which belong to the mouth-parts can
be shown in one drawing.

152 Blatta orientalis, ental aspect of the ventrocaudal part of hy popes
showing the cuticular pouches, salivial bars and salivos.

153 Blatta orientalis, lateral aspect of parapharynx.

154 Blatta orientalis, sagittal section of head showing the lateral aspect of
prepharynx.

155 Melanoplus differentialis, sagittal section of head showing the lateral
aspect of prepharynx.

156 Melanoplus differentialis, lateral aspect of parapharynx.

304

HEAD, MOUTH-PARTS, ORTHOPTERA EUPLEXOPTERA PLATE 8

HACHIRO YUASA

Ci |
mt pinen In ™

155
305

PLATE 9

EXPLANATION OF FIGURES

157 to 163 Mouth-parts, arranged in the same way as in figure 151.
157 Gryllus pennsylvanicus.

158 Orchelimum vulgare.

159 Stenopelmatus sp.

160 Tettix arenosus.

161 Mantis religiosa.

162 Melanoplus differentialis.

163. Diapheromera femorata.

306

HEAD, MOUTH-PARTS, ORTHOPTERA EUPLEXOPTERA PLATE 9
HACHTRO YUASA

162

307

Resumen por los autores, William A. Kepner y Frank Helvestine.
Universidad de Virginia.

La faringe de Microstoma caudatum.

1. La faringe de este animal esta provista de un disco chupador
que le sirve para procurarse el alimento; la faringe es mucho mas
distensible que lo que se ha creido previamente. 2. A la funcién
prehensil de la faringe contribuye el ciego anterior del enteron.
3. Las secrecciones de las glindulas faringeas paralizan a las
Hydra. Esta pardlisis es local, estando limitada a las partes del
cuerpo de la presa que han sido ingeridas y no es permanente, re-
cobrando la presa su actividad cuando es devuelta al exterior.
4. El epitelio que forra a la faringe contiene nucleos esparcidos y
es relativamente bajo. 5. La faringe aparece después que se ha
formado la parte fundamental del sistema nervioso central, es-
tando completamente diferenciada antes que se separen los
zooides de un Microstoma en vias de divisién. 6. De los 6rganos
diferenciados, el sistema nervioso central mantiene la conexi6n
mas intima con los alimentos ingeridos por el padre, durante el
desarrollo del nuevo zooide. 7. En el fondo de la faringe en vias
de desarrollo aparecen células que corresponden a las ‘‘Schlies-
senzellen’’ de las planarias; tales células forman la transici6n
entre los epitelios faringeo y entérico.

Translation by José F. Nonidez *
Carnegie Institution of Washington

AUTHOR'S ABSTRACT OF THIS PAPER ISSUED
BY THE BIBLIOGRAPHIC SERVICE, JANUARY 19

PHARYNX OF MICROSTOMA CAUDATUM

WM. A. KEPNER AND FRANK HELVESTINE, JR.
University of Virginia

ONE TEXT FIGURE AND THREE PLATES

Microstomum caudatum is frequently confused with Steno-
stoma leucops. The sexually immature individuals of these gen-
era greatly resemble each other. The chief differences lie in
their alimentary canals. The alimentary canal of Microstoma
caudatum consists of two regions—the enteron and the stomo-
daeum. The elongated, sac-like enteron extends anteriorly over
the dorsal side of the pharynx (fig. 1, B, and fig. 6, A.S.). This
blind end or caecum of the enteron, lying over and projecting
beyond the pharynx, constituted the chief diagnostic feature of
Microstoma caudatum. The stomodaeum is represented by a
short, glandular-walled tube—the pharynx (fig. 1, A). This is a
simplex pharynx. It is rather more highly refractive than the
other organs of the body and can, therefore, be readily recog-
nized as the animal turns on its side or back. It is not definitely
limited by a membrane, so that its glands stand out discrete pro-
jecting into the surrounding mesenchyme (fig. 1, 4). When a
sexually mature specimen lies on its back in a hanging drop, two
openings of the body are conspicuous on the midline. One of
these, the female genital pore, lies at the anterior limit of the
posterior third of the body (fig. 1, C); the other, the mouth of
the pharynx, lies at the posterior limit of the anterior fifth of
the body (fig. 1, MW). Von Graff (09) describes and depicts
the mouth as a longitudinal, slit-like opening but suggests no
other details. Martin (’08) shows in his figure 10, plate 14, an
open mouth, guarded by a circular disc-like lip. This is the near-
est approach of any student to our observation of the details of
this mouth. A living specimen, caught resting dorsal side down

309

JOURNAL OF MORPHOLOGY, VOL. 33, NO. 2

310 WM. A. KEPNER AND FRANK HELVESTINE, JR.

in a hanging drop of water, will show that the mouth is provided
with a flattened, circular lip which has many fine radiating lines
upon or in it, thus presenting the appearance of a sucking dise
(figs 1D):

Certain observations have been made upon the functions of
the pharynx with its disc-like mouth. The pharynx functions in
a twofold manner, a) as a prehensile and, b) as a secreting organ.
Martin (’08, p. 268) says that when Microstoma attacks Hydra
“it fixes itself for a short time by its posterior end in the neigh-
borhood of the Hydra, and everts its pharynx to its full extent.”
He then refers to his figure 10 to illustrate a pharynx fully ex-
tended (text figure). This figure does not show adequately the

C2

Text-figure Ventral aspect of anterior end of Microstoma to show mouth dis-
tended. From Martin (’08).

degree to which the pharynx of Microstoma may be distended.
We starved-a specimen for five days and then presented it with
half of an oligochaete worm. The worm was at once attacked
near its middle, the Microstoma’s pharynx was distended in such
fashion that the body assumed the contour of a laterally com-
pressed funnel or cone. As the mouth of the rhabdocoele was
thus distended, the prey, though wriggling much at its extrem-
ities, was sucked into the lumen of the enteron (fig. 2). After
the prey had been drawn down to assume a U-shape, it was next
handled in such manner as that one arm of the U was gliding
from the mouth of Microstoma as the other was being dragged
into it as indicated by arrows in figure 3. When the ingested

PHARYNX OF MICROSTOMA CAUDATUM 311

worm lay head-on within the enteron, ingestion of it was renewed
and continued until completed, although the Microstoma was so
gorged that the pharynx of the posterior zooid, not yet being in
communication with the enteron, was everted (figs. 4 and 5).
Thus it is seen that the mouth of the pharynx is distensible to a
surprising degree and that the pharynx operates as a prehensile
organ which can bring considerable force to play upon prey that
is being ingested. The mechanism by which this forcible in-
gestion is accomplished is shown in figures 6 and 7. Figure 6
shows a sagittal section of an animal with its mouth nearly closed.
The passage-way from pharynx to enteron is closed by a region
of slightly peculiar cells. The anterior sac of the enteron is
conspicuous and lies well up within the ‘head’ (fig. 6, A.S.). In
forming a sucking apparatus or piston, the lips of the enteron are
everted into the pharyngeal cavity. This eversion involves drag-
ging the anterior sack of the enteron ventrally and posteriorly.
The widened pharyngeal cavity now contains an everted knob
of enteric tissue (fig. 7, H.K.), which can be drawn back into its
resting position and thus cause a vacuum within the pharyngeal
cavity. It is by the repeated everting and drawing back of this
region of the alimentary canal, together with an alternate dis-
tending and contracting of the pharynx, that objects can be quite
forcibly crowded into the enteron of Microstoma.

This prehensile function of the pharynx is supplemented by the
work of the glands. Martin (’08) kept some Hydras in a solu-
tion of neutralroth, and “the vacuoles of the ectoderm of the
hydras, which had been stained a pink color by the neutralroth,
took a yellowish-brown color under the action of the digestive
fluids, indicating that the secretion (of Microstoma) was of an
alkaline nature, and possibly allied to trypsine.” Martin said
that under the conditions of ingestion the “tentacles of Hydra
do not grasp the Microstoma, but remain extended almost par-
allel with its body, and it would appear as though the pharyngeal
secretion has a paralyzing action on the Hydra.” He further
says that ‘in many cases, after a time, the Microstoma leaves
its prey, and in such a case the Hydra does not seem much the
worse for the attack”’ (p. 268).

312 WM. A. KEPNER AND FRANK HELVESTINE, JR.

That Microstoma can come up and tear off a tentacle of Hydra
without the other tentacles or any other parts of the victim’s
body being disturbed has been frequently observed in this labora-
tory. When the entire Hydra is being ingested, the body of the
prey contracts to a form shown by contour A, figure 8. Some-
times a Hydra that has been thus ingested is egested. Such a
rejected polyp remains quiet, neither expanding nor further con-
tracting, as if both its superficial or longitudinal muscles and its
deeper or circular muscles had been paralyzed. This condition
of complete paralysis is passed within an hour. Within that
period the recovery is complete. An interesting observation in
one case showed that the longitudinal muscles were not completely
paralyzed. For in this case, throughout the process of ingestion
the Hydra has a shape approximately like that indicated by con-
tour A, figure 8. As soon, however, as the polyp was crowded
into the enteron by the side of an oligochaete, that had been pre-
viously ingested by the Microstoma, it became spherical. Here,
then, the longitudinal muscles had the power to contract further
when they were more severely stimulated by the secretions of
the enteron. The partial paralysis of an animal ingested is
local and confined to the part of the body which has been taken
into the pharynx. That this is the case is indicated by a second
observation. A Microstoma was ingesting a Hydra from its
aboral end. The polyp, during the process, had a shape indicated
by contour A, figure 8. The rhabdocoele gave up and left its
prey after having held the aboral half of the Hydra in its pharynx
for nearly a half-hour. The Hydra, after being egested, showed
the power to move and to elongate only the oral half of its body.
For nearly an hour the oral end would elongate, ply to and fro,
contract and expand again; but through all of this activity the
aboral end that had been ingested remained quiet, so that the
Hydra, when its oral half was distended, had the form indicated
by contour B in figure 8. Here it is apparent that the paralyzing
effect of Microstoma had influenced the deeper or circular mus-
cles of Hydra’s body. Thus it is indicated that both the outer
(longitudinal) and the inner (circular) muscles of Hydra are para-
lyzed by an attack from Microstoma, if it be sustained long

PHARYNX OF MICROSTOMA CAUDATUM ale

enough. This paralysis, however, is confined only to the re-
gions of the body taken into the pharynx, and is not complete,
for within an hour the animal had fully recovered.

The observations last cited indicated a striking difference of
reaction on the part of the Hydra, depending upon the region of
the polyp which was attacked by Microstoma. We have fre-
quently seen Microstoma snip away a tentacle without disturb-
ing other regions of the body than the tentacle involved. How-
ever, when the attack is made upon the aboral end of a Hydra
(lying free—not fixed), the response of the polyp is general—the
entire polyp (body and tentacles) contracting. This suggests an
interesting line of speculation. Parker (17 a) found that the
tentacles of actinians were but ‘slightly sensitive,’ while the ab-
oral or pedal regions were highly sensitive to mechanical or con-
tact stimuli. On the other hand, the tentacles of these polyps
were very sensitive to chemical stimuli (juice of mussels) while
the aboral or pedal regions were but slightly sensitive to such
stimulation. In the ight of Parker’s observations, it is suggested
that there may be in Hydra, as in actinians, paths of ‘specialized
transmission.’ If it be that the tentacles of Hydra are more
sensitive to chemical stimuli and the aboral end is more sensitive
to mechanical stimuli, then our observations indicate that the
preliminary phase of Microstoma’s attack upon Hydra is mechan-
ical, the chemical phase ensuing only after the prey lies within
the pharynx and enteron.

The histology of the pharynx indicates that it is prepared to
function in a chemical manner, for it is highly glandular. The
pharynx, in its resting condition, is spindle shaped, with a lumen
in the form of an oblate spheroid. The lumen is lined with a
strongly ciliated, sparsely nucleated epithelium of low cells (fig.
9, Kp). This epithelium is frequently interrupted by the ducts
of the many unicellular glands which radiate into the mesenchyme
from all sides of the pharyngeal wall. There are many indefinite
layers of these gland-cells. Those nearest the lining epithelium
are the smallest, while those lying at the periphery of the phar-
ynx are the oldest and largest (fig. 9, A,B,C). The smallest
gland cells show no sign of secretion formation, having neither

314 WM. A. KEPNER AND FRANK HELVESTINE, JR.

conspicuous vacuoles nor secretion products within the cyto-
plasm (fig. 9, A). In the older cells vacuoles appear within the
cytoplasm, become larger and more crowded with secretion prod-
ucts, until they occupy the greater part of their respective cells
(fig. 9, B). The cytoplasm of such cells is conspicuous only at
the fundus of the cell body. In this basal mass of cytoplasm is
the nucleus (fig. 9, B). It appears that only these large cells at
the periphery discharge their contents into the lumen of the
pharynx, for no empty vacuoles are found in the cells of inter-
mediate size. Many empty large cells occur at the periphery of
the pharyngeal wall (fig. 9, C). The staining reaction of the
empty cells is wholly basic, taking the acid dyes. Bordeaux red,
for example, or eosin stains all the details of these empty cells
red. This is in marked contrast to the reaction of the young
and secreting cells. In these the nuclei are highly acid in their
reaction, while the secretion granules show a great affinity for
the basic dyes.

The origin of the pharynx is readily studied in dividing speci-
mens. Animals are not infrequently found which show two
planes of division, thus presenting a chain of three zooids, while
specimens with two zooids are very common at any season of
the year. |

The division of a Microstoma to form two zooids involves the
division of the enteron, the formation of a new ‘brain,’ new cili-
ated pits, gonads, and pharynx. We are concerned here only
with the origin of the pharynx; but it is an interesting fact that
the developing ‘brain’ of the posterior zooid remains in contact
along its anterior dorsal surface with the enteron until the phar-
ynx has become well established and even connected with the
enteron (figs. 10, 11, and 12, Br.).. This ‘brain’ makes its appear-
ance earlier than does the anlage from which the pharynx is dif-
ferentiated. The pharynx arises as an invaginated sac of ecto-
dermal epithelium. At first the wall of the young pharynx is
composed of but a simple, ciliated columnar epithelium without
any glandular differentiation (fig. 11). Soon, however, a three-
fold differentiation of the cells of the ectodermal sac sets in. This
differentiation involves, a) a lining epithelium of the pharynx;

PHARYNX OF MICROSTOMA CAUDATUM 315

b) the gland cells which leave the epithelium to sink radially
into the mesenchyme, and, (c) a few cells at the fundus of the
young pharynx. These last cells appear to be modified as tran-
sitional cells between the epithelium lining the pharynx and the
enteric epithelium. We may therefore call them transitional
cells (figs. 11 and 12, 7.C.). These transitional cells increase in
number and form a conspicuous plug protruding into the lumen
of the pharynx at the time communication between pharynx
and enteron is established. Both in position and histological
detail these cells suggest the ‘Schliesszellen’ which Mattiesen
(04) describes for the developing pharynx of a planarian embryo.
The region of the transitional cells never becomes extensive. The
most extensive mass of cells in the pharynx is that of the gland
cells. Here the differentiation is rapid and very many gland
cells are formed before the young pharynx communicates with
the lumen of the enteron. Indeed, many of the gland cells of
an advanced pharynx are filled with secretion granules before the
lumina of pharynx and enteron are continuous. No empty gland
cells, however, have been found in a pharynx that does not lead
into the enteron, though the fission of the animal had advanced
to where the parent enteron had completely divided. The pos-
terior zooid is thus provided with a pharynx whose glands are
charged with secretion products, ciliated pits and a ‘brain’ which
lies quite near the cleavage plane. The new zooid is completed
with the formation of a rounded anterior extremity.

SUMMARY

1. The pharynx is provided with a sucking disc which serves
the animal in securing food. The pharynx is much more highly
distensible than previously described.

2. The pharynx is aided in its prehensile functioning by the
anterior caecum of the enteron.

3. The secretions of the glands of the pharynx paralyze Hydras.
This paralysis is local, confined to the parts of the prey’s body
that have been ingested and is not permanent, the prey recover-
ing if egested.

316 WM. A. KEPNER AND FRANK HELVESTINE, JR.

4. The lining epithelium of the pharynx is sparsely nucleated
and relatively low.

5. The pharynx arises after the fundament of the central
nervous system has made its appearance. The pharynx is
fully differentiated before the zooids of a dividing Microstoma
separate.

6. Of the differentiating organs the central nervous system
maintains the most intimate connection with the parent food
supply throughout the development of the new zooid.

7. Cells corresponding to ‘Schliesszellen’ of Planaria appear at
the fundus of developing pharynx. These are transitional cells
between the pharyngeal and enteric epithelia.

LITERATURE CITED

Grarr, L. von. 1909 Die Siisswasserfauna Deutschlands. IV. Turbellaria,
Strudelwiirmer. I. Teil: Allgemeines und Rhabdocoelida. Jena.

Martin, C. H. 1908 The nematocysts of Turbellaria. Quart. Jour. Mic. Se.,
vol. 52.

MatriEsEN, ErtcH 1904 Ein Beitrag zur Embryologie der Siisswasserdendro-
célen. Zeitschr. f. wiss. Zoologie. Bd. 77.

Parker, G.H. 1917 a Nervous system in Actinians. Jour. Exp. Zool., vol. 22.
1917 b The movements of tentacles in Actinians. Jour. Exp. Zodl.,
vol. 22.

RD
=
Sl
<q
~
A.

317

PLATE 1

EXPLANATION OF FIGURES

1 Ventral aspect of sexually mature specimen. A, pharynx; B, anterior sack
of enteron; C, opening of vagina; D, sucking disc; M, mouth. X 75.

2 Specimen loaded with nematocysts from Hydra attacking a living half of
an oligochaete. 50.

3 Oligochaete, partly ingested, being handled in such manner as that one end
is moving out of mouth of Microstoma, while the other end is being dragged into
mouth as shown by arrows. X 50.

4 Prey lying end on and half ingested. X 50.

5 Prey almost ingested. The Microstoma so badly gorged that the pharynx
of posterior zooid (Ph’) is everted. X 50.

6 Sagittal section of specimen with closed mouth. Anterior sac of enteron
conspicuous (A.S.). O, oocyte. X 75.

318

PLATE 1

PHARYNX OF MICROSTOMA CAUDATUM

, JR.

STINE

WM. A. KEPNER AND FRANK HELVE

319

PLATE 2

EXPLANATION OF FIGURES

7 Sagittal section of specimen with mouth open. The enteric epithelium of
anterior sac and of floor of ‘stomach’ is everted at H.K. to form a sort of piston
within the lumen of partly distended pharynx. At fixation specimen ruptured
along fission plane (Ff). O and O’, oocytes of two zooids. XX 75.

8 Hydra that had been half ingested from aboral end. During ingestion
polyp had contour A. When egested Hydra could distend and move oral half,
but aboral half of body was paralyzed. The polyp, with paralyzed aboral half,
had contour B. X 50.

9 Part of pharyngeal epithelium (Hp) with gland-cells. A, young gland-
cell; B, gland-cell at maximum secretion phase; C, emply gland-cell; D, ducts of
gland-cells passing through lining epithelium of pharynx. X 1500.

10 Frontal section of two zooids. Parent enteron completely divided, but
‘brain’ of posterior zooid yet les in intimate contact with the enteron of anterior
zooid. Br, ‘brain’ of posterior zooid; H, enteron of anterior zooid. XX about
100.

320

PLATE 2

OF MICROSTOMA CAUDATUM

PHARYNX

NER AND FRANK HELVESTINE, JR.

WM. A. KEP

Bee’ Sis

a.
“A;

ei

bee

an

ale

‘

PLATE 3

EXPLANATION OF FIGURES

11 Section of a young pharynx showing beginning of differentiation of gland-
cells, G.C., and a transition cell differentiated, T.C. Br, ‘brain;’ E, enteric
epithelium. X 1400.

12 Section of an older pharynx than one shown in 11. Two transitional
cells shown, J.C. Many gland-cells present, G.C. Br, ‘brain;’ E, enteric
epithelium. X 1400.

PLATE 3

A CAUDATUM
ESTINE

M

STO

PHARYNX OF MICRO

JR.

NK HELV

A

AND FR

WM. A. KEPNER

Resumen por el autor, H. D. Reed.
Universidad Cornell, Ithaca.

La morfologia del aparato transmisor del sonido en los anfibios
caudados y su significacion filogenética.

En los anfibios dotados de cola existen dos tipos morfolégicos
de aparato transmisor del sonido. En el tipo mas generalizado,
la columela y el opéreulo son distintos, en lo referente a la fusién
de una con el otro. En el otro tipo los representantes de la colu-
mela y el opérculo estan fusionados entre si, formando de este
modo una placa fenestrada. Esta particularidad hace posible
la divisidn del conjunto del érden en dos legiones: una que in-
cluye a ios Amblystomidae, Cryptobranchidae, Salamandra,
Triton y Diemictylus, y los Sirenidae; la otra que comprende a
los Necturidae, Amphiumidae, Typhlomolgidae, Plethodontidae
y Desmognathidae. El aparato definitivo generalizado es util
solamente en el medio ambiente terrestre y debe haberse originado
en conexidn con este medio. Las formas que actualmente son
acudticas han adquirido secundariamente este modo de vida.
Otras tienden al modo de vida acudtico, mientras que algunas han
retenido las costumbres terrestres mas primitivas con la corre-
spondiente estructura y modo de desarrollo.

Translation by José F. Nonidez
Carnegie Institution of Washington

AUTHOR’S ABSTRACT OF THIS PAPER ISSUED
BY THE BIBLIOGRAPHIC SERVICE, JANUARY 19

THE MORPHOLOGY OF THE SOUND-TRANSMITTING
APPARATUS IN CAUDATE AMPHIBIA AND
ITS PHYLOGENETIC SIGNIFICANCE

H. D. REED

Zoological Laboratory, Cornell University

EIGHTEEN TEXT FIGURES AND SIX PLATES

INTRODUCTION

In earlier papers Kingsbury and Reed (’08 and ’09) pointed out
the existence of two structures composing the sound-transmitting
apparatus of the tailed amphibians. One of these structures they
called columella which is extraotic in origin and is connected,
either directly or by a ligament, with the suspensorium of the
lower jaw. In the typical state it functions as the organ of sound
(jar) transmission during larval life. This condition is probably
the one which prevailed in primitive Caudata. The other ele-
ment was designated operculum which functions during adult
(terrestrial) life. It makes its appearance at the period of trans-
formation in such forms as Amblystoma and, upon completion,
becomes connected with the suprascapula through the M.
opercularis. The operculum arises as a circular plate of cartilage
‘cut out’ from the walls of the ear capsule caudad of the primary
fenestra which is occupied by the columella and is, therefore,
otic in origin. The secondary fenestra which it occupies is
formed at the same time and by the same method as the element
itself. As the operculum completes the formative period and
assumes a functional role, the columella gradually fuses with the
cephalic margin of the fenestra and probably ceases to function, at
least, to any extent.

In these papers the authors discussed the various morphological
relations of the columella and operculum and pointed out the
evidence which seemed to favor the hyomandibular homology of

325

JOURNAL OF MORPHOLOGY, VOL. 33, NO. 2

326 H. D. REED

the columella. Comparisons were also made with the sound-
transmitting apparatus of the Salientia.

There remain to be considered certain points that are con-
cerned with the morphology of the fenestral elements within the
various groups of the tailed amphibia themselves and the zo-
ological significance of these structures. ~The Amblystomidae and
Salamandra possess the two elements mentioned above. This is
the most generalized state found among the terrestrial species.
All other salamanders in the adult state possess only a single ele-
ment filling the foramen vestibuli. Such forms are: Crypto-
branchidae, Proteidae, Plethodontidae, Desmognathidae, Am-
phiumidae, Sirenidae, and Triton and Diemictylus. These
groups include the majority of all living species. In certain of
these forms the morphology of the single fenestral plate was
demonstrated in the papers already mentioned. In Triton and
Diemictylus the single element possesses only the characteristics
of the amblystomid operculum; that is, it is without stylus and
suspensorial connections, but possesses a perilymphatic promi-
nence, between which and the supraclavicle the M. opercularis
extends. Development showed that the single element in Die-
mictylus and Triton is the operculum or that which corresponds
to the caudal of the two elements in the Amblystomidae and
Salamandra. It further showed that the columella is present,
but disappears as such at an early period through complete
fusion with the cephalic lips of the fenestra (fig. 21). In a similar
manner it was revealed that the single element of the Crypto-
branchidae represents the columella of the amblystomid forms,
it being wholly of extraotic origin and possessing only the sus-
pensorial connections.

With regard to the other forms, however, definite information
was not forthcoming at that time to account for the presence of a
single element in the fenestra. In the Plethodontidae and Des-
mognathidae, for example, the single fenestral element possesses
at one and the same time the connections and relations of both
columella and operculum (fig. 22). Through a stylus and liga-
ment the cephalic end of the plate is connected with the suspen-
sorium, and in its caudal portion there is a well-defined peri-

SOUND-TRANSMITTING APPARATUS OF CAUDATA 327

lymphatic prominence which is placed in connection with the
supraclavicle by the M. opercularis. More recently, the present
writer (Reed, ’15) has interpreted certain aspects of the problem
bearing upon the morphology of the single fenestral plate in
Necturus and has pointed out that in its nature it differs from
the single element in both the Triton-Diemictylus group and
the Cryptobranchidae, although apparently agreeing with the
latter in skeletal connections, form, and general estate. A brief
review of these results will appear later in the present paper.

For the present study there were available series of both larvae
and adults of most species considered, but some series were found
lacking in important stages. Of those which were chosen to
serve as type studies for a given taxonomic group an attempt
was made to obtain developmental stages as numerous and as
close together as possible. The results follow: Spelerpes bis-
lineatus, larvae 10, 15, 17, 19, 20, 21, 23, 25, 28, 31, 34, 37, 43,
and 55 mm., respectively; Desmognathus fusca, larvae of 10, 26,
27, and 30 mm.; Amphiuma means, embryos of 30 and 33 mm.,
a mature larva and a newly transformed adult; Necturus em-
bryos of 11, 12, 15, 16, 17, 18, 19, and 20 mm. and larvae of 21,
22, 23, 24, 25, 26, 35, 40, 43, 44, 48, and 70 mm. in length.

THE PLETHODONTIDAE

Spelerpes bislineatus was chosen as the type study for the
family Plethodontidae, chiefly because of the relative ease of
procuring material and partly because it was considered a gen-
eralized member of the family. Asis usual in the Plethodontidae,
the ear capsule is fully ossified in the adult except the lips of the
fenestra and those points where connection is made with the
processes of the palatoquadrate. Both the fenestra and the fen-
estral plate are relatively elongate, but narrow in the vertical
diameter, as shown in figure 26. The stylus, a slender rod, is
attached to the cephalic portion of the plate and extends for-
ward and slightly upward where it joins the ventral edge of the
squamosum. In adults as large as 57 mm. there is no connec-
tion of stylus and os quadratum unless a very slender and

328 H. D. REED

doubtful strand of mesenchymal tissue extending between the
two is to be so considered. The ossification of the fenestral
plate is characteristic of the whole family Plethodontidae. The
central portion is completely ossified while in the periphery
cartilage persists between the outer and inner plates of bone.
This is quite in contrast to the amblystomid type where an
inner and outer shell of bone enclose cartilage at all levels.
The central portion of the plate is relatively thin, and ossifica-
tion begins here. The reason for the thinness of the plate at
its center and its early and complete ossification is to be found
in certain developmental conditions with a morphological sig-
nificance, as well as the tendency in these forms to a reduction
of the chondrocranium beyond that found in the Amblystomidae.
This point will be discussed later.

One of the characteristic features of the fenestral plate of
Spelerpes bislineatus is in the relation of the stylus to the plate
itself. As stated above, the stylus joins the plate in its cephalic
portion, but, even in the adult, the fusion is not so complete
that the identity of the former is lost. In young adults the
entire stylus is composed of a shell of bone surrounding a few
cartilage cells. In its caudal extremity, where it joins the fenes-
tral plate, the bony shell increases in thickness with a resulting
diminution of cartilage within. The stylus, after first touching
upon the plate, extends caudad a very short distance, in some
cases less than 1 mm., but it is not completely incorporated
with the plate substance. The cylindrical sheath of bone can
be made out distinctly at all levels, although an anchylosis of
the two structures takes place (fig. 1). Ata level of 50 u caudad
of that of figure 1, the stylus disappears and only here does
there seem to be any fusion between the two elements. The
significance of this relation is revealed only through develop-
ment. The connection of the definitive fenestral plate with the
ear capsule is in its ventrocephalic margin where the cartilage
of the two structures is continuous (fig. 22). This connection at
all stages is relatively narrow, and in this, as well as position, it
differs from any otic connection of fenestral elements in the
amblystomid forms where the connection is more extensive and

SOUND-TRANSMITTING APPARATUS OF CAUDATA 329

represents a secondary relation. From the morphological view-
point this connection is one of the most important structures of
the whole sound-transmitting apparatus in the Plethodontidae,
Desmognathidae, and others. For this reason and for the sake
of easy reference later, it will be termed the isthmus fenestralis.

Fig. 1 Transection through the fenestra vestibuli of an adult Spelerpes bis-
lineatus at the level of the stylus columellae. C.l., canalis lateralis; Col., stylus
columellae (columella); Hc., ear capsule; Fe.m., fenestral membrane; Op., fenes-
tral plate representing the operculum; Sq., squamosum.

330 H. D. REED

In the caudal portion of the plate is a perilymphatic promi-
nence, in a depression of which the M. opercularis is attached
(fig. 22).

From the preceding paragraph it is obvious that Spelerpes
and other genera have a structure which combines the charac-
teristics of both columella and operculum of the amblystomid
type. The natural inference is that in these forms the single
fenestral element represents some sort of fusion of columella and
operculum. With limitations, this statement is true, but the
significance of ‘fusion’ in this relevancy: becomes evident only
after the examination of numerous developmental stages.
Structures which are here considered to be the homologues of
the columella and operculum are present, as was mentioned
briefly in an earlier communication (Reed, ’14) and in the nature
and order of appearance of these structures in Spelerpes there is
identity with Amblystoma; that is, the columellar representa-
tive appears first, followed by an element of otic derivation which
may be compared with the operculum.

The proton of the columella appears first as a cord of cells
extending from the squamosum to the region of the fenestra,
while the plate filling this opening appears later. The develop-
ment of these structures in Spelerpes is much retarded as com-
pared with Amblystoma. In the latter, embryos 4 mm. long
possess a well-defined columellar cord in its typical position and
relations, while in Spelerpes bislineatus embryos 8 mm. long
this whole region is filled with undifferentiated mesenchyme. In
embryos 10:mm. in length the columella is discernible as a dense
cord which bears a significant relation to the hyomandibular
cleft which is seen as a very pronounced continuation of the
oral epithelium reaching beyond the ventral border of the ear
capsule (fig. 2), but the double folds do not separate to form a
cavity. Just above the dorsal end of this cleft the proton of the
columella may be seen as a dense mass of cells which, farther
caudad, extends ventrad toward the fenestra, where it ends
abruptly. In all of its relations it occupies the typical position
above the facial nerve and between the artery and vein of this
region. The columella at this stage is a dense segregation of

SOUND-TRANSMITTING APPARATUS OF CAUDATA 331

cells which comes into close relation with the caudal side of the
hyomandibular cleft, a relation which is not lost until late larval
life. The ear capsule is clearly indicated by distinct and closely

are U TORE

Ay oR
OY

[ono os

‘ a

5.50

WES

Fig. 2 Transection of the fenestral region of an embryo Spelerpes bislineatus
10mm. long. A.c., arteria carotis interna; A.e., auditory epithelium; Br., brain;
Ep., epidermis; H.c., hyomandibular cleft; O.c., oral cavity; O.ep., oral epi-
thelium; V.p.l., vena petrosolateralis.

associated cells and the facial nerve and its branches may be
distinctly traced.

In larvae which have reached a length of 15 mm. the columellar
cord of cells extends caudad to the cephalic part of the fenestral
membrane, against which it rests, but to which it is not closely

ee H. D. REED

connected. From embryos 10 mm. long to the time chondrifi-
cation of the columellar rod begins, it undergoes a decided re-
duction in diameter. It, however, retains its connection with
the vestige of the hyomandibular cleft through a sheet of fascia
which spreads out fan-shaped for attachment to the lining of the
oral cavity. The dense rod of undifferentiated cells, so appar-
ent in embryos of 10 mm., gives rise to both the columella and
the sheet of fascia just mentioned. The two are distinct only
as regards the more segregated nature of the cells which form
the columellar proton.

In specimens 17 mm. long only slight changes have taken
place over those conditions which obtain in the 15-mm. stage.
The columellar cord is slightly thicker and lies at times a little
closer to the fenestral membrane. Nothing, by way of struc-
ture or relations at this period of development denotes any
morphological difference between the sound-transmitting appa-
ratus of Spelerpes and that of Amblystoma.

Larvae from 19 mm. to 23 mm. in length are important as
showing the first step in the formation of the fenestral plate,
although considered apart from older stages, they are without
significance and indeed might prove misleading. The whole
history of development and morphology of these structures is
evident only after the consideration of a complete and carefully
selected series of stages. In the 19-mm. stage the fenestral lips,
in the ventrocephalic extent, through proliferation and growth
of their own cells, extend out into the fenestral membrane in the
form of a triangle, the apex reaching the level of the columellar
cord of cells. This growth of cells becomes the isthmus fenes-
tralis which, as mentioned above, serves as the connection be-
tween the ear capsule and the definitive fenestral plate, and, as
will be pointed out presently, constitutes the proton of the plate
itself (figs. 23 and 24).

From this stage to those 21 mm. long the only noticeable
changes are in the continued growth of the cells of the isthmus .
into the membrane and the elaboration of a cartilaginous ma-
trix. The columellar cord has not yet begun to chondrify and
there is no indication of a fusion of the two elements. The posi-

SOUND-TRANSMITTING APPARATUS OF CAUDATA Bos

tion, extent, and direction of growth of the isthmus should be
noted, in comparison with the columellar fusion with the cephalic
lips of the fenestra in Amblystoma. Figures 20 and 22 illus-
trate the differences. In Amblystoma the columella, which is
formed outside the ear capsule, comes to lie against the fenestral
membrane, grows toward and secondarily fuses with the entire
cephalic margin of the fenestra. In this case the columella
(fenestral plate) may be said to be the active element in the
fusions and the connections looked upon as extraotic tissue. In
Spelerpes a narrow region of fenestral lips grows into the fenes-
tral membrane, forming the isthmus fenestralis, which differs
from this fusion in Amblystoma in three respects: a) in extent
and location; b) the fenestral lips represent the active elements,
and, c) the tissues forming the connection are strictly otic in
source. The two, then, are in no respect alike. Figure 3 rep-
resents a section through the apex of the isthmus. Caudad of
this level it suddenly decreases in height to the normal level of
the fenestral lips.

In larvae 23 mm. long chondrification has occurred in the
columellar cord at the level of the apex of the isthmus. It rests
close against the fenestral membrane, but is not included within
its tissues. This represents the initial step in the formation of
the stylus columellae, which later, through chondrification, ex-
tends to the edge of the squaamosum. The isthmus, through
further growth into the fenestral membrane, reaches the dorsal
level of the stylus, and there are indications that a few cells at
the growing edge are about to extend further into the mem-
brane beyond the level of the stylus and independent of it. The
matrix of the two elements comes into contact. An important
observation in this respect is that the cells invading the fenestral
membrane from the isthmus lie entad of the stylus, and it is
due to the growth of the former that the final connection is
established. Thus here, as in the formation of the isthmus, the
otic tissue is to be considered the aggressive element. At this
stage the columellar cord and that portion which has chondri-
fied as stylus occupy those relations to the fenestral plate which
are maintained throughout life. It never enters more exten-

334 H. D. REED

sively in these relations, although increasing in size along with
the growth of the animal. The further development of the
fenestral plate is concerned with the growth and extension of the
original isthmus into all parts of the fenestral membrane. Fig-
ures 23, 24, 25, and 26 show the early mode of invasion. When
first formed, the dorsal growth of the isthmus is triangular, with
the apex reaching dorsally toward the stylus columellae, entad
of which it has a tendency to pass. In the 28-mm. larva this
cartilage has extended along the chondrified stylus, both caudad
and cephalad, until the triangle is reversed in position (fig. 24).
During this same period it has invaded the membrane entad
of the stylus, against which the latter lies, and in one specimen it
reached a level dorsad of that of the stylus.

The plan of growth is still more clearly outlined in the 31-mm.
stage where the fenestral plate has spread for a considerable
distance into the membrane in the dorsal and caudal directions
(fig. 25). When the stylus joins the plate the cartilage of the
two, in the posterior extent of the stylus, comes in contact, and
fusion takes place, though both may be distinguished by the
size of the cells or by staining reactions or both. While new
cartilage is being formed all about the free edge of the plate,
growth is not uniform. There are two points, one above, the
other below the stylus, where growth is most active. This
results in two chondrified rod-like structures extending caudad
into the membrane, as shown in figure 25. In this particular
specimen, these rods had a tendency to enclose an unchondrified
area of the fenestral membrane. In their caudal growth the
rods have nothing to do with the stylus or with the cells pro-
liferating from it. This condition is perhaps better illustrated
by transections. The growth from the original invading isthmus.
itself is shown in figure 4 (d.g.) of a section taken at a level
marked s in figure 25. It also shows the independence of the
columella and fenestral membrane at this level. A_ section
through the caudal half of the fenestra (fig. 5), at a level marked
c.s. in figure 25, shows the curved lower bar of the developing
plate composed of cartilage cells within the fenestral membrane,
which contrasts with the location of the cells of the columella in

SOUND-TRANSMITTING APPARATUS OF CAUDATA 335

Fig. 3. Transection through the cephalic portion of the fenestra vestibuli of
a larval Spelerpes bislineatus 20 mm. long. C.l., canalis lateralis; d.g., lips of
the fenestra vestibuli growing dorsad into the fenestral membrane to form the
isthmus fenestralis and to contribute to the formation of the fenestral plate; Ec.,
ear capsule; F.em., fenestral membrane; Hy., hyoid; O.c., oral cavity; V.h.c.,
vestige of hyomandibular cleft.

Fig 4 Transection through the fenestra vestibuli of a larval Spelerpes bis-
lineatus 31 mm. long. Col., columellar stylus; d.g., advancing edge of the isth-
mus fenestralis; Ec., ear capsule; Fe.m., fenestral membrane; 0.c., oral cavity ;

_O.ep., oral epithelium; V.h.c., vestige of hyomandibular cleft.

H. D. REED

336

SOUND-TRANSMITTING APPARATUS OF CAUDATA 337

Amblystoma upon the outer surface of the membrane at a cor-
responding stage of development (fig. 7). Continuous growth of
the isthmus and the resulting bars produce the entire definitive
plate. Frequently isolated centers of chondrification appear
which fuse with other centers and all finally with the plate proper
to produce the definitive structure. With regard to the growth
of the fenestral plate into the membrane, one observation is
important: the advancing edge is thin and the newly formed
cartilage is always within the membrane and usually at its very
middle (fig. 6).

The original invading bars of cartilage and isolated centers
gradually extend in all directions and eventually meet and fuse.
In this way there is produced, in larvae of 43 mm., the con-
tinuous plate shown in figure 26 where the chief difference in
extent from that in the adult is its failure as yet to fill the whole
fenestra. The stylus columellae has the appearance and rela-
tions of a rod which is secondarily applied to the surface of the
fenestral plate rather than primarily a part of the latter’s own
substance. Even in the older stages where both elements are
ossified, the distinction is evident at certain levels through the
loose relations between the two elements (fig. 1).

The perilymphatic prominence is formed by the outpocketing
of the fenestral membrane long before it becomes chondrified,
but the M. opercularis is not in evidence until the transformation
period arrives.

Fig. 5 Transection through the fenestra vestibuli of a larval Spelerpes bis-
lineatus 31 mm. long. This section is caudad of that in figure 4. A.c., arteria
carotis interna; C.l., canalis lateralis; Hc., ear capsule; Fe.m., fenestral membrane;
Op., fenestral plate (operculum) forming within the fenestral membrane; V.p.l.,
vena petrosolateralis.

Fig. 6 Transection through the caudal portion of the fenestra vestibuli of
a larval Spelerpes bislineatus 31 mm. long. A.c., arteria carotis interna; Cl.,
canalis lateralis; Hc., ear capsule; Fe.m., fenestral membrane; Op., fenestral
plate (operculum); V.p.l., vena petrosolateralis.

Fig. 7 Transection through the fenestra vestibuli of a larval Amblystoma
punctatum 35 mm. long. A.c., arteria carotis interna; C.l., canalis lateralis;
Col., columella, plate-like and spreading over the outer surface of the fenestral
membrane; Ec., ear capsule; Fe.m., fenestral membrane; V.p.l., vena petroso-
lateralis.

338 H. D. REED

The further development of the fenestral plate is concerned
with its ossification and has little or nothing to do with its mor-
phology. As mentioned above, the stylus first chondrifies at
those points where it comes into contact with the fenestral
plate, and from here cartilage gradually extends cephalad until
the long dense cord of the columellar proton is transformed into a
cartilaginous rod, except for a short ligament which effects the
attachment to the squaamosum. Ossification in the stylus begins
at its fenestral end and proceeds toward the suspensorial attach-
ment, as did the chondrification, forming, at first, a shell of bone
about a cartilaginous core which gradually diminishes as the
bone increases in thickness. In the oldest adults studied car-
tilage still persists at the center.

Ossification of the fenestral plate begins on the ectal surface
at the base of the stylus. From this as a center bony tissue
spreads toward the periphery and finally the cartilage is entirely
replaced by bone, the formation of which gradually extends
from the ectal, through the plate, to the ental surface. In
Amblystoma the ectal and ental plates of bone are formed
independently. ,

From the foregoing paragraphs the morphology of the fenestral
elements in Spelerpes bislineatus is evident. A brief summary
of the morphology and development of these structures in
Amblystoma will serve as a basis of comparison in the two
forms. In Amblystoma the columella arises as a dense strand of
tissue, extending between the ventral edge of the squamosum
and the fenestral opening, opposite which the end of the cord
of cells expands as the first step in the formation of a plate-like
columella which is later completely to fill the fenestral opening.
Throughout the developmental period and late into larval life
its position outside the fenestral membrane is evident, as shown
in figure 7. In late larval life the cephalic and dorsal margins
of the plate become included within the membrane, prepara-
tory to fusion with the ear capsule. Whether this fusion is
effected by the active growth of the columella or of the lips of
the fenestra, has nothing to do with the morphology of the ele-
ment. Chondrification begins early, and gradually the plate of

SOUND-TRANSMITTING APPARATUS OF CAUDATA 309

newly formed cartilage is pressed against the fenestral mem-
brane and, through growth at its periphery, extends over the
ectal surface of the membrane and fills the fenestra. The pri-
mary fenestra being thus occupied at transformation, the second
element or operculum is cut out from the walls of the ear cap-
sule, which process forms and fills at one and the same time the
secondary fenestra. The stylus in Amblystoma begins as a
conical projection from the plate proper and extends toward the
squamosum, following the original cord of cells, the unchondri-
fied portion of which becomes the ligamentum squamoso-colu-
mellare. A comparison of the fenestral structures in Amblystoma
and Spelerpes is facilitated if two points are borne in mind:
1) that the columella of the former is entirely extraotic in origin
and, though plate-like, the whole structure is formed through its
own growth without the addition of tissue from other sources;
2) the fenestra is small and almost completely filled in the
mature larva by the columella. This influences, without doubt,
the method of development of the operculum which is a part of
the tissue of the ear capsule cut out into a distinct element.

In the method of the formation of the plate itself in Spelerpes
lies the explanation of the homology of this fenestral element in
the Plethodontidae and others to be mentioned later. The
primitive cord of cells extending from the ventral edge of the
squamosum to the fenestra possesses the same relations to facial
nerye and to the arteria carotis and the vena petrosolateralis as
in Amblystoma. This structure represents the columella of
Amblystoma, but does not spread out into a plate when it
comes in contact with the fenestral membrane. It remains a
eylindrical rod taking no part in the formation of other struc-
tures in the sound-transmitting apparatus. It alone is extraotic
in origin, and therefore represents the columella of Amblystoma.
During development it becomes attached to the fenestral plate
secondarily. The plate itself results from the growth of the
isthmus fenestralis into the membrane and is, therefore, com-
posed of cells derived from the ear capsule, and hence represents
the operculum of the Amblystomidae.

340 H. D. REED

While the development of the sound-transmitting apparatus in
Spelerpes bislineatus shows many differences of detail from that
of Amblystoma, it conforms, nevertheless, to the statement
made by Kingsbury and Reed regarding the nature of this
apparatus in urodeles generally. That is, there are two distinct
elements present. The growth of the fenestral plate into the
membrane from the lips, or its independent formation there,
does not argue against its interpretation as operculum. In this
connection one observes that in Triton and Diemictylus the
operculum is formed partly by the cutting-out process and
partly by growth of the cartilage into the membrane. The
caudal part of this plate in Spelerpes possesses the same rela-
tions to parts of the internal ear as the operculum of Ambly-
stoma. Furthermore, some time before the formation of car-
tilage in the caudal portion, the membrane bulges out in a
fashion characteristic of the perilymphatic prominence of the
operculum. It should be noted, too, that the extreme cephalic
portion of the fenestra is not filled by the plate. This is evi-
dent in the drawing of the model (fig. 26). Perhaps a distine-
tion should be made between the cephalic and caudal halves of
the plate, but since it is composed entirely of tissue that belongs
to the ear capsule itself, the term ‘operculum’ will be employed
for the entire structure less the stylus which is morphologically
‘columella,’ as stated above. To the extent that the stylus at
its caudal end becomes joined to the fenestral plate, this single
element in Spelerpes represents a-fusion of columella and oper-
culum. A fusion of these elements in Amblystoma would result
in an equal contribution to the plate on the part of both columella
and operculum, while in Spelerpes the columella takes no part in
the formation of the plate whatever.

There is a peculiar relation between the sound-transmitting
apparatus and the hyomandibular cleft in Spelerpes bislineatus
which should be mentioned. This relation exists from the very
beginning of the columella to the assumption of terrestrial life.
Mention has already been made of the dorsal extension of the
hyomandibular cleft throughout embryonic life and its relation
to the columellar proton. With the beginning of larval life the

SOUND-TRANSMITTING APPARATUS OF CAUDATA 341

dorsal extension of the cleft is reduced to an open and slight
evagination of the oral epithelium toward the margin of the fenes-
tra, where it takes up a position between the artery and vein
and comes into actual contact with the perichondrium of the
developing isthmus fenestralis and the fenestral membrane (figs.
8 and 9). At the same time it is connected by a thin sheet of
fascia with the columellar proton. In older stages it loses a
close connection with the fenestral plate, but retains its rela-
tions with the stylus columellae which has become chondrified
(fig. 4).

A comparison of Amblystoma and Spelerpes larvae with re-
gard to the relations of the fenestral elements and the hyoman-
dibular cleft appears as a favorable argument for the belief that
in Spelerpes the persistence and relations of the cleft are closely
associated with a function, aside from any morphological sig-
nificance which they may have. Without close observation and
experimentation it is difficult to form a definite opinion of what
that function may be. Judging from the nature of the rela-
tions, it might be of use, either as an aid in the apprehension
and deglutition of food, or in the detection of disturbances in
the water. Comparative evidence favors the latter. In Ambly-
stoma larvae no such relations between oral epithelium and
fenestral structures exist. Correlated with that condition it is
noteworthy that the columella in amblystomid forms not only
arises, but chondrifies, early in development, so that it is able
to function as soon as an active free life begins. On the other
hand, in Spelerpes the fenestral structures, especially the stylus,
are tardy in their development. The larvae are active free-
swimming organisms long before a well-defined and functional
columella places the inner ear in communication with the sus-
pensorium. ‘This, together with the early connection of the cleft-
vestige with the end of the growing isthmus and its later relation
to the stylus, are significant. Of further interest in this con-
nection are the observations of Bruner (’14) which would indi- —
cate that the water in the mouth forms a means for the trans-
mission of disturbances between the environment and the
inner ear. The larval period of this species is, at least, two

JOURNAL OF MORPHOLOGY, VOL. 33, NO. 2

Fig. 8 Transection through the fenestra vestibuli of a larval Spelerpes bis-
lineatus 23 mm. long. A.c., arteria carotis interna; E.c., ear capsule; Fe.m.,
fenestral membrane; O.ep., oral epithelium; Op., fenestral plate (operculum) ;
Sq., squamosum; V.h.c., vestige of the hyomandibular cleft; V.p.l., vena petroso-
lateralis.

Fig.9 Transection through the cephalic half of the fenestra vestibuli of a
larval Spelerpes bislineatus 19 mm. long. Cl., canalis lateralis; Ec., ear capsule;
Fe.m., fenestral membrane; Hy., hyoid; O.c., oral cavity; O.ep., oral epithelium;
V.h.c., vestige of hyomandibular cleft.

Fig. 10 Transection through the cephalic half of the fenestra vestibuli of an
adult Gyrinophilus porphyriticus. Cl., canalis lateralis; Col., stylus columellae
(columella); He., ear capsule; Fe.m., fenestral membrane; J.f., isthmus fenes-
tralis; Op., fenestral plate (operculum); Sq., squamosum.

342

SOUND-TRANSMITTING APPARATUS OF CAUDATA 343

years as compared with a few weeks in Amblystoma. During
this period they inhabit cool streams, with more or less current,
rather than stagnant pools, and are more buoyed up by the
water. During late larval life the relation of stylus and cleft
is gradually lost, so that at transformation all traces of former
connections have disappeared.

Spelerpes ruber. This species has not been studied with the
same detail as 8. bislineatus. Larvae from 41 mm. to adults
indicate that the relations and morphology of the fenestral ele-
ments are identical, except for certain specific variations. In
this species the stylus is placed farther dorsad and not quite so
firmly joined to the plate, a condition which foreshadows the
relation of parts in Gyrinophilus to be discussed next.

Gyrinophilus porphyriticus. Both the fenestral plate and the
fenestra itself in Gyrinophilus are nearly circular, in decided
contrast to the elongate structures of Spelerpes. The pletho-
dontid mode of ossification is followed. Although larval stages
were considered, an examination of older larvae and adults alone
is conclusive in the light of the development of these parts in
S. bislineatus. There is no pronounced perilymphatiec promi-
nence as such. The whole fenestral plate represents an out-
pocketing away from the fenestral lips which, on the dorsal side,
form a conspicuous ledge. The M. opercularis is attached at
the middle of the plate, from which there is a conical projection
into the muscle (fig. 32). The cephalic portion of the fenestra is
not completely filled, a similarity of detail which this genus
bears to Spelerpes. The space between the dorsal edge of the
plate and the lips of the fenestra is filled to a certain extent by
the dorsal growth of the plate itself, but to a greater extent by
the development of the above-mentioned ledge. The downward
and backward growth of the squamosal conceals some of the
space which persists in this region.

The relation of the stylus to the plate is a much less close
one than in any of the Spelerpes group. In the adult it is a rod
lying against the plate, and sections show that, although it
touches the plate, there is no fusion until the very tip end of the
stylus is reached. Figures 32 and 10 illustrate this point.

344 : H. D. REED

Although the general relations of the stylus are the usual ones
among urodeles, the course of the facial nerve deserves notice,
since it varies in certain details from those in other species, and
this variation is significant when compared with forms to be
discussed later. This nerve issues below the stylus, which is
normal. Figure 19 is a diagrammatic representation of the
course of the ramus jugularis VII in Amblystoma. It leaves
the main trunk just underneath the stylus and maintains a
horizontal course across the lower third of the columella and
operculum. In general, in its relation to fenestral structures, it
may be said to occupy a ventral position. The course of the
jugular branch in Gyrinophilus is illustrated in figure 22. Here
the R. jugularis VII leaves the main trunk in a dorsal direction
and keeps to a course along the dorsal edge of the fenestral
elements.

A complete description of the development of the sound-
transmitting apparatus in Gyrinophilus would be a needless
repetition after what has already been written of Spelerpes.
A larva 82 mm. long exhibits all of the essential features for an
understanding of the morphology of the parts under consideration.
There are shown by a comparison of figure 25 of Spelerpes and
figure 31 of Gyrinophilus. The latter shows the isthmus fenes-
tralis as present and in its normal position. The estate of the
plate as a whole at this stage leaves no doubt as to the method
of cartilaginous invasion of the fenestral membrane from the
isthmus. A dorsal-arm (D.a.) extends toward the stylus and a
ventral arm (V.a.) extends caudodorsad. The indented mar-
gins and fenestrae within the plate point to its formation by
growth and extension of the original invading arms and their
fusion with isolated areas of independently formed cartilage
within the fenestral membrane itself. The loose relation of the
stylus and plate in the definitive state is here accounted for;
only the extreme caudal end of the stylus comes into contact
with the plate early enough to admit of fusion, both because of
its morphological distinctness and an unchondrified area in the
fenestral membrane underneath it which persists into late larval
life. The long larval period here, as in Spelerpes, is associated
with tardiness in the development of these parts.

SOUND-TRANSMITTING APPARATUS OF CAUDATA 345

The morphology of the sound-transmitting apparatus in
Gyrinophilus is obviously the same as in Spelerpes.

Manculus quadridigitatus. In this species, so far as one can
judge from a study of the adult only, the plethodontid type of
sound-transmitting apparatus prevails. It appears that the
dorsal arm from the isthmus extends scarcely dorsad of the
stylus and that independent islands of cartilage do not form
during the developmental period, so that the membrane is
somewhat free in its dorsal extent.

Hemidactylium is similar in all respects to Manculus. The
stylus, although firmly coossified with the fenestral plate, exhibits
a loose morphologic relation, recalling those in Gyrinophilus.
The two elements touch each other for a short distance and
bony tissue forms between them, but the identity of each is clear
at all levels.

Batrachoseps. Among all the Plethodontidae examined Ba-
trachoseps is unique in its sound-transmitting apparatus. Of
the three characteristic features of this apparatus in the Ple-
thodontidae only one is evident in the adult. There is not the
slightest suggestion of a stylus or of a suspensorial connection
through a special cord of fascia. The isthmus fenestralis is
absent, leaving the plate freely suspended in the membrane.
There is, however, a well-developed M. opercularis. Further-
more, the plate is relatively short in its horizontal diameter.
Except for features of ossification in the capsule and plate itself,
the whole region might easily be identified as belonging to an
amblystomid form in which only the operculum is present.
Judging the structure and relations of this element from the adult
only, it appears to represent the definitive operculum of Ambly-
stoma. If this be true, its whole estate should be looked upon
as the parallel of what occurs in Triton and Diemictylus. The
available evidence points to such a conclusion. The relation
of this element to other cranial structures favors its designation
as operculum, although these relations might vary with such
changes as the elongation, flattening, or shortening of the head.
One relation which is here to be more relied upon is that with
parts of the internal ear. A survey of the position of the colum-

346 H. D. REED

ella and operculum, or their corresponding parts, in the whole
series of caudate forms reveals a fairly uniform relation of the
sound-transmitting organs to parts of the membranous ear. The
operculum occupies a position directly opposite the caudal ele-
ments of the inner ear. It never extends farther cephalad than
the caudal half of the lagena cochleae, thus including the extreme
caudal extent of the sacculus only. Cross-sections in some cases
do not contain the sacculus at all. The whole plate in Batra-
choseps attenuatus is entirely caudad of the sacculus. It appears
evident that the columellar element is absent in this form, and
that which is present represents the operculum of other forms.
The uniform conditions and mode of development of the fenes-
tral structures in the Plethodontidae would not suggest a mode
of development and morphology different in fundamentals for a
particular genus. Taking this view, it appears that the columella
(stylus) has failed to develop or at least to reach the definitive
state. The slight relations of the stylus and plate in all pletho-
dontids are in line with such an explanation. Batrachoseps is a
strictly terrestrial species. If, as is the case with others of its
family living under similar conditions, the larval state is passed
within the egg, there is no need of a columella, and quite natu-
rally it should disappear. In the adult a strong cord of tissue
extends from the ear capsule between the artery and vein of the
region to the squamosum which may represent the vestigial
columella. The absence of the isthmus points to cartilage
formed within the fenestral membrane as the source of the plate
which becomes functional in the adult.

If these deductions prove true, then Batrachoseps represents ~
the extreme in specialization of sound-transmitting apparatus
among the Plethodontidae.

FAMILY DESMOGNATHIDAE

Desmognathus fusca, of all the plethodontid types studied,
shows most clearly the independent origin and definitive state
of the stylus and fenestral plate. In the morphology and rela-
tion of parts there is perfect agreement with Spelerpes, and the
development is such that no doubt is left with regard to the

SOUND-TRANSMITTING APPARATUS OF CAUDATA 347

significance of structure. In certain minor details Desmognathus
resembles Gyrinophilus more than other Plethodontidae, but in
others it reflects conditions found in Plethodon. Concerning
Desmognathus, Kingsbury and Reed wrote: ‘“‘An examination of
young larvae and embryos which would determine the origin or
origins of the stylus and fenestral plate has not been undertaken.
The mode of insertion of the stylus upon the fenestral plate,

might suggest that the stylus alone developed out-
side the otic capsule as the description of Parker would indicate.”’
A study of the development substantiates this statement and
proves the correctness of Parker’s views. As detailed a study
as possible of Desmognathus was undertaken, because, at the
time the work was begun, it was believed to represent a distinct
family. A careful consideration of the group is justified, since
it has strengthened conclusions regarding the relationships of
this family, to be mentioned later, and aids in interpreting the
general nature of these parts in Spelerpes, where they are found
in amore generalized state.

An examination of the insertion of the stylus upon the plate
gives the impression of a knob pressed into plastic material to
which it adheres. In medium-sized adults the two structures
are easily distinguished in sections, as shown in figure 11. The
columella (stylus) arises in the typical way as a cord of cells in-
dependent of the ear capsule. It is not yet chondrified in larvae
26 mm. long. The cord of cells, however, is very distinct (fig.
12) and lies against the fenestral membrane for about 40 u, when
it ends abruptly. In this stage the isthmus fenestralis has ex-
tended some distance into the membrane, and isolated centers
of chondrification, consisting of scattered cells, appear in the
middle and caudal parts of the membrane. These cells are
formed within the membrane, with no relations to extraotic ele-
ments. Figure 13, from a transection 40 » caudad of the end of
the columellar cord and through the apex of the isthmus, shows
the position and relation of the cells just mentioned. Still fur-
ther caudad chondrification has begun at several distinct places
(fig. 14). The areas of cartilage are not yet associated with
others at any level, but show growth in every direction and the

348 H. D. REED

SOUND-TRANSMITTING APPARATUS OF CAUDATA 349

“formation of a matrix. In larvae 27 mm. long the stylus is
chondrified for nearly its whole length and is loosely joined to the
_eartilage of the rapidly forming plate.

The origin of the elements in Desmognathus is identical with
those of the Spelerpes group, but the réle of the isthmus is not
-so important. Figure 30 illustrates the mode of development.
The isthmus, instead of extending directly upward and sending
_out both dorsal and caudal arms, thus effecting a union between
-it and the stylus, sends out only the caudal arm which grows
. diagonally upward and backward, never coming in contact with
‘the stylus at. all. Sooner or later it meets isolated centers of
_cartilage with which it fuses, and these centers in turn fuse with
each other, thus establishing a band of cartilage between the orig-
inal isthmus and the independently formed stylus. This band,
-when finally completed by the fusion of isthmus and isolated
centers, extends to the caudal margin of the fenestra, where it
bends upon itself, sending a bar of cartilage cephalad to join the
end of the stylus as mentioned above. The place of fusion is
marked F in figure 30. The fenestral bar is well outlined in
larvae 26 mm. long, but an unchondrified space exists between
it and the columellar cord of cells. Gradually these spaces are
filled by growth, but certain areas remain open until filled by
bony tissue.

By way of summary, it may be stated that in Desmognathus
the plan of development and morphology of parts conform strictly

Fig. 11 Transection through the fenestral plate and stylus of an adult Des-
mognathus fusca. Col., stylus columellae (columella); Op., operculum (fenestral
plate).

Fig.12 Transection through the fenestra vestibuli of a larval Desmognathus
fusca 26 mm. long. Col., columellar proton; Ec., ear capsule; Fe.m., fenestral
membrane; Sqg., squamosum. :

Fig. 13 Transection through the middle of the fenestra of a larval Desmog-
nathus fusca 26 mm. long. Ec., ear capsule; Fe.m., fenestral membrane; J./.,
isthmus fenestralis; Op., isolated cartilage cells which through growth and fusion
with others form the fenestral plate or operculum.

Fig. 14 Transection through the fenestra of a Desmognathus fusca larva at
a level slightly caudad of that of figure 13. Length of specimen, 26 mm. Ec.,
ear capsule; Fe.m., fenestral membrane; Op., fenestral plate (operculum) formed
by the fusion of isolated areas of chondrification.

350 H. D. REED

to those of Spelerpes, except that the fenestral plate is formed
in a less degree from the invading isthmus and more from car-
tilage appearing independently within the fenestral membrane
itself. Other genera of the family Plethodontidae have been
studied and found to agree in all fundamentals with Spelerpes.
As comparisons with Desmognathus and Spelerpes the observa-
tions of Peter (98) are both interesting and significant. His
studies were made upon Ichthyophis glutinosus, one of the Gym-
nophiona. He concluded that the sound-transmitting apparatus
in this species represents two distinct components, one otic, the
other extraotic in origin. The latter is first laid down as a con-
tinuous and dense cord of cells extending from the quadrate
cartilage to the fenestral region. Chondrification begins at the
fenestral end and proceeds toward the quadrate. The fenestral
element in the stages figured by Peter is joined, to the stylus,
although in earlier stages the two are distinct according to de-
scriptions. In the adult state, as shown by the descriptions and
figures of the Sarasins (’87 to 793), the plate completely fills the
fenestra. Peter concludes that the element in the fenestral mem-
brane is to be likened to the operculum, and that the lateral pro-
jection or stylus represents the columella of urodele types. With
regard to the presence of two elements in Ichthyophis and their
independent origin, there is complete agreement with the Caudata.

FAMILY AMPHIUMIDAE

The fully formed fenestral element in Amphiuma is of the
single-plate type with a large stylus. A typical isthmus fenes-
tralis is present. Kingsbury and Reed described this structure
in the adult, and according to material available at that time,
were unable to make any statement regarding homology other
than that the plate appeared to represent the columella. More
recently additional material has been examined revealing new
facts which should be presented. For the nature of the plate in
the definitive state, see figure 29.

Embryos of a given stage in development of this species are
relatively much longer than those of other urodeles, and the
length of an embryo or larva does not serve as an index of devel-
opment when compared with other urodeles.

SOUND-TRANSMITTING APPARATUS OF CAUDATA 351

The youngest stage studied was an embryo 30 mm. long, in
which the ear capsule is completely chondrified. The sound-
transmitting apparatus presents a condition not met with in
other urodeles. ‘The stylus is present and chondrified through-
out its whole length while as yet no fenestral plate is formed (fig.
27). <A section through this region (fig. 15) shows the chondri-

Fig. 15 Transection through the fenestra vestibuli of an embryo Amphiuma
means 30 mm. long. Ac., arteria carotis interna: Cl., canalis lateralis; Col.,
stylus columellae (columella); Hc., ear capsule; Fe.m., fenestral membrane;
Vp.l., vena petrosolateralis.

fied stylus lying along the fenestral membrane, which here, as at
all levels, entirely lacks cartilage. The progress of chondrifi-
cation, so far as direct observation is concerned, remains un-
known, since stages of the proper age were not available. The
stylus extends only a few microns along the fenestra. Two
series of embryos 33 mm. long (which vary as regards advance
in development) give a clue as to one mode of plate formation,

Suz, H. D. REED

that of the growth of cartilage from the stylus out upon the mem-
brane of the fenestra. Growth is confined entirely to the dorsal
side of the stylus and its tip end. A comparison of figures 15
and 16 illustrate this point. In the former is the very beginning

Fig. 16, Transection through the fenestra vestibuli of an embryo Amphiuma
means 30 mm. long, but more advanced than that from which figure 15 was drawn.
Ac.,farteria carotis interna; Col., stylus columellae (columella) spreading slightly
over the outer surface of the fenestral membrane; Ec., ear capsule; Fe.m., fenes-
tral membrane; Sq., squamosum; V.p./., vena petrosolateralis.

of this growth} where, in the section sketched, only one cell is
shown pushing out’ toward the membrane. Figure 16, which is
more advanced, shows that the diameter of the stylus is much
increased, all of which is due to the extension of its own substance
over the surface of the membrane. To what extent the fenestral

SOUND-TRANSMITTING APPARATUS OF CAUDATA aoe

plate is formed by such growth cannot be definitely stated, but
certain limits for its contribution may be judged by two obser-
vations upon a young adult 85 mm. long in which the plate is
not yet completed. The first point to be noted is that the stain-
ing reaction of the stylar and fenestral cartilage is different and
apparently consistent in each. From this it appears that only
a small portion of the plate surrounding the end of the stylus is
formed by the spreading of the stylar tissue over the fenestral
membrane and this only about the very end of the stylus. The
second observation of importance is that concerned with the
connection of the fenestral plate and otic capsule. An isthmus
fenestralis is present, typical in location and relations.

In urodeles there are only two different connections between
the fenestral plate and the margin of the fenestra. One of these
is typified by the Amblystomidae, the other by the Plethodont-
idae, both described above and determined by development. In
the amblystomids the connection is produced by growth from the
plate toward the ear capsule, in the latter by growth from the
ear capsule into the fenestral membrane. The two are distinct
as regards location and mode of development and are consistent
within the groups in which they obtain. The evidence, so far as
it may be considered as such, favors the conclusion that the isth-
mus in Amphiuma is of the plethodontid type. Further evi-
dence for this view is found in the incomplete state of the plate
in the young adult studied. The band of cartilage below the
stylus (fig. 28, V.a.) represents the growing ventral arm from the
isthmus, while that above is to be considered the dorsal arm as
in Spelerpes. Except for the few developmental observations
actually made and the conclusions deduced from comparisons
with other urodeles, the columellar nature of the fenestral plate
in Amphiuma would not be questioned. All that has been gained
from the present study forces the conclusion that the sound-
transmitting apparatus of this species resembles the plethodontid
type more than the amblystomid type, and in this respect Am-
phiuma must be viewed as intermediate between Necturus and
the Plethodontidae. |

354 H. D. REED

FAMILY NECTURIDAE

The sound-transmitting apparatus has been discussed at suf-
ficient length in another communication (Reed, 715); therefore,
only a summary is given as an aid to comparisons made later
in this paper. The columella appears in the usual fashion out-
side the ear capsule and the proton comes to lie for a short dis-
tance along the fenestral membrane. As chondrification advances,
the tip of the stlyus spreads over the fenestral membrane, but
to a very limited extent as compared with Amblystoma. Ap-
proximately a third of the plate, located in the cephalic part of
the fenestra, is formed in this manner. This area is triangular
in outline, the base being applied to the cephalic lips of the fen-
estra while the apex is on a level with, and extends slightly be-
yond the stylus. Other parts of the plate are formed by carti-
lage, produced within the membrane itself, and during growth
it becomes joined to that proliferating from the stylus (fig. 33).
The freedom of the definitive plate from the lips of the fenestra
and its formation partly from extraotic and partly from otic
tissues stamp it very decidedly as an intermediate in comparison
with the amblystomid and plethodontid types.

FAMILY TYPHLOMOLGIDAE

The general estate of the sound-transmitting apparatus in
Typhlomolge has been described by Kingsbury and Reed. The
serial sections of the specimen 95 mm. long have been carefully
reexamined in the light of what has been gained through develop-
mental studies of other species. The presence of an isthmus fen-
estralis indicates that the fenestral plate is of the plethodontid
type. In connection with this, as the best available evidence,
certain features are mentioned here for whatever value they may
have in a significant way. These features could not be depended
upon were it not for the consistent rdle of the isthmus in all
cases where it has been possible to trace its development. The
isthmus, projecting into the fenestral membrane, very soon ex-
tends underneath a bony plate to which it becomes attached
through cartilage cells, as shown in figure 17. The cartilage of

SOUND-TRANSMITTING APPARATUS OF CAUDATA 355

the isthmus is within the membrane, while that of the small
plate rests against it. These are the relations which should ob-
tain, respectively, for elements of otic and extraotic origin. In
following sections caudad, this relation is found to persist. The
extension of the extraotic part of the plate in the dorsal direction
is only slightly above the base of the stylus. Figure 18 shows
this feature and also that the stylus appears to spread out

Fig. 17 Transection through the fenestra vestibuli of an adult Typhlomolge
at the level of the stylus columellae. C.l., canalis lateralis; Col., stylus columel-
lae (columella); Hc., ear capsule; Fe.m., fenestral membrane; Op., fenestral plate
(operculum); Sg., squamosum.

Fig. 18 Transection of the ear region of Typhlomolge, passing through the
fenestra vestibuli caudad of the level of the stylus columellae. C.l., canalis
lateralis; Ec., ear capsule; Fe.m., fenestral membrane; J.f., isthmus fenestralis,
continuous with the lips of the fenestra; Op., fenestral plate (operculum), the
free membrane between it and the dorsal margin of the isthmus representing the
space into which the columella failed to spread; Sq., squamosum.

slightly as a funnel-shaped element. Taking together what evi-
dence there is, both direct and deduced, it appears that the fen-
estral plate is double; the stylus and a small portion of the plate
being extraotic, and, therefore, columella, while the isthmus and
bulk of the plate represents tissue formed within the membrane
and, therefore, otic in nature. In principle it agrees with the
plethodontid type.

356 H. D. REED

FAMILY SIRENIDAE

The possible conclusions regarding the fenestral elements in
Siren are based to a minor degree upon circumstantial evidence,
which, however, carries weight with one who has reviewed these
structures in the entire series of caudate amphibia. The known
facts may be briefly summarized. ‘There is only a single element
in the fenestra, and this is not connected with either quadrate
or squamosal. In individuals up to 215 mm. in length the plate
is connected with the ear capsule in the dorsal part of the fen-
estra (fig. 34). This connection is different from the cephalic
connection of the columella in the Amblystomidae (fig. 20) and
the ventrocephalic relations of the plate in the Plethodontidae
(fig. 22). In a specimen 440 mm. long (undoubtedly a fully ma--
tured individual) the fenestral plate is free from any cartilaginous
or bony connections with the-ear capsule whatever (fig. 35).
Cope (’88), writing of this structure, observes: ‘‘In -Siren the
stapes is osseus. Its columella is replaced by the stapedius
muscle which extends posteriorly.”’ In view of these facts, the
origin and nature of the fenestral element in Siren becomes clear.
Its freedom from the ear capsule in the definitive state, its lack
of connections with the suspensorium, and the presence of the
M. operculare (stapedius muscle of Cope) identify it as the homo-
logue of the operculum of the amblystomid forms. Not only
does it possess the relations of the operculum, but it is formed
from the walls of the ear capsule by the ‘cutting out’ process as
described by Kingsbury and Reed for this element in Ambly-
stoma. In a specimen 133 mm. long (fig. 34) the ‘uncut’ por-
tion of the opercular margin is coextensive with its width, and
there are only slight relations with the M. operculare. Serial
sections of the head of a specimen 215 mm. long show the uncut
portion very much reduced, with the M. operculare firmly at-
tached to the side of the plate along with the strong ligamentum
hyocolumellare.

There is no evidence as to the fate of the columella in this
species. In others, where this element has become functionless
in adult life, it has fused with the cephalic lips of the fenestra,

SOUND-TRANSMITTING APPARATUS OF CAUDATA are

though retaining its stylus. In those where total disappearance
obtains it has come about through loss of stylus and complete
incorporation with the ear capsule. Inferentially, this accounts
for the complete effacement of the columella in Siren. Leaving
inference, however, out of the question, and drawing upon the
known facts only, it becomes obvious that the fenestral plate in
Siren is identical with the functional operculum of the adult
Amblystoma and, being such in its morphology and the apparatus
as a whole lacking the columellar portion, it argues for the simi-
larity of the sound-transmitting apparatus of Siren to that of
Triton and Diemictylus.

SUMMARY

A general survey of the sound-transmitting apparatus in the
tailed Amphibia reveals the existence of two morphological types.
One has been mentioned as the amblystomid type since it is
found in its most generalized state in the Amblystomidae. The
other is typified by the Plethodontidae and may, therefore, be
termed the plethodontid type. In the perfect amblystomid type
two distinct elements are present in the completed apparatus.
One of these is columella which is extraotic in origin. It is com-
posed of a stylus which joins the suspensorium at its cephalic
end and at its caudal end spreads out over the fenestral mem-
brane, forming a plate. This structure serves during larval life.
At transformation the columella fuses with the ear capsule, and
a new element, the operculum, is cut from the walls of the ear
capsule to function during adult life. It occupies a position
caudad of the columella and comes into connection with the ceph-
alic end of the M. opercularis. The’variation from the perfect
or generalized type results from a loss of function of the colum-
ella when the terrestrial existence is assumed. The Amblystom-
idae and Salamandra possess this type in its most perfect state.
The identification of the type resides in the operculum, it being
the constant and consistent one of the two elements. In the di-
rection of Triton and Diemictylus the columella develops, but
soon completely disappears through fusion with the ear capsule,
leaving only the operculum as a distinct element. So far as there

JOURNAL OF MORPHOLOGY, VOL. 33, NO. 2

358 H. D. REED

is any evidence, the only element in Siren is the operculum, the
general state of affairs resembling those of Triton. The single
element in Cryptobranchus is the columella, formed by the
growth of the stylar portion over the fenestral membrane in true
amblystomid fashion. The failure of the operculum to appear
in this form is due probably to its aquatic life.

That which has been taken as the plethodontid type of sound-
transmitting apparatus is exemplified by Spelerpes. The plate
filling the fenestra is single and possesses three extrinsic connec-
tions: a) one with the suspensorium through the stylus; b) one
with the shoulder-girdle through the M. opercularis, and, c) one
with the ear capsule through the isthmus fenestralis. The plate
itself is formed by both the growth of the isthmus into the fen-
estral membrane and the independent formation of cartilage
within the substance of the membrane which meets and fuses
with that of the invading isthmus. Figures 23, 24, 25, and 26
illustrate the mode of formation. The stylus is the only repre-
sentative of what in the amblystomid type is columella. Varia-
tions from this type in its perfect state, as Just described, are
found in Necturus, Typhlomolge, and Amphiuma, where the
stylus expands, forming a varying portion of the plate itself.
In Batrachoseps, where the stylus (columella) has disappeared
entirely, the isthmus is likewise absent, leaving a plate which is
free from the ear capsule and which represents, apparently, one
derived from independent cartilage formed in the membrane of
the fenestra.

The characteristic features are the distinctness of the two ele-
ments, columella and operculum, in the amblystomid type and
the fusion of the two in the plethodontid type as well as a differ-
ence in the exact mode of origin.

THE PHYLOGENETIC SIGNIFICANCE OF THE SOUND-
TRANSMITTING APPARATUS

The following discussion is presented with a twofold object:
first, to outline the history of the tailed amphibia as one reads it
in the morphology of the sound-transmitting apparatus, and,
second, to emphasize the zoological bearing of the subject as a

SOUND-TRANSMITTING APPARATUS OF CAUDATA 359

means of interpreting the morphology of the various systems of
organs in these animals. The present discussion is not an
attempt, primarily, to meddle with the classification.

It has been the aim in most instances to employ the same tech-
nical designation for the various groups and species of tailed
amphibians as were used in previous publications upon this sub-
ject. An attempt on the part of systematists, however, to estab-
lish a stable nomenclature through the application of the rule
of priority, together with views gained from more recent studies
regarding the affinities of groups, have wrought many changes
in names. In order to avoid the possibility of confusion, a com-
parative table of former names and those now considered correct
is here introduced.

FORMER NAME

Proteidae
Pleurodelidae

Diemictylus

Amblystomidae

Amblystoma

Amblystoma punctatum Cope

Chondrotus tenebrosus Cope
Spelerpes bilineatus Cope

Autodax lugubris Cope
Desmognathidae

NOW EMPLOYED

Necturidae

Included with the Salaman-
dridae

Notophthalmus

Amblystomidae

Amblystoma

Amblystoma maculatum
(Shaw)

Amblystoma tenebrosum
Baird and Girard

Eurycea bislineata (Green)

Aneides lugubris (Hallowell)

Included with the Pletho-
dontidae

The studies embodied in the present paper, as well as its fore-
runners, have emphasized, in the mind of the writer, three
points: a) that much of the confusion which has existed respecting
the sound-transmitting apparatus of the Urodela has resulted
from fragmentary studies, and that a true morphological inter-
pretation of the structures in question is to be arrived at only
through a complete developmental study of each distinct type

360 H. D. REED

of auditory element found to exist in each taxonomic group; b)
that the possibility of neoteny, as the explanation of certain
features in such forms as Necturus, Cryptobranchus and others,
cannot be overlooked, and, c) that the morphology of these organs
has a significant bearing upon the relationships and descent of
the principal groups of caudates.

It is contended by some that structures so intimately associ-
ated in function with the habits of the animal are likely to be
affected by the environment and, therefore, unreliable in showing
affinities or tracing descent. It is true that these structures are
not only affected by the environment and needs of the animal,
but show a decided adaptation in this respect. There are prob-
ably few internal structures in animals which are not ultimately
affected by environment, and the differences of form and rela-
tions of internal parts are, to a considerable extent, a reflection of
the life and habits of the animal itself. Form and relation of
parts alone, therefore, do not always constitute reliable criteria
for passing judgment, either with regard to morphology or line-
age. It is exactly to this error that some of the confusion men-
tioned above can be traced. It has been apparent from the be-
ginning that, so far as this system of organs is concerned, the only
safe basis for homology resides in the principle that two struc-
tures, however much they may resemble each other in form and
function, are different, unless comprising the same combination
of elements. For example, the fenestral plate and stylus of Nec-
turus and Cryptobranchus, in form and skeletal relations, appear
identical. Development shows them to be entirely different in
the elements which combine to form them, and they are, there-
fore, not homologous structures and not indicative of close affin-
ity of the animals possessing them.

It is mentioned in the summary of the first part of this paper
that two morphologic types of sound-transmitting apparatus
occur in the tailed amphibia. When these types in the various
groups are reviewed with the above-mentioned principle in mind,
it will be found that the different families of Caudata become
grouped into two legions, each of which represents a line of de-
scent, judged by the nature of the variations of the combining

SOUND-TRANSMITTING APPARATUS OF CAUDATA 361

elements to form the definitive sound-transmitting apparatus.
The forms included within a given legion possessing a sound-
transmitting apparatus involving the same architectural prin-
ciples are considered as more closely related to each other than
to those of another legion. |

Since the M. opercularis occurs in more than half of the uro-
dele families, regard'ess of legion, and bears a direct relation to
the mode of life of the animal, it is to be considered as of physio-
logical rather than morphological import. This being its status,
it is obviously of no significance in a strictly phyletic consider-
ation. The whole question of descent and relationships of the
urodeles, so far as concerns the sound-transmitting apparatus,
hinges upon the elements composing the fenestral plate or plates
and their mode of origin. Thus, in the Amblystomidae, Sala-
mandra, Triton, and Diemictylus one finds the constant and in-
dependent operculum of the amblystomid type. They all agree
in this particular among themselves and differ from all other
groups. They may, therefore, be looked upon as of kin or having
descended from a common ancestral stem in which this morpho-
logical feature was well established and survived the vicissitudes
of change in such a way as to preserve its caste. Similarly con-
stant in this legion is the tendency of the columella to spread over
the outer surface of the fenestral membrane and form a plate
which is continuous with the stylus columellae. The variations
in the state of the columella in this legion appear to be correlated
with the extent to which the various forms have become terres-
trial. The tendency of the columella to fuse with the ear capsule
expresses the trend of modifications with regard to this element
in this legion. It is very apparent that the more terrestrial a
species becomes, or the longer the period in its descent during
which it has occupied the terrestrial zone, the more completely
is the columella fused with the ear capsule. The extreme in
this direction is found in Triton and Diemictylus, where complete
effacement of the columella has taken place. The tritons, it
will be noted, are terrestrial except at the breeding season. The
aquatic abode of Diemictylus viridescens in the mature state has
been secondarily acquired in more recent times, following a ter-

362 H. D. REED

restrial period of existence, as pointed out by Gage (’91). The
other extreme (the less modified) is found in the Amblystomidae
and Salamandra, the latter in many respects exhibiting features
which foreshadow the Triton state. The columella in this le-
gion, although fusing with the ear capsule in varied degrees,
shows, nevertheless, its characteristic spreading over the fenes-
tral membrane during development. The plate thus formed does
not fuse with the operculum or contribute to it in any way what-
soever. In no other caudates does this state obtain.

Although the columellar element in Siren is unknown, the mor-
phology of the operculum is sufficient for the inclusion of this
family among those of legion I. There are, however, certain
features of habit and morphology which seem to be at variance.
Thus it appears that the type of sound-transmitting apparatus.
found only in terrestrial urodeles is here present and well defined
in an aquatic species. It seems desirable, therefore, to intro-
duce whatever there may be of evidence upon this point gained
from other studies upon this species. Cope (’85) was the first to
point out that the present aquatic abode of Siren is secondary,
following a period of terrestrial existence. His conclusion was
based upon a tendency of the gills to disappear and become func-
tionless in specimens of a certain age, reaching full development
again only in large adult specimens. He writes: ‘The only ex-
planation appears to me to be that the present sirens are the de-
scendants of a terrestrial type of Batrachia, which passed through
a metamorphosis like other members of their class, but that
more recently they have adopted a permanently aquatic life, and
have resumed their branchiae by reversion.” In a later publica-
tion, Cope (’88) expressed the belief that he had found confirma-
tory evidence in the support of this view in the structure of the
ossicula auditus. The morphology of the sound-transmitting
apparatus as deciphered in the present study supports: Cope’s
belief for an operculum, of whatever mode of formation, in com-
munication with the shoulder-girdle, is associated only with a
terrestrial existence, and, if its function has been correctly inter-
preted, is useful in such an environment only. The period of
terrestrial existence of the antecedents of the present Siren must

SOUND-TRANSMITTING APPARATUS OF CAUDATA 363

have been extended and pronounced, for the columellar element,
which is useless on land, has disappeared without leaving a trace
in the adult. On the other hand, there are structural features
which argue that this secondary aquatic period has been long
enough to admit of certain readjustments of the sound-trans-
mitting apparatus to this type of abode. Extending between the
operculum and the hyoid is the extremely dense and large liga-
mentum hyo-operculare which places the inner ear in commu-
nication with the exterior as perfectly as could the columella
itself. This ligament is by far the largest and most pronounced
of any ligament in this region in any urodele. Although it is
attached to the operculum, and is given a name which indicates
its relations, it is quite likely that it represents the ligamentum
hyocolumellare of other urodeles. The extent and relations of
the general sheet of fascia in which it is formed admits of no
other conclusion. Siren is the only urodele in which the oper-
culum has such relations with the hyoid. The only interpretation
of its relations here seems to be that of compensating the loss of
the columella in transmitting disturbances from the surrounding
water to the inner ear.

It seems probable that the morphology of the sound-trans-
mitting apparatus in the Tritons and Sirens and their past history
are identical; but this does not necessarily argue for a close re-
lationship of these forms within the legion itself. Norris (’13),
in his work upon the cranial nerves of Siren, refers to strong sim-
ilarities between it and other urodeles, which is significant in
the present consideration in two connections: one bears evidence
in support of the view that Siren is not a primitive form, while
the other points to a close relationship with those urodeles which
comprise legion I as here constituted. Bearing upon this point
in particular, in his summary Norris writes: ‘The contribution
of maxillaris and buccalis fibers to the profundus palatine anasto-
mosis has such a closely corresponding arrangement in Triton
(Coghill) and also in Salamandra, if von Plessen and Rabino-
viez’s figures be correct, that it can hardly be explained as inci-
dental.” ;

364 H. D. REED

It has been stated above that the complete absence of the col-
umella in Siren points to an extended period of terrestrial exist-
ence further back in its phylogeny. A study of the general
anatomy of this form by H. H. Wilder (’91) led him to the same
conclusion. In summarizing his work he observes concerning the
phylogenetic relations of Siren: “I am fully convinced that it
has once possessed a terrestrial existence and been driven back
to an aquatic life during the struggle for existence, similarly to
the case of the Axolotl . . . But unlike the Axolotl, which
has simply repressed the later stages and represents still a fairly
typical larva, the Siren-form has been modified by the influence
of external conditions during a much longer period of time.

. .’ While the position assigned Siren in plate 6 is tentative,
although indicated by all of the available structural evidence,
its position within the legion seems unquestionable.

The case of the Cryptobranchidae, so far as the sound-trans-
mitting apparatus is concerned, must be adjusted by the evi-
dence offered by the columella alone, since the operculum is
wanting. The fenestral plate is single, possesses only suspen-
sorial connections, and is formed by the spreading of extraotic
material over the outer surface of the fenestral membrane, in
which respect it agrees with the columella of the amblystomids,
tritons, and Salamandra, and is at variance with every other
known group of urodeles. This feature, being in such sharp
contrast between the two great groups of urodeles, it very clearly
allies the Cryptobranchidae with the amblystomid division or
legion I. The position of this family within the legion is inter-
preted as a direct offshoot of the amblystomid stem.

The fenestral plate of the Cryptobranchidae with its suspen-
sorial connections only, is of the type found in aquatic species
or during the larval period of terrestrial forms and does not of
itself indicate whether these animals are primitively or second-
arily simple. During the transitional period from the fish-hke to
the terrestrial amphibians, the skeletal remains, and especially
the restorations, denote a lumbering mode of locomotion in
which the body was scarcely elevated above the substratum
upon which it probably rested during inactive intervals. What

SOUND-TRANSMITTING APPARATUS OF CAUDATA 365

these movements must have been may be appreciated by recall-
ing the waddling gait of a walking fish and the crawling gait of
a modern salamander. The transitional amphibian must have
possessed a mode of locomotion which would fall between these
two extremes, in which case the jaws and branchial apparatus
rested upon the substratum for a large portion of the time. The
lateral line sense becoming functionless as the animal left the
water, and hearing, if it existed in a refined state, certainly be-
coming much impaired, left these transitional amphibians with
no special means of communication between the inner organism
and disturbances which might occur in the surroundings. The
relation of the jaws to the substratum, through contact, and to
the auditory capsule, through the hyomandibular and its liga-
ments, formed a natural pathway for the transmission of vibra-
tions. Thus, for physical reasons alone, it can be understood
how the columella represents a refined hyomandibular and how
the fenestra vestibuli may have come about. The structural
supports for this view and the importance of such hyomandibular
relations as are found in the notidanid sharks were discussed by
Kingsbury and Reed (’09).

Accepting the general view that the larval period represents
a more recent interpolation in the life-cycle of amphibians, one
observes that the gait and relations of the body to the sub-
stratum of a modern larva is undoubtedly like that of the earliest
amphibian forms, and that the demands upon the columella of
the recent larvae are unquestionably no different from those made
upon this structure in the primitive estate when amphibians were
slowly evolving from fish-like forms. Since the columella was
the first of the sound-transmitting elements to appear in phy-
logeny, it is quite natural that it should persist and function dur-
ing the larval period of recent salamanders, although this period
is a later addition to the life-cycle. It follows, then, that, in
aquatic species such as the Cryptobranchidae the columella
alone does not indicate the phyletic relations of its possessor,
and one must look further into the morphology of these animals
in order to determine their true rank.

366 H. D. REED

Various studies of the Cryptobranchidae contribute informa-
tion which is suggestive in its bearing upon the rank and kinship
of this group. Versluys (’09), in summarizing the structure of
the Cryptobranchidae, observes that they retain many features
of a larva, and, at the same time, acquire many of adult sala-
manders. Thus the skull is that of the adult, while the circula-
tory organs approximate those of the larva. Versluys concludes
with the statement that the Cryptobranchidae are to be looked
upon as partly transformed larvae and an offshoot of the ambly-
stomid stem. That they are arrested in transformation is the
conclusion of Bruner (’14), based upon a study of the respiratory
mechanism. Wiedersheim (’77) and Driiner (’01, ’02) find that
both the skull and branchial apparatus of the Cryptobranchidae
bear striking resemblances to these structures in Raniceps and
Hynobius, which argues for an amblystomid alliance of this fam-
ily as shown by Wiedersheim in his phyletic arrangement of fam-
ilies. Herrick (’14), in a comparative study of the cerebellum
of urodeles, discovered some suggestive points which I interpret
as designating the Cryptobranchidae an offshoot of the ambly-
stomid stem rather than the reverse of these conditions. In one
instance Herrick writes: ‘‘The cerebellum of Cryptobranchus
occupies an intermediate position between those of Amphiuma
and Amblystoma.”’ The position given the Cryptobranchidae
in plate 6 is in accord with Herrick’s observation.

A review of the important features of morphology, develop-
ment, and distribution supports the view that the Cryptobranch-
idae are not primitive amphibians, but, as Smith (’12) has pointed
out in his conclusions, are terrestrial forms which have secondarily
become aquatic, in consequence of which certain larval features
are retained, although these forms advance far enough toward
their former state to actually begin transformation which is
arrested. The conclusions arrived at from a consideration of
the sound-transmitting apparatus are not in harmony with any
view which allies the Cryptobranchidae with any other than the
amblystomid group.

The sound-transmitting apparatus of the amblystomid legion
seems to the mind of the present writer to echo the past history

SOUND-TRANSMITTING APPARATUS OF CAUDATA 367

of all its members. During the early transitional period, when
the head must, for the greater part of the time, have rested upon
the substratum, the columella became perfected and served as
the communicating element between inner ear and jaws. As
amphibians became more and more terrestrial the head and
anterior end of the body was elevated above the substratum, in
consequence of the perfection in the use of the arms in terrestrial
locomotion. ‘This elevation of the head rendered the columella
functionless, and, a second time in their evolution, these animals
were left without direct communication between the outer world
and the inner ear. As a compensation for this loss a second
auditory element, the operculum, was cut out from the ear cap-
sule behind the columella; this appropriated a slip from the ad-
jacent musculature, thus coming into communication with the
shoulder-girdle and establishing a new coupling between the
inner ear and the substratum along which disturbances might
travel. The columella, being no longer of use, gradually fused
with the ear capsule, leaving only the operculum freely suspended
in the fenestra as the functional organ. The Amblystomidae
either live in their original type of habitat or have departed
only slightly from the terrestrial habitat of their ancestors, so
that more of the primitive features of the sound-transmitting
apparatus persist than in any other group. The Cryptobranch-
idae must have separated from the amblystomid stem fairly early,
since sufficient time has elapsed for the complete suppression of
the operculum. If this view be correct, the sound-transmitting
apparatus of the Cryptobranchidae is secondarily simple and the
Amblystomidae are, so far as the auditory structures are con-
cerned, the most primitive of living Caudata. It might be argued
that the Cryptobranchidae returned to an aquatic environment
before the operculum had become a functional part of the sound-
transmitting organs, in which event they would necessarily be
looked upon as primitive urodeles. The nature of their partial
transformation, however, and numerous features of structure do
not encourage such a conclusion.

After the separation of the modern Cryptobranchidae and Am-
blystomidae from the main stem of this legion, all the other uro-

368 H. D. REED

deles grouped here exhibit unmistakably the impression of ter-
restrial existence upon the sound-transmitting apparatus. The
state of these organs in Salamandra is a parallel for that in the
Amblystomidae, although a bit more inclined toward the terres-
trial type. The Tritons, Diemictylus, and Siren, whatever their
present-day habits, possess a type of sound-transmitting organ
that could have become perfected only in the terrestrial zone.
The columella has been a functionless structure for such a con-
tinued period that, in recent forms, it fails to produce a stylus or
a permanent communication with the suspensorium, and at a
very early period of development is effaced completely through
fusion with the ear capsule, leaving the operculum only to com-
municate with the exterior through the M. opercularis and the
arm. Diemictylus and Siren have returned to an aquatic abode
so recently as still to retain the sound-transmitting apparatus in
its former highly specialized state. They are, therefore, con-
sidered as having departed in this respect most widely from prim-
itive conditions, and accordingly represent the culminating
branches of the amblystomid stem or legion.

Legion IJ, the plethodontid group. The arrang:ment of this
group is not fraught with as many difficulties as the former, since
the morphological nature of the sound-transmitting organs is
such that the generalized and specialized states are easily de-
tected. This minimizes the difficulties of phyletic arrangement.

The distinctive peculiarities of the sound-transmitting appa-
ratus of this legion are found in the presence of a single, but com-
pound, fenestral element under all conditions of life and at all
stages of development. The stylus is always present and is con-
nected with the suspensorium. The variable element is the M.
opercularis, which is present or absent, accordingly as the animal
is terrestrial or aquatic in habit. The state of this muscle is
precisely that which obtains among the forms of legion I. In
the amblystomid legion the two elements, columella and oper-
culum, are always distinct so far as fusions between them are
concerned. In legion II, or that which is conveniently termed
the plethodontid group, representatives of the columella and
operculum fuse with each other in varying degrees, resulting in

SOUND-TRANSMITTING APPARATUS OF CAUDATA 369

a compound fenestral plate which is quite in contrast to the two
distinct plates of species composing legion I, where the columella
invariably fuses with the ear capsule when its function is reduced.
This compound relation of elements represents one which is ob-
viously a derivative from the more primitive estate of two dis-
tinct and simple elements and one which is reminiscent of a
pronounced terrestrial life far back in phylogeny.

That the Necturidae are neotitic forms, and that they rank
with others of the urodele order, has been pointed out by Kings-
bury (09), Norris (11), Bruner (714), and others. The morphol-
ogy of the sound-transmitting apparatus harmonizes with such a
view. This family is adjudged the most primitive of the legion
since the fenestral plate is an exact intermediate between others
of its group and those composing the amblystomid legion. The
columella, during development, spreads out over the surface of
the fenestral membrane so as to fill considerably less than half
of the window, and fuses with opercular tissue formed within the
fenestral membrane itself. This mode of formation reflects a
former terrestrial existence for the Necturidae and may be ex-
plained by a brief reference to the mode of development of fen-
estral elements in Amblystoma and Triton. In the former the
‘cutting out’ process in the production of the operculum is a
necessity resulting from the complete filling of the primary fen-
estra by the columella, which, in this form, comes into full func-
tion and proportions. In Triton the columella is so small be-
cause of its functionless state and it fuses so early with the lips
of the fenestra that it forms merely a rim of cartilage, thus leav-
ing a great portion of the primary fenestra free for the later in-
vasion of cartilage, or for its formation within the free membrane.
Consequently, in Triton, the operculum is produced, in part, by
the cutting-out process, and, in part, by the growth of cartilage
from the edges of the window into the fenestral membrane.
The ultimate effect of this trend of modification, then, is the
increase of free space in the primary fenestra, within which the
opercular element may develop, with no necessity of ‘cutting
out’ from the walls of the ear capsule. Thus one may account
for the mode of development and morphology of the fenestral

370 H. D. REED

plate in the Necturidae. As stated above, it points to a former
terrestrial existence for these forms, in consequence of which the
columella became reduced and fused with the rapidly forming
operculum, rather than with the ear capsule. The loss of the
M. opercularis is to be associated with the recently acquired aqua-
tic habit. There results, in the Necturidae, a single fenestral
plate which is perfectly free from the ear capsule and which bears
the outward form of that structure in the Cryptobranchidae,
but morphologically is quite different. The two families, judged
by these organs, are not closely related. The Necturidae are
considered as the most primitive of the plethodontid legion since
the columella spreads out over the fenestral membrane to a
greater extent than in any of the others of the group. This con-
dition is interpreted as reminiscent of the earliest type of sound-
transmitting apparatus. The family is, therefore, placed in plate
6 as an offshoot of the plethodontid stem, intermediate between
others of that group and the amblystomid legion. Such a re-
lationship is in harmony with the presence of lungs in the Nectur-
idae which they should not possess were they closer of kin to the
Plethodontidae. Especially significant in this connection are
Norris’ (’11) observations upon the cranial nerves, which are
said, in their entirety, to reach a rather high degree of speciali-
zation, and, in their arrangement, to resemble both the pletho-
dontid and amblystomid types and especially the former.

The other families of the plethodontid group represent branches
which originate from a main stem leading back toward the place
of departure. of the Necturidae. In these families there is a
gradual reduction of the columella from a state where it spreads
slightly over the fenestral membrane, to one where, as in the
Plethodontidae and Desmognathidae, it is represented by stylus
only and takes no part whatever in the formation of the plate,
to the surface of which it becomes joined. The extent to which
the columella extends over the fenestral membrane is taken as
the index of specialization. Applying this criterion, the Amphi-
umidae stand nearest the Necturidae, agreeing with them and
differing from the others in the possession of lungs.

SOUND-TRANSMITTING APPARATUS OF CAUDATA 371

The Amphiumidae have been variously placed with regard to
their kinship. Cope held tenaciously to the view that they rep-
resent a group intermediate between urodeles and the Apoda.
The cousins Sarasin (’87 to ’90) proceed a step further, and in-
clude the Amphiumidae among the Apoda. Kingsley (’02) made
a critical study of the various supposed similarities of these
groups and observes that it ‘would appear that some of them
[resemblances] are of minor value, some are based upon imperfect
knowledge or misconception, while some are false.’”’ Kingsley’s
study seems to have settled for all time the urodele affinities of
the Amphiumidae. With that much granted, the sound-trans-
mitting apparatus evinces an alliance with the plethodontid le-
gion, because of the single compound fenestral plate and the pro-
nounced isthmus fenestralis. Since the columella enters into the
formation of the fenestral plate to a lesser degree than in the
Necturidae, they must be considered an offshoot of the pletho-
dontid stem, between the Necturidae and those still more spe-
cialized with regard to this feature. Furthermore, the greater
reduction of the columellar element as a component of the fen-
estral plate in the Amphiumidae bespeaks a longer terrestrial
period for this family than probably existed in the case of the
Necturidae.

The Typhlomolgidae are unquestionably of the plethodontid
legion, as indicated by the absence of lungs and by their similar-
ity to Spelerpes larvae in most of their structural peculiarities,
as pointed out by Emerson (’05) in her study of the anatomy of
Typhlomolge. The sound-transmitting apparatus is decidedly of
the plethodontid type, with a still smaller amount of the colu-
mella entering into the fenestral plate than in the Amphiumidae.
Emerson expresses the opinion that the Typhlomolgidae should
be included with the Plethodontidae. So far as the morphology
of the sound-transmitting apparatus can be made out from the
adult alone, it does not warrant such a union of families. A
noticeable amount of the columella contributes to the plate part
of the apparatus, which is not the case in any of the Plethodont-
idae. While not to be included with the Plethodontidae, Typh-
lomolge appears to have been derived in very close relations with
them.

AA : H. D. REED

The Plethodontidae and Desmognathidae may be considered
together, since the sound-transmitting apparatus in these fam-
ilies is identical. Here the columella is reduced to that extreme
that it takes no part in the formation of the fenestral plate,
merely becoming joined to it as the stylus columellae. In these
families the identity of the columella is not lost, even after its
attachment to the side of the plate. The differences between the
sound-transmitting apparatus of the two families are slight, cer-
tainly of not more than generic value, which argues for the inclu-
sion of the two groups in the same family, as was suggested by
Moore (’00) in his study of the vertebrae and later adopted by
Dunn (17). Moore’s point is well taken. The vertebrae of
urodeles are acentrous. In such a shell of bone formed by the
descent of the dorsal elements, a cavity necessarily results and
its later partly or completely filled state, which resembles an opis-
thocoelian vertebra, is without significance in this connection. ~

As stated above, it seems clear that the type of sound-trans-
mitting apparatus which obtains in the plethodontid legion could
have come about originally only under the influence of or in
adaptation to a terrestrial abode. For the existence of the isth-
mus fenestralis no reason suggests itself, unless it be that dis-
cussed in connection with Spelerpes in the first part of this paper
or the persistence of the earlier cutting-out process of the oper-
culum, which is here chiefly that of growth of the fenestral lips
into the membrane.

The plethodontid legion, as a whole, has descended from ter-
restrial forms, the Necturidae, Amphiumidae, and Typhlomolg-
idae having returned to an aquatic abode. The extreme spe-
cialization of the sound-transmitting apparatus in these second-
arily aquatic species leads to the belief that the legion has lived
in a terrestrial environment from a very remote period. The
unusually long larval period of Spelerpes and Gyrinophilus
should be interpreted as a tendency of these modern species to
return to an aquatic life as Necturus has done. The short larval
period of Desmognathus, therefore, represents the normal pe-
riod, or one in which the tendency is toward a more strictly ter-

SOUND-TRANSMITTING APPARATUS OF CAUDATA 373

restrial existence. The absence of a free larval period in Pleth-
odon and others is to be looked upon as further progress toward
a strictly terrestrial existence.

The relations and estates of the columella and operculum in
urodeles, when coupled with other studies, lead to the conclusion
that these animals, as a group, have not found favorable sur-
roundings in the terrestrial zone. One after another has re-
turned to the water permanently, and the relative duration of
this secondary aquatic period is reflected in the structure and
development of the animal. Others are now in the course of
their regressive radiation, while a few, such as Plethodon, Auto-
dax, and Hemidactylium, because of their small size and secre-
tive habits, have succeeded in the terrestrial struggle and exhibit
features of structure and life-cycle which show no regression, but
rather an advance in adaptation to the dry zone.

SUMMARY

1. In all caudate amphibia two elements, columella and oper-
culum, are present in the sound-transmitting apparatus.

2. In the most generalized state these elements exist inde-
pendent of each other as in Amblystoma, Triton, Diemictylus,
Siren, and Salamandra. The columella, being useful in aquatic
life only, fuses in part with the ear capsule at transformation
(Amblystomidae, Salamandra) or completely (Triton, Diemicty-
lus, and probably Siren). The adult Cryptobranchidae, having
failed to complete the metamorphosis, has the sound-transmitting
apparatus in an arrested state of development, the columellar
element alone being present.

3. In all of the other families (Necturidae, Amphiumidae,
Typhlomolgidae, Plethodontidae, Desmognathidae) the repre-
sentatives of the columella and operculum fuse to form a single
plate. In this fusion there is, throughout the series of families,
a gradual reduction of the columellar element from a state where
it forms a portion of the fenestral plate, as in Necturus, to one
where it becomes stylus only, as in Desmognathus.

4. The morphology of this apparatus shows the affinities and
descent of the families as indicated in plate 6.

JOURNAL OF MORPHOLOGY, VOL. 33, NO. 2

374 H. D. REED

5. The nature and relations of the sound-transmitting appa-
ratus indicate that these structures came into their present state
in a terrestrial environment. Results gained from this study
combined with others tend to confirm the view that the present-
day aquatic species are secondarily so; that some species give
evidence of just beginning a return to an aquatic abode; that
others are still strictly terrestrial and likely to remain so.

LITERATURE CITED

Bowers, Mary A. 1900 Peripheral distribution of the cranial nerves of
Spelerpes bilineatus. Proc., Am. Acad. Arts and Sci., vol. 36, p. 177.

Bruner, H. L. 1914 The mechanism of pulmonary respiration in amphibians
with gill clefts. Morphol. Jahrb., Bd. 48, p. 68.

Corr, E. D. 1885 The retrograde metamorphosis of Siren. Am. Nat., vol. 19,
p. 1226.
1886 On the structure and affinities of the Amphiumidae. Proc., Am.
Phil. Soc., vol. 23, p. 442.
1888 The ossicula auditus of the Batrachia. Am. Nat., vol. 22,
464.

Driner, L. 1901-’02 Studien zur Anatomie der Zungenbein-Kiemenbogen und
Kehlkopf Muskeln der Urodelen. Zool. Jahrb., Bd. 15, s. 435.

Dunn, E. R. 1917 The salamanders of the genera Desmognathus and Leurog-
nathus. Proc., U. S. Nat. Mus., vol. 53, p. 393.

Emerson, ELLEN Tucker 1905 General anatomy of Typhlomolge rathbuni.
Proc., Bost. Soc. Nat. Hist., vol. 32, p. 43.

Gaaes, 8. H. 1891 Life-history of the vermilion-spotted newt, Diemictylus viri-
descens Raf. Am. Nat., vol. 25, p. 1084.

Hereick. C. Jupson 1914 The cerebellum of Necturus and other urodele Am-
phibia. Jour. Comp. Neur., vol. 24, p. 1.
1914a The medulla oblongata of Amblystoma. Jour. Comp. Neur.,
vol. 24, p. 348.

Krinassury, B. F. 1905 The rank of Necturus among tailed Amphibia: Biol.
Bull., vol. 8, p. 67.

Kinessury, B. F., anp Reep, H. D. 1908 The columella auris in Amphibia.
Anat. Rec., vol. 2, p. 81.
1909 The columella auris in Amphibia. Jour. Morph., vol. 20, p. 549.

Kinestey, J. S. 1902 The systematic position of the Caecilians. Tufts Col-
lege Studies, vol. 1, p. 323.

Moorgz, J. P 1900 Postlarval changes in the vertebral articulations of
Spelerpes and other salamanders. Proc., Acad. Nat. Sci. Phila., vol.
52, p. 613.

Norris, H. W. 1908 The cranial nerves of Amphiuma means. Jour. Comp.
Neur. and Psychol., vol. 18, p. 527.

SOUND-TRANSMITTING APPARATUS OF CAUDATA oe

Norris, H. W. 1911 The rank of Necturus among the tailed amphibians as
indicated by the distribution of its cranial nerves. Proc., lowa Acad.
SGie; WOlle ish; jos BMC
1913 The cranial nerves of Siren lacertina. Jour. Morph., vol. 24,
p. 245.

PeTer, Kart 1898 Die Entwicklung und funktionelle Gestaltung des Schddels
von Ichthyophis glutinosus. Morphol. Jahrb., vol. 25, p. 555.

Reep, H. D. 1914 Further observations on the sound-transmitting apparatus
in urodeles. (Proc. Amer. Anat. Assoc., Dec., 1913) Anat. Rec., vol.
Sp 112:

1915 The sound-transmitting apparatus in Necturus. Anat. Rec.,
vol. 9, p. 581.

SARASIN, P., anD F. 1887-1890 Ergebnisse naturwissenschaftlicher Forschungen
auf Ceylon, Bd. II, Zur Entwicklungsgeschichte und Anatomie der
ceylonesischen Bliindwuhle Ichthyophis glutinosus. Wiesbaden.

VersLuys, J. 1909 Die Salamander und die urspriinglichsten Vierbeinigen
Landwirbeltiere. Naturwiss. Wochensch., (N. F.), Bd. 8.

WIEDERSHEIM, Ropert 1877 Das Kopfskelet der Urodelen. Morph. Jahrb.,
Bd. 3, s. 352 and 459.

Wiper, H. H. 1891 A contribution to the anatomy of Siren lacertina. Zool.
Jahrb. Anat. Abt., Bd. 4, s. 653.

Winstow, G. M. 1898 The chondrocranium in the Ichthyopsida. Tufts Col-
lege Studies, vol. 1, p. 147.

PLATE 1

EXPLANATION OF FIGURES

A series of schemas to show the relations of the different morphological types
of fenestral elements in caudate amphibians. The cross-hatched areas represent
columella or portions derived outside the ear capsule. The elements derived
from ear capsule are unshaded and represent operculum. Broken lines indicate
a fusion of fenestral element and ear capsule.

19 Larval Amblystoma punctatum.

20 Adult Amblystoma punctatum.

1 Adult Diemictylus and Triton.
22 Adult Gyrinophilus representing the Plethodontidae.

ABBREVIATIONS
Ec., ear capsule P.q., palatoquadrate
I.f., isthmus fenestralis Sq., squamosum
Op., operculum or functional fenestral St.c., stylus columellae
plate of the adult VII., nervus facialis
M.op., musculus opercularis r.j.VII., ramus jugularis facialis

Pl.c., plate portion of columella

576

SOUND-TRANSMITTING APPARATUS OF CAUDATA PLATE 1
H. D. REED

377

PLATE 2

EXPLANATION OF FIGURES

Drawings of wax models of the ear capsule and sound-transmitting apparatus
of Spelerpes bislineatus.

23 Larva 25 mm. long.

24 Larva 28 mm. long.

25 Larva 34 mm. long.

26 Mature larva 34 mm. long.

ABBREVIATIONS
Col., stylus columellae (columella) I.f., isthmus fenestralis
C.S., level of text figure 5 Op., fenestral plate (operculum)

D.a., dorsal arm of isthmus fenestralis S., level of text figure 4
growing into the fenestral membrane Sq., squamosum
E.c., ear capsule Va., ventral arm of growing isthmus

378

SOUND-TRANSMITTING APPARATUS OF CAUDATA PLATE 2
H. D. REED

295 26

379

PLATE 3

EXPLANATION OF FIGURES

Drawings from wax models of the ear capsule of Amphiuma means.
27 Embryo 30 mm. long.

28 Young adult 85 mm. long.

29 Adult 265 mm. long.

ABBREVIATIONS

Col., stylus columellae (columella) Ps., parasphenoid

I.f., isthmus fenestralis Sq., squamosum

Op., operculum (fenestral plate) V.a., ventral arm of isthmus fenestralis

380

SOUND-TRANSMITTING APPARATUS OF CAUDATA PLATE 3
H. D. REED

381

PLATE 4

EXPLANATION OF FIGURES

Drawings of wax models of the ear capsule.
30 Larval Desmognathus fusca, 21 mm. long.
31 Larval Gyrinophilus porphyriticus 82 mm. long.

32 Adult Gyrinophilus porphyriticus.

33 Larval Necturus 48 mm. long.

ABBREVIATIONS

C.i., isolated area of cartilage formed
in the fenestral membrane

Col., stylus columellae (columella)

Da., dorsal arm of invading isthmus
fenestralis

F., level of the fusion of the stylus col-
umellae and cartilage formed inde-
pendently in the fenestral membrane

I.f., isthmus fenestralis

Op., fenestral plate (operculum)

Pl.c., portion of columella taking part
in the formation of the fenestral plate

Ps., parasphenoid

Sq., squamosum

Va., ventral arm of isthmus fenestralis

VII, nervus facialis

r.j.VII, ramus jugularis facialis

382

PLATE 4

SOUND-TRANSMITTING APPARATUS OF CAUDATA
H. D. REED

383

PLATE 5

EXPLANATION OF FIGURES

Drawings from wax models of the ear capsule of Siren.
34 Specimen 133 mm. long.
35 Specimen 215 mm. long.

ABBREVIATIONS

Op., operculum, which in the younger specimen is not yet completely cut out.
Sq., squamosum.

384

SOUND-TRANSMITTING APPARATUS OF CAUDATA PLATE 5
H. D. REED

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Resumen por el autor, G. W. Tannreuther.
Universidad de Missouri.

El desarrollo de Asplanchnia ebbesborni (Rotiferos).

La formacién y segmentacién de los évulos puede seguirse
paso a paso dentro del animal vivo a causa de su transparencia.
La segmentacién, en muchos puntos, tales como la direccién,
orden y marcha de la misma, presenta el mismo caracter que la
de los anélidos, formandose tres generaciones de ect6meros. La
regidn ventral de los ect6meros corresponde al futuro extremo
anterior, mientras que la que ocupan los macrémeros, A, B y C
corresponde mas al extremo posterior. Durante la gastrulacién
el macrémero grande 3D pasa al centro del embrién. A, B y C
permanecen en la superficie del extremo posterior del embrién
y sus derivados estén relacionados mas directamente con la pro-
duccion del voluminoso pié embrionario. 38D (E) origina el sis-
tema reproductor con unas cudntas fibras musculares y todo el
sistema digestivo, con la excepcién del stomodaeum. Los deri-
vados de los ect6meros producen las demas estructuras. Todos
los 6rganos, (con excepcidn del sistema digestivo del macho) son
funcionales y estan bien desarrollados en ambos sexos. El ani-
mal produce 6vulos machos, hembras y otros en estado de reposo.
Todos los 6vulos, excepto los en reposo, son transparentes; los
ultimos poseen abundante vitelo. Los machos y las hembras no
son originados nunca por el mismo animal, mientras que los em-
briones machos y 6vulos en reposo son producidos por la misma
hembra. Los évulos machos y los ultimamente mencionados
difieren en estructura y solamente los en reposo son susceptibles
de fecundacién. La naturaleza y marcha del desarrollo indican
que los rotiferos no son larvas troc6foros de los anélidos, que han
persistido, sino mas bien que los rotfferos y anélidos se han origi-
nado a expensas de una misma forma ancestral, habiendo alcan-
zado los anélidos un estado mas elevado en la historia de su evo-
lucidn.

Translation by José F. Nonidez
Carnegie Institution of Washington

AUTHOR’S ABSTRACT OF THIS PAPER ISSUED
BY THE BIBLIOGRAPHIC SERVICE, FEBRUARY 2

THE DEVELOPMENT OF ASPLANCHNA EBBESBORNII
(ROTIFER)

GEORGE W. TANNREUTHER

Zoological Laboratory, University of Missouri

TWENTY-ONE TEXT FIGURES AND SEVEN PLATES

CONTENTS
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INTRODUCTION

The size of the adult rotifer, in many instances, has made the
study of development a difficult problem. A great descriptive
mass of literature has grown up around this group of animals,
but with the exception of a very few detailed accounts of their
structure, the writers have contented themselves with the
description of the external form.

389

JOURNAL OF MORPHOLOGY, VOL. 33, NO. 2

390 GEORGE W. TANNREUTHER

The morphology of the Rotatorian family Flosculariidae was
worked out by T. H. Montgomery (’03) in considerable detail.
Zelinka (’92) published an account of the development of Calli-
dina ruseola. Jennings (’96) traced the cell lineage of Asplanchna
herricki, for a few generations, but did not correlate the cells of
the early embryo with the adult structures.

Some rotifers are very transparent; this makes possible,
not only a detailed study of the position and relation of the
various structures, but an investigation of the origin, growth,
and cleavage of the eggs. Where complete development occurs
within the parent, a sequence of the different stages from the
first appearance of the egg to the time of birth can be followed
with a considerable degree of accuracy. The extreme trans-
parency of the adult Asplanchna ebbesbornii, makes possible
a detailed study of the successive stages in development.

The more important points in development may be summarized
as follows:

1. The cleavage is unequal and regular. A small cleavage
cavity is present. The gastrula is formed by a modified epiboly.
The blastopore occurs at the posterior end of the developing
embryo.

2. The first cleavage plane is at right angles to the future
median longitudinal axis of the adult. It divides the egg into
two very unequal cells. The smaller cell is ectodermal and
mesodermal, while the larger cell contributes to the three germ
layers.

3. The gastrula consists of an outer layer of epithelial cells
enclosing an inner cell mass. The outer layer includes all of
the derivatives of A, B, and C and the three cells d!, d?, and d?
derived from D. The inner cell mass includes all of the remain-
ing cells.

4. The outer layer or ectoderm gives rise to the cuticle, hypo-
dermis, brain, excretory system, trochal disc, cilia, buccal
pouch, and musculature. The inner cell mass derived from
3D produces the remaining parts of the digestive system and
glands, the reproductive system, and a few of the muscles which
control it.

ASPLANCHNA EBBESBORNII (ROTIFER) 391

5. The origin of the various organs cannot be traced back
to any definite cell or cells of the early cleavage stages, but can
be directly associated with definite regions of the early gastrula.
It is true, however, that the products of any one cell A, B, C,
or D, in the production of the ectoderm, can be definitely followed
and localized in the gastrular ectoderm, but there are no struc-
tural differences to mark off these regions. The ectodermal
cells of any region possess the same potentiality in producing
muscle fibers, regardless of their origin. It is impossible to tell
definitely the differentiation of the digestive and reproductive
systems until late cleavage.

6. The derivative of the blastomere B form the ventral, those
of D the dorsal, those of A the left lateral, and those of C the
right lateral ectoderm of the adult animal.

7. The brain on the median dorsal side is derived from the
ectoderm at the anterior end. The urogenital sinus is formed
by a solid ingrowth of the ectoderm on the median dorsal side
at the posterior end above the base of the embryonic foot. Its
position is considered as being ventral in the adult, due largely to
the disappearance of the foot in later embryonic life. The
embryo in its early development is curved ventrally, making the
dorsal side appear quite long in comparison to that of the ventral.
The mouth is ventro-anterior.

NATURAL HISTORY

The rotifer Asplanchna ebbesbornii is not very abundant in
this immediate locality. The material was collected from small
rain pools and placed in aquaria, filled with tap-water. This
particular rotifer was first observed in one of the freshly prepared
cultures in January, 1916. They persisted about two weeks and
disappeared. They reappeared in the following March and
continued for two weeks. The rotifers appeared again in the
following May. In each instance about two months elapsed
between the times of their appearance. This periodicity con-
tinued until December, 1918, when this paper was completed.
In each cycle of appearance males, females, and resting eggs were
formed in about the same proportions. The cultures were kept
indoors at laboratory temperature.

392 GEORGE W. TANNREUTHER

The January and March cycles were not studied very exten-
sively. The studies proper were begun with the May cyele,
1916, in tracing out the cell lineage. The adults are extremely
transparent, and by holding them in any desired position under
a supported cover-slip the cleavage of the individual cells can
be traced step by step. Two distinct kinds of adult females
exist, which are structurally similar as far as can be determined
under a magnification of 100 to 150 diameters. The one repro-
duces females parthenogenetically, and the other males parthen-
ogenetically or resting eggs, which carried the cycle of one period
to their next reappearance. The resting eggs pass through their
early stages of cleavage before deposition. ‘There are, however,
two kinds of resting eggs: a very thin-shelled one, with a single
shell membrane, and a thick-shelled egg, with a double shell
membrane. The thin-shelled egg develops with the same
rapidity as the parthenogenetically produced individuals and
hatches out immediately after deposition. Two polar bodies
are formed in each kind of resting egg, which do not develop
unless fertilized.

Females and resting eggs or females and males are never
produced by the same individual. The parthenogenetically
produced males and females are sexually mature at birth. The
uterus of the young females often contains embryos in the late
cleavage stages at the time of their birth. The two kinds are
practically the same size at birth. Copulation occurs almost
immediately after the birth of the males. The male may copu-
late with either kind of female. The uterus and the oviduct of
the parthenogenetic producing female often contain sperm, but
neither males nor resting eggs are produced by this particular
individual. On the other hand, the uterus and oviduct of the
male-producing parent may contain an abundance of sperm, and
yet produce males only, or again, they may produce both males
and resting eggs. There is no definite sequence in the production
of males and resting eggs by the same parent. A single male
may be produced and all the remaining become resting eggs, or
vice versa. Or, on the other hand, it is not unusual to find the
two alternating. The following is a good example: the sequence

ASPLANCHNA EBBESBORNII (ROTIFER) 393

was as follows: two resting eggs, one male, one resting egg, one
male, one resting egg, one male.

In case of the female which produces the resting eggs the
vitellartum undergoes a marked change after impregnation.
The yolk spherules become larger and more abundant and give
the vitellarium a very dark color. The yolk is first produced at
the point where the oviduct takes its origin. This process
continues until the vitellarium is completely filled with yolk.
Where males and resting eggs are produced by the same parent,
the yolk is produced at intervals just before the resting egg
begins its growth in the ovary. The male eggs are free from the
dark yolk and remain transparent. Impregnation has no effect
on the vitellarium of the female-producing individual in the
production of yolk. Figures 7 to 11 show the single thin-shelled
and the double thick-shelled resting eggs. In figure 12 the
spermatozoa are shown in different stages of development.

In many instances the sperm of the sexually mature male
(before birth) would escape from the testis and become deposited
in the uterus of the parent and bring about the production of
resting eggs. In other cases the male was little more than a
large sperm sac. The male embryos developed normally until
the early differentiation of the reproductive organs. At this
stage of development all of the cleavage cells, except those directly
concerned in the production of the sperm, ceased dividing, took
on a vesicular appearance, gradually deteriorated, and functioned
as food for the developing sperm. The sperm, when. mature,
escaped through the egg membrane into the lumen of the uterus.
This condition accounts, to some extent, for the few free-swim-
ming males in the cultures.

The males are structurally degenerate at birth. ‘The digestive
tract is very rudimentary (figs. 5 and 6) and never opens to the
exterior. ‘The males are very short-lived, and few free-swimming
individuals are present at any one time. The males do not
increase in size after birth (figs. 5 and 6). On the other hand,
the females increase to at least four to six times their size at the
time of birth (fig. 1). The embryology of the male and the
female developing individual is practically the same throughout

394 GEORGE W. TANNREUTHER

the different stages of development. One polar body is formed
in the female egg, while in all others two polar bodies are formed.

The individuals hatching from the resting eggs are always
parthenogenetic females. But the next generation is of two
kinds, one producing females parthenogenetically and the other
producing males parthenogenetically or resting eggs.

MATERIAL AND METHODS

The rotifers were removed from the different cultures with a
pipette and placed in watch crystals. The excess water was
then removed and the fixing fluid added. SBouin’s fluid gave
the best results. The animals were preserved in 70 per cent
alcohol until used. If allowed to stand indefinitely in alcohol
the individuals turn brown and are not very satisfactory for
study. For whole amounts Delafield’s haematoxylin and eryth-
rosin were used. Either stain gave good results. The changes
from alcohol to xylol and from xylol to the mounting medium
must be made very gradually. A few drops of carbo-xylol and
clove oil will help considerably in the process. The cuticle is
very resistant and will cause considerable shrinkage and dis-
tortion if the change be made too quickly. The same caution
must be taken in clearing specimens that are to be imbedded
and sectioned. Iron-alum haematoxylin and erythrosin gave the
best results for sections. In the case of whole mounts, the cover-
slips should be supported.

ORIGIN AND FORMATION OF OVUM

The reproductive organs are composed of the vitellarium,
ovary proper, oviduct, and uterus. The uterus opens into the
urogenital sinus. The vitellarium is somewhat U-shaped (fig. 4),
very transparent when free from yolk, and contains many large
nuclei. -The ovary (figs. 1 to 4) occupies a very small area at
the base of the vitellarium.

Parthenogenetic ova: The ova are very small, and asingle ovum
at regular intervals begins its growth. The contents of the
growing egg is derived directly from the vitellarium. The

ASPLANCHNA EBBESBORNII (ROTIFER) O90

passage of cytoplasmic and yolk granules into the egg is visible
under a low power. When the egg reaches the end of its growth
period, it is separated from the ovary (fig. 4), and enters the
upper end of the oviduct, which, in reality, encloses the greater
part of the ovary. A single polar body is formed. In most
cases immediately after this maturation, the following egg begins
its growth. Two eggs may begin their growth at the same time,
but this is very unusual. In the case of the male-producing
eggs, two polar bodies are formed, the first of which often divides.
The origin, formation, size, and development of the female and
the male-producing eggs are identical (figs. 2 to 4).

Sexual or resting ova: The origin and growth of the resting
eggs are similar to that of the parthenogenetic eggs. The vitel-
larium, however, is very dark from the presence of a rich supply
of yolk. The yolk passes directly from the vitellarium into the
growing egg. Immediately after maturation and fertilization, a
very thick inner shell is formed from the cytoplasm. The contents
of the double-shelled egg cannot be studied except in sections
In many of these resting eggs no inner shell membrane is formed.
They are about the same size as the thick-shelled eggs, but are
more transparent and contain less yolk. Their cleavage stages
can be followed without the aid of sections. The number of
resting eggs in the uterus at any one time varies from one to
eight. In case of the female or male-producing parent, there
may be as many as sixteen embryos in the oviduct and uterus
at the same time, ranging from the early cleavage stages to the
mature young (fig. 2).

CLEAVAGE
1. Designation of the cleavage cells

The nomenclature adopted in the designation of the cleavage
cells is a modification of the system used by previous investigators
on cell lineage. The first four cells (macromeres) are designated
by the capital letters A, B, C, and D. The generations of micro-
meres (ectomeres) by the small letters a, b, c, and d. The first
index number indicates the generation to which the ectomere

396 GEORGE W. TANNREUTHER

belongs. Thus a', b!, c!42, or d'1+, all belong to the first genera-
tion; c?, b?+, or d?’, belong to the second generation, and a},
b322, ¢32, or d33, ete., belong to the third generation.

On account of the peculiar shifting of the macromeres A, B,
C, and D in the formation of the first quartette, A, B, and C
take a position more anterior and D posterior, instead of at the
vegetal pole, as in annelids. When a cell divides, the product
receives the designation of the parent cell with the addition of a

. 2.1
further index number; thus, 4 mae The cell D, after the for-
mation of d? and d?, is designated by the capital letter E; it gives
rise to the reproductive system and all of the endoderm (digestive

system) except the stomodaeum and the pharynx.

2. Nature of cleavage

First cleavage: Immediately after maturation the nucleus
passes from the surface toward the center of the egg, but nearer
the anterior end. The first cleavage spindle is formed about
thirty: minutes after maturation (figs. 13 to 16), the time varying
somewhat with external conditions. Low temperature retards
the rate of cleavage. The first cleavage spindle occurs in the
plane of the long axis of the egg (fig. 7). It passes through the
region of the polar body or bodies and divides the egg into two
very unequal parts, AB and CD (figs. 15 to 17). The smaller
cell, AB, is anterior and the larger cell, CD, is posterior. The
cleavage furrow at first is deep and the cells are rounded, but
before the second cleavage occurs the cells flatten at their point
of contact and the egg becomes more elliptical with the first
cleavage plane scarcely visible.

The granular content of the cells is uniform, with very few
yolk bodies visible. The region immediately surrounding the
nucleus is almost free from cytoplasmic granules and makes it
possible to follow the nuclear activities in the process of division
in the living egg. The first cleavage in the femaleé- and the male-
producing egg is the same. It occurs at right angles to the future
longitudinal axis of the adult. The second cleavage plane occurs

ASPLANCHNA EBBESBORNII (ROTIFER) 397

at an angle of about 45° with that of the first. The two cells
divide at different times. These two cleavages combined corre-
spond to the second cleavage as it occurs in many of the annelids
and molluses. The cell CD divides first into two very unequal
parts (figs. 18 and 19). The division of AB is nearly equal
(figs. 18 to 20). Shortly after the second cleavage, a slight
shifting occurs as the cells flatten (figs. 19 to 24). The largest
cell, D, is posterior, B median anterior, C right, and B left (fig.
21), with reference to the median axis of the future animal.
The largest cell, D, always divides first in the formation of the
quartettes. The orientation of the four-celled embryo is very
simple and agrees with that of the annelids at the same stage of
development. From this point forward, however, the position
taken by the resultant cleavage cells is no longer comparable
with that of the annelids, but the sequence of cleavages in the
following stages is very similar.

Third cleavage: In the formation of the first generation of
ectomeres (d!, b!, c', at), the cell D divides first, the new cell is
budded off in a dorsal anterior direction on the median dorsal
side of A, B, and C (text fig. b and figs. 23 and 24), making a
five-cell stage. In this process the polar body is carried forward
to the anterior end with the cell d'. While the cleavage spindle is
forming for the production of the micromere d!, the cells A, B, and
C elongate in an anteroposterior direction (text fig. ec and figs.
24 and 25), so that the cleavage plane of A, B, or C is not hori-
zontal, but in a dorsoventral direction at right angles to the
long axis of the embryo (figs. 26 to 31). Thus, instead of having
the micromeres above the macromeres as in many forms, the
cells a1, b!, c1, and d! are on the same level with A, B, and C (figs.
31 and 32). The division of the macromeres A, B, and C is
nearly equal in the formation of the first generation of ectomeres.
They do not divide simultaneously, but in the invariable order
C, B, A. Thus there occurs successively a six-, seven-, and
eight-cell stage (figs. 830 to 32). In the formation of the eight-
cell stage B and b! are pressed ventrally by d! (figs. 24 and 31).
In the eight-cell stage d' is median dorsal, B and b! median
ventral, C and c! right, A and a! left, and D posterior extending

398 GEORGE W. TANNREUTHER

dorsoventral (figs. 31 to 33). The micromeres form the anterior
end. In a few instances, C, B, and A divided in a nearly hori-
zontal plane, thus placing the micromeres above the parent cells
instead of on the same level with them (text figs. d and e and
fig. 33). This mode of division, however, is very unusual, but
is comparable with that of the annelids and polyclades.

At the completion of the eight-cell stage, the embryo often
assumes the shape of the one-cell condition and the cleavage
furrows are scarcely distinguishable. The embryo at the dif-
ferent stages of development is very plastic and may asssume
almost any shape under abnormal pressure. If the egg be
removed from the reproductive organs, with the egg membrane
intact, after cleavage has begun, normal development will
continue. By slight pressure the individual cells of the eight-cell
stage can be separated. The isolated cells seldom continue to
divide, but begin to deteriorate almost immediately.

Fourth cleavage (sixteen-cell stage): A nine-cell stage is reached
by the formation of d? from the large cell D on the median dorsal
side, in an anterior direction (figs. 34 and 35). As d? is formed,
d! is carried around the dorso-anterior end. Next, d! divides in
an anteroposterior direction (fig. 36). Following this, the
macromeres A, B, and C and their micromeres divide in an
anteroposterior direction. The division occurs in the order C
and c!, B and b!, A and a', thus producing a twelve-, fourteen-,
and sixteen-cell stage, respectively (figs. 34 to 38). The embryo
is now composed of four rows of cells with four cells in each row
(figs. 38 to 44).

The fifth cleavage: The derivatives of D divide in the follow-
ing order: d?, di, d'1, thus producing a seventeen-, an eighteen-,
and a nineteen-cell stage (figs. 45 to 48). The cleavage spindles
in the C, B, and A rows indicate the direction of the cleavages
in passing from the nineteen- to the thirty-one-cell stage (figs.
45 to 48). In a few of the embryos, as in annelids, D budded off
a third cell, d* (figs. 46, 47, 49, and 50). This extra cell, when
produced, has no special significance in the future development
of the individuals which bear it; d* and its derivatives will not
be considered in the further description of the cleavage stages.

ASPLANCHNA EBBESBORNII (ROTIFER) 399

At the close of the fifth cleavage, the quadrants A, B, and C are
composed of two rows with four cells each, and the quadrant D
of two rows with three cells each, including the large entoderm
cell D, making thirty-one cells in all. The comparative sizes of
the cells are shown in the different figures. During the fifth
cleavage the cells withdraw toward the exterior (fig. 55) and

Fig. a Four-cell stage, left side.

Fig. b Four-cell stage, left side, showing the cleavage spindle in the forma-
tion of the ectomere d!.

Fig. ¢ Six-cell stage, ventral side; shows the anteroposterior extension of
cells.

Figs. dande_ LEight-cell stage, ventral and dorsal sides; shows the position
of the first quartette of ectomeres similar to that of annelids. This condition is
unusual.

Fig. f Seventeen-cell stage, left side.

400 GEORGE W. TANNREUTHER

form a cavity, which later is occupied by the large cell D. Before
the fifth cleavage is complete, the anterior end of the cell D is
partially covered by the cleavage cells immediately in front of it
(figs. 45 to 50).

Sixth cleavage: After the formation of the small cell d+, the
cell d!11 (fig. 51) divides. Next a small cell d*° is formed from
D. The first cell of the sixth cleavage to divide is d2!, ete. The
sequence of cell formation in the sixth cleavage is similar to that
of the fifth, the derivatives of the D quadrant dividing first, then
those of C, B, and A. The cell lineage of any one quadrant can
be followed indefinitely. Figure 51 represents the beginning of
the sixth cleavage. From this stage the surface cells will not
be labeled, as it does not contribute to the understanding of the
further development. The sixth cleavage doubles the number of
cleavage cells on the surface. It does not increase the number
of rows in each quadrant, but the number of cells in each row is
doubled. The embryo at the end of the sixth cleavage is com-
posed of the following cells: the D quadrant contains two rows
of six cells each, and d‘ and d*. The C, B, and A quadrants
each contain two rows of eight cells each. With the large
entoderm cell D, there are thus sixty-three cells in all.

3. Gastrulation

Gastrulation, which begins during the close of the fifth cleavage,
adheres more strictly to the epibolic type. Immediately after
the formation of d2, the surrounding cells at the anterior end of
D begin to extend over its surface (fig. 48). The embryo when
viewed from the posterior end (figs. 52 and 53) shows the position
of the surface cells with reference to D. The spindle indicates
the direction in which d‘ is formed. Figures 52 and 54, later
stages during the sixth cleavage, show the method of overgrowth
on the surface of D (E). Gastrulation is a double process; while
the surface cells are extending posteriorly over the surface of E,
the large cell itself is migrating into the interior, a result of the
pressure of the surrounding cells, and the cavity within the
embryo, which began its formation before the sixth cleavage
started.

ASPLANCHNA EBBESBORNII (ROTIFER) 401

During gastrulation the embryo shortens and increases in
width, as shown in figures 55 to 57. The formation of the central
cavity (figs. 57 and 58) and gastrulation occurs very rapidly.
The entire process requires about fifty minutes and can be
demonstrated in the living egg. Figures 58 and 59, a sixty-four-
cell stage, viewed from the dorsal and ventral sides, respectively,
show the embryo at the end of gastrulation. The blastopore,
situated at the macromere end of the embryo, is rather large at
first and is surrounded by eight cells, two belonging to each of
the four quadrants (figs. 52 to 54).

Gastrulation brings about a fundamental change in the re-
lation of E to the remaining cells of the embryo. At first it
formed the posterior end of the cleavage cells, but at the end of
gastrulation it occupies the central region of the growing em-
bryo and is completely enclosed by the surrounding cells (figs. 58
and 59). The large cell E is now designated as the mesentoblast,
and, on account of its new position is destined to assume a new
rdle in the development of the embryo. The embryo at the end
of gastrulation contains about 200 cells, which are divided into
two distinct regions; the mesentoblast including d‘ and d*, and
the epithelial ectoderm, which, at the anterior end, shows the
beginning of a double layer (figs. 58 and 59).

SEGREGATION OF THE GERM LAYERS

In Asplanchna ebbesbornii a distinct segregation of the germ-
layers begins with the formation of the cell d? or d*, the nine- or
seventeen-cell stage. The cell d? is not usually formed, hence
the variation in the number of cells at the time of segregation.
The large posterior cell D is destined to become entomesodermal,
and all of the remaining cells are ectomesodermal.

1. Ectoderm

The dorsal part of the ectoderm is derived from the quadrant
D, the right, the left, and the ventral parts from C, A, and B,
respectively. The three cells of the quadrant D, d!, d?, and dt,
are the first to divide in the formation of the ectoderm during

402 GEORGE W. TANNREUTHER

the fifth cleavage. Each cell divides unequally. The anterior
cell divides vertically, while the other two divide transversely
and parallel to the long axis of the body (fig. 48). For further
account of the cleavage process in the formation of the early
ectoderm, see description given for the later cleavage stages.
Figures 55 to 57 represent the earlier stages in which the cleavage
cells become arranged into a definite epithelial layer. The cavity
shown in these figures is in reality the early body cavity, since
the epithelium later becomes the definitive body wall. A later
condition of the ectoderm is shown in figures 67 and 68. From
this point onward the division of the epithelial cells is very
irregular and their products become differentiated into the
definitive ectoderm and its various derivatives. At first the
definitive ectoderm is represented by an epithelial layer com-
posed of large nucleated cells, and is in immediate contact with
the organs forming within (figs. 77 to 90). But as the different
organs reach their complete development, the body cavity
becomes more and more marked and the cells of the ectoderm
are drawn out into a very thin epithelial layer, with the boundary
of the cells no longer visible (figs. 96 to 104). The cuticle is a
very thin layer formed by a secretion from the ectoderm shortly
before birth. The cuticle is usually free from markings and has
a smooth surface. Figure 104 represents the condition of these
various structures at the time of birth.

2. Mesoderm

In the late gastrula stage (figs. 63 to 70), the outer epithelial
layer of the embryo at different regions begins the proliferation
of cells on the inner surface. These cells contribute directly to
the formation of the mesoderm, which later becomes differen-
tiated into the muscular system. It is impossible to distinguish
in the embryonic ectoderm the cells which directly give rise to
the mesoderm. Apparently all parts of the ectoderm possess
the same potentiality in the process. These cells, in their for-
mation, remain connected with their point of origin. Later these
proliferated cells show an apparent connection or fusion with the

ASPLANCHNA EBBESBORNII (ROTIFER) 403

inner cell content (the invaginated stomodaeum and the deriv-
atives of the mesentoblast) (text figs. g, h, and q). Other rows
of proliferated cells at either end are connected with the ectoderm
(text fig. k). Many of these cell processes have several attach-
ments (text fig. h), but in their further development some of
these points of fusion are lost and the developing muscle remains
attached to the later formations, which they are destined to
control. The muscle fibers, in their early formation, are com-

Figs. g and h Early muscle cells connecting ectoderm with inner structures.

Figs.iandj Early and definitive stages of muscle-fiber development.

Figs. k andl Shows method of attachment in the early and late development
of muscle fibers.

Fig. m A completely formed muscle, showing attachment to brain and
ectoderm.

Fig. n A network of muscle fibers extending across body cavity.

ABBREVIATIONS
b.c., body cavity m.c., muscle cell
br., brain m.f., mauscle fiber
cp., corpuscle tr., trochus

ect., ectoderm w.ph., wall of pharynx

404 GEORGE W. TANNREUTHER

posed of cellular processes (text fig. k), which, later, as develop-
ment progresses, lose all traces of cell boundaries, with a few
nuclei persisting (text figs. 1 tom). The method of attachment
in one of the completely formed muscles is represented in text
figurel. Itis attached anteriorly to the corona and at its opposite
end it is anchored to the ectoderm. Some of these muscle fibers
show a distinet cross or longitudinal striation with few nuclei.
In the mature embryo (text fig. n), many of the muscle fibers are
drawn out and form a delicate network, which extends through
the different regions of the body cavity. Some of these fibers
are mere lines and are hard to distinguish. Many of the apparent
migratory cells within the fluid of the body cavity are directly
connected with very delicate processes (text figs. m and n),
while others are mere floating corpuscles, which are highly
vacuolated. The various structures within the body, as well as
the trochal disc, are kept in constant motion by the activities
of the various muscle fibers. No attempt was made to represent
the position of the different muscles in the various figures drawn.

3. Entoderm

During the early part of the seventh cleavage, the blastopore
becomes completely closed (figs. 59 and 60). The cells desig-
nated as the entoderm include the large cell E and the two small
cells d* and d*, which are formed from E during the sixth and
seventh cleavages of the embryo. Figures 58 to 60 show the
first stages in the cleavage of E, which corresponds to the eighth
cleavage. This cleavage is unequal and separates a smaller cell,
E!, from a larger cell, E?, posteriorly. Figure 60 is a dorsal
view of the embryo and shows the condition of the ectoderm
and entoderm at the beginning of the eighth cleavage. The
ectodermal cells during the ninth cleavage divide very rapidly
and are difficult to follow. Immediately after the first cleavage
of E, the cell E? divides equally and transversely, or at right
angles to its first cleavage. Figures 61 to 64 show the different
stages in the process of division. The embryo, as a whole, is
very plastic and becomes more spherical during the cleavage of

ASPLANCHNA EBBESBORNII (ROTIFER) 405

E? (fig. 62). Figure 63 represents a 250-cell stage, and by turn-
ing the embryo in different positions all of the cells can be recog-
nized, but it is impossible to tell the exact boundary of the cells
derived from any one quadrant. The derivatives of D always
take the initiative in division. Figure 63 represents the eighth
cleavage complete and the beginning of the ninth. In figure 64
the entodermal cells do not completely fill the central cavity of
the embryo. Next, the anterior entodermal cell E! divides very
unequally and forms E!! and e!? (fig. 65). The smaller cell,
after a few divisions, is difficult to follow. Immediately after the
division of the anterior cell E', the cells E?! and E?? divide
equally. The spindles of these divisions are shown in figure 65.
The division is equal and takes place in an anteroposterior
direction. The entoderm is now composed of five large cells and
three smaller ones.

Figure 66 represents an embryo with the five large entodermal
cells viewed from the left side. Spindles are present in each of
the cells for the following cleavage. Figure 67 represents an
optical section a little earlier than the preceding, viewed from
the right side. The ectodermal cells are somewhat contracted
and do not fill the central cavity. The cells of the embryo at the
dorsal posterior end multiply. very rapidly, extend backward
over the blastopore, and, in a later stage, contribute to the for-
mation of the temporary foot. At the next division each of the
five entodermal cells divides dorsoventrally and forms two
layers of five cells each, as shown in an optical section in figure
68, with the ectoderm removed. Figure 69 shows the same
stage with the ectoderm intact. At the next division each of
the five upper and the lower entodermal cells divide equally.
The cleavage occurs in an anteroposterior direction and produces
twenty large entodermal cells, as shown in figure 70, an upper
view. From this point forward no attempt was made to follow
the individual cleavage cells. Figure 71, a ten-hour embryo,
shows about the same stage as the preceding from the right side.
The position of the entoderm is indicated by a dotted outline.
The beginning of the foot and the first stage in the formation of
the stomodaeum is evident from the ventral side. Figure 72, a

JOURNAL OF MORPHOLOGY, VOL. 33, NO. 2

406 GEORGE W. TANNREUTHER

little later stage than the preceding, from the right side, shows
the position of the central entodermal mass and its relation to the
embryo as a whole. The ectodermal cells at the anterior end
of the embryo now divide very rapidly and later contribute in
part to the formation of the stomodaeum and the pharynx (figs.
73 to 75). The central mesentodermal mass of cells becomes
differentiated into two distinct regions (figs. 76 and 77), the
entoderm proper, which produces the stomach, oesophagus, and
digestive glands; and the part which produces the reproductive
organs with a few of its controlling muscle fibers.

FORMATION OF THE TROCHAL DISC

The developing embryo is divided into three distinct regions;
the body, the head bearing the trochal disc, and the foot. The
foot is an embryonic structure and is absorbed before the birth
of the individual.

The trochal disc begins as ectodermal prominences or growths,
due to a proliferation of cells on the ventro-anterior region.
Figure 73, viewed from the ventral side, shows a small lateral
fold on either side. <A stage little later than the preceding is
shown in figure 74 from the right side. These ectodermal folds
extend toward the posterior end, but only the anterior ends
contribute to the trochal disc. This figure shows the first steps
in the differentiation of the animal into three distinct regions
(head, body, and foot). The folds are more developed in figure
78, and the large central depression between the lateral folds is
open at either end. Later stages are represented in figures 81
and 83. The position of the mouth is visible in figure 81. The
embryo at this stage of development is considerably curved and
the ectodermal folds posterior to the stomodaeum have begun
their development. ‘The extreme anterior folds later become the
dorsal part of the trochal disc. The position of the mouth is
now more definite. The lateral, anterior, and posterior folds are
now continuous (fig. 89). This figure shows the mouth close to
the posterior prominence, which, later, when the embryo
straightens out, becomes ventro-anterior. Figures 91 to 93 show
the foot at its maximum development, with its two pointed toes.

ASPLANCHNA EBBESBORNII (ROTIFER) 407

It projects forward over the anterior end of the embryo and
obscures a part of the fold which contributes to the formation of
the disc. These figures show how the disc is depressed posteri-
orly on the ventral side. The dorsal part of the disc is indicated
in figure 92. The entire disc in its final stage of development
with the outer row of cilia, is represented in figure 99, ventral
view. ‘The trochal disc is very retractile, and its activities are
regulated by a number of well-developed muscle fibers. The
cilia extend into the buccal pouch, but not into the pharynx.
The ciliary wreathes serve for locomotion and feeding.

DIGESTIVE SYSTEM

The major portion of the digestive system is derived from the
large mesentoblast cell E, the stomodaeum and pharynx from
the ectoderm. The early stages in the formation of the anterior
end of the digestive tract begin as an invagination on the ventro-
anterior end (figs. 71 to 76). The development of the head region
in the formation of the trochal disc is closely associated with the
origin and growth of the anterior end of the digestive tract, and
the prominence of the trochal folds is accentuated by the
stomodeal invagination. Figures 76 and 77 represent optical
sections from transparent whole mounts in the early stages of
differentiation in the region from which the pharynx develops.
The embryos are folded ventrally, making the dorsal side appear
abnormally long. These figures show that two distinct regions
are recognizable—an outer epithelial portion, which gives rise to
the definitive ectoderm and the musculature, and an inner
slightly differentiated region, which produces part of the enteric
canal and the entire reproductive system with a few muscle
fibers directly connected with it. As the cells invaginate to form
the pharynx, the products of the mesentoblast are forced more
and more posteriorly (figs. 75 to 77). The cells on the posterior
face of the invaginated cavity become several layers thick and
form the ventral and posterior walls of the pharynx, from which
the jaws are later developed. The anterior and dorsal walls of
the pharynx seldom become more than one layer of cells in thick-
ness. Rudimentary salivary glands are often present in connec-
tion with the walls of the pharynx (fig. 102).

408 GEORGE W. TANNREUTHER

The inner cell mass, as shown in figure 77, becomes differen-
tiated into the parts which produce the pharynx, the oesophagus,
stomach, and the gastric glands, and those which form the repro-
ductive structures. A later differentiation is indicated in figure
79, where the lumen of the stomach is present and the pharyngeal
wall has become several layers thick on its posterior face. Figures
80, 82 and 88 represent horizontal sections of the preceding
figure at different levels. The space which corresponds to the
body cavity is evident at different points. These three sections
as a whole show little differentiation and are rather hard to
interpret unless directly compared with the figure from which
they were taken. In the further growth and curvature of the
embryo the upper wall of the pharynx fuses with the entodermal
cells (figs. 85 to 87) forming the region which later is differen-
tiated into the oesophagus and the gastric glands. In a medium
longitudinal section of an embryo (fig. 87) corresponding to
figure 86, a large concavity is evident on the ventral side. This
is true of all embryos, when the invagination in the formation of
the pharynx has reached its maximum extent in a dorsoposterior
direction. Figure 89 (ventral view) represents the condition of
the embryo, when the pharynx has reached its maximum devel-
opment. The anterior and posterior ends of the embryo are in
immediate contact and the mouth is in the center of the embryo
when taken as a whole. An optical section of the same stage
(fig. 90) shows the position of the mesentodermal cells as they
are forced nearer the posterior end. This shifting is due to the
invagination of the ectoderm in the formation of the anterior
end of the enteric canal. The above figure shows the connection
of the pharyngeal and the entodermal cells.

The embryo at this stage of development has reached its
maximum curvature and growth in length (figs. 91 to 93), but
does not resemble the adult rotifer. The embryo now begins to
straighten out (figs. 94 and 95), the foot is gradually absorbed,
and the mouth is carried more anteriorly (fig. 96) during the
process. The invaginated cavity is differentiated into the
buccal cavity and the pharynx proper. ‘The embryo during its
period of maximum curvature has a segmented appearance (fig.

ASPLANCHNA EBBESBORNII (ROTIFER) 409

97), which is due to the folds in the body wall. The pharynx
and stomach are now united by a distinct tube, the oesophagus
(figs. 97 and 98) and the digestive system is now completely
formed and differentiated into the following regions: buccal
pouch, pharynx, oesophagus, and stomach. ‘The trophi or jaws
are formed from the ventroposterior wall of the pharynx (fig. 100).
The gastric glands are formed at the junction of the oesophagus
and the stomach (figs. 103 and 104). A few of the muscles which
are directly connected with the different organs are represented
in figures 98, 101, and 102. Figures 96 to 101 show the gradual
straightening of the embryo, and the migration of the mouth to
its definitive position. A small part of the foot still persists.

The trochal disc with its cilia (fig. 99) is completely formed.
Its relation to the buccal pouch is represented in figure 100, a
horizontal section of the same stage. The invagination of the
ectoderm to form the urogenital sinus (figs. 98 and 101) corre-
sponds to the proctodaeum when an intestine is present. It is
formed on the dorsal side of the foot near its base, but when the
foot is completely absorbed the opening is considered as being
on the ventroposterior end. Figures 102 and 103 represent
horizontal sections of figure 101, taken near the ventral and dorsal
sides, respectively. The body cavity at this stage becomes very
prominent and the different organs within reach their definitive
condition. Figure 104 represents an embryo which has reached
its distinct adult condition, represented as a transparent object
from the dorsal side. All of the more important organs are
shown. The digestive tract has reached its complete develop-
ment as it occurs in the parthenogenetic female. The cells of
the ectoderm lose their boundaries and become a definite syncytial
layer, and the ectoderm with its delicate smooth cuticula con-
stitutes the body wall.

The early stages in the development of the digestive tract of
the male and female are similar, but when the male embryo has
reached the condition represented in figures 79 and 87, there is
a temporary fusion of the cells of the wall of the pharynx with
those of the entoderm as in the female. The cells which, in the
female, produce the oesophagus, cease to divide in the male

410 GEORGE W. TANNREUTHER

embryo and have more the appearance of yolk cells. These
cells gradually recede from the pharyngeal wall (fig. 5), and
finally take up a position on the dorsal side of the embryo (figs.
2, 6). Figure 6 represents the adult condition of the digestive
tract in the male at birth. The stomach in the female rotifer
Asplanchna ebbesbornii ends blindly. ‘There is no indication
of an intestine in the early stages of embryonic life.

REPRODUCTIVE SYSTEM

The origin of the reproductive organs is directly associated
with the entoderm and they arise from the derivatives of the
mesentoblast. Thus, the digestive and the reproductive organs
have a common origin. The differentiation of the reproductive
system is very rapid, especially in the male, since they are sexually
mature before birth. Practically all of the derivatives of the
large cells E?!1 and E*! of the five large mesentodermal cells
(fig. 67) contribute to the formation of the reproductive system.
About the first indication in the differentiation of the digestive
and reproductive regions is the appearance of darker granules
in the reproductive portion. This differentiation is well marked
during the early formation of the pharynx (figs. 76, 77, 79, and
87). These cells are indicated by heavy stippling.

The reproductive cells continue to divide and later become
specialized into two groups or regions (fig. 90), one forming the
vitellarium and the other the oviduct and uterus, which becomes
continuous with the urogenital sinus (figs. 95 to 97). The
differentiating vitellarium is at first a spherical mass of cells, but
later sends out an arm right and left and finally becomes U-shaped
(figs. 95 to 104). The vitellarium produces the yolk and supplies
the egg with its granular and yolk content. Yolk production is
well marked in those individuals that produce thick-shelled
resting eggs. A small portion of the vitellarium, in the region of
the oviduct becomes specialized into a rudimentary ovary (figs.
100 to 104), which contains a number of very minute cells.
These cells enlarge, one at a time (occasionally two develop
simultaneously), and become the mature ova. In the older
female, at the close of the reproductive period, the ovary isvery

ASPLANCHNA EBBESBORNII (ROTIFER) 411

indistinct and finally is indiscernible. No vitellarium is formed
in the male, but instead the cells are differentiated into the testis
and the vas deferens, which are continuous with the urogenital
sinus, which in the male is ciliated. These cilia are very active
and aid in the passage of the sperm from the vas deferens into
the uterus. In the female the upper end of the reproductive
tube is very narrow and can be designated as the oviduct. The
urinary bladder is formed by an evagination of the urogenital
sinus.

THE EXCRETORY SYSTEM

The excretory system is ectodermal in origin. It arises from
special cells, which become separated from the outer embryonic
epithelial layer on its inner surface at the ventroposterior end
(figs. 77, 79, 87, and 90). These cells act like teloblasts and, by
a rapid proliferation of cells in an anterior direction, produce on
either side of the ventral median line a mass of cells (text figs.
o and p) from which the excretory tubules are formed. The
cells of each band are arranged in definite rows. Later cavities
appear within the cell rows; these become continuous from cell
to cell and give rise to the lumen of the future forming tubules
(text figs. q andr). The cells directly concerned in this process
contribute to the tubule walls with a few persisting nuclei.
Later, each band becomes differentiated into a system of tubules
with nucleated walls (text fig. s). Text figure u represents the
condition of the embryonic excretory tubules of the right side of
the embryo, shortly before birth. Either half is composed of
one main and two recurrent tubules. The three tubules are
united at either end. The manner in which this union occurs
varies in different individuals. Each tubule has a distinct, non-
ciliated lumen with a nucleated wall. A number of small club-
like evaginations are formed on the main tubule of either side.
Their number and size vary. The maximum number found on
any one tubule was fifty. The free ends of many of these evagi-
nations are enlarged and contain cilia, which exhibit a constant
flickering motion. These club-shaped organs constitute the so-
called tags or flame cells. In their early formation the free ends

412 GEORGE W. TANNREUTHER

often show protruding cilia and communicate directly with the
body cavity (text fig. s). In the later stages, however, the open
tags are rare and exhibit closed ends with pending cilia (text fig.
u). Tags are never formed on the recurrent tubules.

The systems of excretory tubules on either side do not com-
municate at their anterior ends, but at their posterior ends they
connect with the urinary bladder. The urogenital sinus, as
stated above, arises as a solid ingrowth of the ectoderm at the

ae

ener
cs

rs Gu)
oe se

= Pee Yo
Bers
ABQ GIO

Ce)

Oe .
f at
Oe

Figs. o and p Median longitudinal sections, to show the early formation of
the excretory system; note the nephridial bands on the ventral side within the
ectoderm.

Fig. q Horizontal section of the same stage as above; the section shows the
rows of nephridial cells and their connections with the extoderm at different
intervals; these connections become the controlling muscle fibers.

Fig. r A horizontal section of a later state; shows the early lumen in the
formation of the excretory tubules.

ABBREVIATIONS
a., anterior . n.b., nephridial band
‘b.c., body cavity ph., pharynx
d, dorsal sto, stomodaeum
m.c., muscle cell ur.s., urogenital sinus
m.f., vauscle fiber v, ventral

mo, mouth

ASPLANCHNA EBBESBORNII (ROTIFER) 413

dorsal posterior end (text fig. r and fig. 98). The urinary bladder
(pulsating vacuole) is formed by an evagination and enlargement
of the inner end of this invagination (text figs. s and t). The
walls of the urogenital sinus on either side, in its early differen-
tiation, fuse with the posterior ends of the group of cells which
form the excretory rudiments (text figs. r and s), and finally a
direct communication is established between the pulsating

Fig. s Horizontal section of embryo, to show fomration of the excretory
system, tags, urogenital sinus, and bladder.

Fig. t Median longitudinal section, to show the relation of parts.

Fig. u Completely formed excretory tubules from the right side of embryo
at the time of birth.

ABBREVIATIONS
bl, bladder oe, oesophagus
br, brain ov, ovary
b.w., body wall r.t., recurrent tubules
c, cilia st. stomach
d.g., digestive. glands ta, tags (flame cells)
ft, foot u, uterus
k.t., kidney tubules vt, vitellarium

lu, lumen

414 GEORGE W. TANNREUTHER

vacuole and the tubules. At first the urinary bladder shows a
distinctly nucleated wall (text fig. s), but later, as enlargement
continues, the cells are no longer distinguishable and the wall
becomes more membranous, as in the adult. Text figure t, an
optical section of a fourteen-hour embryo from the left side,
shows the relation of the different parts of the excretory system.

The movement of a finely granular substance within the lumen
of the tubules is visible under high magnificatién. This flow of
substance is due to the action of the cilia within the flame cells
and the activities of the delicate fibers in the tubule walls. The
constant rhythmical contraction and expansion of the urinary
bladder contribute to the process of elimination of the excretory
products. In the formation of the excretory system on the
ventral side, its early rudiments do not lie free in the body
cavity but are directly connected with the ectoderm at different
points by means of cells, which originated from the ectoderm
(text figs. q and r). These cell connectives later develop into
the muscles, which connect the forming tubules with the body
wall. These tubules at first are on the ventral side of the body
cavity, but later, as their associated muscle fibers develop, the
excretory tubules may occupy almost any part of the body
cavity, depending upon the contraction and expansion of their
controlling muscles.

NERVOUS SYSTEM

The brain is formed by a solid ingrowth or proliferation of
cells from. the ectoderm at the anterior end of the embryo, dorsal
to the mouth (figs. 96 and 98). A small portion of the brain on
the dorsal side gives rise to the rudimentary eye, which appears
as ared pigmented body. The different intensities in the pigment
formation of the eye can be recognized in the developing embryo.
The origin of the nerve fibers can be distinguished in section (fig.
103). This figure shows the origin of the lateral and anterior
nerves, which innervate the trochal disc. Each of the lateral
trunks gives off a branch which runs nearly the entire length of
the animal. These nerves are hard to demonstrate except in
the close proximity to their origin.

ASPLANCHNA EBBESBORNII (ROTIFER) 415

Sense organs: The antennae are tubular outgrowths of the
ectoderm with number of sense hairs projecting from the apex
of each. In their early development, these hairs show a slight
vibratory action, but when fully developed they are stiffer and
firmer and serve as organs of touch. Delicate nerves extend
from the brain to the different antennae. There are four an-
tennae present, two at the anterior end and one on either side
of the body in a dorsolateral position, nearer the posterior end
(figs. 1 to 3, and 6).

POSITION OF ROTIFERS IN THE ANIMAL KINGDOM

The position of rotifers has been a subject of considerable
controversy. Huxley (’51) suggested that they represent a
primitive form and are preserved, with modification, in the
larvae of molluscs, annelids, and other forms. Lankester main-
tained a similar view and considered the trochophore of worms
and molluscs, when compared with rotifers, as possessing close
relationships. Hartog held the view that the structure of the
rotifers brings them into close relationship with the lower flat
worms and with the more primitive larvae of the Nemerteans,
the Pilidium, and that there is a striking resemblance in the
structure and function of the different parts, when carefully
compared. Thus the rotifers, he says, may be considered as a
group apart, but probably representing an early offshoot from a
free-swimming plathelminths (Rhabdocoele) with minor change.

Zelinka endeavored to prove that the course of development
in the rotifer Callidina russeola, as well as other rotifers, shows
affinites with the trochophore larvae of Annelida and Mollusca,
and that the adult rotifers are in a sense persistent trochophore
larvae. Balfour states that the trochophore larva is found in
rotifers where it is preserved in the adult state, and that there
is every reason to believe that the types with trochophore
larvae, viz., the Rotifera, the Mollusca, the Chaetopoda, and
the Polyzoa, are descended from a common ancestral form, and
that it is also fairly certain there was a remote ancestor common
to these forms and to the Plathelminthes. Other investigators

416 GEORGE W. TANNREUTHER

interpret the trochophore as representing a simplified form, an
ancestor which, were it living to-day, would be classified as a
ctenophore, and that the three distinct larval types, namely,
Miillers larva, the Pilidium, and the trochophore, all represent,
with more or less change of form, a group of ancestral Cteno-
phora, from which sprang the Polyclada, and through them all
the plathelminthes, the Nemertfans, and the annelids.

Considering the life history of the different groups in question
from the standpoint of resemblances and differences, the above
phyletic scheme becomes a tenable one. If the rotifers repre-
sent a primitive or ancestral type, and are preserved, with modi-
fications, in the larvae of annelids and other forms, what position
in the phyletic scheme do they occupy, more especially when —
considered from the standpoint of cleavage and early develop-
ment? The larvae of the annelids not only showresemblances
to that of the rotifers, but, in addition, they possess in concen-
trated form the rudiments of the future adult annelid body.
Also the mesodermal structure in the trochophore must un-
doubtedly represent the mesoderm of the ancestral type. The
point at issue is, however, not so much what the completely
formed larvae of annelids or the rotifers as such possess, but is
there any parallelism in their development in reaching this point,
which is of any phyletic value?

If cell homology have any significance, according to some
writers we must conclude that the cells whose products are
homologous must be themselves homologous, even though they
may have the same or different origin and position in cleavage.
Light on the systematic position of rotifers may be gained by
comparison of their development with that of other groups. The
studies on cell lineage no doubt are invaluable in determining
phyletic relationships, as has been proved by results along this
line of investigation. Characters of forms, as manifested during
cleavage, in many instances are as constant as are anatomical
characters in later stages, and must therefore be as truly in-
herited. And since coenogenetic changes may be supposed to
affect the later stages of development first, we may expect to
find earlier stages retaining longer their primitive characters. It

ASPLANCHNA EBBESBORNII (ROTIFER) 417

is true that the early development cannot be regarded as in-
fallible in determining relationships in every case, yet it may
be called to aid in solving genetic relationships of questionable
forms.

It is with the above points in view that the early stages in the
development of’ the rotifers, as compared with other forms, are
emphasized as being of considerable phyletic importance in the
determining of relationships.

In Asplanchna ebbesbornii, the early development shows such
striking and accurate resemblances to that of the annelids, es-
pecially that of the fresh-water Bdellodrillus, that it seems
almost impossible to think that such minute similarities could
have arisen independently in their evolutionary history.

The shape of the eggs, the position of the polar body or bodies, '
and the early cleavage stages are almost identical. In the be-
ginning with the one-cell stage the position of the cleavage spindle
and the direction of the cleavage are the same, dividing the egg
into two very unequal cells (figs. 7, 8, 16, and 17). The first
_ cleavage plane occurs at right angles to the median longitudinal
axis of the future adult. In the formation of the four-cell stage,
the large cell divides first and very unequally, while the smaller
cell divides equally. These two cleavages, taken together repre-
sent the second cleavage as it occurs in the annelids. The
large cell D is posterior, B anterior, C right, and A left, with
reference to the median axis of the future individual. The
large cell D and its derivatives always divide first. The position
of the four macroneres, as in Bdellodrillus, determine the orienta-
tion of the future adult organs of the rotifer (figs. 20 to 24).

The sequence of cleavage stages corresponds to that of the
polyclades, nemerteans, and the annelids. Three generations of
micromeres are formed, which contribute to all of the definitive
ectoderm and the ectomesoderm (larval mesoderm of the anne-
lids). After the formation of d*, 3D gives rise to all of the en-
toderm and the mesentoderm, including the reproductive organs.
In the rotifers, however, the entoderm and mesoderm are sepa-
rated at a later stage in the cleavage of 3D, also 3A, 3B and
3C remain on the surface in the region of the blastopore and do

418 GEORGE W. TANNREUTHER

not invaginate with the large cell 3D, and thus take no part in
the formation of the future gut, but, by a rapid proliferation,
their products grow posteriorly and form the major portion of the
foot, which is absorbed before birth. In the polyclade Planocera,
as in the rotifers, 3D has the same fate; likewise, 3B, 3C, and 3A,
with their derivatives, take part in the invagination, but do not
contribute to the gut, and are later absorbed as food.

As stated, the sequence of cleavage in the formation of the
quartettes in the rotifers is comparable to those of the poly-
clades and annelids, but the position taken by the resultant
generations of cells is different. This difference, however, is
adaptive. The cells, instead of remaining in a more spherical
mass, are drawn out in an anteroposterior direction. The
cleavage cells take up this early position in accordance with
their later formation and the needs of the future animal. The
rotifers, in their early development, possess characters which
are common to both the polyclades and the annelids. In ad-
dition, however, they possess characteristics which are peculiar
to the polyclades and others which are distinctly annelidian.

When rotifers are compared with the adult Dinophilus, some
very striking points of resemblance are recognizable. In both
the bands of cilia, which are the free-swimming organs of loco-
motion, represent the prototroch or remnants of it. The adult
Dinophilus remains to a certain extent at the stage of the an-
nelid larva or a stationary larva which has become sexually
mature. Here the trochophoral characteristics persist in common
with the worm like-form of the annelids.

In the rotifers the trochophore stage persists, and among
some of the rotifers at least, it is the end or climax stage in
development. Many of the rotifers, however, possess in ad-
dition a worm-like body and are capable of an annelid creeping
motion, independent of the trochal cilia, which indicates to some
degree a specialization in a definitely directed line. As pre-
viously stated, there is a distinct parallelism in the development
of the two forms. The sequence of cleavage is the same, but
there is a dissimilarity in the position taken by the cleavage
cells. This difference is, however, adaptive. In the rotifers the

ASPLANCHNA EBBESBORNII (ROTIFER) 419

entire animal (trochal region, body, and foot), develop simul-
taneously, hence the position taken by the early cleavage cells to
meet the future needs in the formation of the adult structures.

In Dinophilus the larval or trochophoral development is ac-
centuated, and the future adult body at first exists in a concen-
trated form in a certain cell or group of cells at the future
posterior end of the animal. The end result in either case is a
distinctly formed animal, which exhibits many points that are
common to both. The development of Dinophilus, however, is
more like that of the annelids than of the rotifers, and could be
considered as intermediate between the two groups of animals.
Further, when considered from the standpoint of development,
the rotifers exhibit many points of resemblance to the annelid
larva, and no doubt represent a primitive type preserved as
modifications in the annelid larva, but they cannot be regarded
as an ancestral type from which the annelids sprung, but rather
a form which represented the annelids at one period in their
phylogeny.

Hence from cleavage and development of the various forms in
question, the conclusion is reached that the polyclades, annelids,
and rotifers must have originated from a common ancestral
form comparable to that of the ctenophore.

The rotifers, although retaining their primitive condition, have
departed somewhat from the common ancestral form and to some
degree have become a specialized group. The annelids, on the
other hand, have reached a much higher level in their evolu-
tionary history and show affinities to the ancestral type during
their larval development.

GENERAL SUMMARY

The undivided egg of Asplanchna ebbesbornii is nearly oval.
Its median longitudinal axis, passing through the polar body or
bodies, corresponds to the median longitudinal axis of the
future adult. The first cleavage plane is at right angles to this
axis and divides the egg into two very unequal cells. The
second cleavage occurs at an angle of about 45° to that of the
first. It divides the smaller cell equally and the larger very
unequally. The larger cell divides first.

420 GEORGE W. TANNREUTHER

In the four-cell embryo, the large cell, D, is posterior, B
anterior A left, and C right. The ectoderm is separated from the
four macromeres by a series of three cleavages. The three quar-
tettes of ectomeres share in the formation of the ectomesoderm.
The macromere 3D gives rise to all of the entoderm, the repro-
ductive bodies, and some of their directly associated muscle
fibers. The macromeres 3A, 3B, and 3C, do not invaginate
with 3D, but remain on the surface in the region of the blasto-
pore and, by a rapid proliferation of cells, give rise to the major
portion of the temporary foot, which is absorbed before birth.

Three kinds of eggs are formed: The female-producing and
the male-producing, both of which develop parthenogenetically,
and the thin- and thick-shelled resting eggs, which are fertilized
and always produce females. There are two kinds of adult
females, one produces females parthenogenetically and the other,
males parthenogentically and resting eggs. Males and females
or resting eggs and females are never produced by the same
parent. The male and resting eggs have two polar bodies.
When conditions are favorable, it requires about seventeen hours
from the time of the formation of the polar body to the time of
birth of the parthenogenetically produced males and females.
The males and females are sexually mature at birth. The female
increases four to eight times its size at time of birth. The males
do not increase in size after birth, but perish almost immediately
after copulation. Very few free-swimming males are found at
any one time in the culture. The males are structurally de-
generate. The development of the males and females is similar
up to the late period in embryonic life. The cells of the stomach
and the invaginated pharynx become continuous, but the
oesophagus is never formed.

Gastrulation is epibolic. A central cavity is formed, which
begins at the close of the formation of the third quartette of
micromeres. The cavity is due to the shortening of the cells in
the epithelial ectoderm, and is later occupied by the large cell,
3D. The gastrula is composed of a single outer epithelial layer,
enclosing 3D, d‘, and d® with their later derivatives.

ASPLANCHNA EBBESBORNII (ROTIFER) 421

The digestive system is composed of the mouth, buccal pouch,
pharynx, stomach, and the attached digestive glands. The
stomach is strongly ciliated. There is no evidence of an intes-
tine in embryonic life. The trochal dise is formed from early
proliferated ectodermal folds at the ventro-anterior end. The
embryo, at its greatest flexure, is completely bent on itself, and
the embryonic foot extends anteriorly over the stomodaeum. The
brain is formed by a solid ingrowth of ectoderm at the anterior
end, above the buccal pouch.

The excretory system is formed on the ventrolateral sides of
the median line from excretory bands or rows of cells. These
bands begin at the ventroposterior end by the proliferation of
special cells derived from the ectoderm. The adult system on
either side consists of one main and two recurrent tubules. The
main tube has a number of small evaginated tags bearing cilia
on their interior.

I take this opportunity to express my gratitude to Mr. George
T. Kline, the biological artist, for his helpful suggestions and
execution of the drawings and lettering.

Columbia, Missouri, January 1, 1919

JOURNAL OF MORPHOLOGY, VOL. 33, NO. 2

422 GEORGE W. TANNREUTHER

LITERATURE CITED

Hupson, C. T., anp Gossz, P. H. 1889 Rotifera or wheel-animalcules. 2 vols.
London.

JenninGs, H.S. 1896 The early development of Asplanchna herricki. Bull.
Mus. Comp. Zool. Harvard, vol. 30.
1894 Rotatoria of the Great Lakes and some of the inland lakes of
Michigan. Bull. Mich. Fish Comm., no. 3.
1902 Rotatoria of the U. S. II. A monograph of the Ratulidae.
Bull. U. 8S. Fish Comm.

Monrcomery, T. H. 1903 On the morphology of the Rotatorian family Flos-
cularidae. Pro. Ac. Nat. Sci. Phila.

Netson, A. J. 1904 The early development of Dinophilus. Proc. Ac. Nat. Sei.
Phila.

Surrace, F. M. 1907 The early development of a polyclade Planocera in-
quilina. Proc. Ac. Nat. Sci. Phila.

TANNREUTHER, G. W. 1915 The embryology of Bdellodrilus philadelphicus.
Journ. Morph., vol. 26.

ZELINKA, C. 1892 Studien iiber Radertiere III. Zeitschr. fiir wis. Zool.,
Bd. 53.

PLATES

EXPLANATION OF PLATES

All drawings were made with the aid of the camera lucida under a magnification
of 130 diameters. All whole-mount drawings were made from either the fixed or
living egg and checked. Variations in the size of the surface views is due to a

difference in the size of the eggs.

The sections were not uniformly magnified.

Stippling has been adopted for the sake of clearness.

REFERENCE LETTERS

a., anterior

a.a., anterior antennae
b.c., body cavity

bl., bladder

br., brain

bu.c., buecal cavity
b.w., body wall

c., cilia

c.c., cleavage cavity
cin., cingulum

c.l., cerebral lobes
coe., coelom

cut., cuticula

d., dorsal

d.a., dorsal arms
d.g., digestive glands
e., eye spot

€.cp., egg capsule
ect., ectoderm

emb., embryo

ent., entoderm

ft., foot

gn., ganglion

7.e.sh., inner egg-shell membrane
k.t., kidney tubule
l.a., lateral antennae
lu., lamen

m., muscle

m.c., muscle cell
mes., mesoderm
m.f., muscle fiber
mo., mouth

n.b., nephridial band

oe., oesophagus

0.e.sh., outer egg-shell membrane

ov., ovary

ovd., oviduct

p., posterior

p.b., polar body

ph., pharynx

pr., proctodaeum

p.s., polar spindle

r.t., recurrent tubules

st., stomach

sto., stomodaeum

ta., tag (flame cell)

tr., trochal dise (trochus)

tro., trophi

ts., testis

u., uterus

ur.s., urogenital sinus

v., ventral

v.m., vitelline membrane

vt., vitellarium

A, left macromere

B, anterior macromere

C, right macromere

D, posterior macromere

a}, b', c}, d', d'-!, ect., first generation
of ectomeres

a?, b?, c?, d?, a®-!, etc., second genera-
tion of eetomeres

a®, b3, c3, d3, a3.1, ete., third generation
of extomeres

E, entoderm (mesentoblast)

PLATE 1

EXPLANATION OF FIGURES

1 Dorsal view of a female-producing individual.

2 Dorsal view of a male-producing individual, with nine male embryos at
different stages of development.

3 Young female at time of birth, viewed from the left side.

4 Reproductive organs of a young female, dorsal view.

5 Male embryo, from dorsal view before birth; note the position of the
digestive system (d.s.).

6 Male at time of birth, from right side; compare the final position of the
degenerating digestive system with the preceding figure.

7 to 9 Thin-shelled resting egg, one- to four-cell stage; note the irregular
structure of the egg membrane.

10 and 11 Thick-shelled resting egg; note the porous condition of the inner
egg membrane.

12 Sperm cells in different stages of development.

424

ASPLANCHNA EBBESBORNII (ROTIFER) PLATE 1
GEORGE W. TANNREUTHER ;

PLATE 2

EXPLANATION OF FIGURES

13 Parthenogenetic egg with polar spindle.

14and15 Same as the preceding after maturation.

16 Two-cell stage, dorsal view.

17 Two-cell stage, upper view, with asters for second cleavage.

18 Two-cell stage, ventral view, with CD dividing.

19 Four-cell stage, upper pole.

20 Four-cell stage, ventral pole.

21 Four-cell stage, dorsal view, showing the cells contracted.

22 and 23 Four-cell stage from left and dorsal sides.

24 Little later than figure 23, with d! cleavage-spindle forming; note the
anterior extension of D in the process.

25 Five-cell stage, left side, to show lengthening of cells.

26 to 28 Five-cell stage, left and dorsal sides; note the abnormal flattening
of cell d! in figure 28.

29 Five-cell stage, dorsal pole; shows the position and the rounded condition
of the cells in preparation for the next cleavage.

30 Six-cell stage, dorsal view; note the position taken by d! and c! in the
early third clevage.

31 Seven-cell stage, dorsal view.

426

PLATE 2

ASPLANCHNA EBBESBORNII (ROTIFER)

TANNREUTHER

GEORGE W.

PLATE 3

EXPLANATION OF FIGURES

32 Eight-cell stage, ventral view.

33 Hight-cell stage, dorsal view; the ectomeres are above instead of anterior
to the parent cells as in figure 32.

34 Early nine-cell stage, upper pole (dorsal view).

35 Late nine-cell stage, ventral view.

36-37 Ten-cell stage, ventral and right sides.

38 Sixteen-cell stage, ventral view; the embryo is extended in an antero-
posterior direction.

39 and40_ Sixteen-cell stage, the left side; D side is dorsal and dorso-posterior.

41 Sixteen-cell stage, right side.

42 Sixteen-cell stage, ventral view; all of the cells are represented; note the
shape of the cells.

43 and 44 Sixteen-cell stage, dorsal view; the D derivatives are considerably
elongaged transversely.

45 Nineteen-cell stage, right side; d'!, d?, d'-! having divided; the eggs at
this stage are transparent and the position of all the cells can be recognized.

46 Twenty-cell stage, right side; the extra cell is d* from D; the formation
of d* is unusual.

47 Twenty-nine-cell stage, upper view; a continuation of figure 46 during the
fifth cleavage; the A row of cells has not divided.

425

ASPLANCHNA EBBESBORNII (ROTIFER) PLATE 3
GEORGE W. TANNREUTHER

PLATE 4

EXPLANATION OF FIGURES

48 Nineteen-cell stage, dorsal view, showing the exact position of all the
cleavage cells.

49 Thirty-three-cell stage, dorsal view; the end of fifth cleavage; d* is present
and has divided; the large cell D (E) has begun to invaginate.

50 Thirty-five-cell stage, dorsal view; the minute cells d‘ and d® are the
additional cells formed.

51 Thirty-four-cell stage, ventral view.

52 Thirty-four-cell stage, posterior end of the embryo, to show the position
of the closing blastopore.

53 Thirty-three-cell stage; same view as preceding figure.

54 Later stage in the closure of the blastopore from the posterior end.

55 to 57. About fifty-cell stage, showing the invagination of the large cell E
the central cavity into which E passes is indicated by fine stippling.

58 Sixty-four-cell stage, dorsal view, showing the first cleavage spindle of E.

59 Same as the preceding from ventral view.

60 Later stage in which E has divided.

430

PLATE 5

EXPLANATION OF FIGURES

61 Late cleavage, dorsal view, showing the preparation of E? for division.

62 A little later than preceding, showing three large entodermal cells; the
embryo is considerably contracted and is more spherical.

63 Two-hundred-and-fifty-cell stage, dorsal view.

64 Same as preceding, in a contracted condition.

65 Embryo with the dorsal ectoderm not represented, to show E and its
derivatives; the two posterior cells are in preparation for cleavage.

66 Stage little later than last; shows the five large interior cells; e!-? not
represented.

67 Embryo viewed from the right side, the ectoderm not shown; the interior
cells are slightly rounded and do not completely fill the space; the dorsal side is
to the right of figure; the blastopore is closed.

68 Dorsal view of embryo with overlying ectoderm not shown; ten large
entodermal cells are present, five upper and five lower; the cavity represented is
the future body cavity.

69 Same as the preceding, contracted, with the ectoderm represented.

70 An embryo showing the division of the five upper and five lower large
entodermal cells; a few of the spindles are shown.

71 Late embryo from the right side showing the early formation of the
stomodaeum and the temporary foot.

72 An optical section of embryo little Jater than last.

'

432

ASPLANCHNA EBBESBORNII (ROTIFER) PLATE 5

GEORGE W » TANNREUTHER

433

PLATE 6

EXPLANATION OF FIGURES

73 Embryo, ventral view, showing position of stomodaeum and the be-
ginning of the trochal folds (ectodermal proliferations).

74 Same as figure 73, right side; the unshaded portion represents the trochal
prominence, especially near the ventral edge.

75 A median longitudinal section of the preceding figure.

76 A median longitudinal section of a twelve-hour embryo, to show the
early formation of the foot and the differentiation of the inner cell mass in the
formation of the digestive and reproductive systems.

77 A longitudinal section to side of median line, to show further differen-
tiation.

78 Ventral view of whole embryo to show formation of foot and trochal
folds.

79 Longitudinal section of preceding figure; the lumen of stomach is formed.

80 Horizontal section of figure 79, taken at a-a.

81 Embryo, ventral view, to show the approximation of the two ends; the
lateral folds later become part of the trochal disc.

82 Horizontal section of figure 79 taken at b-b.

83 Embryo from right ventral side.

84 Same as the preceding, dorsal view, showing the condition of the ecto-
dermal cells; this figure represents a three-hundred-cell embryo.

85 Embryo, ventral view, showing further differentiation of the foot and
development of the lateral trochal folds.

86 Embryo, ventral view, showing the anterior and posterior folds of the
trochal disc; note position of mouth.

87 Longitudinal section of embryo corresponding to figure 86.

88 Horizontal section of figure 79 taken at c—c.

°

434

ASPLANCHNA EBBESBORNII (ROTIFER) PLATE 6
GEORGE W. TANNREUTHER

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PLATE 7

EXPLANATION OF FIGURES

89 Ventral view of embryo, showing the curvature in a ventral direction;
the trochal disc is completely formed, but is very irregular; the ventral view
at this stage corresponds to the ends of the embryo.

90 Longitudinal section of embryo represented in figure 89.

91 and 92 Embryos, ventral view showing the overlapping of ends; the foot
at this stage is forked.

93 Embryo showing maximum curvature.

94 Embryo contracted into spherical condition.

95 Longitudinal section of figure 93 taken to the side of the median line.

96 A median longitudinal section of embryo passing through the brain region;
note the position of the mouth; the embryo has begun to straighten.

97 Embryo from left side, showing maximum development of foot; the seg-
mented appearance is due to the folded condition of the ectoderm.

98 Longitudinal section of figure 97; the digestive system is completely
formed.

99 Embryo showing the further separation of the two ends and the shortening
of the foot; the ciliated trochal dise is formed.

100 An optical horizontal section passing through the floor of the pharynx
and the reproductive organs; note condition of body cavity and body wall.

101 Median longitudinal section of embryo later than preceding; the mouth
has reached its definitive position at the ventro-anterior end.

102 and 103 Horizontal sections of preceding figure through dorsal and
ventral sides, respectively.

104 An optical section of embryo at time of birth, showing the relative
position and relation of different organs.

436

ASPLANCHNA EBBESBORNII (ROTIFER) PLATE 7
GEORGE W. TANNREUTHER

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JOURNAL OF MORPHOLOGY, VOL. 33, NO. 2

Resumen por el autor, Henry Carrol Tracy.
Escuela de Medicina Marquette.

E] crdneo cupleoideo y su relacién con el diverticulo de la vejiga
natatoria y el laberinto membranoso.

El diverticulo precelémico de la vejiga natatoria de los cuplei-
dos envia a la base del crdneo, a cada lado de la cabeza, una rama
que ocupa un canal en los huesos exoccipital y proético, y termina
en dos vesiculas dilatadas, una de las cuales esta situada en una
cdpsula 6sea del hueso pterdético, y la otra en una capsula 6sea del
proético. Los rasgos especializados del craéneo cupleoideo son
los siguientes: El orificio auditorio situado entre los huesos basi-
occipital, pterdtico y prodtico; el orificio temporal colocado entre
los huesos frontal, parietal, epidtico y pterdtico, ocupado por una
voluminosa expansion del canal de la linea lateral; el receso lateral
(que communica interiormente con la cavidad craneal) esta colo-
cado entre los huesos esfen6tico, pterdtico y prodtico y el ala late-
ral del frontal; la capsula 6sea esférica del hueso prodtico se abre
interiormente por medio de una ventana en forma de hendidura;
una vesicula fusiforme en el hueso exoccipital; una cépsula désea
esférica en el hueso pterético. Una parte del canal de la linea
lateral esta situada en el receso lateral, que sirve de canal libre
para la transmisién de la presién ejercida por el agua desde el
exterior a los espacios situados alrededor del laberinto. El gan-
glio trigémino-facial esta situado en un receso del borde anterior
del hueso prodtico, en la superficie de la cApsula 6sea de dicho
hueso. Las ramas recurrentes de los nervios facial y vago for-
man un plexo intracraneal que parece inervar los canales de la
linea lateral del receso lateral y el orificio temporal.

Translation by José F. Nonidez
Carnegie Institution of Washington

AUTHOR'S ABSTRACT OF THIS PAPER ISSUED
BY THE BIBLIOGRAPHIC SERVICE, FEBRUARY 23

THE CLUPEOID CRANIUM IN ITS RELATION TO
THE SWIMBLADDER DIVERTICULUM AND
THE MEMBRANOUS LABYRINTH

HENRY C. TRACY

Department of Anatomy, University of Kansas

THREE FIGURES AND FIVE PLATES (TWELVE FIGURES)

CONTENTS
Iii SROCCHIOCING.. 5. tro Rots he SE GOB BES ple OB Oa RPI es och Clea anne ets eer ee 439
Historical summary of the literature on the ear-swimbladder relation in
CiIOTaVENClEES. 3 Sewolen pct notes Gite OIG eek Rea Ea Siok ioe nen eS aces mre 442
ermine O Cy pee eter heer ae eens ioe AS Fk Sis's SSE he, See aero ceric: 448
IMI EERETENON 5 5 0g ci OB deg Gao alo DRG CISC aoso 6 Sic rs aa aie eR REN RO Seen: clone 448

Genera! relations of the swimbladder and the precoelomic diverticulum.... 449
The skull in its relation with the precoelomic diverticulum and the mem-

pranoussralbyninGhtanss ary asa S ors, oe ee Ee hich Sok foes ASAE 453
iPhetbasiocerpitalWlo OMe Nema cr cco )ocs.5 arc SSeS hs sacs ee eto eet ae 453
DIN ney CECOLK ETON! LOONEY; Gey done we oe Ce a ee OMe tid ocd vinw Be co bok 454
Bae INE FOROOUNG [aout s.4%.6.ci0 & dare Bale Ree nee SO Eos onic conic an nite © 455
AMIN ea LETOUICEOOMCrari see wy tae oe ee. ss a ch a'e M ola eRe ele eer 459
Bye INVES RYO AVE AVOV HOS CLOVE se moro do 6 CAS cr Bae Oe eI San Oe Sine cae cla de.c 461
Beer eslasinpHenOiGsOOMer et ale tg ICE Sites. 2°. « Sn, Sich oein a eat 461
(exh evopisthotieno One: ements eres oie nec «sos 0g. 3 « grorate deeto ene sacle 461
SaebhedeteralawincwOotmchemmontalWponewer.. a. .- sac ceeeeeemomee cee 461
FaRemainsiofsuhercartllaginouswenaniiimmse) . 14. asics cree eee 462

lOmeihetlatendlinecessue wear sere meta cr s.s- doe sais Sisine Cae oes knee 463

Hikebiestemporaleloranienwer pws. yes iar. = Sher. sce s/o. c.e eaete eho oe nel 463

ESCH amialls WERViCSer, me eenee ae RATS PY A Lt to svc ae aos Gaon oa Ree Bios 464

DIS CISS TOM pee eae re ee end ME IN ys ss ora. es k % a Ns Rie eee rua 466

SummManyan beeen seen eh CO Sg. oOo RECS ERIE TE PIO DE Lomo bcc be aceee 471
INTRODUCTION

The remarkable relation existing between the ear and the
swimbladder in certain groups of fishes has been known since the
publication of the classic monograph of Weber (’20). The
greater part of his account holds good even to the present day,
and most of the current text-book statements and figures on

439

4AQ HENRY C. TRACY

the subject have their source in his thorough and accurate
description.

Weber shows that the ear-swimbladder relation is structurally
very different in different cases; in the five groups of fishes in
which he found this relation, he described three entirely dif-
ferent anatomical types. In later times, the list of species has
been extended in which the swimbladder is known to be in rela-
tion to the membranous labyrinth; yet no one has been able to
add any distinctly new types to those described by Weber.
These three types may be briefly summarized as follows:

1. The primitive type. This type is essentially a relation of
simple apposition of a precoelomic diverticulum of the swim-
bladder to the base of the skull. In many cases there is a
membrane-covered foramen in the cranial bones between the
swimbladder diverticulum and the membranous labyrinth. There
are many varieties of this type. It is found in widely separated
groups of fishes, e.g., Megalops, Notopterus, Sparidae, and
species of Serranidae and Gadidae. Probably it occurs in other
species not yet investigated.

2. The clupeoid type. In this type a diverticulum of the
swimbladder in the form of a minute capillary tube extends into
the head on each side, enters the skull, and ends in two large
expanded vesicles which occupy an extensive and complicated
cavity in the bones of the lateral and basilar region of the skull.
The anterior vesicle comes into a definite relation with the
utriculus. This arrangement, so far as is known, is found only
in the Clupeoids and nearly related families.

3. The Weberian mechanism. In this type there is an articu-
lated chain of small bones developed from certain anterior
vertebrae, which serve to connect the anterior end of the swim-
bladder with the perilymph cavity. The two sacculi of the
opposite sides are connected by a duct to which is attached a
medium unpaired sac. This mechanism is found in the families
Cyprinidae, Siluridae, Characinidae, and Gymnonoti. The ex-
istence of this specialized apparatus in these families suggests a
common descent and has led to the formation of the order
Ostariophysi.

THE CLUPEOID CRANIUM AND THE SWIMBLADDER 441

The first of these types as it exists in different species has
been described by various observers from the point of view of
gross structure. The third type, that is, the Weberian appa-
ratus, has been thoroughly investigated and, on its anatomical
side, is comparatively well known.

Our knowledge of the second (i.e., the clupeiod) type of the
ear-swimbladder relation has not greatly advanced since the
work of Breschet (’38). Tysowski (’09) and de Beaufort (’09)
are the only investigators to apply modern methods to the
study of this mechanism. De Beaufort’s work, however, is
concerned with the whole group of Malacopterygii, and he re-
ports little that is new regarding the essentials of this structure.
Tysowski’s paper is brief, and although it considerably advances
our knowledge of certain features of the ear-swimbladder rela-
tion in Clupeoids, it contains certain morphological conceptions
which are based on inadequate knowledge of the cranial struc-
tures in these fishes.

This investigation has been undertaken for two reasons,
namely, that the American Clupeiods have been little studied
and that many elementary features in these structures seem to
be still matters of controversy. This paper will deal only with
the bony structure of the skull in its relations to the swimbladder
diverticulum and the membranous labyrinth. Certain details
regarding the relations of the cranial nerves to these structures .
are also described.

Upon investigation it was found that the ear-swimbladder rela-
tion is essentially the same in all of the American Clupeoids
examined. Therefore, in this paper, those parts of the cranium
related to the swimbladder and the membranous labyrinth are
described as found in a representative American species (Pomol-
obus pseudoharengus), with only incidental reference to the
other members of this group. Another paper, soon to follow,
will describe the membranous labyrinth and its relations with
the swimbladder diverticulum.

The drawings for this paper were made by Mr. Leo Masso-
_ pust, the department artist at Marquette School of Medicine.

449 HENRY C. TRACY

HISTORICAL SUMMARY OF THE LITERATURE ON THE EAR-
SWIMBLADDER RELATION IN CLUPEIDAE

According to Weber’s description (1820), the anterior end of
the swimbladder in Clupea harengus bifurcates and sends a small
diverticulum into each side of the head. It enters the occipital
region of the skull and passes anteriorly along a canal inside the
bones at the base of the skull, and ends in two expanded vesicles
each enclosed in a capsule of bone (globulus osseus). One of
these, the anterior, lies in the basilar part of the ‘temporal’ bone,
the other, in the lateral part of the ‘temporal’ bone.

The anterior bony capsule is nearly filled by the membranous
bulla, but from the inner side it also receives a diverticulum
from the vestibule of the membranous labyrinth which extends
into it from the cavum cranii. The opening of the capsule,
through which this diverticulum of the vestibule enters, is a
large transverse fissure which opens into the cavum cranii.
Inside the bony capsule the air-filled membranous vesicle of the
swimbladder flattens against the surface of the distal end of the
diverticulum and fuses with it. By the apposition of these two
surfaces a septum is formed which stretches across the cavity
of the capsule like a tympanic membrane and divides it into two
parts. The edge of this septum is fixed to a ‘cartilaginous’
ring which is attached to the inner surface of the capsular wall.
The posterior of the two bony capsules admits no diverticulum
from any part of the membranous labyrinth, but is entirely
filled by the membranous vesicle of the swimbladder.

Weber believed that he demonstrated an endolymphatic canal
which runs under the brain and connects the vestibules of the
two sides, (subcerebral canal). He also noted that in these
fishes the wall of the membranous labyrinth is much thicker
than the same structure in other fishes.

Breschet (’38) studied the ear-swimbladder relation in Clupea
alosa. He describes accurately, and in more detail than Weber,
the membranous labyrinth, the bony capsules, the cartilaginous
tubes which enclose the swimbladder diverticulum, and the
general relations of these structures to the rest of the skull. -

THE CLUPEOID CRANIUM AND THE SWIMBLADDER 443

He observed the lateral recess of the skull (which has been almost
overlooked by later investigators) and its relation to the mem-
branous labyrinth internally, and to the lateral line system
(canaux excréteurs) externally. That this unusual relationship
might have a functional significance, he evidently recognized,
though the theory which he proposed is hardly consistent with
modern physiological knowledge.

Breschet’s conception of the perilabyrinthine spaces was re-
markably accurate, considering the crude technical methods of
investigation available in his time. He describes the ear as
composed of tissue ‘‘ni absolument membraneux, ni absolument
cartilagineux,” fragile, a little elastic, and of the softness of
rubber. He speaks of two ‘commissures,’ made up of the same
tissue as the rest of the organ which hold the two ears together.
One of. these ‘commissures’ he describes as passing above the
brain and connecting the utricular sinuses of the opposite sides;
the other ‘commissure’ is the structure that Weber described as
an endolymphatic connection between the two utriculi. Bres-
chet, however, describes these commissures as not hollow like
the membranous labyrinth, but composed of a ‘tissu foliace,’
or a membrane ‘pliée ou roulée,’ which is only a thickening of
the tissues of the vestibule. He says: ‘“‘La commissure in-
fériéure n’offre point de véritable canal dans son interieur, pas
plus que la commissure supérieure. . . . . La substance de
la commissure s’indentifie avec les parois du vestibule, et ne
doit étre considérée que comme une expansion membraneuse de
ces derniéres.”” Modern technique proves Breschet to have
been essentially correct in his conception of these structures.
Nevertheless, certain investigators long after his time persisted
in considering these structures as endolymphatic canals.

Breschet describes what he called an ‘accessory bulb’ attached
laterally to the utriculus under the ampullae of the horizontal
and anterior semicircular canals. He describes the utriculus as
merely resting on the anterior capsule, thereby denying the
existence of Weber’s vestibular diverticulum.

Hasse (’73) investigated the ear and swimbladder diverticulum
in Clupea:alosa and Clupea harengus. He carries Breschet’s

444 HENRY C. TRACY

conception somewhat further in stating that the thickening of the
tissue of the vestibule, that is, the ‘knorpelartige Fassergewebe,’
is only a dense connective tissue and is to be considered asa
localized thickening of the dura mater. He denies the existence
of the utricular diverticulum in the anterior bony capsule. He
describes with more accuracy than previous writers the form and
relations of the perilabyrinthine spaces. He is in error, how-
ever, in assuming that the endolymphatic duct of the two sides
passes through the supracerebral canal to connect the two
sacculi. In agreement with Breschet; he affirms the existence of
the accessory bulb, but denies that it is supplied with a nerve
twig or connected with the ear. He also mentions the relation
between the tissue of the perilabyrinthine spaces and the lateral-
line canals.

Retzius (’81) briefly describes the relation of the ear to the
swimbladder in Clupea harengus. His work essentially agrees
with that of Hasse, except that he was inclined to the opinion
that the endolymphatic ducts end blindly as in other fishes.
He also considers the supracerebral canal as merely a thickening
of the dura mater. He denies the existence of the utricular
diverticulum in the anterior bony capsule.

Mathews (’86), in an investigation of the skeleton of the
British Clupeoids, has accurately described the bony canals and
capsules in the herring (Clupea harengus), the pilchard (Clupea
pilchardus), and the shad (Clupea alosa). He finds the pos-
terior capsule in the pilchard differing from that of other species
in that it is divided by a constriction. By cutting serial sections
through the whole length of the swimbladder diverticulum of
the herring, he demonstrates the tube to be open throughout.

Ridewood (’91) describes the ear-swimbladder relation in the
British species, viz.: herring, pilchard, shad, sprat (Clupea
sprattus), thwaite (Culpea finata), and anchovy (Engraulis en-
crasicholus). It is unnecessary to mention here the minor varia-
tions in structure which he found in these different species.
Ridewood by this paper set back the course of investigation of
the ear-swimbladder relation in the Clupeoids by reviving cer-
tain older conceptions which investigators were gradually show-

THE CLUPEOID CRANIUM AND THE SWIMBLADDER 445

ing to be untenable. For example, he describes a utricular
diverticulum in the anterior bony capsule, the existence of
which no investigator since Weber has accepted; he describes a
caecum of the utriculus (‘bulbe accessoire’ of Breschet), al-
though Hasse had stated that this structure is not connected
with the membranous labyrinth; he revives Weber’s conception
of the subcerebral canal, although no investigator since Weber
has admitted it to be an endolymphatic connection.

In papers on the osteology of the skull in various lower groups
of Teleosts, Ridewood (’04 a, b,) describes the form and rela-
tions of the bony capsules of the swimbladder in several genera
of Clupeidae and related families. Essentially the same rela-
tions are found not only in all species of Clupeoids, but also in
Chatoesus, Chirocentius, Dussumieria, Engraulis, Coila, Pellona,
Pellonula, Pristingaster, and Hyperlophus. The posterior cap-
sule is wanting in Clupea sprattus, and both anterior and posterior
capsules are absent in Chanossalmoneus. In Coilia nasus there is
also a large exoccipital capsule corresponding, in Clupea, to the
fusiform enlargement of the exoccipital part of the bony tube.
Megalops has a structure in the opisthotic bone which perhaps
lodges a swimbladder vesicle. Ridewood also states that ves-
icles occur in the skull of Hyodon, Notopterus, and in the
Morymoids.

The structure of the skull and its relations to the swimbladder
in these last three genera are more fully described by Ridewood
(04) and by Bridge (’00). The ear-swimbladder relation in
these forms is unlike that of Clupeoids; it belongs with the first
or primitive type as described above.

All the writers above discussed are to be classed together so
far as their method, technique, and results are concerned. They
relied entirely on dissection and injection methods and made
little or no attempt to investigate minute relations. The next
two writers, however, applied more modern and accurate
methods.

Tysowski (’09) shows that both the subcerebral and supra-
cerebral connections between the membranous labyrinths of the
two sides are not endolymph tubes, but channels in the tissue

446 HENRY C. TRACY

around the labyrinth. He demonstrates the fact that the utric-
ulus does not send a diverticulum into the anterior bony capsule,
and that the caecum of the utriculus (Ridewood) or the ‘bulbe
accessoire’ (Breschet) is merely a three-sided thickening which
projects from the wall of the utriculus.

Tysowski discusses at length the morphology of the dense
tissue and the channels in it which surround and connect the
membranous labyrinth of the two sides. He states that the most
characteristic feature of the labyrinth in Clupeoids is the much
thickened connective tissue of its walls. Structurally, this tis-
sue is “eine fast homogene Grundermasse mit dunkleren fibrosen
Streifen und den fiir das Labyrinthgewebe so characterischen
Spindelzellen.” This dense tissue is well developed on the upper
and inner walls of the utriculus from which it goes over to the
anterior wall of the sacculus. It is not limited to the wall of the
labyrinth, however, but continues off from it quite independ-
ently; it extends under the brain, it bridges the space between
the two anterior capsules, and becomes continuous with the
corresponding tissue of the other side; it also crosses over the
cerebellum from one superior sinus of the labyrinth to the other.
Hasse had made out enough of these relations to enable him to
suggest that this tissue is a condensation of the dura mater.
But since Hasse’s time Sterzi (’01) has shown that the dura
mater is first differentiated in the Amphibia, and that in fishes
the covering of the brain consists merely of an undifferentiated
meninx primitiva surrounded by a perimeningeal tissue. Ty-
sowski thinks that the true explanation of this dense tissue is
that it represents a transitional stage in differentiation from the
perimeningeal tissue into the envelope of the membranous laby-
rinth. The spaces in this tissue he considers as belonging to the
membranous labyrinth, and hence properly called perilymphatic
spaces. In these spaces are strands of elastic tissue which stain
by the Weigert method; they doubtless develop by transforma-
tion of connective tissue. If we imagine these spaces developing
around the whole labyrinth, we shall have spaces actually
homologous with the perilymphatic spaces of higher forms.

THE CLUPEOID CRANIUM AND THE SWIMBLADDER 447

An interesting structure which was entirely overlooked by all
previous investigators except Weber, is a septum which divides
the cavity of the anterior bony capsule into two unequal chambers,
the inferior of which contains the membranous vesicle of the
swimbladder. Tysowski describes this septum as a “‘fibrose,
stark elastische Membran, deren deutlich lings verlaufende
Fasern aus sonderbaren Kanialchen in der Knochenwand der
Kapsel hervorzugehen scheinen.” The periosteum from the in-
side of each chamber is reflected on to the corresponding surface
of the septum.

To de Beaufort (09) we are indebted for a comprehensive
investigation of the swimbladder in the Malacopterygii. Of
these fishes, the following genera present the clupeoid type of
relation between the labyrinth and swimbladder: Clupea, Pel-
lona, Opisthonema, Sardinella, Chatoessus, Engraulis, Dussu- ~
miera, Spratelloides, Coilia, and Chirocentrus. In this group,
Pellona is the only genus in which the ear-swimbladder mechan-
ism differs from the clupeoid type in any important details.

By applying modern technique, he was able to demonstrate
definitely the non-existence of the utricular diverticulum ex-
tending into the anterior bony bulla (as described by Weber
and Ridewood). He states that in sections the forward part of
the utriculus with the macula acustica rests upon the opening
of the bulla ‘“‘davon durch den perilymphatische Raum getrennt
wird.” This relation showed in all his sections, both of older
larvae and of young fishes. He also denies positively any direct
connection between the two utriculi. The subcerebral canal is
only a perilymphatic space. With regard to the supracerebral
canal, he says: ‘‘Ich darf jedoch mit Bestimmtheit erkliren,
dass auch diese Verbindung nicht besteht, ebensowenig wie von
Hasse beschriebene Verbindung des beiden Ducti endolympha-
tica.”’

Although de Beaufort adds little to our previous conceptions,
he was the first to have a view of the structure of the Clupeoids
and allied forms comprehensive enough to discuss the com-
parative anatomy of the clupeoid type of the ear-swimbladder
telation. His discussion need not be repeated here, but we

448 HENRY C. TRACY

may mention his suggestion that forms like Megalops may be
transitional stages between the primitive type and the clupeoid
type. As de Beaufort remarks, however, it is evident that the
connection between the ear and swimbladder has developed in-
dependently in different groups of fishes. Apparent resemblances
in these structures, as found in existing genera, indicate little
regarding relationship or even the phylogenetic development .of
the ear-swimbladder relation in any given group of fishes.

De Beaufort describes stages in the embryonic development of
these structures in the herring. He shows that the bony cap-
sules are formed not by a ‘hollowing out’ process of the bones.
in which they are enclosed in the adult, but by a new process of
bone development from connective tissue around the mem-
branous vesicle of the precoelomic diverticulum. .

TERMINOLOGY

The following terms are used throughout this paper in re-
ferring to the anatomical relations of the cranial structures,
exoccipital (for occipitale laterale), epiotic, prootic (for petrosal) :
pterotic (for squamosal), opisthotic (for intercalar), and sphe-
notic (for postfrontal or postorbital ossification). Median
designates the midvertical plane or structures lying within it;.
medial indicates the opposite of lateral, i.e., toward the middle.
In referring to the channels in the tissue around the mem-
branous labyrinth and the brain, the term ‘perilabyrinthine’ is
used instead of perimeningeal or perilymphatic.

MATERIAL

This investigation is based on the study of the more common:
American clupeoid fishes. Adult specimens of the following
species were examined: Alosa sapidissima (shad), Pomolobus.
pseudoharengus (alewife), Pomolobus aestivalis (summer her-
ring), Pomolobus mediocris (hickory shad, fall herring), Bre--
voortia tyrannus (menhaden). The shad were bought in local
markets; the specimens of the other species were obtained from
the Marine Biological Laboratory, where they had been pre-—-

THE CLUPEOID CRANIUM AND THE SWIMBLADDER 449

served in 10 per cent formalin. Sections of embryonic stages of
Stolephorus mitchilli were also available.

The cranial structure in all these species is essentially the
same, but the skull of P. pseudoharengus is less specialized than
in the other species. Hence it may be used asa type form. All
descriptions in this paper refer to this species, unless otherwise
stated.

GENERAL RELATIONS OF THE SWIMBLADDER AND THE PRE-
COELOMIC DIVERTICULUM

The swimbladder, in the species investigated, has the form and
relations typical of the Clupeoids. It consists of a fusiform,
tubular organ of small caliber, which runs the length of the
dorsal part of the visceral cavity. Posteriorly it tapers rather
suddenly, and either ends blindly or opens to the exterior by
communicating with the anus. Anteriorly it usually tapers
slightly and terminates in a rounded end in the region opposite
the third or fourth vertebra; cephalad to this it sends up into
the base of the skull a bifurcated precoelomic diverticulum
which brings the swimbladder into direct relation with the
membranous labyrinth. The pneumatic duct is open in these
fishes; it arises near the middle of the swimbladder and opens
into the blind end of the V-shaped stomach sac.

The only important variations from the typical form in the
species under consideration are in Stolephorus. In this genus a
constriction near the middle of the swimbladder divides it into an
anterior part which retains the typical tubular form and a
posterior part which expands into a thin-walled chamber. The
pneumatic duct springs from near the anterior end of this cham-
ber. Similar relations are found in Pellona (de Beaufort, ’09).

Through the greater part of its extent the swimbladder is
covered on its ventral surface by peritoneum; dorsally, it is in
contact with the body wall and separated from the vertebral
column by the aorta and kidney; areolar tissue, in some places
containing a large amount of fat, connects it with the strong
aponeurosis which bridges across the intercostal spaces.

450 HENRY C. TRACY

The relations of the anterior part of the organ are somewhat
different. As it passes over the pericardial cavity, it comes to
lie between the kidneys of the two sides. Here the intercostal
aponeurosis sends off two strong layers, one of which runs
ventral to the swimbladder and the other dorsal to it; each
connects with the corresponding layer of the other side. This

f Uj,
sg ul,
ye il Y Y,

y

by TT
», ‘| N|
un

Fig. a Drawing to show relations of the anterior end of the swimbladder and
the cartilage canals containing the precoelomic diverticulum and its branches
(Alosa sapidissima). The structures are shown from the ventral side and in
cross-section. Semidiagrammatic. Abbreviations on page 4738.

part of the organ, then, is enclosed in an aponeurotic sheath of
nearly tubular form, which runs anteriorly and attaches itself
to the cartilage tube which encloses the precoelomic diverticu-
lum (fig. a). The intercostal aponeurosis itself continues cepha-
lad, to clothe the ventral surface of the musculature covering
the occipital and basilar parts of the skull ; other extensions of
it pass dorsally to be attached to the vertebral column.

The precoelomic diverticulum is a tubular prolongation of the
tunica interna of the swimbladder. There is some variation in

THE CLUPEOID CRANIUM AND THE SWIMBLADDER 451

the details of this structure in different species. In Alosa (fig. a)
and Stolephorus it passes forward a few millimeters from the
anterior end of the swimbladder and enters a cartilage canal in
which it almost immediately bifurcates. In Pomolobus, the
branches come off directly from a slight bulbous enlargement of
the tapering end of the swimbladder. Each of the branches,

cc

Fig. b. Drawing to show relations of the membranous vesicles of the pre-
coelomic diverticulum to the bones at the base of the skull. Based on figure 8.
The ventral segments of the bony capsules are represented as having been cut
out. Diagrammatic. Abbreviations on page 473.

enclosed in a continuation of the cartilage canal, runs forward,
one on each side, under the occipital portion of the skull and
enters a canal in the exoccipital bone (fig. 2). The canal bulges
somewhat here (P. pseudoharengus) to form a fusiform enlarge-
ment; it then passes forward to the angle where the exoccipital
prootic and pterotic bones meet (fig. b). At this point, it
gives off a lateral branch which enters a nearly spherical cavity
in the pterotic bone which it expands to occupy, thus forming
the pterotic or posterior membranous vesicle. The part of the
pterotic bone which encloses this vesicle is the posterior or ptero-

452 HENRY C. TRACY

tic osseous capsule, or bulla. The main branch of the diver-
ticulum, from the angle between the bones above mentioned,
continues forward in a canal in the prootic. In this bone the
diverticulum ends by forming a membranous vesicle (anterior
or prootic). The bone around the vesicle forms the anterior,
or prootic bulla, or osseous capsule.

The cartilage canals in which the extracranial portions of the
diverticulum are enclosed form a Y-shaped structure (fig. a).
The stem of the Y is a median, unpaired structure; in Alosa it
is a short, thick cylinder with its posterior surface forming a
vertical transverse plane and facing the anterior end of the
swimbladder; this surface is smooth and circular, and around
its edge is beset with several large blunted spines. In Pomolo-
bus, the posterior surface is concave. To these spines and
around the margin of its smooth face is attached the aponeurotic
sheath which invests the anterior end of the swimbladder. An-
teriorly, this cylinder-like structure is continued off into two
cartilage tubes which diverge from each other to form the arms
of the Y which embrace the aorta between them. These tubes
gradually diverge from each other, pass obliquely over the sur-
face of the parasphenoid wings just in front of their tips, then
over the aponeurosis which clothes the occipital musculature
and extend cephalad to the side of the exoccipital bone. They
are slightly flattened as they are applied to the surface of the
aponeurosis; one of these in a shad measured 0.38 mm. in one
dimension and 0.5 mm. in the other.

On the surface of the exoccipital bone is a groove (fig. 5)
which begins toward the lower posterior angle of the bone and
slants upward and forward. The groove gradually deepens as it
passes upward, and finally ends in the opening of the canal
through which the diverticulum passes. The ends of the car-
tilage tubes are very oblique and are applied to the sides of the
groove and the edge of the mouth of the canal into which it
leads. Thus the groove in the bone is made into a complete
canal by the application to it of the oblique end of the cartilage
tube, and through this canal the diverticulum of the swimbladder
enters the exoccipital bone.

THE CLUPEOID CRANIUM AND THE SWIMBLADDER 453

THE SKULL IN ITS RELATION WITH THE PRECOELOMIC DIVER-
TICULUM AND THE MEMBRANOUS LABYRINTH

In this connection, the sides and base of the cranium are of
particular importance; description of other parts of the clupeoid
skull may be found in the papers of Mathews (’86) and Ridewood
(704).

The clupeoid cranium, although highly specialized as to cer-
tain features, conforms in its more essential osteological char-
acteristics to the general structure of the skull of the lower
teleostean groups (pl. 1). The axis of the posterior part of the
skull is formed by the basioccipital, which articulates posteriorly
with the vertebral column, anteriorly with the medial plates of
the prootic of the two sides, and laterally with the exoccipital
bones of the two sides. Lateral to the exoccipital and the
prootic bones, and forming the most lateral part of the occipital
region of the cranium, is the pterotic bone. The anterior
medial cranial floor, formed chiefly by the two prootics, is
completed by the basisphenoid (figs. 7 and 8) with the hypo-
physial foramen intervening between that bone and the median
articulation of the medial plates of the two prootics. The ex-
treme lateral part of the cranium anteriorly is formed by the
sphenotic which articulates with the lateral edge of the prootic
bone. The posterior osseous capsule, containing the posterior
membranous vesicle of the swimbladder diverticulum, is con-
tained in the pterotic bone; the anterior osseous capsule con-
taining the anterior membranous vesicle forms a considerable
part of the mass of the prootic bone (fig. 5).

These bones may now be described in detail.

1. The basioccipital bone

The condylar end of this bone is circular in form where it
articulates with the first vertebra. The body of the bone is
hollowed on its lateral surface in such a manner that it consists
merely of a thin median plate separating two lateral concavities;
small lateral wings slightly deepen these concavities (figs. 4 and

JOURNAL OF MORPHOLOGY, VOL. 33, NO. 2

454 HENRY C. TRACY

12). A conspicuous notch in the edge of these wings anteriorly
forms part of the margin of the foramen auditivum. The lateral
concavities in the sides of the bone form the median and ventral
walls of the occipital portion of the saccular cavities (fig. 3).

The ventral side of the body of the bone forms a narrow,
longitudinal, slightly concave surface bounded on each side by a
broad ventrally projecting plate of bone (figs. 8 and 11). . This
surface and the ventral plates form the roof and part of the sides,
respectively, of the occipital portion of the eye-muscle canal.

The external surface of the bone is formed chiefly by the ex-
ternal surface of the ventral plates and to a slight extent by the
ventral surface of the lateral wings, joined by a few roughened
trabeculae of bone.

2. The exoccipital bone

For descriptive purposes, we may consider this bone as com-
posed chiefly of two flattened masses; one forms a part of the
vertical posterior face of the skull, at the sides of the foramen
magnum (fig. 4), the other forms the part of the ventral surface
of the skull next to the basioccipital (fig. 5). These are con-
tinuous posteriorly with a thin, curved plate of bone which is
applied to the lateral and dorsal sides of the condylar part of the
basioccipital. The vertical part of the bone has laterally on its
outer surface a rounded projecting ridge which is continuous
above with a similar ridge on the epiotic (fig. 1). This ridge is
the prominent angle where the posterior surface of the skull meets
the ventral and lateral surfaces, and it lodges the posterior semi-
circular canal of the membranous labyrinth.

Arising obliquely from the cerebral surface of the posterior
portion of the exoccipital is a thin triangular plate (TPEXO, ~
fig. 4), which passes medially to meet the corresponding plate
from the other side just over the dorsal edge of the median,
vertical plate of the basioccipital where a synchondrosis unites
the three contiguous edges of bone (fig. 12). Anteriorly the
plate tapers to a point which ends in the middle line just back
of the prootic bone (fig. 3); posteriorly it extends to the condylar

THE CLUPEOID CRANIUM AND THE SWIMBLADDER 455

part of the basioccipital. It thus forms the roof of the hinder
part of the saccular cavity. The plates of the two sides exclude
the basioccipital completely from the foramen magnum and the
cerebral cavity, and form a trough in which the brain stem
rests. Near the anterior edge of the triangular plates are two
oblique foramina (fig. 4); one, anterior to the other and much
the smaller, opens to the upper lateral part of the saccular cavity
and gives passage to the ninth nerve; the other is the entrance
to a canal which is directed obliquely backward and outward
and forms the exit of the tenth nerve. Posteriorly are the exits
of the two occipital nerves.

The ventral part of the exoccipital bone forms a considerable
portion of the floor and sides of the saccular cavity. Its most
conspicuous feature is a small fusiform bulla through which the
diverticulum of the swimbladder passes. Dorsolateral to that
structure is the large oval opening of the canal of the tenth
nerve; medial to it and almost at the edge of the auditory fora-
men is a small opening for the ninth merve (fig. 5).

3. The prootic bone

The conspicuous feature of the prootic bone is the large spher-
ical bulla or osseous capsule which lodges the anterior vesicle of
the swimbladder diverticulum. Looking first at the cerebral
surface of the bone (figs. 6 and 7), one observes that it is thick
and massive medial to the bony capsule (medial plate) and
forms with the corresponding part of its fellow a rounded ridge
of bone which extends transversely across the floor of the an-
terior part of the cerebral cavity like a raised threshold. The
sixth nerve passes vertically through this part of the bone to the
eye-muscle canal below (figs. 6 and 8). Posteriorly, on the
cerebral surface of the bone, just behind the capsule, is a deeply
concave depression which, in the articulated skull, is continuous
with the dorsolateral concavity of the basioccipital and com-
pletes the saccular cavity anteriorly.

Lateral to the osseous capsule, two bony plates are given off
(fig. 6). The lower plate, the lateral wing of the prootic, is con-

456 HENRY C. TRACY

fluent with the posterior part of the bone around to the saccular
cavity; it extends horizontally and ends in a crescent-shaped
margin, forming part: of the floor of the lateral recess which will
be described later. The upper plate of bone is divided into two
laminae; it extends backward, upward, and outward from the lat-
eral surface of the bulla itself. From its shape it may be des-
ignated as the falciform process. It is continuous in front with
the lateral wing of the prootic, with which it makes a round open
angle; its posterior edge is free; its dorsal edge misses reaching the
pterotic bone by a very narrow interval (pl. 2) which, in the fresh
skull, is bridged over by the lateral mass of cartilage retained
from the primordial cranium (fig. 8). From the under surface
of this process, forming with it a rounded angle along its line of
origin, the lower lamina of bone is given off; this may be called
the inferior lamina of the falciform process (IZF). Posteriorly,
this lamina articulates with a plate of bone from the pterotic,
but, below, is free and curved so as to resemble half of a low
broad arch (fig. 4). Between the two laminae are included the
inferior end of the anterior semicircular canal and its ampulla.

Anterior to the osseous capsule, the prootic extends forward
to articulate with the alisphenoid. Near the center of this part
of the bone is a deep notch (fig. 6), which extends down to the
rounded surface of the bulla, and which, when articulated with
the alisphenoid, forms the foramen for the exit of the trigemino-
facial nerve complex.

The anterior or orbital surface of the prootic is formed by the
smooth surface of the osseous capsule surrounded by projecting
bony processes in such a way as to form a broad shallow fossa
(ganglionic fossa). The capsule retains its spherical form; the
bony processes around it have the appearance of being molded
on to it. The fossa contains the ganglia of the trigeminofacial
complex. Inferiorly, a sharply projecting ridge separates the
orbital from the ventral surface of the skull. Medially, a pillar
of bone bounds this surface and forms the lateral margin of the
opening of the eye-muscle canal. A deep recess extends from the
fossa behind the pillar; from this recess the ramus palatinus VII
gains access to the eye-muscle canal (fig. 8, RP VIJ), through a

THE CLUPEOID CRANIUM AND THE SWIMBLADDER 457

small, ventrally directed foramen; from it also the ramus sympa-
theticus and the ramus pretrematicus for the pseudobranch pass
in a bony canal over the anterior medial surface of the bony
capsule to the ventral surface of the skull (fig. ¢).

Lateral to the ganglionic fossa a rather thick process of bone
articulates with the sphenotic. The orbital surface of this proc-
ess has a large groove for the maxillomandibular nerve. The
base of the process is penetrated by a canal which passes laterally

Fig. ec Ventral surface of the middle region of the skull, showing the relations
of the trigeminofacial ganglia and their branches to the prootic bone. Based on
figure 7. Diagrammatic. Abbreviations on page 473.

over the anterolateral surface of the osseous capsule from the
ganglionic fossa to the ventral surface of the skull. Through
this canal the hyomandibular ramus of the facial nerve is con-
ducted to the inside of the gill cover.

The relations of the ganglionic fossa recall the ‘trigemino-
facial chamber’ of Allis (’03) and suggest the possibility of the
homology. of the capsule with a portion of that chamber. Knowl-
edge of the development of the clupeoid skull is necessary, how-
ever, before this suggestion can profitably be considered.

On the ventral surface of the prootic bone, the medial plate is
separated from the remainder of the bone by the broad, thin

458 HENRY C. TRACY

ventral plate (VPRO), which articulates posteriorly with the
ventral plate of the basisphenoid and with the posterior wings of
the parasphenoid below, to form the lateral wall of the eye-
muscle canal (fig. 8). This plate passes back from the medial
pillar on the orbital surface of the bone across the ventral
surface of the bony capsule in such a way that a small segment
of the capsule is exposed to the eye-muscle canal (fig. 8). The
foramen for the entrance of the carotid artery hes between the
ventral plate of the prootic and the parasphenoid wings. The
articulated medial plates of the prootics are joined with the
basisphenoid in front and the basioccipital behind, and thereby
form the roof of the eye-muscle canal. The ventral surface of
the remainder of the prootic bone forms a large part of the ven-
tral surface of the cranium. It is mainly taken up by a segment
of the osseous capsule which protrudes from the middle of its
surface. On each side of the front part of the capsule is a
foramen through which the branches of the facial nerve men-
tioned above make their exit.

It is evident from the above description that a segment of the
prootic capsule projects into all the cavities of which the prootic
bone forms a part. About a third of the surface of the capsule
contributes to the cerebral surface of the cranial floor; a segment
faces the orbit where it forms the floor of the ganglionic fossa;
about one-fifth of its surface protrudes from the ventral surface
of the cranial floor; a small segment is presented to the lateral
recess and another to the eye-muscle canal. A portion of the
cerebral segment limits the saccular cavity anteriorly. The
curvature of these ségments is very little altered by its attach-
ment to the parts of the bone in which it lies.

There are two openings in this capsule; the posterior (fig. 5)
is the mouth of the canal through which the swimbladder diver-
ticulum enters the interior of the capsule. The prootic portion
of this canal begins at the posterior lateral angle of the prootic
and proceeds obliquely through the thick part of the bone
lateral to the saccular portion of the auditory recess (fig. 10).

The other opening or fenestra is in the cerebral segment of the
capsule in that part of its curvature which looks posteriorly

THE CLUPEOID CRANIUM AND THE SWIMBLADDER 459

(figs. 3,4 and 6). In consists of a narrow transverse slit, slightly
crescent-shaped in some species. Its edges, at its lateral end
and sides, protrude upward slightly to form a raised lip around
its margin. This lip gradually becomes lower toward the
medial end of the fenestra where it is flush with the surface of
the surrounding bone. The utriculus rests on the edges of this
lip to which it is attached by delicate connective-tissue fibers.

The cavity of the capsule is subdivided into an upper and a
lower chamber by a nearly horizontal septum of elastic tissue,
the elements of which are in the form of flattened plates (figs. 5
and 9, SC). Each surface of this septum is covered by a re-
flection of the periosteum from the inner surface of the bone of
the corresponding chamber. The septum is closely attached to
the bone by means of radial connective-tissue fibers which pass
from the elastic plates of the septum into minute canals in the
bone substance. The lower chamber contains the anterior mem-
branous vesicle of the swimbladder. The upper chamber was
erroneously supposed by Weber and Ridewood to be occupied by
a diverticulum from the utriculus; it is merely a tissue space
which communicates with the subcerebral perilabyrinthine canal
through the fenestra.

4. The pterotic bone

This bone forms the lateral part of the posterior portion of the
cranial wall. It is a relatively large, massive bone in which is
imbedded the posterior osseous capsule (figs. 3 and 11) con-
taining the posterior vesicle of the swimbladder diverticulum.
The lateral surface of the pterotic is partly covered anteriorly
by a thin scale of bone which is an extension of the lateral wing
of the frontal bone (fig. 1, LWF); posteriorly, the lateral surface
is deeply indented by the lower portion of the epiotic fossa
(EPF). The posterior osseous capsule protrudes into the epiotic
fossa and also exposes its surface slightly on the face of the bone
below the fossa. The lateral semicircular canal encircles the
bony capsule near its equator.

460 HENRY C. TRACY

The ventral surface of the pterotic is triangular and exhibits a
small segment of the posterior osseous capsule (fig. 5, POC)
lateral to which there is a shallow groove (deepened by cartilage
in the fresh skull) which forms the posterior half of the hyoman-
dibular articulation.

The greater part of the medial surface of the bone is deeply
beveled so as to face nearly medially, and roughened for the
attachment of the lateral cartilage plate (figs. 4 and 10). The
bone articulates posteriorly with the upright posterior part of the
exoccipital. A small vertical surface of the bone lies anterior
to this articulation and makes an open rounded angle with the
confluent surface of the exoccipital; through this angle the
superior sinus of the membranous labyrinth passes vertically.
A deep recess below extends back between the two parts of the
exoccipital and lodges the inferior end of the posterior semi-
circular canal and its ampulla. ‘This recess is separated medially
from the saccular cavity by the canal for the vagus nerve. In
front of the vertical medial surface is a cone-shaped recess which
contains the ampulla of the lateral semicircular canal.

Anteriorly, the body of the bone juts out over its base to meet
the backward projecting laminae of the falciform process of the
prootic. ‘To the angle between these laminae, the pterotic pre-
sents an obliquely excavated surface and thus forms a chamber
for the reception of the lower end of the anterior semicircular
canal and its ampulla. The floor and outer wall of this chamber
is formed by the apposition of a thin curved plate of the pterotic
bone to the inferior lamina of the falciform process. Superiorly,
as has been mentioned above, the edge of the falciform process
does not quite meet the pterotic: the irregular slit thus formed
(figs. 3, 4 and 6) is closed by the lateral mass of cartilage which
is penetrated by the anterior semicircular canal. Below the
chamber the free edge of the projecting part of the pterotic is
continuous with the edge of the inferior lamina of the falciform
process and forms with it a low arch under which the utricular
part of the auditory recess communicates laterally with the
lateral recess.

THE CLUPEOID CRANIUM AND THE SWIMBLADDER 461

5. The sphenotic bone

This pyramid-shaped bone forms the extreme lateral part of
the anterior portion of the cranium (figs. 1, 2, and 9, SPH).
The ventral part of the bone articulates posteriorly with the
pterotic. Its upper part does not meet the pterotic posteriorly
and the interval between them is bridged by the lateral wing of
the frontal (fig. 3). Medially, the sphenotic bone articulates
with the prootic and alisphenoid; laterally and dorsally, it is
overlapped by the lateral wing of the frontal bone. The bone
is deeply excavated posteriorly, so that a cavity occupies a large
portion of its body. This cavity shares in the formation of the
lateral recess. .

6. The basisphenoid bone

This bone is a thin, slightly curved, median plate of bone,
articulated between the front parts of the two prooties (fig. 7,
BSP). It forms most of the floor of the hypophysial fossa and
its posterior edge is the anterior margin of the hypophysial
foramen. A deep notch in the posteriolateral margin of each
side is converted into a foramen by articulation with the prootic
and gives passage to the oculomotor nerve. The anterior edge
of the bone forms the posterior margin of the optic foramen.

7. The opisthotic bone «

This bone is almost rudimentary in the Clupeoids. It is a
somewhat rectangular scale-like bone overlying the articulation
between the exoccipital and pterotic bones (figs. 5 and 11, OPT).
It has no relation to the membranous labyrinth or swimbladder
diverticulum.

8. The lateral wing of the frontal bone

This is a thin scale of a bone extending down over the lateral
face of the skull (fig. 1, LWF). Near its origin from the main
part of the frontal, the posterior edge of the lateral wing forms
the anterior margin of the temporal foramen; the anterior edge

462 HENRY C. TRACY

of the wing joins the orbitosphenoid and alisphenoid. The
distal end of this process flattens to form a thin lamella of bone
which fits closely over a part of the adjacent surfaces of the
pterotic and sphenotic bones on the lateral face of the skull.
By bridging over the interval between these bones it helps to
form the lateral wall of the lateral recess.

The lateral wing of the frontal bone serves chiefly as a bony
sheath of the supra-orbital canal of the lateral line system.
In the distal third of the wing, the inner wall of the bony sheath
is deficient, so that here, at its origin, the supra-orbital canal
is neither on the surface nor enclosed in a bony sheath, but
lies in the lateral recess of the cranium (fig. 9, LLC). Two
large foramina in the extreme distal end of the bone give passage
to the infra-orbital and hyomandibular canals.

9. Remains of the cartilaginous cranium

Ixxtensive unossified remains of the chondrocranium are
retained in the adult, particularly in the orbital and nasal regions.
There is also a thick triangular plate in the cranial roof and a
large lateral plate which lines the cranial wall above the auditory
recess. In the basal and occipital regions cartilage is limited to
the synchondroses of the various articulations.

The lateral cartilage plate is an important structure in the
auditory wall in Pomolobus pseudoharengus but is limited to the
neighborhood of the anterior semicircular canal in most other
species. Anteriorly, it lines the lateral wing of the frontal bone
in its proximal part; further back it overlies the beveled medial
surface of the pterotic (fig. 10) and nearly the whole medial surface
of the epiotic (fig. 8, LPC). Owing to a deficiency in the
lateral part of the epiotic bone, the plate contributes to that
part of the exterior surface of the skull which lies at the bottom
of the epiotic fossa (fig. 11). It completes the roof of the
chamber containing the ampulla of the anterior semicircular
canal by bringing across the interval between the falciform
process and the opposed edge of the pterotic bone. The anterior
semicircular canal passes through this interval and traverses a

THE CLUPEOID CRANIUM AND THE SWIMBLADDER 463

canal in the cartilage plate until it reaches the edge of the epiotic
where it passes under a hook-like process from that bone and
emerges to the cranial cavity. The cartilage plate also con-
tributes a small part to the roof of the lateral recess.

10. The lateral recess

This is an almost isolated cavity between the sphenotic,
pterotic, and prootic bones and lateral wing of the frontal. It
communicates medially with the utricular portion of the auditory
recess through the low arch under the falciform process. The
only other openings to this recess are through the foramina for
the lateral-line canals in the lateral wing of the frontal bone, i.e.,
through the foramina for the exit of the suborbital and hyoman-
dibular lateral-line canals and through the channel for the supra-
orbital canal as it arches up over the eye. By means of these
canals it communicates immediately with the exterior. The
recess contains a large sac-like expansion of the lateral-line canal
from which the above-mentioned canals are given off. The
rest of the recess is filled with a reticular-like connective tissue
which is continuous through the arch with the perimeningeal
tissue in the auditory recess. In this tissue is a large perilaby-
rinthine space limited by a well-defined marginal membrane
(fig. 9, PS). The tissue is traversed by a recurrent lateral-line
branch of the facial nerve which gains access to the recess through
articulation between the prootic and sphenotic bones (fig. 3).

11. The temporal foramen

This is a characteristic feature of the clupeoid skull. It is a
very large foramen in the upper part of the temporal fossa of the
skull between the frontal, parietal, epiotic, and pterotic bones
(fig. 1, 7F). It is filled with adipose tissue which is continuous
with, and apparently a part of, the perimeningeal tissue within
the cranium. Previous writers have overlooked the fact that
imbedded in this tissue, and nearly coterminous with the margin
of the foramen, is a large bay-like expansion of a lateral-line
canal.

464 HENRY C. TRACY

THE CRANIAL NERVES

The relations of the cranial nerves in clupeoids correspond in
general to those existing in other teleosts. The branches of the
nerves from the third to the tenth correspond closely to those
described by Herrick (’99) for Menidia. There are, however,
interesting special relations of certain branches of these nerves.

The third nerve makes its exit from the cranium through the
foramen between the basisphenoid and prootic bones; the fourth
nerve penetrates the center of the alisphenoid bone (fig. 8); the
sixth nerve gains access to the eye-muscle canal by penetrating
the medial plate of the prootic bone.

The ganglia of the trigeminofacial complex lie in the ganglionic
fossa and give off branches which pass through the prootic bone
across the surface of the bulla (fig.3). In addition to the branches
mentioned above (p. 457), a recurrent branch is given off from
the lateral part of the ganglion. It immediately divides into
two rami which pass laterally to the back of the ganglionic fossa
and through the articulation between the prootic and sphenotic
bones, and enter the lateral recess of the skull. The more lateral
of these rami courses obliquely across the floor of the recess to
its posterior lateral corner where it supplies the hyomandibular
lateral-line canal (fig. 3, RVJZ). The medial ramus curves
gradually upward through the recess and penetrates the lateral
plate of cartilage, thus entering the perimeningeal tissue (fig. 9)
of the cranial cavity; it continues upward through this tissue,
slanting slightly backward, and appears to form a plexus with
the recurrent branch of the vagus. It also contributes to the
supply of the sac-like expansion of the lateral line canal which
occupies the temporal foramen. The medial branch has a course
similar to that of the ramus recurrens facialis which Herrick
describes in Menidia as made up of communis fibers.

In sections of the head of adult Pomolobus, I was able to trace
branches to the bay-like expansions of the lateral-line canal as
described above. ‘These bays are so large that they may not
function exclusively as lateral-line structures. The termination
in them of the recurrent facial branches is therefore not neces-

THE CLUPEOID CRANIUM AND THE SWIMBLADDER 465

sarily inconsistent with Herrick’s account of these branches in
Menidia.

The auditory nerve pierces the subcerebral plate of perilaby-
rinthine tissue (fig. 8, VJZJI) and divides into a saccular and a
utricular division. The saccular division passes backward under
the triangular plate of the exoccipital bone to the inner side of
the sacculus. Under the edge of this plate it gives off a branch
which passes over the sacculus to the posterior ampulla. The
utricular division divides into branches which supply the ampullar
organs of the anterior and horizontal semicircular canals and
the three divisions of the macula acustica utriculi. The two
branches to the ampullae are long; between them lie the two
short branches to the posterior and middle divisions of the
macula; the anterior division of the macula is supplied from the
anterior ampullar branch.

The glossopharyngeal and vagus nerves leave the medulla
together. They emerge from the skull through the exoccipital
bone, though by different routes. The vagus passes through the
vagus canal; the glossopharyngeal nerve passes through its
foramen in the triangular plate of the exoccipital (fig. 4), traverses
the upper part of the saccular portion of the auditory recess,
passes under the posterior ampulla, and penetrates the skull
just above the margin of the auditory foramen. The fusiform
bulla lies between the exitsof these nerves. The glossopharyngeal
nerve gives a large communicating branch to the vagus as it
passes through the auditory recess and receives one from it
outside the skull. The vagal branch to the second branchial
arch has a separate ganglion.

Just before entering its canal, the vagus gives off a branch,
the course of which is entirely intracranial. Its ganglion is at
its origin and gradually tapers off along its course. It passes
vertically upward in the perimeningeal tissue, at first Just behind
the superior sinus of the labyrinth, but it gradually passes some-
what medial to it and in front of the supracerebral perilaby-
rinthine canal. In the perimeningeal tissue under the cranial
roof it divides into severalbranches. Ihave not traced all these
to their destinations; one contributes to the nerve supply of the

466 HENRY C. TRACY

lateral-line enlargement which occupies the temporal foramen;
another appears to go to the commissural lateral-line canal;
probably others contribute to the plexus formed by the ramus
recurrens facialis.

Two occipital nerves leave the skull through the upright
portion of the exoccipital bone.

DISCUSSION

The relations of the bony elements of the skull to each other
and to the precoelomic diverticulum of the swimbladder and to
the membranous labyrinth which have been described in this
paper are essentially the same for all the other American clupeoid
species which I have examined. They also seem to correspond
to those of the European species in which the details of the ear-
swimbladder relation have been investigated. Certain of the
more conspicuous features in which other American species differ
from P. pseudoharengus may now be briefly summarized. ©

In general, differences in skulls of the other species seem
dependent chiefly upon differences in the degree of ossification.
The lateral cartilage plate is more or less completely ossified in
the adult skulls of the other American species; in Brevoortia and
Alosa, about the only remains of this structure is that part
which contains the anterior semicircular canal. In these forms
also the hook-like process of bone from the epiotic around the
upper end of the anterior semicircular canal forms a complete
ring.

On the external surface of the skull of Pomolobus mediocris,
the posterior osseous capsule is only slightly visible; in Brevoortia
tyrannis the surface of this capsule is completely covered with
bone except for an area in the epiotic fossa so small as to be
visible only with a microscope; in Alosa sapidissima the surface
of this capsule is entirely obliterated by the development of bone.
In most of these species, also, the bone development in the
anterior cranial floor is much more extensive than in P. pseudo-
harengus. The lateral parts of the basisphenoid bone are
thickened into massive processes and the sharply projecting edge

THE CLUPEOID CRANIUM AND THE SWIMBLADDER 467

of bone which separates the orbital from the ventral surface of
the prootic is greatly developed (fig. 2). As a result of these
differences in the relative bone development in this region, the
third and fifth nerves of B. tyrannis and of A. sapidissima appear
to exit from the skull through canals instead of mere foramina as
is the case in P. pseudoharengus.

The morphology of the relation of the. swimbladder diver-
ticulum to the cranium is a difficult problem. The suggestion
of de Beaufort has been discussed above. ‘Tysowski has de-
veloped an elaborate theory, the factual basis of which seems to
be the relation of the septum in the anterior bony capsule to
the bone to which it is attached. This septum, being in inti-
mate connection with the bone of the capsule—its structure, in
fact, continuous with it—and being covered by a reflection of
‘periosteum from the bone surface, is (according to Tysowski’s
view) morphologically a part of the bone itself.

These facts persuaded Tysowski that the swimbladder diver-
ticulum does not pierce the floor of the skull and lie next to the
labyrinth in a cavity hollowed out in the prootic bone, but that
the septum is a part of the cranial floor, and the swimbladder
vesicle in the chamber below it is as much outside the skull as in
other forms. Similarly the wall of the bony capsule is not a
simple structure, but is of double origin, the part above the
septum developing differently than the part below. He con-
‘siders that the floor of the skull in the basisphenoid region gives
off a process dorsally, which bends over medially and so forms
the upper chamber which he says is comparable with the saccular
«cavity. The inferior chamber is formed similarly by a lateral
process (presumably from the basisphenoid bone) which bends
ventrally to enclose the anterior membranous vesicle of the
-swimbladder.

Tysowski’s theory outlined above presents an interesting
hypothesis in that the ear-swimbladder mechanism in the Clu-
peoids can easily be correlated with the simpler, more primitive
_ type. With this theory as a basis, we might assume a ‘nearly
complete series of transition stages from the primitive relation of
apposition (as in the case of the ear and swimbladder of Sparidae

468 HENRY C. TRACY

and Gadidae) through forms like Hyodon and Notopterus, up to
the highly elaborated mechanism in Clupeidae. But, unfortu-
nately, Tysowski’s morphological conceptions are quite incon-
sistent with well-known facts regarding the bony structure of
the clupeoid skull, concerning which there is no essential disa--
greement on the part of other investigators.

In support of his theory, he uses the following argument.

Auf Grund meiner Befunde wiirde es mir schwer fallen, die Bullae
osseae anteriores als ein Erzeugnis der Prootica anzusehen, da ich
Prootica, ahnlich wie Opisthotica, infolge der breiteren Gestaltung des-
Basioccipitale und Basisphenoideum mehr nach aussen liegend, wie
gewohnlich mit der vorderen und dusseren Ampulle und dem Ausseren
Bogengang in Beziehung treten sehe, iibrigens lasst auch die starke
Ausbildung des Corpus basisphenoidei vermiiten, dass er von den
Knochenlamellen der Prootic nicht iiberbriickt sein kann und die
kapselartigen Gebilde eher ihm den Prootic angehoéren.

The relation of these bones to each other and the location of
the bony capsules I have demonstrated to be quite different
from the relations as Tysowski conceives them. That the
anterior osseus capsule, in its entirety, is a part of the prootic
bone in the adult cannot be questioned. It has been so described
by all writers on the subject; my own preparations also demon-
strate it. The general relations of the fifth and seventh nerve
complex to the capsule and to the bone in which it lies are the
same as the relation of these nerves to the prootic in the typical
teleost cranium.

In investigating these relations I made use of dried preparations —
of the skull, as well as freshly cleaned specimens cleared in xylol,
some of which are stained with alizarin, to bring out the cartilage
lines of articulation. Specimens prepared by the latter method
were particularly satisfactory when examined under the binocular
microscope by transmitted light. The lines of demarkation of
the bony elements were so clearly visible as to leave no room for
doubt as to their identity. My results agree with the descriptions
of the clupeoid skull as given by previous workers on the subject
and are also consistent with the osteological relations of other
teleostean groups as described by Sagemehl (’91), Allis (797),
Brooks (’84), and others. In view of these considerations, it

THE CLUPEOID CRANIUM AND THE SWIMBLADDER 469

cannot be true that the two chambers of the capsule are formed
by processes of the basisphenoid bone, as Tysowski has assumed,
and therefore his whole morphological theory falls to pieces.
This conclusion is substantiated by de Beaufort’s observations on
the embryology of the precoelomic diverticulum. His figures
show that the anterior membranous vesicle actually develops
inside of the chondrocranium immediately under the mem-
branous labyrinth. This agrees with my previous published
statements, regarding the developing stages of Stolephorus
mitchilli (Tracy, ’11).

It is another question to explain the anterior bony capsule as
“ein Erzeugnis der Prootica”’ in view of de Beaufort’s obser-
vation that the bony capsule is formed by a process of bone
development from the connective tissue surrounding the mem-
branous vesicle.

Tysowski questioned Ridewood’s identification of the so-called
‘posterior wings of the parasphenoid’ as a part of the para-
sphenoid bone. Relative to this, it is pertinent to refer to one
of the most characteristic morphological features of the teleostean
skull, viz., that the parasphenoid wings form the walls of all
except the upper part of the eye-muscle canal. The para-
sphenoid wings in Clupeoids conform to this relation. It can
easily be seen in dissections, and also in cross-sections of the skull,
that the rectus eye muscles arise along the whole medial surface
of these wings as far back as their extreme posterior end. ‘There
is nothing to indicate that these wings differ in any essential
respect from the corresponding bones in other fishes, except in
their unusual ventral and posterior extension.!

Tysowski’s denial of the existence of the auditory foramen
(which is described in all previous papers on the clupeoid skull)
is also probably without substantial basis. It is true that on
careful dissection a thin scale can be demonstrated over this
foramen, but it is transparent and does not resemble bone in

1 Tysowski compares the wings of the parasphenoid bone with the processus
pharyngealis in Ostariophysae. Sagemehl (’91, p. 516) has suggested that the

processus pharyngealis is formed by the ossification of a ligament passing from
the posterior end of the base of the skull around the aorta to the swimbladder.

JOURNAL OF MORPHOLOGY, VOL. 33, NO. 2

470 HENRY C. TRACY

appearance; furthermore, examination of sections of the skull in
this region show a dense membrane which apparently is not true
bone. What this membrane may be morphologically can only
be determined by a developmental study of this region of the
cranium, but the existence of the auditory foramen in the adult
ean hardly be denied.

The lateral recess has heretofore received scant attention.
This is all the more remarkable when we consider that by means
of this recess the exterior is brought into direct relation through
the lateral-line canals with the fluid under the utriculus and so
with the fluid through the whole system of the perilabyrinthine
canals. These relations were first noted by Breschet, whose
descriptions and figures show them somewhat vaguely. Most of
the writers since have also referred to them, but only incidently
and briefly. The work of Ridewood (’04 a) indicates that the
lateral recess is a characteristic feature of the Clupeoid skull.
He briefly refers to the cavity in Engraulis encrasicholus. ‘‘The
lateral temporal groove is broad and shallow. Removal of its
floor exposes a fairly large cavity opening laterally by two
apertures. . . . This cavity is roofed by the frontal and is
bounded in front by the postfrontal, prootic and squamosal,
behind by the squamosal and below mainly by the prootic.”’
In Coilia nasus, he describes the recess in similar terms and
refers to the prootic bulla as projecting ‘‘upward into a lateral
vacuity of doubtful homology.” He states that the squamosal
bulla (posterior bony capsule) is just visible in the hinder part
of this cavity. This description corresponds to the relations of
this cavity as described in this paper. Ridewood, however, did
not mention the recess in connection with the other species which
he studied. Matthews appears to have completely overlooked it.
De Beaufort merely quotes Ridewood’s statement.

The relations of the lateral recess are such that changes in
pressure as the fish swims from one water level to another may
be transmitted directly from the outside by way of the lateral-
line canals and the loose tissue in the lateral recess to the fluid in
the perilabyrinthine canals. Changes in hydrostatic pressure are
thus conveyed directly to the walls of the utriculus. The struc-

THE CLUPEOID CRANIUM AND THE SWIMBLADDER 471

ture of the macula acustica utriculi and its functional significance
in connection with these mechanical relations will be discussed
more fully in the paper to follow.

SUMMARY

1. The precoelomic diverticulum of the swimbladder divides
into two branches which are conducted in a cartilage tube to the
exoccipital bone on each side of the skull.

2. In canals in the bones at the base of the skull the diver-
ticulum bifurecates; one branch passes laterally to the pterotic
bone where it expands to form the posterior membranous vesicle
contained in a spherical bony capsule; the other branch passes to
the prootic bone where it forms the anterior membranous vesicle
also surrounded by a bony capsule.

3. The osteological structure of the clupeoid cranium conforms
to the type of the lower teleosts. Specialized features of the
clupeoid cranium are:

a. The deep saccular recess partially covered -by a triangular
plate of the exoccipital bone.

b. The auditory foramen between the basioccipital, pterotic,
and prootic bones.

c. The temporal foramen between the frontal, parietal, epiotic,
and pterotic bones, occupied by a bay-like expansion of a lateral-
line canal.

d. The lateral recess between the sphenotic, pterotic, prootic
bones and the lateral wing of the frontal bone.

e. The falciform process of the prootic bones.

f. The great ventral and posterior extension of the wings of
the parasphenoid bone.

g. The spherical osseus capsule in the prootic bone which
communicates with the cavum cranii through a slit-like transverse
fenestra.

h. The spherical osseus capsule in the pterotic bone.

7. The fusiform bulla in the exoccipital bone.

j. The inner wall of the lateral-line canals belonging to the
lateral wing of the frontal bone is absent; the canals therefore
lie in the loose tissue of the lateral recess.

AG? HENRY C. TRACY

4. The lateral recess communicates with the exterior through
the lateral-line canals, and with the cavum cranni through the
arch under the falciform process of the prootic bone. Hence
there exists a free channel for the transmission of changes in
water pressure from the outside to the fluid in the perilaby-
rinthine spaces.

5. The cranial nerves, from the third to the tenth, correspond
in the general course and distribution of their branches to the
nerves as described for Menidia. The branches of the trigemino-
facial nerve complex make their exit from the skull in close
relation to the surface of the prootic bony capsule. There is an
intracranial plexus of nerves in the perimeningeal tissue supplied
by recurrent branches of the seventh and tenth nerves. These
recurrent branches also supply the expansions of the lateral-line
canals in the lateral recess and in the temporal foramen. From
the utricular branch of the eighth nerve, three branches are
given off to supply the three divisions of the macula acustica
utriculi.

BIBLIOGRAPHY

Auuts, E. P., Jk. 1897 Morphology of the petrosal bone and of the sphenoidal
region of the skull of Amia clava. Zool. Bull., vol. 1.

1903. The skull, the cranial and first spinal nerves in Scomber scomber.
Jour. Morph., vol. 18.

De Beavurort, L. F. 1909 Die Schwimmblase des Malacopterygii, Morph.
Jahrbuch, Bd. 39.

BrescHet, G. 1838 Recherches anatomiques et physiologiques sur l’ organs de
? ouie des poissons. (Mémoires presente par divers savantes a
l’ Academie Royale des Sciences, T. 5, Paris).

Bripae, T. W. 1900 The air bladder and its connections with the auditory
organ in Notopterus borneensis. Jour. Linnean Soc., vol. 27.

Brooks, Str. J. 1884 Osteology and arthrology of the haddock (Gadus aegle-
finus). Proc. Roy. Soc. Dublin, vol. 4, N.S.

Hasse, C. 1873 Beobachtungen itiber die Schwimmblase der Fische. Ana-
tomische Studien, Bd. 1, Leipzig.

Herrick, C. J. 1899 Cranial and first spinal nerves of Menidia. Jour. Comp.
Neur., vol. 9.

Matuews, J. D. 1886 Structure of the herring and other clupeoids. Fifth
Rep. Fish. Bd., Scotland, Appendix F, Sessional Papers; House of
Commons, 21, 1887.

Retzius, G. 1881 Das Gehérorgan der Wirbeltiere. I. Gehérogan der Fische
und Amphibien. Stockholm.

THE CLUPEOID CRANIUM

RipEwoop, W. G.

AND THE

SWIMBLADDER 473

1891 The air bladder and ear of British clupeoid fishes.

Jour. Anat. and Physiol. (Lond.), vol. 26 (N. s. vol. 6).

1904 a On the cranial osteology of the clupeoid fishes.

Soc. Lond., vol. 2.

1904 b On the osteology of the Elopidae and Albulidae.

Soc. Lond., vol. 2.

Proe. Zool.

Proe. Zool.

1904 ¢ On the cranial osteology of the families Mormyridae, Notop-

teridae, and Hyodontidae.
SAGEMEHL, M.
Cranium der Cyprinoiden.
Sterzi, G.

meningi., P. I. Meningi midollari.

Scienza.
RmAcy He C:
Anz., vol. 38.
TysowskI, M. A.

zur Schwimmblase bei den Clupeiden.

covie, 8. 1.
WEBER, E. H.

1911 The morphology of the swimbladder of teleosts.

1820 De aure et auditi hominis et animalium.

Jour. Linnean Soc., vol. 24.
1891 Beitrige zur vergl.
Morph. Jahrbuch, Bd. 17.

1901 Ricerche intorno alla anat. com. ed all’ontogenesi delle

Anatomie der Fische. IV. Das

Atti del R. Instit. Veneto di

Anat.

1909 Zur Kenntnis des Gehérorganes und seiner Beziehungen

Bull. Intern. Acad. Sei. Cra-

Pars I. De aure

animalium aquatilium. Lipsiae.

PLATES

ABBREVIATIONS

A, aorta

AF, auditory foramen

AMV, anterior membranous vesicle

AOC, anterior osseous capsule

AP, aponeurosis

ASC, anterior semicircular canal

ASP, alisphenoid bone

BR, brain

BSO, basioccipital bone

BSP, basisphenoid bone

CC, cartilage canal of the swimbladder
diverticulum

CF, carotid foramen

CSD, canal of the swimbladder diver-
ticulum

DC, opening of canal for swimbladder
diverticulum

EPF, epiotic fossa

EPO, epiotice bone

ETH, ethmoid bone

EXO, exoccipital bone

F, fenestra

F ITI, foramen for oculomotor nerve

F V, foramen for trigeminofacial com-
plex

FB, fusiform bulla

FCC, foramen for cartilage canal

FHM, foramen for hyomandibular
nerve

FP, falciform process

FR, frontal bone

FSP, foramen for sympathetic and pre-
trematic VII

GF, ganglionic fossa

GL V, ganglion V nerve

GL VII, ganglion VII nerve

HMA, hyomandibular articulation

HY, hypophysis

HYF, hypophysial foramen

ILF, inferior lamina of the falciform
process of prootic

K, kidney

LLC, lateral line canal

LPC, lateral cartilage plate

LR, lateral recess

LSC, lateral semicircular canal

474

LSCA, ampulla of lateral semicircular
canal

LW, lateral wing of prootic

LWF, lateral wing of frontal

M, muscle

MPRO, mesial plate of prootic

NC, nasal cartilage

OBSP, orbitosphenoid

ON 1, first occipital nerve

ON 2, second occipital nerve

OPF, optic foramen

OPS, ramus ophthalmicus superficialis

OPT, opisthotic bone

PF, parietal bone

PMV, posterior membranous vesicle

PN, branch of VII nerve to ampulla of
posterior semicircular canal

POC, posterior osseous capsule

PRO, prootic bone

PS, perilabyrinthine tissue space

PSC, supracerebral perilabyrinthine
canal

PSD, posterior semicircular canal

PSDA, ampulla of posterior semicir-
cular canal

PSP, parasphenoid bone

PSPW, posterior wings of parasphe-
noid

PT, pterotic bone

R VII, recurrent ramus of VII nerve

RE, rectus eye muscles

RHM, ramus hyomandibularis

RMM, ramus maxillomandibularis

RP VII, ramus palatinus of VII nerve

RPT, ramus pretrematicus

HENRY C. TRACY

RSY, ramus sympatheticus

S, sacculus

SB, swimbladder

SBP, synchondrosis between basioc-
cipital and prootic

SC, septum of anterior osseous capsule

SCA, prootic portion of saccular cavity

SCP, subcerebral plate of perilabyrin-
thine tissue

SLF, superior lamina of faleiform pro-
cess of prootic

SN, saccular division of VIII nerve

SOC, supraoccipital bone

SOL, supraorbital lateral line canal

SPH, sphenotic bone

SR, saccular recess

SS, superior sinus of utriculus

SSC, saccular subcerebral canal

TF, temporal foramen

TPEXO, triangular plate of exoccipital

VM, vomer

VT, vertebra

VBSO, ventral plate of basioccipital

VPRO, ventral plate of prootie

U, utriculus

USC, utricular subcerebral canal

ITI, oculomotor nerve

IV, trochlear nerve

V, trigeminal nerve

VI, abducens nerve

VIII, acoustic nerve

IX, glossopharyneal nerve

X, vagus nerve

* foramen for ramus palatinus VII

x, foramen for hyomandibular nerve

PLATE 1

EXPLANATION

1 Lateral view of skull of Pomolobus pseudoharengus.
2 Ventrolateral view of posterior part of skull of Alosa sapidissima.

OF FIGURES

x 4.5.
X 2.

PLATE 1

THE CLUPEOID CRANIUM AND THE SWIMBLADDER

HENRY C. TRACY

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475

PLATE 2
EXPLANATION OF FIGURES

3 Bones of the floor and lateral wall of the skull viewed from above (Pomolo-
bus pseudoharengus). A part of the lateral wing of the frontal bone has been
removed to expose the lateral recess; the vertical, posterior part of the exoc-
cipital has been cut away to show the floor and side of the saccular recess. X 9.

4 Bones of the floor and lateral wall of the skull viewed from the median
side (Pomolobus pseudoharengus). The articulating surface of the prootic bone
(PRO) is in the medial plane; the lateral surface of the basioccipital is shown
(BSO); the frontal and sphenotic bones are removed. X 9.

THE CLUPEO1D CRANIUM AND THE SWIMBLADDER PLATE 2
HENRY C. TRACY

i ee =

<i

\

PLATE 3

EXPLANATION OF FIGURES

5 Ventral surface of the cranium (Pomolobus pseudoharengus). On the
right half of the figure the cartilage canal is shown; the ventral segment of the
prootic osseous capsule (AOC) has been cut away to expose the septum and
opening of the canal for the precoelomic diverticulum. %X 5.4.

6 The cerebral surface of the prootic bone (Pomolobus pseudoharengus).
X 6. é

7 The ventral surface of the middle of the skull (Pomolobus pseudoharengus).
xX 5.

8 Medial surface of the lateral half of the cranium, showing the relations of
the lateral cartilage plate, the perilabyrinthine canals, the nerves, and the eye-
muscle canal (Pomolobus pseudoharengus). XX 5.

478

THE CLUPEOID CRANIUM AND THE SWIMBLADDER PLATE 3
HENRY C. TRACY

479

PLATE 4

EXPLANATION OF FIGURES

9 Cross-section through the head just behind the orbital region to show the
bony structure of the side and base of the cranium. The section is oblique
with their left side in advance; on the left side, the section passes through the
ganglionic fossa, on the right through the lateral recess (Pomolobus pseudo-
harengus).

10 Cross-section through the head at the articulation between the prootic
and basioccipital bones. On the left side the section passes through the front
part of the posterior osseous capsule, on the right side, just in advance of that
structure (Pomolobus pseudoharengus).

480

PLATE 4

THE CLUPEOID CRANIUM AND THE SWIMBLADDER

HENRY C. TRACY

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PLATE 5

EXPLANATION OF FIGURES

11 Cross-section of the head through the posterior osseous capsule and
auditory foramen (Pomolobus pseudoharengus).

12 Cross-section through the occipital region of the head. The section is
oblique with the right side in advance (Pomolobus pseudoharengus. )

482

THE CLUPEOID CRANIUM AND THE SWIMBLADDER PLATE 5
HENRY C. TRACY

Resumen por el autor, O. W. Hyman.
Universidad de Tennessee.

El desarrollo de Gelasimus después de abandonar el huevo.

El autor ha obtenido de los huevos de hembras ovigeras de
Gelasimus pugilator, G. pugnax y G. minax, los primeros estados
de zoés. Las larvas zoés de estas especies son tan semejantes
entre si que los esfuerzos para determinar la especie en los ejem-
plares capturados por medio de la sonda han sido estériles. Se
criaron cuatro de los cinco estados de zoé por que pasan estos ani-
males; las restantes formas fueron obtenidas mediante la sonda
o en la playa, dejindolas sufrir una muda durante la observaci6n.
También ha estudiado y descrito los cambios que tienen lugar
durante la metamorfosis de las zoés, megalops y el cangrejo j6ven
hasta el momento de la diferenciacién sexual. Los apéndices ab-
dominales del estado megalops son estructuras larvarias. Los
apéndices abdominales del adulto son formaciones nuevas que .
aparecen al adoptar el animal la forma de cangrejo. Algunas de
las costumbres de las hembras ovigeras, zoés, megalops y cangre-
jos jOvenes se han descrito incidentalmente.

Translation by José F. Nonidez
Carnegie Institution of Washington

AUTHOR'S ABSTRACT OF THIS PAPER ISSUED
BY THE BIBLIOGRAPHIC SERVICE, FEBRUARY 2

THE DEVELOPMENT OF GELASIMUS AFTER
HATCHING

O. W. HYMAN

University of Tennessee College of Medicine

TWELVE PLATES (HIGHTY-EIGHT FIGURES)

During the summers of 1915 and 1916 at the U. S. Fisheries
Biological Station at Beaufort, North Carolina, while engaged in
an endeavor to rear crustacean larvae under artificial conditions,
I had an opportunity to study the habits and developmental
stages of Celasimus. While this study was only incidental to °
the experiments in hand, I found the material so abundant and
other conditions so favorable that I have been enabled to review
the development in considerable detail: During the progress of
the study I have been aided greatly by the criticism and guidance
of Dr. H. V. Wilson and Mr. W. P. Hay. The work done at
Beaufort has been made a pleasure by the generous cooperation
unfailingly extended by Mr. 8. F. Hildebrand, director of the
laboratory.

OCCURRENCE OF THE ADULTS

Of the many decapods at Beaufort, the three species of Gelasi-
mus are perhaps the most numerous. Celasimus pugilator, the
common gray sand-fiddler or fiddler crab, is present almost every-
where, but is most abundant on the islands and shores where a
sandy beach is exposed at low tide. Conditions are especially
favorable if the beaches have a fringe of sedges which are in the
water at high tide. The crabs find a ready refuge in these when
frightened.

Celasimus pugnax is not so abundant as G. pugilator, but is
rather common. It is most often found in marshes, especially
where there is a considerable estuary formed at high tide, but where
the soft boggy marsh is exposed when the tide is out. One such

485

JOURNAL OF MORPHOLOGY, VOL. 33, NO. 2

A486 oO. W. HYMAN

estuary leads north from Taylor’s creek, a few hundred yards
west from Lenoxville Point. This species is also abundant on
the banks of the Beaufort end of the Inlet Canal from Pamlico
to Beaufort.

The third species, Gelasimus minax, is not common. It seems
to prefer higher ground bordering a marsh, and frequently occurs
at some distance from salt water. It occurs along the banks of
the Inlet Canal from Pamlico to Beaufort, and especially in one
of the large estuaries on the Shackelford Banks east of the Mullet
Pond.

METHODS OF STUDY

In studying the larval history of these forms, the first zoeae
were hatched in the laboratory. The ovigerous females of G.
pugnax and G. minax were secured readily by digging them from
their burrows in the marshes. Egg-bearing females of G. pugi-
lator at first were very hard to find. I have dug the crabs from
their burrows for hours and searched the hundreds at the water’s
edge at ebb tide without finding a single ovigerous female. Quite
by accident I discovered a method of securing all the eggs of this
species to be desired. Landing on Horse Island just at dusk one
evening, I noticed that a number of the fiddlers hurrying to their
burrows were egg-bearing females. Investigation showed that a
large percentage of the females carried eggs. Thereafter I had
no difficulty in securing all the eggs I wished by going to some
favorable spot at dusk during an ebb tide. The crabs were taken
to the laboratory and kept in crystallization dishes partially filled
with water, until the eggs hatched. Cenerally the eggs hatched
within a few days. The approximate age of the embryos can be
determined at a glance. When the eggs are newly laid they are
a deep purple—almost black. As the embryo develops it be-
comes lighter and distinctly purple. It continues to lose its color,
becoming a dirty gray when it is nearly ready to hatch. The
eggs of all three species hatch soon after 7 p.M., that is, at about
dusk. This fact probably explains the presence of the females
at the water’s edge at that time.

DEVELOPMENT, GELASIMUS AFTER HATCHING 487

Rearing the larvae. During the summers of 1915 and 1916 I
made many unsuccessful attempts to rear the larvae secured by
hatching the eggs while they were still on the female. I carried
one solitary specimen through three molts, several through two
molts, and many through one molt, but the great majority of
the zoeae died before their first molt. The only method by
which I succeeded in carrying any beyond the first molt was as
follows: Small floats, 6 inches by 8 inches by 2.5 inches, with
sides of fine bolting cloth, were made. The zoeae were placed
in these and these bolting-cloth floats then placed in crab floats for
protection from the rippling of the surface water. The difficulty’
with this method was that debris rapidly collected on the float
and clogged its meshes. The bolting cloth rotted quickly and it
was difficult to recover the zoeae.

Larvae from the tow. The fifth zoea and the megalops and crab
stages were not reared from the egg. The method of tracing the
development through these stages was to collect specimens of
some known stage from the tow and keep them in the laboratory
until the next molt. The zoeal stages could rarely be kept alive
through more than one molt. On the other hand, the megalops
and crab stages could be kept alive mdefinitely if they were fed
and protected from the cannibalistic tendencies of their fellows.

The stages for study were obtained as follows. The first, sec-
ond, and third zoeal stages were abundant in the surface tow from
July 1st to September 15th. The tow might be taken anywhere
in the harbor or outside. The stages could be separated with
some difficulty and placed in glass bowls. The next molt gener-
ally occurred in a few days.

The fourth and fifth zoeal stages seldom appeared in the sur-
face tow. These stages, however, could be obtained readily from
the bottom tow. The method of towing on the bottom was as
follows: A bolting-cloth sac was made to fit inside a small heavy,
bottom dredge. This was then dragged carefully along with
just enough rope to let it touch the bottom. Care must be exer-
cised to keep it from digging. The only successful tows of this
kind were taken over a sandy bottom. The best place was found
to be along the southwest edge of Bird Island Shoal.

A488 O. W. HYMAN

Megalops and crab stages. The specimens of megalops were
taken most abundantly in surface tow from the ocean just out-
side the inlet. They were also taken in numbers from crevices
in rotting boards exposed at low tide, under oyster shells and
stones, or from the bark on pilings. The crab stages do not
appear in the tow. They may be picked from the old boards,
with the megalops stages, or obtained during ebb tide on a sandy
beach (where they are very hard to see) or in the debris in the
marshes. After the crab measures about 2 mm. across the cara-
pace, it digs its own burrow and may then be collected by dig-
ging. All of the crab stages are found mingled with the adults.

HISTORY OF THE DEVELOPMENT

From the foregoing facts the following history of the develop-
ment may be deduced with reasonable assurance. The egg-
laden females remain hidden in their burrows during the day,
probably because the egg mass retards their movements to such
an extent as to endanger their existence. At dusk, when the
eggs are ready for hatching, the females approach the water’s
edge and the eggs are’ hatched in the water. The larval skin
with which the embryo is covered is shed in hatching.

The young zoea wobbles off on the surface of the water, being
carried along largely by the tide. Its own efforts, however, serve
to keep it at the surface, all the zoeal forms being positively pho-
totropic. After about four days the first molt occurs. The
second zoea behaves like the first—keeps itself near the surface
of the water by the rapid beating of the maxillipeds and is swept
along by the tides. After four or five days a second molt occurs.
The third zoea is the form most rarely found in the tow. This
indicates the possibility that it swims at an intermediate depth,
the maxillipeds not being strong enough to sustain at the surface
the increased weight of the body.

After the molt to the fourth zoeal form, the zoea sinks. It
does not lie or crawl on the bottom, but is swept along by the
current and at short intervals drives itself upward by the rapid
beating of the maxillipeds. As soon as the maxillipeds cease
beating it falls slowly to the bottom again on account of its

DEVELOPMENT, GELASIMUS AFTER HATCHING 489

weight. After about a week the next molt occurs. The fifth
zoea lives near the bottom like the fourth. It is still more dis-
proportionately heavy and correspondingly clumsy. During the
last day or so of this stage the animal is almost entirely at the
mercy of the tide. The next molt occurs at the expiration of a
week or ten days.

When the zoea molts to the megalops stage, its mode of life
suddenly changes. The animal is now provided with powerful
swimming organs, the pleopods, so situated as to be most ef-
ficient. Its chelae serve as an excellent means of securing its
prey, which now consists, partly at least, of smaller crustaceans.
Its organs of equilibration are suddenly well developed and its
other sense organs are more nearly perfect. ° The animal rapidly
ascends to the surface and darts swiftly about. The megalops
stage probably lasts a long time, and there is only one such stage.
Megalopa were kept in the laboratory as long as three weeks be-
fore molting to the crab stage. All of those that molted became
crabs at the first molt. After swimming about at the surface for
three or four weeks, the megalops seeks some protected place,
such as the crevices in a rotting board near the shore, and there
undergoes the molt to the first crab stage.

The young crab clings closely to its refuge or crawls about at
the water’s edge, especially among the exposed roots of sedges.
It is very clumsy and very weak. At the end of three days it
molts to the second crab stage. After four or five days a second
molt occurs. After this molt the little crab runs about quite
freely and may dig its first burrow. It now measures about 2
mm. across its carapace at its broadest point. Its mode of life
from now on is like that of the adult.

IDENTIFICATION OF THE ZOEAE

The zoeal forms of Gelasimus may be identified readily. They
have prominent anterior and dorsal spines, but have no lateral
spines on the carapace. This distinguishes them at once from
all the other common zoeae of the Beaufort region, except those
of the two species of Sesarma. From these the zoeae of Gelasi-

490 O. W. HYMAN

mus may be distinguished in two ways: the length of the antenna
does not equal that of the anterior spine in the case of Gelasimus,
but does in Sesarma; the first maxillipeds of Sesarma have
pigment spots at the proximal ends of the protopodites, while in
Gelasimus the first maxillipeds have pigment spots at the distal
ends of the protopodites.

The pigmentation of all the zoeal stages of Gelasimus is re-
markably constant and serves as a ready means of establishing
the identity of the form. The pigment spots are jet-black when
contracted. In the expanded condition they vary in color, being
black or olive or red-brown or orange or combinations of these
colors. The distribution of the spots is as follows: on the cara-
pace, a spot posterior to the base of the dorsal spine, a spot on
each lateral flange of the carapace near its posterior angle, a
median spot between the eyes, a large spot on the front of the
base of the anterior spine, a spot between the bases of the first
and second maxillae; on the appendages, a spot.on the labrum,
one on each mandible, and one on the distal border of the proto-
podite of each of the first maxillipeds; on the abdomen, a pair of
dorso-lateral spots between the first and second segments, a pair
of ventral spots on the second and third segments, and lateral
spots on each side of the posterior borders of the fourth and
fifth segments.

DISTINGUISHING CHARACTERISTICS OF THE ZOEAL STAGES

The first and second zoeal stages of the three species were ob-
tained with certainty by hatching and rearing them in the lab- ©
oratory. The distinctions between equivalent stages of the three
species, however, all proved to be relative differences of such slight
degree that I was never sure that I could separate certainly the
specimens obtained from the tow. Some specimens had the
characteristic broad-based, evenly tapering frontal spine of G.
pugilator and others the slender constricted spine of G. pugnax,
but many of them seemed intermediate, and I gave up the at-
tempt to distinguish the species. The different developmental
stages of the zoeae were easily distinguishable from each other,
however, as is indicated in the descriptions below.

DEVELOPMENT, GELASIMUS AFTER HATCHING 49]

First zoeal stage of G. pugilator (figs. 1 and 2). These zoeae are
relatively small (length from head between the eyes to tip of
telson, 1 mm.). They swim by means of the first and second
maxillipeds, and, so far as was observed, swim in only one direc-
tion, upward and slightly forward. When at rest the maxillipeds
are habitually carried in the position shown in figure 1. In
swimming, these are raised to the sides of the carapace and
driven downward. When not swimming, the larva is nearly
always actively writhing about, chiefly by lashing the abdomen.
The first and second maxillae beat regularly and rapidly in such
a way as to drive a current toward the mouth opening.

The carapace is slightly flattened from side to side. It bears
the usual anterior and dorsal spines, but shows no traces of lat-
eral spines. The anterior spine rises from the anterior margin -
of the carapace between the eyes and passes ventrally almost at
right angles to the long axis of the body. It is about 0.2 mm.
long, straight, smooth, and evenly tapering from a slightly swol-
len base. The dorsal spine arises in the mid-dorsal line posterior
to the eyes and just above the heart. It is shorter than the an-
terior spine and curved posteriorly. There are constantly pres-
ent a pair of setae which arise on each side of the carapace, an-
terior and lateral to the base of the dorsal spine. The lateral
ventral borders of the carapace show the usual anterior and
posterior lobes.

The eyes are sessile and immovable. The facets are clearly
indicated, but are not perfectly marked on the surface. The an-
tennule (fig. 20) is 0.07 mm. long and conical. From its tip arise
two or three long olfactory hairs and one or two short, sharp-
pointed setae. The antenna (fig. 28) is 0.11 mm. long and biseg-
mented. The proximal part of the basal segment is thick and
cylindrical. At its distal end its inner half is produced into a
stout serrated spine about twice as long as the proximal portion.
The outer half of the tip of the basal portion bears the distal
segment, which is small and cylindrical. From its tip arise two
setae, one long, which seems to be a continuation of the segment,
and a short outer one.

492 O. W. HYMAN

The mandible is short, stout, and unsegmented. Its edge has
the usual teeth for tearing and grinding. The first maxilla (fig.
45) is bisegmented. The basal segment is bilobed and thickly
lamelliform. The medial lobe bears one movable smooth spine
on its median border, and, at its tip, three macerating spines.
The lateral lobe bears similar spines arranged in two series.
From the distal border of the lobe arise two or three strong.
spines, and from its inner face, near the border, arise three weaker
spines. The distal segment is cylindrical and bears four tactile
hairs at its tip.

The second maxilla (figure 54) is a lamellar appendage, its
median border produced into four lobes and a hairy process ex-
tending laterally. The three median lobes represent the basal seg-
ment or segments. Of these, the most median bears two series
of smooth spines. The middle and lateral lobes of the basal seg-
ment are each armed with three macerating spines at their tip
and one on their inner surface near the tip. The fourth lobe rep-
resents the distal segment. ' It bears three tactile hairs at its tip.
The outer plate represents the epipodite. It consists of a proxi-
mal lamelliform portion which bears four finely plumose hairs
along its lateral border, and is produced posteriorly into a proc-
ess which tapers to a blunt end. The process is covered with
fine hairs over most of its suface.

The first maxilliped (fig. 62) is the best developed of the ap-
pendages at this time. It is 0.25 mm. long without its terminal
hairs. It is composed of a basal portion, an endopodite, and
exopodite. The basal portion is unsegmented, compressed, and
of approximately uniform circumference. The endopodite, slen-
der and slightly longer than the basal portion, is composed of
five segments. The terminal segment bears three tactile hairs at
its tip and a single plumose seta from its median superior surface.
The exopodite is unsegmented, cylindrical, and about equal in
length to the endopodite. It bears four long plumose hairs which
are jointed near their middle. The length of the hairs is from
0.16 to 0.20 mm.

The second maxilliped (fig. 62) is like the first in all respects ex-
cept its endopodite. This is much shorter and is trisegmented.

DEVELOPMENT, GELASIMUS AFTER HATCHING 493

The two basal segments are very short, the terminal segment is
like the terminal segment of the endopodite of the first maxilliped.

The abdomen is composed of five movable segments. Each
of the first four is cylindrical and of approximately the same
diameter. They increase slightly in length from the first to the
fourth. The second, third, and fourth segments are produced
backward and laterally into an angular process which slightly
overlaps the next succeeding segment. The posterior border of
each of these segments bears a median seta dorsally. The sec-
ond segment bears a short blunt lateral spine which curves for-
ward. It is so placed that its curvature serves as a groove into
which the posterior border of the carapace fits. The third seg-
ment also bears a spine which curves backward on each side and
is less conspicuous. The terminal segment represents the sixth
abdominal segment fused with the telson. It is crescent-shaped
with the horns elongated. The anus lies on the ventral surface
of this segment. It is surrounded by very tumid, movable lips.
which may form a protuberance as in figure 1. From the median
surface of each horn near its base, three setae arise which are
plumose with short stout hairs. The length of the segment with
its horns is 0.22 mm.

First zoeal stage of G. pugnax (figs. 3 and 4). The first zoea of
(. pugnax differs from that ‘of G. pugilator only in size. It is
smaller in all dimensions. The anterior and dorsal spines are
shorter and slenderer. Otherwise there is the most absolute
identity in pigmentation and conformation of the appendages—
even to the number and kind of hairs found on each.

First «oeal stage of G. minax (figs. 5 and 6). The first zoea of |
G. minax is distinguishable from that of G. pugnax with the
greatest difficulty. It is slightly smaller, but shows the same
‘slender spines of the carapace.

The first zoeal stage of Gelasimus (figs. 1 to 6) is most readily
distinguished by the four plumose hairs of the exopodites of the
maxillipeds. The caudal portion of the scaphognathite is a single
elongated conical process thickly beset with fine hairs.:

The second zoea (figs. 7 and 8) has increased in length to 1.175
mm. The eyes are on stalks and are slightly movable. The

494 oO. W. HYMAN

lateral hairs on the scaphognathite are now five and the posterior’
conical portion is tripartite distally (fig. 56). The exopodites of |
the first and second maxillipeds become bisegmented and the
number of hairs at their tips is increased to six.

The third zoea (figs. 9 and 19) has increased in length to 1.5
mm. ‘The eyes are freely movable. A broad, low tubercle ap-
pears between the spine of the basal segment of the antenna and
the base of the distal segment (fig. 30). This is the anlage of the
flagellum of the permanent antenna. The distal portion of the
first maxilla (fig. 48) has two segments and has become separated
from the basal portion by a joint. In the second maxilla (fig.
57) the scaphognathite now bears six hairs along the lateral-
border of its anterior portion. The exopodites of the first and
second maxillipeds bear eight hairs. The third maxillipeds are
clearly distinguishable for the first time as minute buds just be-
hind and somewhat median to the second pair. The buds of
four pairs of periopods are distinguishable and those of the
cheliped are somewhat larger and show an identation at their
tips indicating the position of the chela. Blunt protuberances
from the ventral surfaces of the second to sixth abdominal
segments are the beginnings of the pleopods. On the telson (fig.
84) are two minute spines medial to those earlier present.
There is a deep groove between the sixth abdominal segment and
the telson, but no joint has yet developed.

The fourth zoea (fig. 11) has increased in length to2mm. The
antennule has increased in size somewhat and a lateral hair is
present near its tip (fig. 24). The tubercle of the antenna is
greatly enlarged and the former distal segment now appears as
a lateral appendage (fig. 32). The second maxilla shows several
changes (fig. 58). The palp is separated from the basal portion
by a joint and the median lobes are more pronounced. The
scaphognathite appears as a single plate and bears eighteen hairs
along its lateral border. The first and second maxillipeds have
nine or ten hairs at the tips of their exopodites. The bud of the
third maxilliped is notched at its tip, indicating a division into
two rami. The buds of all the periopods appear, and those of
the chelipeds are clearly bifurcated. The buds of the pleopods

_ DEVELOPMENT, GELASIMUS AFTER HATCHING 495

have become more prominent. The border of the carapace bears
a series of straining hairs which serve to keep foreign particles from
getting under it.

The fifth zoea (figs. 12 and 13) has increased in size to a length
of 2.25mm. The antennule (fig. 25) shows the following changes:
a rather deep constriction divides the distal portion from the
slightly enlarged basal portion; on the distal portion the number
of hairs of the second series is increased from the single one to
three. When this stage is nearing its molting time, the distal
portion shows indistinctly two or three constrictions where the
joints of the next stage will appear. In the antenna (fig. 33)
the flagellum is bisegmented and is marked off by a joint. As the
time for the next molt approaches, the flagellum shows indica-
tions of about twelve constrictions which mark out the joints of
the next stage. The first maxilla shows a few minor, but inter-
esting changes (fig. 50). There is developed on its lateral border
a rounded low prominence which bears a single sparsely plumose
stout hair similar to those found on the coxopodites of the maxil-
lipeds at the base of the epipodite. Between the palp and the
epipodital prominence is a peculiar, densely plumose hair similar
in structure to the hair on the distal segments of the endopodites
of the first and second zoeal maxillipeds and to the so-called ‘audi-
tory hair’ of Mysis. The exopodites of the first and second maxil-
lipeds bear ten hairs (figs. 66 and 67). The endopodite of the
second maxilliped has grown considerably larger. The third
maxillipeds and the periopods are finger-shaped appendages, and
two or three small buds dorsal to their bases are the early gills.
The pleopods are also finger-shaped and show indications of divi-
sion into protopodite, exopodite, and endopodite, although the
endopodite is exceedingly minute. The telson bears four pairs of
plumose spines. During the last day or two of this stage a
number of changes are noticeable in preparation for the next
molt. The soft part is withdrawn from the dorsal spine, until it
is entirely empty, and from the anterior spine until it fills the
basal fourth only. The exopodites of the maxillipeds are
shrunken away from their coverings, thus accounting for the
sluggishness of the larva at this time. All the joints of the perio-
pods are differentiated.

496 O. W. HYMAN

Description of the Megalops (figs. 14 and 15). When the fifth
zoea molts to the form of the megalops, a profound change occurs
in many of the parts. The contours of the cephalothorax and
the abdomen are both changed. Throughout the zoeal stages
the cephalothorax is flattened from side to side. In the megalops
it is flattened dorso-ventrally. The abdomen is cylindrical in
the zoea and now becomes flattened dorso-ventrally also. The
changes in many of the appendages are still more striking. The
animal suddenly becomes well equipped for an active predatory
existence. The sensory structures of the antennule and the
antenna are practically in the adult condition. The chelae are
efficient structures for securing the prey and the maxillipeds are
transformed into masticatory organs. The pleopods are now de-
veloped into powerful swimming organs and the animal darts
swiftly about.

The antennule (fig. 26) is now composed of a large basal portion
and a terminal process of four segments. The basal segment
bears the statocyst which can be distinguished through its walls.
The ultimate and penultimate segments bear from five to seven
olfactory hairs each. The antennule has now reached what is
practically the adult condition. The antenna has undergone a
striking change (fig. 34). The zoeal lateral spine and lateral
segment are absent. The flagellum is composed of eight small
cylindrical segments and is borne at the tip of a basal portion of
three larger segments. The antepenultimate segment of the
flagellum bears four or five long tactile hairs and the terminal seg-
ment two or three.

The mandible (figs. 40 and 41) has reached practically the
adult condition, as it now bears a three-jointed palp. The first
maxilla (fig. 51) shows few changes. Its basal median lobe bears
more spines and is enlarged. The joints of the palp are obscure
and its segments somewhat shriveled. The hairs on the lateral
border of the basal portion have the same form as the hairs on
the epipodites of some of the appendages posterior to it. The
second maxilla (fig. 60) has undergone changes similar to those of
the first. The palp has lost its hairs and joint and appears as a
smooth lobe of the basal portion. The scaphognathite is larger
and has more hairs along its border.

DEVELOPMENT, GELASIMUS AFTER HATCHING 497

The changes in the maxillipeds are profound (figs. 68, 70, and
74). The first and second pairs are transformed from swimming
organs and all three pairs become functional as mouth parts.
The first and third pairs bear well-developed epipodites, and each
of the second pair bears a tiny bud on its lateral surface—the be-
ginning of an epipodite and a gill. In all these appendages the
proximal segment of the exopodite is elongated and slender, while
the distal segment is small and is carried at right angles to the
basal segment. It bears three or four weak, slender, plumose
hairs. The endopodite of the first pair is twisted so that the
lateral edge of its distal portion becomes median. It bears only
a few small hairs. The endopodites of the second and third pairs
are composed of four segments, are stout, and bear numerous
macerating spines.

The gills of the megalops are four on each side—a pleurobranch
between the third maxilliped and the cheliped, two podobranchs
on the cheliped, and one podobranch on the second periopod.

The chelipeds are large and functional as pincers. The sec-
ond, third, and fourth periopods are long and slender with some-
what hooked extremities. They may be used in crawling, but
are used chiefly for clinging to some protecting cover. The fifth
periopod seems to be useless. It is small and has several long
hairs at its tip, and is carried folded over the back of the carapace.

The pleopods (fig. 79) are large, well-developed swimming or-
gans. Each is composed of a basal segment bearing an exopodite
and an endopodite. The exopodite is a flattened lobe bearing
from seven to fourteen swimming hairs around its border. The
endopodite (fig. 81) is small, bears no hairs, but has three shriv-
eled, curled processes at its tip which may represent atrophied
hairs.

The first crab stage (fig. 16). When the megalops molts to the
first crab stage, the cephalothorax is slightly altered, becoming
broader and more depressed. The abdomen shows a great
change. It is permanently flexed under the thorax into a groove
in which it fits, and its appendages, the pleopods, are shriveled
and hidden under it.

498 O. W. HYMAN

The antennules and antennae show only slight changes. The
mandible and first maxilla are very slightly altered also, but the
second maxilla (fig. 61) is changed both in shape and in the rela-
tive size of its parts. The scaphognathite has increased in size;
the two basal lobes are larger and are partly constricted from the
coxal segment; the palp is relatively smaller and is reduced to an
inconspicuous lobe of the lateral basal lobe.

The maxillipeds (figs. 69, 71, and 75) show changes, but none
so marked as in the previous molt. In the first, the change is
largely an increase in size and apparent strength. The endopo-
dite has changed shape and is now a trisegmented, flattened ap-
pendage with the middle segment twisted or folded on itself.
All its segments are hairy. The basipodite and coxopodite ap-
pear as prominent rounded lobes medially, but fuse into an un-
jointed mass laterally. Each lobe has a spiny border. The
epipodite is larger and bears scattered, slender, barbed hairs.
The second maxilliped is not greatly changed. The proximal
segment of the endopodite is relatively larger and bears a row of
stout spines along its median border. The tubercle of the future
gill and epipodite shows a differentiation into minute lobes. The
change in the third maxilliped is largely confined to the endopo-
dite, which is relatively larger, due to an increase in size of the
proximal two segments. The number of hairs is increased on all
segments, but especially on the lateral portions of the protopodite
and the proximal half of the epipodite.

The chelae are further developed. Both the carpus and the
dactyl end in rounded, spoon-shaped points. Both chelae are
identical. The fifth periopods are adapted for walking and cling-
ing. They are small, but have the usual five segments of the
periopods.

The most striking change among the appendages occurs in the
pleopods. These are no longer the well-formed, powerful swim-
ming. organs of the megalops, but are smaller, hairless, and
shriveled. They are hidden between the abdomen and _ the
thorax.

The second crab stage (fig.17). After the next molt the crab
has increased in size from 1.35 mm. long and 1.25 mm. broad to

DEVELOPMENT, GELASIMUS AFTER HATCHING 499

1.5 mm. long and 1.9 mm. broad, thus showing a relative broad-
ening of the carapace. The lateral borders of the carapace are still
lobulated and beaded, but not so prominently as before. The only
changes of note in the appendages are the assumption of adult
form by the antenna (fig. 36) and further reduction of the pleo-
pods. The antenna is now made up of a large basal segment
and a flagellum. The proximal two joints of the flagellum are
distinct, but the others are reduced to surface constrictions,
The pleopods are distinguishable as minute, shriveled appendages
on the second, third, fourth, and fifth abdominal segments, but
are absent from the sixth. The abdomen has begun to broaden
by the development of lateral flanges.

The third crab stage (fig. 18). There is no pronounced change at
the next molt except in the pleopods. They may be entirely
absent in this stage or may be present on the first to the fifth seg-
ments as buds so minute as to be indistinguishable under magni-
fication less than five hundred diameters. Those on the second
segment may be larger than the others. The abdomen has
become broader.

Beginning of sexual differentiation. After the next molt the
young crab attains a width of carapace of 3 mm. This stage
shows the beginning of sexual differentiation; in males one chela
is slightly larger than the other. Abdominal appendages of a
second series make their appearance. These develop into the
genital appendages of the adult. In male specimens appendages
are present as minute buds on the first and second segments.
In the female buds are distinguishable with difficulty on all the
the segments from the second to the fifth.

Description of a 4-mm. crab. When the crab reaches a width of
4 mm. across the carapace, the sexual differentiation is pro-
nounced and other important changes have occurred. The
carapace now has the adult shape with straight sides. Numer-
ous very brushy hairs have appeared on its anterior surface below
the orbits. The abdomen is still further flattened and its segments
seem to be more or less completely fused except at their lateral
borders. The telson, however, is freely movable, being joined
to the rest of the abdomen by a membranous joint. The whole

500 O. W. HYMAN

abdomen fits tightly into its groove on the thorax. Its lateral -
borders are beset with numerous straining hairs. The hairs
around the telson are numerous and brushy.

The eyes (fig. 19) have now reached their adult condition.
They are bisegmented and the terminal segment bears the com-
pound eye facets over its distal and lateral faces. The eyes are
carried erected over the carapace in this stage, but may be lowered
into their imperfect orbits for protection.

No changes have occurred in the first five pairs of appendages
except slight changes in the relative sizes of some of their parts
and a multiplication of the hairs on each.

The first and third maxillipeds show no change of importance.
They are more hairy and some of their hairs have developed into
so-called ‘comb hairs.’ On the second maxilliped (fig. 73) the
gill and the epidodite are now developed, although both are
quite small. The gills present in this stage are as follows: a
podobranch on the second maxilliped; two arthrobranchs or
pleurobranchs between the third maxilliped and the cheliped;
two pleurobranchs at the base of the cheliped, and one pleuro-
branch at the base of the second period.

In the female the chelipeds are not differentiated, but both
remain small with spoon-shaped extremities (fig. 78). In the
male, one of the claws is considerably enlarged, is thicker, and is
adapted to cutting and pinching (fig. 77). The spoon-shaped
chelae are especially adapted for scooping up the fine sand from
which the animals get their food.

The abdominal appendages are now modified to form sexual
organs. In the male, the appendages of the first and second
abdominal appendages only are present. Each consists of two
segments. The appendage of the first segment is composed of
a rather broad basal portion and a rod-like distal segment. The
distal segment is grooved along its median border. The appen-
dage of the second segment is much smaller than that of the first,
but has the same enlarged basal segment and rod-like distal seg-
ment. The distal segment, however, is cylindrical. In the fe-
male, appendages appear on the second to the fifth segments.
Each is composed of a basal portion and two rami. Both rami:

DEVELOPMENT, GELASIMUS AFTER HATCHING 501

are cylindrical and the endopodite is bisegmented. None of the
parts are separated by distinct Joints as yet. The appendages
of the second segment are the largest, the others becoming pro-
gressively smaller from before backward.

BIBLIOGRAPHY

Bats, C. Spence 1879 Report on the present state of our knowledge of the
Crustacea. Part IV. On development. Rep. Brit. Asso. Adv. Sci.
for 1879.

Faxon, W. 1880 On some points in the structure of the embryonic zoea. Bull.
Mus. Comp. Zool. Harv. Coll., 6.

PackarD, A. 8., Jr. 1874 Life-histories of the crustacea and insects. Amer.
Nat., vol. 9.
1881 Notes on the early larval stages of the fiddler crab and of Al-
pheus. Amer. Nat., vol. 15.

SmitH, 8. I. 1873 The metamorphosis of the lobster and other crustacea. In-
vert. Animals of Vineyard Sound, etc. (Verrill and Smith), Rep. U. 8.
Fish. Comm., 1871-72.

JOURNAL OF MORPHOLOGY, VOL. 33, NO. 2

aoourwh rH

PLATE 1
EXPLANATION OF FIGURES

First zoea, G. pugilator, lateral view
First zoea, G. pugilator, front view.
First zoea, G. pugnax, lateral view.
First zoea, G. pugnax, front view.
First zoea, G. minax, lateral view.
First zoea, G. minax, front view.

502

DEVELOPMENT, GELASIMUS AFTER HATCHING PLATE 1
O. W. HYMAN

a .
Be oe >, 4

503

PLATE 2

EXPLANATION OF FIGURES

7 Second zoea, G, pugnax, lateral view.
8 Second zoea, G. pugnax, front view.
9 Third zoea, G. pugilator, lateral view.
10 Third zoea, G. pugilator, front view.

504

DEVELOPMENT, GELASIMUS AFTER HATCHING
0. W. HYMAN

PLATE 2

505

PLATE 3
EXPLANATION OF FIGURES

11 Fourth zoea, G. pugilator, lateral view.
12 Fifth zoea, G. pugilator, lateral view.
13 Fifth zoea, G. pugilator, front view.

506

DEVELOPMENT, GELASIMUS AFTER HATCHING PLATE 3
0. W. HYMAN

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19
20
21
22
23
24
25
26
27
28
29
30
jl
32
35)

PLATE 6

EXPLANATION OF FIGURES

Eye, 4-mm. crab, G. pugilator.
Antennule, first zoea, G. pugilator.
Antennule, first zoea, G. minax.
Antennule, second zoea, G. pugnax.
Antennule, third zoea, G. pugilator.
Antennule, fourth zoea, G. pugilator.
Antennule, fifth zoea, G. pugilator.
Antennule, megalops.

Antennule, first crab stage.
Antenna, first zoea, G. pugilator.
Antenna, first zoea, G. minax.
Antenna, second zoea, G. pugnax.
Antenna, third zoea, G. pugilator.
Antenna, fourth zoea, G. pugilator.
Antenna, fifth zoea, G. pugilator.

On
_
iw)

PLATE 6

DEVELOPMENT, GELASIMUS AFTER HATCHING

HYMAN

oO. Ww.

PLATE 7

EXPLANATION OF FIGURES

Antenna, megalops.

Antenna, first crab stage.

Antenna, second crab stage.

Mandibles, first zoea, G. minax.

Mandible, third zoea, G. pugilator.

Mandible, fifth zoea, G. pugilator.

Mandible, anterior surface, with palp pulled off, megalops.
Mandible, from above, megalops.

Mandible, posterior surface, first crab stage.
Mandible, anterior surface, first crab stage.

Tip of mandible, posterior surface, second crab stage.
First maxilla, first zoea, G. pugilator.

First maxilla, first zoea, G. minax.

First maxilla, second zoea, G. pugnax.

First maxilla, third zoea, G. pugilator.

First maxilla, fourth zoea, G. pugilator.

First maxilla, fifth zoea, G. pugilator.

514

DEVELOPMENT, GELASIMUS AFTER HATCHING ; PLATE 7
O. W. HYMAN

51
52
53
54
55
56
57
58
59
60
61

PLATE 8

EXPLANATION OF FIGURES

First maxilla, megalops.

First maxilla, first crab stage.

First maxilla, 4-mm crab stage.

Second maxilla, first zoea, G. pugilator.
Second maxilla, first zoea, G. minax.
Second maxilla, second zoea, G. pugnax.
Second maxilla, third zoea, G. pugilator.
Second maxilla, fourth zoea, G. pugilator.
Second maxilla, fifth zoea, G. pugilator.
Second maxilla, megalops.

Second maxilla, first crab stage.

516

DEVELOPMENT, GELASIM US AFTER HATCHING
O. W. HYMAN

PLATE 8

517

JOURNAL OF MORPHOLOGY, VONh. 33, NO. 2

62
63
64
65
66
67

PLATE 9

EXPLANATION OF FIGURES

First and second maxillipeds, first zoea, G. pugnax.
First maxilliped, first zoea, G. minax.

Second maxilliped, first zoea, G. minax.

First and second maxillipeds, third zoea, G. pugilator.
First maxilliped, fifth zoea, G. pugilator.

Second maxilliped, fifth zoea, G. pugilator.

518

DEVELOPMENT, GELASIMUS AFTER HATCHING PLATE 9
0. W. HYMAN

519

68
69
70
71
72
73

PLATE 10

EXPLANATION OF FIGURES

First maxilliped, megalops, without coxal segment.

First maxilliped, first crab stage, without coxal segment.
Second maxilliped, megalops.

Second maxilliped, first crab stage.

Tip of comb hair.

Second maxilliped, 4-mm. crab stage.

520

PLATE 10

DEVELOPMENT, GELAS{MUS AFTER HATCHING

Oo. W. HYMAN

PLATE 11
EXPLANATION OF FIGURES

Third maxilliped, megalops.
Third maxilliped, first crab stage.
Left chela, megalops.

Left chela, 4-mm. crab stage.
Right chela, 4-mm. crab stage.
Second pleopod, megalops.

Fifth pleopod, megalops.

522

PLATE 11
DEVELOPMENT, GELASIMUS AFTER HATCHING
0. W. HYMAN

PS eS eS LETS Pe

523

81
82
83
84

85°

86
87
88

PLATE 12

EXPLANATION OF FIGURES

Endopodite, second pleopod, megalops.
Telson, first zoea, G. pugilator.
Telson, first zoea, G. minax.

Telson, third zoea, G. pugilator.
Telson, fourth zoea, G. pugilator.
Telson, fifth zoea, G. pugilator.
Telson, megalops.

Telson, first crab stage.

524

PLATE 12

DEVELOPMENT, GELASIMUS AFTER HATCHING

Oo. W. HYMAN

Resumen por la autora, Louise Smith.
Colegio Smith, Northampton, Massachusettts.

El aparato hiobranquial de Spelerpes bislineatus.

La autora ha hecho un estudio morfolégico del aparato hio-
branquial, esqueleto y mtisculos, en diferentes estados del desar-
rollo de Spelerpes bislineatus comparandole con las mismas partes
de otros urodelos. Los métodos empleados han consistido en
cortes seriados, preparaciones tefidas en masa con azul de meti-
leno y disecciones. El aparato larvario esta caracterizado por una
firmeza y rigidez considerables, en parte debidas a a presencia de
una placa branquial formada por la fusidn de los primeros cera-
tobranquiales con el segundo basibranquial. La presencia de esta
placa es un fendmeno universal en las larvas de los Salamandri-
dos, pero su existencia no se ha reconocido generalmente. En el
adulto, los musculos y cartilagos de la lengua, que es libre y
boletiforme, han sido objeto de una investigacion especial en el
presente trabajo. El aparato en conjunto es muy delicado y
delgado y capaz de una gran complejidad de movimientos, es-
tando adaptado al modo de respiracion y a la captura de las pre-
sas. La transicién de la estructura de este aparato desde la
larva al adulto ha sido seguida durante la metamorfosis, habiendo
trazadola autora un cuadro normal. La transformacidn del apara-
to hiobranquial tiene lugar no por una mera absorcién de las
partes destinadas a desaparecer y el simple cambio de posicién
de otras, sino por un proceso complicado que implica la degenera-
cidn y pérdida de ciertas partes a consecuencia de fagocitosis, y la
formacion de nuevos tejidos en la posicién ocupada por el aparato
del adulto.

Translation by José F. Nonidez
Carnegie Institution of Washington

AUTHOR’S ABSTRACT OF THIS PAPER ISSUED
BY THE BIBLIOGRAPHIC SERVICE, JANUARY 19

THE HYOBRANCHIAL APPARATUS OF SPELERPES
BISLINEATUS

LOUISE SMITH
Department of Zoology, Smith College, Northampton, Massachusetts

FORTY-SEVEN FIGURES

CONTENTS
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INTRODUCTION

1. Purpose and scope

The study of the hyobranchial apparatus of Spelerpes bis-
lineatus! was undertaken in the hope that its results might prove
of some significance to the broader investigation which Mrs. I.
W. Wilder is carrying on in regard to the metamorphosis of this
animal. As the whole process of metamorphosis is a series of
adaptations to the transition from a wholly aquatic to a semi-
terrestrial life, and, as especially important adaptations come in

1In their Checklist (’17) Stejneger and Barbour have replaced the generic
name Spelerpes by Eurycea, a Rafinesquian name. As Spelerpes has been uni-
versally accepted in morphological literature for nearly seventy years, there
seems to be no necessity for adopting the change here.

527

528 LOUISE SMITH

response to the necessity for a changed mode of respiration and
a changed diet at that time, an anatomical description of the
apparatus associated with these phenomena may be of interest.

It is of course the hyobranchial skeleton which supports the
external gills of the larva and its muscles that move them in
the many varied and characteristic ways. The first basibranchial
and ceratohyals also support the very primitive larval tongue,
which is merely a fold in the mucous membrane filled in with
connective tissue and scarcely more pronounced than the lateral
oblique folds in the floor of the mouth that are an adaption to
the movement of the gill arches. Whatever motion this larval
tongue, so like that of the fishes, is capable of, is also brought
about by the hyobranchial muscles.

With the loss of the external gills at metamorphosis, the
function of the visceral skeleton as a support for the organs of
respiration is minimized, though its muscles still bring about the
characteristic movements of the pharynx that accompany bucco-
pharyngeal respiration. Its main purpose now seems to be to
support and move the very specialized tongue which has devel-
oped for capturing and disposing of the more rapidly swimming,
jumping and flying prey of the adult. The adult tongue is a
mushroom-shaped organ capable of a great variety of motion.
Its stalk is formed by the basibranchial cartilage and the pair of
abdominohyoideus muscles, and its very glandular disk is
supported by the tip of this basal piece and associated cartilages
and regulated by fibers of the hyoglossal muscle (fig. 2). It is
capable of being withdrawn wholly into the mouth, with the
disk parallel to the stalk, and the mucous membrane of the
latter folded back like a semi-inverted glove finger; of being
extended, with the disk 3 or more millimeters beyond the
mandible and moved through an angle of 90°, so that it is per-
pendicular to the stalk, or of assuming any of the innumerable
positions between these two.

The purpose of this paper is to give the anatomical basis for
further investigation of the physiological phenomena related to
the functions of respiration and capturing and swallowing of
prey, and more particularly for understanding these activities
during the period of transformation.

HYOBRANCHIAL APPARATUS OF SPELERPES 529

In the carrying out of this plan I shall attempt to give a
complete description of the anatomy of the hyobranchial skeleton
and of its muscles, without reference to their innervation, in
the larva and in the adult, with some reference to the mor-
phology of the parts, and shall then try to trace the method of
development of the larval apparatus into that of the adult,
through the period of metamorphosis. Correlation of the facts
found with physiological observations, in the main, fall outside
the scope of the present paper.

2. Material and technique

I have used a large number of specimens of Spelerpes bislineatus,
larval, metamorphic, and adult, many of which had been pre-,
viously observed for external physiological phenomena in the
living state. I prepared about twenty-five or thirty of these for
study of the skeleton by Van Wijhe’s methylen-blue method for
cartilage. This method was particularly good for the larvae
after killing in 10 per cent formalin, but did not give quite such
successful results with the adults and metamorphic specimens.
Figures 4, 5, 17, 18, and 28 are drawn from such preparations.

I could dissect the adults very successfully under the binocular
microscope, especially if stained first in methylen-blue. Figures
19, 20, and 21 are based upon such dissections. For the more
detailed work, however; serial sections were the surest method.
Several larvae of about 20 to 24 mm. were sectioned for the
typical larval condition, and several adults for comparison with
my dissections. For the metamorphic phenomena I made series
of some eleven specimens, which I afterward found fell into about
four distinct stages. My sections are 15 and 20 ,» thick and
mainly transverse, with a few horizontal and sagittal series for
comparison. Delafield’s haematoxylin after the picric acid of
the decalcifying fluid was the most useful stain.

Reconstructions were made with millimeter paper (the drawing
of the larval muscles (fig. 6) is made from one of these) and
certain details I reconstructed with wax plates. But for the
main part I relied on rough reconstruction in plastiline, and on

530 LOUISE SMITH

comparison with methylen-blue. preparations, dissections, and
other series of this work.

For comparison, dissections were made of Necturus and
Cryptobranchus; methylen-blue preparations of Diemyctylus
viridescens, Amblystoma opacum, and Spelerpes ruber; dissec-
tions under the binocular of Spelerpes ruber, adult Amblystoma
opacum, and adult Salamandra maculosa; serial sections of a
Salamandra larva, Necturus embryos, a small Cryptobranchus,
an Axolotl, Typhlomolge rathbuni, and of several stages otf
Desmognathus fusca were studied.

At this point, I wish to express my sincere thanks to Dr. H.
H. Wilder for his valuable advice and criticism; to Mrs. Wilder
for many helpful suggestions and for the use of her specimens
and notes on metamorphosis, and to Mr. E. R. Dunn for material
from his amphibian collection.

LARVAL CONDITION
1. Hyobranchial skeleton

The hyobranchial apparatus in the larva of Spelerpes bislin-
eatus consists of the hyoid and three branchial arches (figs. 4
and 5). The individual arches are not clearly defined, however,
as in the lower fishes, for in some instances certain cartilages are
missing and in others the exact morphology of parts is doubtful.
The apparatus consists, in the main, of paired cartilages which
are hung, directly or indirectly, on a single median basal piece.
This basal piece or first basibranchial, a rather small, more or
less cylindrical cartilage which lies well forward in the midline
of the floor of the pharynx, is thus of considerable physiological
importance. Anterior and lateral to it, is the pair of ceratohyals,
the sole remnants of the hyoid arch. The ceratohyal is a long,
heavy cartilage, the largest in the whole apparatus. At its
proximal end, it articulates with the anterior end of the first
basibranchial and projects posterolaterally to be suspended from
the skull by connective tissue at its distal end.

Ventral to the posterior half of the first basibranchial and also
median, is a somewhat triangular, dorsally concave branchial

HYOBRANCHIAL APPARATUS OF SPELERPES 531

plate with thickened lateral edges. It is notched at its apex, and
fits around the middle of the shaft of the first basibranchial to
form a more or less freely movable articulation. Posteriorly, as
shown in cross-section (figs. 9 and 10), it seems to hold the basi-
branchial and proximal ends of the second epibranchials within
its coneavity, as in a trough. Its lateral thickenings are pro-
longed into short, stout shafts which undoubtedly represent the
distal ends of the first ceratobranchials and with which the long,
heavy first epibranchials are articulated. In addition to the
lateral projections is a median, posterior one (probably the
second basibranchial,? ending in the widening of the cartilage
which, in adult life, becomes ossified to form the os thyreoideum.
Thus, it is evident that the plate is, in reality, a fusion of three
cartilages, the first ceratobranchials and the second basibranchial.,
This makes it difficult to determine just what elements are
actually involved in the first branchial arch.

The second branchial arch, however, quite plainly consists of
a pair of cerato- and a pair of epibranchials. The second cerato-
branchial is a short, slender cartilage which articulates, proxi-
mally, with the posterior end of the first basibranchial and,
distally, with the proximal end of the long, delicate second
epibranchial.

The third branchial arch is represented by a delicate epi-
branchial alone, which articulates proximally with the median
edge of the second epibranchial; the fourth arch, merely by a
raphé in the muscles as in Necturus, and the fifth, represented
in the lunged forms by the arytaenoids, has disappeared entirely.

The branchial plateis the most significant feature of the visceral
skeleton of the larva. Although it is apparently a universal
phenomenon among larval Salamandrids* and is unmistakably

2In calling this cartilage the second basibranchial I follow Wiedersheim,
whose nomenclature of the hyobranchial skeleton I have used throughout this
paper. As it is not absolutely proved that this is morphologically the second
basibranchial, many zoologists, noticeably followers of Gegenbaur, prefer to be
non-committal and eall it ‘copula.’

3 Tn retaining the term ‘Salamandrid’ as a convenient method of designating
the Urodeles that undergo complete metamorphosis, I am aware that recent
systematists no longer make this distinction.

532 LOUISE SMITH

shown by a study of both sections and preparations in toto (figs.
4, 5, 9, and 10), it has escaped the notice of most authors, who
picture the first ceratobranchials and the second basibranchial
as quite separate from each other and articulating with the first
basibranchial in the same plane with the second ceratobranchials.
In fact, in the literature I find only two references to the plate.
Gaupp (706, p. 705) incidentally mentions that in the visceral
skeleton of Triton taeniatus larva, ‘‘Der ventrale Teil hat die
Form einer breiten dreieckigen Platte, die hinten in einen langen
medianen Fortsatz, den Copulastiel (=second basibranchial),
auslaiift,’’ and pictures it plainly (p. 706, fig. 35). Mrs. Wilder
has carefully worked out the anatomy of the hyobranchial
skeleton in Desmognathus fusca and shows, without any shadow
of a doubt, that “. . . . the second basibranchial cartilage

during laa life forms one continuous chondrifi-
pation with the first pair of ceratobranchials” (713, p. 320).
This she pictures clearly (p. 321, fig. 25 (a)) by a drawing of a
methylen-blue preparation.

Parker (’80) and Driiner (’02) just miss showing it for Sala-
mandramaculosa. The former pictures a section a little posterior
to the position of the actual continuity of the cartilages and notes
that the first ceratobranchials lie ventral to the second, and the
latter comes even nearer the truth, for he not only says that the
first ceratobranchials “nicht in derselben Frontalebene wie die
Hypobranchiale 2 (= second ceratobranchials) sondern ventral
verschoben liegt”’ (p. 471), but also shows a ventral piece (Taf.
25, fig. 3) which corresponds to the second basibranchial plus the
median portion of the plate, and with which he makes the first
ceratobranchials articulate. If he had not shown this articu-
lation, which I am convinced by study of a series of cross-sections,
does not exist, he, too, would have pictured the plate.

I have stated that the plate is probably a universal phenomenon
among the larval Salamandrids only, because it is not present
even in very young stages of Necturus or Cryptobranchus or in
Siren lacertina,‘ but it is present in important representatives of

4 In Siren a transitional form may be present, as here the first ceratobranchials

and second basibranchials are slightly ventral to the rest of the apparatus (cf.
He Wilder, 91, pl: 295 fig. 7).

HYOBRANCHIAL APPARATUS OF SPELERPES 533

each of the three families of Urodeles that undergo complete
metamorphosis. Thus, among the Plethodontidae, Desmog-
nathus fusca, Typhlomolge rathbuni,> and Spelerpes ruber and
bislineatus; among the Salamandridae, Triton taeniatus and
cristatus, Salamandra maculosa, and Diemyctylus viridescens,
and among the Amblystomidae, Amblystoma opacum and
punctatum, and the axolotl, all have this fusion of the cartilages.
Had time and material permitted my carrying this investigation
further, I have no doubt I should have found similar results
throughout the group, for the plate seems to have an important
physiological function in these larvae, serving for the basis of
attachment of powerful muscles which have to do with changes
of position of much of the hyoid region, including movements of
the lower jaw. i

2. Hyobranchial muscles

The hyobranchial muscles of the Spelerpes larva correspond
rather closely to those of other larval Urodeles, including such
forms as Necturus. They are shown from the ventral aspect in
figure 6 and in sections, figures 7 to 16.

The most superficial are the intermandibulares anterior and
posterior. These are thin, sheet-like muscles which cover the
whole ventral surface of the lower jaw just beneath the skin.
The former extends between the two halves of the anterior three-
fourths of the mandible; the latter, in its attachments, is rather
more complex, as it takes some fibers from the mucous membrane
of the pharynx, some from the lateral edge of the distal end of
the ceratohyal, some from a fascia which covers the ceratohyoid-
eus externus muscle, and some from the midventral surface of
the distal end of the first epibranchial, on both sides.

In young larvae there is but little indication of the median
raphé separating the two halves, which is present in metamorphic
specimens and adults (see below) as it is in most Urodeles. Out

5 Stejneger and Barbour (717) do not include Typhlomolge among the Pletho-
dontidae. The presence of the plate seems to me, however, to be only an ad-
ditional proof that this animal is in reality a larval Spelerpes, as Miss Emerson
so clearly shows (05).

JOURNAL OF MORPHOLOGY, VOL. 33, NO. 2

534 LOUISE SMITH -

of about ten more or less complete series of larvae I found a few
sections that showed a definite, though narrow raphé (fig. 1, a)
and a few more, where careful focusing brought out a minute
division across the middle of what otherwise appeared to be a
single fiber (fig. 1, b), but in the majority of cases the fibers were
quite unbroken across the midline (fig. 1, ¢).

The geniohyoideus, a long, narrow muscle, arising from the
symphysis of the mandible, extends posteriorly at either side of
the midline and is inserted into the anterior edge of the lateral
projection of the second basibranchial.

The thoracicohyoideus is very large and powerful. Its main
portion arises from the pectoral girdle and the whole segmental
trunk musculature, passes forward around the _ pericardial

Fig. 1 Representative fibers of the M. intermandibularis drawn from a trans-
verse series.

chamber, and is inserted into the whole dorsal surface of the
plate, the posterior end of the first basibranchial and proximal
end of the second epibranchial. In addition, a superficial slip
arises from a myocomma opposite the proximal end of the third
epibranchial, and is inserted into the posterior edge of the lateral
projection of the second basibranchial.

The ceratohyoideus externus, the largest of the intrinsic muscles
of the hyobranchial apparatus, arises from the middle of the
ventral surface of the ceratohyal along almost its entire length,
and curves: around posterolaterally to be inserted, together with
a slip from the dorsal surface of the head, probably the levator
arcus primus, into the dorsal surface of the distal end of the first
epibranchial.

The ceratohyoideus internus is very much smaller than the
externus and is entirely covered by it. It arises from the dorso-

HYOBRANCHIAL APPARATUS OF SPELERPES 535

medial surface of the ceratohyal and converges to a narrow
insertion on the ventral surface of the proximal end of the first
epibranchial.

The adductor arcuum is a ribbon-like muscle which is un-
doubtedly a fusion of the adductores arcuum secundi et tertii of
other forms. It arises from the lateral projection of the second
basibranchial, curves around the thoracicohyoideus in a postero-
lateral direction and divides into two slips, the larger of which is
inserted into the ventral surface of the second epibranchial and
the smaller into the ventral surface of the third epibranchial.

The two constrictores arcuum (interbranchiales of Driiner) are
small muscles, parallel to each other on the gill arches. The first
arises from the ventral surface of the first epibranchial, posterior
to the insertion of the ceratohyoideus internus, and passes in an
oblique posteromedial direction over the insertion of the first slip
of the adductor arcuum to be inserted into the ventral surface of
the third epibranchial, a little posterior to the insertion of the
second slip of the adductor. The second constrictor lies just
posterior to the first. It arises from the ventral surface of the
second epibranchial and is inserted into the ventral surface of
the third.

Five levatores arcuum are represented in the Sperlepes larva,
all of which arise from a fascia just beneath the dorsal integument.
The first, as mentioned above, is inserted into the first epi-
branchial along with the ceratohyoideus externus. The second
and third are small muscles, inserted into the dorsal surface of
the distal ends of the second and third epibranchials, respec-
tively. The fourth (the dorsal half of digastricus pharyngeus
of Géppert and dorsobranchialis 4 of Wilder) is a larger muscle,
inserted into the tendinous raphé which represents the fourth
epibranchial, and the fifth, the dorsotrachealis of lunged forms,
is inserted with its fellow in the midline dorsal to the pericardium.

Two depressors arcuum, the third and fourth, occur. The
depressor arcus tertius (anterior half of hyotracheales, or pharyngo-
branchialis 3) arises from the medial border of the third epi-
branchial and is inserted with its fellow in the midline dorsal to
the pericardium. The fourth (posterior half of hyotrachealis,

536 LOUISE SMITH

ventral half of digastricus pharyngeus or pharyngobranchialis 4)
arises from the rudimentary fourth epibranchial and is inserted
like the third, in the midline. The fifth, like other parts associ-
ated with the larynx, is lacking altogether.

ADULT CONDITION
1. Hyobranchial skeleton

In response to its changed functions, the hyobranchial skeleton
of the adult has lost almost all resemblance to that of the larva
(figs. 17 and 18). From a rather heavy structure with all its
parts firmly bound together, it has become a very delicate one
with a lack of firm articulations, and thus well adapted to the
complexity of motion characteristic of it at this tinie. This
great range of motion makes description rather difficult for the
relationships when the tongue is withdrawn are much changed
when it is extruded. In the following discussion, I describe the
skeleton in the normal resting position with the tongue well
withdrawn, unless the contrary is definitely stated.

Though the first basibranchial is still of great importance as
supporting the tongue and the cartilages which govern its motion,
it is no longer the pivot which binds together all the other parts
of the visceral skeleton. Through the loss of the articulation
between the ceratohyals and the basal piece and the breaking
down of the branchial plate into the first ceratobranchials and the
os thyreoideum, the hyobranchial skeleton may be arbitrarily
divided into two portions, a central one associated with the first
basibranchial and an outlying one, consisting of separate pieces.

Of the latter, the os thyreoideum is the most superficial (fig.
17). This is a small, median, crescentic bone, just beneath the
ventral integument, and, despite its minuteness, it is especially
significant as the basis of attachment of three important muscles.
It is formed by the lateral growth and ossification of the free end
of the second basibranchial and is unique as being the only ossified
part of the entire hyobranchial apparatus.

The ceratohyal shows perhaps more change from its larval
appearance than any other single cartilage. It now lies with its

HYOBRANCHIAL APPARATUS OF SPELERPES SAV

broad flattened proximal end quite free in the floor of the mouth
and projecting slightly into the double fold of mucous membrane
dorsal to the tongue stalk (figs. 23 and 24). Posteriorly it
becomes narrowed and more rounded, dorsal to the cerato-
branchials, till a little posterior to the articulation of the mandible
it hooks around, laterally and then anteriorly and has its distal
end firmly attached to the quadrate bone.

Of the cartilages of the central portion, the first basibranchial
is the pivot, as I have already stated. This median cartilage lies
in the tongue stalk and is somewhat curved anterodorsally, with
its anterior end in the substance of the tongue (fig. 2). Its ventral
surface is flat and, for the anterior two-thirds, is broadened by
lateral cartilaginous shelves, formed at metamorphosis. On the

Fig. 2 Median sagittal section of tongue and tongue-stalk of adult.

dorsal surface these shelves help to form rather deep lateral
grooves in which the abdominohyoideus muscles lie. The pos-
terior third is more cylindrical and tapers sharply at the tip like
the basibranchial of the larva.

A pair of ‘little horns,’ such as are quite universally present
among adult Salamandrids, occurs at the anterior end of the
basibranchial. These coruna articulate with the basibranchial
anteriorly and extend laterodorsally into the disk of the tongue.
Between the horns and dorsal to the basibranchial, is a tiny
y-shaped lingual cartilage (figs. 18 and 22) of which I find no
mention in any of the description of the visceral skeleton in
Plethodontidae. From its position, however, and from its
function as the origin of the hyoglossal muscle, it is undoubtedly
the morphological equivalent of the ‘Sehnen-platte’ which Oppel

538 LOUISE SMITH

(00, p. 138) describes in the tongue of Salamandra maculosa and
of the ‘oto-glossal cartilage’ which Cope finds in the Ambly-
stomidae (’87, pp. 87 and 88) and considers a distinctive feature of
that family (’89, p. 33).

The first ceratobranchial articulates proximally with the
basibranchial at the point where the gradual reduction of the
lateral shelf forms an angle in that cartilage; the second cerato-
branchial articulates with the posterior end of the basal piece as
in the larva (figs. 17 and 18). Both are slender rod-like cartilages
which pass posterolaterally to articulate distally with each other
and with the proximal end of the first epibranchial. This last-
named cartilage is another delicate rod which extends latero-
posteriorly and les at the side of the throat in a curious sac
which will be described later under M. ceratohyoideus internus.
Only in its anterior third is the adult epibranchial the same
cartilage as the larval first epibranchial. Its posterior part
consists of a new structure which buds off at metamorphosis, as
described below. The distal end of the larval first epibranchial,
as well as the whole of the second and third epibranchials, have
broken down and become wholly lost.

It may not be extraneous at this point to note the relationships
which it is possible for the cartilages of the central portion to
assume when the tongue is extruded to its greatest extent. At
that time the epibranchials are pushed anteromedially so that
their distal ends lie just in the opening of the sac and their proximal
ends lie in contact with each other at the curve of the mandible;
the ceratobranchials are collapsed like the ribs of a closed
umbrella and lie within the epithelium of the tongue stalk with
all but their posterior tips entirely outside the mouth; ‘and
undoubtedly the ‘little horns’ are then tipped forward in the
plane of the basibranchial as Wiedersheim found to be the case
in Salamandrina perspicillata (’75, pp. 88 and 89), though this
last fact I have not yet verified.

HYOBRANCHIAL APPARATUS OF SPELERPES 539

2. Hyobranchial muscles

The hyobranchial muscles of the adult also show a delicacy
and complexity, well adapted to the great mobility of the region.
I find in them a marked correspondence, in all but a few compara-
tively unimportant details, to the muscles of the European
species Spelerpes (Geotriton) fuscus, as described in 1875, by
Wiedersheim who designated the various muscles merely by
letters, as he had not the morphological data to name them. As
they also resemble the muscles of the adult Salamandra maculosa
described by Driiner in 1904, and as I have traced their devel-
opment through metamorphosis, I now attempt to give them
their morphological names. Figures 19 and 20 show them from
more superficial and from deeper ventral dissections, respec- ,
tively, and figure 21 pictures a pharyngeal (dorsal) dissection.
Figures 22 to 27 show important levels in cross-section.

The intermandibularis anterior arises from the inner surface
of the mandible and is like the larval muscle, except that the
median raphé, which in the larva is scarcely indicated, is now
broad and definite.

In the position of the intermandibularis posterior of the larva
there appear two distinct muscles. Whether these together are
the morphological equivalent of the larval muscle, or whether
only the more anterior one represents it, and the more posterior
is a new muscle which develops at metamorphosis, I cannot at
present state, though the latter view is the more probable. The
more anterior of these muscles, the interhyoideus (interossa-
quadrata of Driiner) arises from the distal end of the ceratohyal
near its attachment to the quadrate bone, but not from the latter
bone, as Driiner’s name suggests; spreads out like.a fan, and is
inserted on the median raphé. ‘The more posterior, (Driiner’s
quadrate-pectoralis) curves around the throat in a posteromedial
direction, from just beneath the dorsal integument and is in-
serted in the gular fold. As I can find in it no relation either to
the quadrate bone or to the pectoralis muscle, and as it seems so
clearly to be an integumental muscle regulating the gular fold,
gularis might be a better name for it.

540 LOUISE SMITH

The adult geniohyoideus consists of two parts, a more super-
ficial geniohyoideus medialis and a deeper one, the geniohyoideus
lateralis. The medialis corresponds closely to the entire muscle
in the larva. It is a rather broad, extremely thin, ribbon-like
band, with its somewhat thicker lateral edge in close association
posteriorly with the ceratohyoideus internus which lies just
beneath it. As in the larval muscle, its origin is the posterior
border of the mandible, lateral to the symphysis, and its insertion,
the anterior edge of the os thyreoideum (= distal end second
basibranchial). The lateralis arises with the medialis (fig. 22),
but lies dorsal and lateral to it, and is inserted along the entire
outer border of the ceratohyal. A very thin sheet of fibers
from this portion also passes posteriorly dorsal to the hyoid
and is inserted in the mucous membrane of the pharynx behind
the posterior end of that cartilage (figs. 24 and 25).

The thoracicohyoideus, as such, has broken down, but is still
partially represented by the sternohyoideus and the two divisions
of the abdominohyoideus. The sternohyoideus is a very thin
superficial muscle arising from the dorsal surface of the sternum,
and inserted on the posterior border of the osthyreoideum. In
its course, it passes latero anteriorly, crossed by two myocommata
to a raphé behind the anterior end of the procoracoid, whence it
continues medio anteriorly to its insertion. Thus the inner edge
of the muscle forms, with that of the other side, a rhomboidal
space over the pericardium (fig. 19).

The abdominohyoideus is a very long, round muscle arising
on the pelvic girdle and extending forward at the sides of the
body, crossed by many myocommata, to a position about behind
the most anterior raphé in the sternohyoideus. From this point
it sends off a small round slip running anteromedially and
inserted on the middorsal surface of the os thyreoideum.* The
main portion also begins at this point to bend a little medially
toward the posterior end of the first basibranchial. There the
fibers diverge; some pass straight forward, ventral to the lateral

®T find no indication that this slip is continued anteriorly. beyond the os
thyreoideum as the part of the geniohyoideus medialis which Wiedersheim letters
‘f? (75, fig. 133) and so describes (ibid., pp. 187-8).

HYOBRANCHIAL APPARATUS OF SPELERPES 541

edge of the basibranchial, for a short distance, but most of them
bend around ventrolaterally and partially enwrap the second
ceratobranchial from the ventral side. The muscle then con-
verges again, dorsal to the first ceratobranchial, and, considerably
diminished in size, continues forward in the dorsolateral groove
of the basibranchial, within the tongue stalk, nearly to the
anterior end of the cartilage. Finally it converges dorsolaterally
within the tongue substance and inserts through a tendon into
the ‘little horn.’

Near this lingual insertion of the abdominohyoideus is a
muscle, not present in the larva and not very fully developed
even in the adult. This is the hyoglossus, which consists merely
of delicate fibers which arise from the lingual cartilage and
anterior tip of the basibranchial and radiate out through the:
tongue substance, some to be inserted on the ‘little horns’ and
some in the mucous membrane of the disk.

The ceratohyoideus externus has disappeared entirely, but the
simple small ceratohyoideus internus of the larva has become, in
the adult, by far the most unique and highly specialized of the
entire group. It now arises from the ventral surface of the
ceratohyal, and for a short distance its fibers run posteriorly in
a straight longitudinal direction. Soon, however, they begin
to curve around from the medial edge of the ceratohyal to a
position lateral to its outer edge, and back again to a medio-
posterio direction. At this point the muscle forms a kind of
pocket which opens medially and in which lie the distal ends of
the first and second ceratobranchials. Posteriorly, the edges of
the pocket converge, and the muscle, added to by new fibers
which develop at metamorphosis, becomes wound around the
epibranchial in a complicated spiral, the more exact structure
of which I have not worked out. The muscle continues a little
posterior to the end of the epibranchial, forming a kind of closed
sac which, on shortening of the spiral muscle, evidently acts as
a bulb and squeezes the epibranchial almost entirely out of the
pocket as a means of pushing out the tongue.

Conspicuous in a dorsal view of the floor of the mouth, when
the posterior edge of the tongue is lifted forward, even before

542 LOUISE SMITH

the mucous membrane is removed, is an extremely delicate but
definitely marked muscle, peculiar to adult free-tongued sala-
manders, which I call the suprapeduncularis (figs. 21 and 28).
It is the muscle which Wiedersheim designates by the letter ‘I’
(75, fig. 1384) and describes as arising from the medial border of
the ceratohyal (ibid., p. 191). I find that in Spelerpes bislineatus,
however, it is quite plainly attached to the anterolateral border
of the dorsal surface of the ceratohyal on each side and stretches
across within the dorsal fold of mucous membrane, with its
anterior edge defining and strengthening the free border of the
orifice through which the tongue protrudes (fig. 2, a).

Of the other larval muscles, most of which were in association
with the degenerated epibranchials (constrictors, levators,
depressors, etc.), only two are represented. A rather deep-lying
stout, muscular band encircles the ventral half of the oesophagus
dorsal to the pericardium. The larger part of this is made up
by the digastricus-pharyngeus, but a small portion is formed by
the dorsolaryngeus. The component parts are not clearly defined
however, but tend to form a continuous pharyngeal sheet, as has
been pointed out by H. H. Wilder (’96, p. 188).

METAMORPHIC PHENOMENA
1. Skeleton

The metamorphosis of the hyobranchial skeleton of Spelerpes
bislneatus shows some very interesting facts which I do not
find mentioned in the literature. I have placed these in the
form of a normal table which presents the details of develop-
ment from the larval apparatus to that of the adult. Cross-
sections of the four specimens described in this table are pic-
tured in figures 29 to 47, and the condition of the skeleton of a
metamorphic animal about like stage III is shown in figure 28.

Perhaps the newest and most striking of the metamorphic
phenomena and that which may give a clue to the whole method
of skeletal metamorphosis is shown in the development of the
adult epibranchial. This cartilage is not merely the larval first
epibranchial with its position slightly changed, as has been

HYOBRANCHIAL APPARATUS OF SPELERPES 543

generally taken for granted, but it is largely a new structure
which has developed from the old in a most curious way. At
the stage which Mrs. Wilder calls ‘incipient premetamorphic,’
when the first signs of approaching metamorphosis appear, I
find on the ventral surface of the first epibranchial for a short
distance posterior to the insertion of the ceratohyoideus internus
and the origin of the constrictor arcuus primi, a slight swelling
of the cartilage and conspicuous thickening of the perichron-
drium with a number of mitoses (figure 32). This is the anlage
of a new portion of the epibranchial. Dorsal to it, a very slight
raggedness of the hyaline matrix is the first indication of the
degeneration soon to take place. The laying down of new car-
tilage by the chondrioblasts and degeneration of the old matrix
continues slowly until the time when the animal enters into the
true ‘metamorphic’ period. By that time the new sprout has
been cut off from the larval first epibranchial for a short dis-
tance and appears as a ventral fork of that cartilage with the
old matrix degenerating behind it (fig. 36). Posterior to the
tip of the new epibranchial, the old one is still quite typically
larval. From now on, these phenomena continue rapidly. The
new epibranchial grows ventroposteriorly, apparently by pro-
liferation of cells from the perichondrium and frequent mitoses
especially near the tip (fig. 3), and the old epibranchial breaks
down by an anteroposterior wave of degeneration. The first
sign of degeneracy is seen in a raggedness of the haline matrix;
then the spaces in the cartilage become filled with leucocytes
(phagocytes) which eventually eat away all the old tissue.
Thus, by the end of the metamorphic period, the new epibran-
chial has very nearly its adult proportions and position, and the
posterior two-thirds of the larval first epibranchial is almost
indistinguishable from a blood-vessel, except for occasional re-
maining bits of hyaline matrix (figs. 42 and 47).

So, throughout the whole visceral skeleton new pieces develop
from old by a proliferation of and subsequent formation of car-
tilage by chondrioblasts; old ones disappear by a degeneration of
matrix and final consumption by phagoctyes, and permanent
cartilages change their shape and position by partial degeneration
and outgrowth of new tissue in the same ways.

544. LOUISE SMITH

Of the wholly new structures the only examples are the ‘little
horns’ and lingual cartilage. They are no exception to the
above rule, but first appear as a collection of chondrioblasts
close to the basibranchial—the former, in stage II, and the
latter, in stage III (fig. 37)—and later develop hyaline matrix.

Of the cartilages which disappear entirely, the second and
third epibranchials are the type. They follow the method of
the larval first epibranchial in degenerating in an anteropos-

else “rt

EE mee 4 ua soneuee

a 5 r
i i

HH : :
In SESE Bi

E Tt Ty ree aE am El

4 Lit i Se ane oan ie a

Fig. 3 Millimeter-paper reconstruction of metamorphosing first epibranch-
ial; lateral aspect. Stars indicate location of mitoses. The degree of degenera-
tion of the remaining portion of the old epibranchial is shown by the amount
of stippling. X 50.

terior wave. First they lose their connection anteriorly with the
rest of the visceral skeleton (fig. 28), and then the disintegration
advances more and more posteriorly until they are wholly lost.

In the breaking down of the branchial plate it is interesting
to note that the first change comes in a deepening of the groove
at which it articulates with the basibranchial, giving the articu-
lation almost an adult appearance as seen in cross-section (figs.
30 and 24), but that the actual degeneration begins in the middle

HYOBRANCHIAL APPARATUS OF SPELERPES 545

and cuts off the second basibranchial, and from that position
radiates until only the first ceratobranchials and the gradually
ossifying osthyreoideum are left.

Almost all of the cartilages show, to some extent, the phe-
nomenon of becoming adapted to their new functions by the
principle of degeneration and new growth. In the case of the
ceratobranchials and the proximal end of the first epibranchial
which is retained through adult life, it is seen in a slight de-
generation around the periphery, to give them the slenderness
characteristic of the adult skeleton.

The principle is even more marked in the case of the basi-
branchial, and most evident of all, in that of the ceratohyals.
The basal piece, during the metamorphic period, loses its anterior
end and attains the flattened ventral surface of the adult by the
process of degeneration of old tissue (figs. 34 and 38), and be-
comes broadened by the lateral shelves which first appear in
stage II as thickenings in the perichrondrium, and gradually
becomes cartilaginous (figs. 38 and 44). The ceratohyals lose
their connection with the basibranchial and assume their more
dorsolateral position and adult form by the degeneration of the
anterior end, and dorsal, medial, and ventral surfaces an-
teriorly, and of the whole periphery more posteriorly, and by
the outgrowth of new tissue laterally, especially at the posterior
end where the ‘hook’ which becomes attached to the quadrate
is almost wholly new.

These processes go on so quickly during the true metamorphic
period that at the end of that time all the changes are indicated
and only final consumption by phagocytes of the already de-
generating matrix, and fuller chondrification of the new parts
already laid down, is necessary before the final adult condition
is reached.

2. Muscles

The study of the metamorphosis of the hyobranchial muscles
shows quite as important results as does that of the transforming
skeleton. I have not yet worked out the more minute details
of the histological phenomena, but in the main, I find results

546 LOUISE SMITH

similar to those which Mlle. Smirnova (’14) found in the his-
tology of metamorphosing frog muscle. For the order of de-
velopment of the various muscles from the larval condition to
that of the adult and for their appearance in sections I would
refer to the same table and plates as in the case of the skeleton.

The general method of muscle metamorphosis is typified in
the transformation of the thoracicohyoideus of the larva into
the abdominohyoideus and sternohyoideus of the adult. The
first indication of any change in the thoracicohyoideus appears
in stage I, when a proliferation and anterior growth of fibers
from that muscle brings the origin on to the dorsal surface of
the first basibranchial, considerably anterior to its articulation
with the plate (fig. 30). These new fibers, which are very small
and so quite easily distinguishable from the old, make up the
‘anlage of the abdominohyoideus muscle. Stage II shows the
origin of what is now quite unmistakably the abdominohyoideus
well forward in the connective tissue of the tongue, and the
beginning of the breaking down of the median portion of the
thoracicohyoideus. In this degenerative process I find, as Mlle.
Smirnova did, that the muscle fibers first appear very large and
devoid of their cross-striations and then are interspersed with
muscle phagocytes which eventually dispose of them. As to
the nature of the phagocytes, however, my opinion differs from
hers, though I have insufficient data to prove my point con-
clusively. She states that they are formed by the muscle cells
themselves and are not leucocytes, but a careful comparison of
them with preparations of fresh human blood shows such a
striking similarity to leucocytes of the polynuclear type that I
fail to see how they can be anything else. At this stage, also,
the differentiation of the ventral slip of the thoracicohyoideus
into the sternohyoideus begins by the degeneration of the more
dorsal fibers of the slip and ventral ones of the muscle proper
(fig. 36). The degeneration of the old and formation of the
new fibers continue anteroposteriorly, so that by stage III the
abdomino hyoideus is quite adult in position anteriorly, but
more posteriorly the whole position of the thoracicohyoideus is .
filled in with degenerating fibers of that muscle, muscle phago-

HYOBRANCHIAL APPARATUS OF SPELERPES 547

cytes, and new fibers of the abdominohyoideus. By stage IV
the entire abdominohyoideus and sternohyoideus muscle are
quite definitely formed and await only final consumption by the
phagocytes of the few remaining degenerate fibers and the filling
out to their adult capacity of the new ones already formed.

While the transformation of the thoracicohyoideus into the
abdominohyoideus and sternohyoideus involves the two prin-
ciples—development of new fibers and the degeneration of old—
the other muscles exhibit the one principle or the other, and not
both. Thus the first alone is found in the geniohyoideus lateralis,
which is also present in stage I as a proliferation of cells from the
dorsolateral surface of the anterior end of the larval genio-
hyoideus, or geniohyoideus medialis as it must now be called.
In this stage the new fibers are very short and are inserted into,
the mucous membrane of the pharynx (fig. 29). They now
grow rapidly in a posterolateral direction, so that by stage HH
they approach the ceratohyal; by stage III are inserted on it
for a considerable distance, and by stage IV have very nearly
their adult insertion. The second principle is found in the
adductor, constrictors, first three levators, and the third de-
pressor, which gradually degenerate, as the epibranchials which
they regulated break down. The fourth depressor, however,
and the fourth and fifth levators, which are hyobranchial muscles
only in their comparative morphology, but are physiologically
pharyngeal muscles used in respiration, change merely by an
increase in size and loss of the identity of their component parts.
‘This last fact brings out quite forcibly the difference between
the raphé separating the fourth levator and depressor which has
been proved to be a vestigial organ (Géppert, 91, H. H. Wilder,
’96), and the raphé separating the moieties of the intermandibu-
lares, which developed secondarily at metamorphosis, evidently
in response to an important physiological need, and which is
therefore not vestigial, but progressive.

Perhaps the most noticeable and quickly carried out of the
changes in the musculature at metamorphosis is furnished by
the ceratohyoideus externus and internus. The former, which
-was by far the largest of the intrinsic larval muscles, but was in

548 LOUISE SMITH

association with the portion of the first epibranchial that breaks
down, begins to show sings of degeneracy at stage II; is much
reduced in size and very degenerate at stage III, and by stage
IV is so far gone as to be almost indistinguishable form a blood-
vessel on the ventral surface of the ceratohyoideus internus that
lies beneath it (figs. 35, 39 and 46). The latter, which in the
larva was small and insignificant, begins at the same time to
proliferate new fibers anteriorly and laterally, so that simul-
taneously with the flattening out of the ceratohyal, the origin
of the muscle migrates from the medial border of that cartilage
to its whole anteroventral surface (figs. 30, 34, 37, 38, 39, 44 and
45). The muscle also increases in size posteriorly and gradually
begins to curve around the ceratobranchials (figs. 35, 40 and 46)
to form the adult pocket. The greatest change of all comes at
its posterior end where, simultaneously with the outgrowth of
the new portion of the epibranchial, there appear muscle fibers
encircling the cartilage (fig. 36). These seem to be a pro-
liferation from the end of the ceratohyoideus, as they are per-
fectly continuous with it; but it is barely possible that they may
be developed from undifferentiated mesoderm cells retained in
the connective tissue through larval life, and thus not strictly a
part of the ceratohyoideus internus. Whatever their origin, they
develop very rapidly, always keeping pace with the growing tip:
of the adult epibranchial, so that by stage III the spiral muscle
is fully formed (figs. 41 and 42) and by stage IV it has even
begun to be filled out as it is in the adult (fig. 47).

The hyoglossus and suprapeduncularis, the two wholly new
adult muscles, are the ones of whose development I am not as
yet quite sure. The former appears in stage II as a mass of
almost undifferentiated cells, but quite distinguishable from the
connective tissue that surrounds them, and in stages III and
IV they have assumed the position of, and a slight resemblance
to, the radiating fibers of that muscle as they spread out from the
anlage of the lingual cartilage. There is no sign of the supra-
peduncularis until stage IV, when a few cells, similar to those in
the analge of the hyoglossus, appear within the short fold of
mucous membrane now formed in the developing tongue-stalk,
and undoubtedly represent the anlage of that muscle.

HYOBRANCHIAL APPARATUS OF SPELERPES 549

So, with the possible exception of the suprapeduncularis, the
same conclusion may be drawn in regard to the hyobranchial
muscles as for the hyobranchial skeleton, namely, that during
the true metamorphic period all changes are indicated and well
on their way to completion, and only a continuation of the
process already started is necessary before the adult condition
is attained.

CONCLUSIONS

In concluding, there are three points which I wish to em-
phasize:

First: that the hyobranchial apparatus of the larva is char-
acterized by a certain firmness and rigidity which is greatly in-
creased by the presence of a branchial plate. This plate is a
universal phenomenon among larval Salamandrids, but has been
generally overlooked in the past.

Second: that the hyobranchial apparatus in the adult has
become very delicate and slender, and capable of a great com-
plexity of motion, adapted to its mode of respiration and the
capturing of prey.

Third: that the transition from the larval condition to that
of the adult is brought about not by a mere breaking down of
the parts that are to be lost and the simple shifting of the posi-
tion of others, but by a complex process which involves the
degeneration and loss of certain parts through phagocytosis and
the formation of new tissues in the adult position.

BIBLIOGRAPHY

Corr, E. D. 1887 The hyoid structure in amblystomid Salamanders. Amer.
Nat., vol. 21.
1889 The Batrachia of North America. Bull. no. 34, U.S. Nat. Mus.

Driner, L. 1902 Studien zur Anatomie der Urodelen. Zool. Jahrb., Bd. 15.
1904 Zungenbein-, Kiemenbogen-, und Kehlkopfmusculatur der
Urodelen. Zool. Jahrb., Bd. 19.

Ducts, A. 1835 Recherches sur l’osteologie et la myologie des Batraciens.
Mem. Savans Etrangers, 6, Paris.

Emerson, ELLEN T. 1905 General anatomy of Typhlomolge rathbuni. Proc.
Boston Soc. Nat. Hist., vol. 32.

Fiscuer, J. G. 1864 Anatomische Abhandlungen iiber die Perennibranchiaten
und Derotremen. Hamburg.

JOURNAL.OF MORPHOLOGY, VOL. 33, NO. 2

550 LOUISE SMITH

Gaupp, E. 1894 Beitriige zur Morphologie des Schiidels; II, Das Hyobranchiai-
Skelet der Anuren und seine Umwandlung. Morph. Arb., Bd. 3.
1906 Die Entwickelung des Kopfskelets; in Hertwig, Entwickelungs-
lehre der Wirbeltiere, Bd. 3.

GEGENBAUR,C. 1898 Vergleichende Anatomie der Wirbeltiere. Bd. 1. Leipzig.
1901 Ibid., Bd. 2.

Gorter, A. 1875 Die Entwickelungsgeschichte der Unke (Bombinator igneus).

Leipzig.

GopreErRT, E. 1894 Die Kehlkopfmusculatur der Amphibien. Morph. Jahr.,
Bde 22:
1898 Der Kehlkopf der Amphibien und Reptilien. Morph. Jahrb.,
Bd. 26.

Hay, O. P. 1890 The skeletal anatomy of Amphiuma during its earlier stages.
Jour. Morph., vol. 4.

Hertwic-MarKx 1892 Text-book of Embryology. London and New York.

Hitton, W. A. 1909 The hyobranchial apparatus of Typhlotriton. Biol. Bull.,
vol. 16.

Horrman, C.K. 1873 Amphibien; in Bronn, Klassen und Ordnungen des Thier-
Reichs. Leipzig and Heidelberg.

Hyrtt, J. 1865 Cryptobranchus japonicus. Vindobonae.

OpreEL, A. 1900 Lehrbuch der vergl. mikroskop. Anat. der Wirbeltiere, Bd. 3.
Jena.

Osawa, G. 1901 Anatomie des japanischen Riesensalamanders. Tokyo.

Parker, W. Kk. 1875 On the structure and development of the skull in the
Batrachia; Part I]. Phil. Trans. London.
1879 On the morphology of the skull in Amphibia Urodela. Trans.
Linn. Soc., ser. Il. Zoology, vol. 2.
1880 On the structure and development of the skull in Urodeles.
Trans. Zool. Soc., London, vol. 11.

Sa:rrnowa, W. 1914 Uber Regenerationsercheinungen des Muskelgewebes bei
der Metamorphose von Rana temporaria. Archiv fiir mikros. Anat.,
Bd. 84.

STEJNEGER, L., AND Barsour, T. 1917 A check-list of North American Am-
phibians and Reptiles. Harvard University Press.

vAN WisHE, J. W. 1902 A new method for demonstrating cartilaginous mikro-
skeletons. Proc. Koniklijke Akad. van Wetenschappen te Amsterdam.

WIeDERSHEIM, R. 1875 Salamandrina perspicillata und Geotriton fuseus. Ann.
Mus. Civico, Genoa, vol. 7.
1877 Der Kopfskelet der Urodelen. Morph. Jahrb., 3.
1°09 Vergl. Anatomie der Wirbeltiere; 7te Aufl., Jena.

Wiper, H. H. 1891 A contribution to the anatomy of Siren lacertina. Zool.
Jahrb., Bd. 4.
1892 Studies in the phylogenesis of the larynx. Anat. Anz., Bd., 7.
1893 Lungenlose Salamandriden. Anat. Anz., Bd. 9.
1895 The amphibian larynx. Zool. Jahrb., Bd. 9.
1896 Lungless Salamanders; second paper. Anat. Anz., Bd. 12.

Vorrper,INezW. 1913 Life-history of Desmognathus fusea. Biol. Bull., vol. 24.
(In preparation.) Growth and metamorphosis in Spelerpes
bislineatus.
(In preparation.) The morphology of metamorphosis.

“Se ee.

Stage II
1914C
Killed Worcester-
Wilder Fixative,
June 28, 1914
Section, 15 » trans.
November, 1917
Stained, haematoxy-
lin

of blood in them

Lifting head out of

water and breath-
ing air

Anlage

Gills still have flow | Folds in mucous

membrane of floor
of mouth, including
larval tongue,
straightened out

of adult
tongue longitudinal
outfolding of mu-
cous membrane
with anterior end
glandular

Articulation with ba-
sibranchial break-
ing down. Ventral
surface slightly;
proximal end, dor-
sal and mediodor-
sal surfaces rapid-
ly, degenerating.
Posterior ‘hook’
budding off later-
ally as new cartilage

Anterior end degen-

erating rapidly.
Ventral surface be-
ginning to degen-
erate

Anlage of lateral
shelves present as
collection of chon-
drioblasts

Plate quite larval,
though anterior
groove rather deep

rather thin
Os thyreoideum be-
ginning to ossify

in the middle
Cb, degenerating
around periphery
especially laterally
Osthyreoideum
somewhat more os-
sified

‘and median portion

Plate degenerating

Geniohyoideus =
€

Ceratohyoideus
externus and internus

Anlage of adult eb;
present as slight
swelling of cartilage
and thickening of
perichondrium
with frequent mi-
toses, at level of
insertion of M.chi
and origin of M.ca
and for about 600 »
posteriorly

Larval epibranchials
degenerating
around periphery
anteriorly

Adult eb; formed as
ventral fork of car-
tilage for about 750
u. Dorsal to it,
larval eb; degen-
erating. Posterior
to it, ebe2 shows
slight degeneracy
and ebs, and eb;
are quite larval

Stage IIT
1915 Js
e=+mm.
Killed Worcester-
Wilder Fixative,
July 3, 1915
Sectioned, 20, trans.
November, 1917
Stained, haematoxy-
lin and eosin

but not curved for-
ward. Beaten

Gills: Pale, bushy, | Outfolding in mouth | Articulation

beginning to as-
sume shape of

muscularly, slightly} disk

and irregularly
Right gill reduced

Quite glandular

Stage IV
1914 B;
g=circum 55 mm.
Killed Worcester-
| Wilder Fixative,
| June 28, 1914
Sectioned, 15 trans.
| December, 1917
Stained, haematoxy-
lin

Gills large and very
ted. Blood mov-
ing steadily
through them.

Crawls out of water
somewhat

Disk about formed.
Beginning of in-
folding of pharynx
beneath it to form
stalk

with
basibranchial gone.
Becoming some-
what flattened
Considerable de-
generation still
taking place
Articulation with

basibranchial quite
gone. Anterior end
still degenerating.
Becoming quite
flattened, though
dorsal and ventral
degeneration till
going on

Posteriorly degener-
ating all around
periphery

Old anterior end
(anterior to horns)
represented by leu-
cocytes and shreds
of cartilage

Ventral surface be-
coming flattened.

‘Shelves’ becoming
cartilaginous

Only a faint sugges-

tion of former an-
terior end left

Ventral surface quite
flat though old out-'
line and leucocytes
present

‘Shelves’ about as in
19165 Jy

Plate broken down,
but first cerato-
branchials still in
ventral plane with
many leucocytes
and suggestion of
perichrondrium be-
tween them

Osthyreoideum
more ossified

Plate almost broken

down but a few
shreds of cartilage
mark its former
position
Osthyreoideum
more ossified

Adult epibranchial
formed for about
12004. Larval eb:
very degenerate.
Consists mainly of
leucocytes and
shreds of cartilage
anteriorly and de-
generating carti-
lage posteriorly.
Ep:, degenerating
rapidly. Very
ragged with many
leucocytes, es-
pecially anteriorly

Anlage
present as collec-
tion of chrondrio-
blasts

Horns

of lingual cartilage
present

Median raphé form-
ed in M.ima except
for the few most
anterior sections.
Raphé formed in M.
imp. anteriorly.
Larval posteriorly

M.ima and M.lmp.

ed throughout

Interhyoideus and
gularis becoming
separate

M.gh budded off
from M.ghm anter-
iorly for about 800
Be Inserts into
mucous membrane
of pharynx

of horns | Medianraphéinboth| M.gh more differ-

entiated from
M.ghm, though
still small, and en-
tered into pharyn-
geal wall. Ap-
proaching cerats-
hyals, however

face of Bb, consid-
erably anterior to
articulation of
plate with it

Origin of M.ah in
connective tissue of
tongue posterior to
anlage of horns.
M.th beginning to
degenerate medially

Sternohyoideus be-
coming differenti-
ated from M.ths

Origin on dorsal sur- | Larval

M.che beginning to| M.aa degenerating | Anlage of hyoglossus Probably early

degenerate, espe-
cially posteriorly.
Insertion larval

M.chi forming new
fibers and increas-
ing in size. Origin
more anterior and
ventral. Growing
posteriorly and be-
coming curved
around Cb;,: and
Eb;. Spiral fibers
forming around new!
eb;

slightly
M.ca; degnerating.
Others larval

present as almost
undifferentiated
cells

becoming | Median raphé form-| M.ghl inserted on
chondrified. Anlage|

gh for considerable
distance

Adult epibranchial
formed for about
1350 ». Larval eb;
practically gone.
Position marked by
collection of leu-
cocytes with occa-
sional shreds of car-
tilage

About as in 1915 J;

About as in 1915 Js

M.ghp inserted on
ch for its anterior
half. Fibers still
small

M.ah adult an-
teriorly. Poster-
iorly, large number
of leucocytes, de-
generating fibers of
M.th and new fibers
of M.ah fill old posi-
tion of M.th

M.sh more differenti-
ated

M.ah practically
adult anterior to
osthyreoideum.
Fibers small and
some degenerate
fibers of M.th. left.
M.sh about as in
1915 Js

M.che still present
but very degener-
ate. Origin on
M.chi (not Ch)

M.chi origin anterior
and ventral. As-
suming adult posi-
tion and shape and
spiral formed but
not filled out

M.che practically
gone

M.chi has almost
adult form but not
fully developed,
though more filled
out than in 1916 J;

M.aa degenerating

M.ca;,; nearly gone

M.Pa;-s degenera-
ting

M.da; degenerating

M.ac degenerate
M.ca,, 2 nearly gone
M.lai-s gone

M.da,; degenerate

ing radiating posi-
tion

Anlage M.hg assum- | Probably late —“

‘metamorphic’

:
:
:
:
:
:
;

morphic,” sp-
Proaching ad-
vanced metamor-
phic. Not very
different from :
1914B;. In condi-
tion of gillsand
visceral skeleton,

Anlage M.hg about | Probably late ‘Meta-

asin J;

Anlage M.sp form-| 1915IV, 1917C17 and —

ing?

morphic’
1915 J, were in
same condition

I Mh eae arte nce oe

Sbobrres. sites itt Lng

ghd maihars i
Raline (arhitet ©
bist pithionia’
Bye shee
sinh dey oualak
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fanettotaa biG)

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ibotaneseest
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gagitim, (nage Y
medial uy iiit1o9
Roowd ©'sarlese:
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Seterny?, “to? inst
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MINS tise devin
blosgigeo ds sah
marnsaaréel kita. ai
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. _ ‘ :
ee ea ee ee ee ae
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sepa

i ve: OR a x
, : : adtasotay .

sr ne aye nn gee, A irre ete wera

fidtey

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ie Sa eR Ne

> snoy Webiperdiged

\. “eating ‘puintonsa’ ie ; cares an

tiyitod Molt Sacha yy
bab.” diehaso bao iy tht
jiste QOitATIGNR | a mI . hoody my re

eee gaitat)

}

| i
ene See ett rete ny ty ee one REEMA ATL fe
Porte HG het fishin, ne nuneetOt, yy

\4
|
t

vi Be

aiihic ciey latikgins reli: to ere Meare i
bhua snare Swile neat iowa Hl

a musi eT gavaab (loa head od Fi it aod { oe in |
i Mp: Y -
1p. we uaa 9 ue n! ; “alate a Be

fy freseh) ota iad
(ahd foe Dag Get be

Way, a Ws ea ae Y ioe aii rey
paiee UR LL ts ee i |
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L THiewsh Vitara d | ! ry

Ae PL SSE A GeO A ea | ee

PLATES

The outlines in plates 1, 5, and 10 were drawn from methylen-blue preparations
with the His embryograph and enlarged with pantograph. In finishing, certain
details were added from reconstruction of sections. Plates 6,7, and 8 were drawn
free-hand from dissections with the camera outlines of plate 4 as the skeletal
basis. The outlines of all the cross-sections were made with the projection
lantern.

ABBREVIATIONS
Skeleton
Bb 1-2, basibranchial 1-2 H, little horn
Bp, branchial plate H*, anlage, little horn
Cb 1-2, Ceratobranchial 1-2 L, lingual cartilage
Ch, ceratohyal L*, anlage, lingual cartilage
Eb 1-3, epibranchial 1-3 Ot, os thyreoideum
Eb*, developing new portion of first
epibranchial
Muscles
aa, adductor areuum gh m, geniohyoideus medialis
ah, abdominohyoideus hg, hyoglossus
ah*, developing abdominothyoideus hg*, anlage hyoglossus
ahs, ventral slip of abdominohyoideus th, interhyoideus
ca 1-2, constrictores arcuum 1-2 ima, intermandibularis anterior
che, ceratohyoideus externus imp, intermandibularis posterior
chi, ceratohyoideus internus la, 1-5, levatores areuum 1-5
da 3-4, depressores arcuum 3-4 Sh, sternohyoideus
g, guliris Sp, suprapeducularis
gh, geniohyoideus th, thoracicohvoideus
gh l, geniohyoideus lateralis ths. ventral slip of thoracicohyoideus

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552

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HOINS TSIN0T
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Yon)

PLATE 2

EXPLANATION OF FIGURE

6 Hyobranchial muscles of larva; ventral view. The lines 7 to 16 show the
levels at which the sections shown in plates 3 and 4, figures 7 to 16, respectively,
were cut. X50.

PLATE 2

HYOBRANCHIAL APPARATUS OF SPELERPES

LOUISE SMITH

1o

ll

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585

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PLATE 6

EXPLANATION OF FIGURE

19 Superficial dissection of hyobranchial muscles of adult; ventral view.
X20.

PLATE 6

HYOBRANCHIAL APPARATUS OF SPELERPES

LOUISE SMITH

563

PLATE 7

EXPLANATION OF FIGURE

20 Deeper dissection of hyobranchial muscles of adult; ventral view. The
mucous membrane of the floor of the mouth has been partially removed to show
its relationship to the tongue and tongue-stalk. X20.

564

_ HYOBRANCHIAL APPARATUS OF SPELERPES PLATE 7
LOUISE SMITH

565

JOURNAL OF MORPHOLOGY, VOL. 33, NO. 2

PLATE 8

EXPLANATION OF FIGURE

21 Dissection of hyobranchial muscles of adult; dorsal view. The dorsal
surface of the tongue has been partially removed, and the posterior edge entirely
so, to show underlying structures. The lines 22 to 27 show approximately the
levels at which the sections shown in figures 22 to 27, respectively, were cut.
These levels cannot be shown exactly, as the position of the apparatus at the
time of fixation was different in the two specimens. X20.

HYOBRANCHIAL APPARATUS OF SPELERPES
LOUISE SMITH

PLATE 8

567

PLATE 9
EXPLANATION OF FIGURES

Cross-sections of the adult. 28

22 Level of anterior end of first basibranchial, showing lingual cartilage
and ‘horn’ in tongue.

23 Level of the origin of M. ceratohyoideus internus, showing M. suprape-
duncularis and the tongue stalk.

24 Level of articulation of the first ceratobranchials with basibranchial.

568

PLATE 9

HYOBRANCHIAL APPARATUS OF SPELERPES
LOUISE SMITH

PLATE 10

EXPLANATION OF FIGURES
Cross-sections of adult. X 28

25 Level of articulation of second cerabranchials with basibranchials, showing
the beginning of the pocket formed by the ceratohyoideus internus muscle.

26 Level of the os thyreoideum and of the articulation of the epibranchial
with the first and second ceratobranchials, where the ceratohyoideus internus
begins to form the spiral.

27 Level of the heart, showing the fourth levator and depressor of the gill
arches, and the spiral muscle toward the posterior region of the epibranchial.

570

HYOBRANCHIAL APPARATUS OF SPELERPES PLATE 10
LOUISE SMITH

eX. A Oa
SS SS Sg — ¥
tir ——-— ai ee x
ETT Pe a Pe me a
1 1 ' ( i 1 y ! :
Cicer atsterat ° are i pee
J b, oh qh ch om Ot gh Che ret on a

PLATE 11

EXPLANATION OF FIGURE

28 Hyobranchial skeleton of metamorphic stage III, ventral view. The
position of cross-sections cannot be shown on this drawing, as the specimen of
stage III, used for sectioning, differed somewhat in development and considerably
in position of cartilages from this one. X27.

572

PLATE 11

HYOBRANCHIAL APPARATUS OF SPELERPES
LOUISE SMITH

PLATE 12

EXPLANATION OF FIGURES
Cross-sections of metamorphic stage I. X28

29 Level corresponding to larva, figure 7, showing presence of M. genio-
hyoideus lateralis anteriorly.

30 Level corresponding to larva, figure 9, showing the deepening of the
groove at which the branchial plate articulates with the basibranchial, and the
anterior growth of the abdominohyoideus muscle.

31 Level corresponding to larva, figure 10, showing development of the
abdominohyoideus, and absence of change in other particulars.

32 Level corresponding to larva, figure 14, showing the anlage of the adult
epibranchial and beginning of degeneration of the larval first epibranchial.

574

HYOBRANCHIAL APPARATUS OF SPELERPES PLATE 12
LOUISE SMITH

:
| \
c fi / | i | | \ \ \
he Che @h th Shim By. Cl Bp Imp Ch
) ae

PLATE 13

EXPLANATION OF FIGURES
Cross-sections of metamorphic stage II. X28

33 Level corresponding to a section of larva between figures 7 and 8, showing
degeneration of anterior end of basibranchial and ceratohyals.

34 Level corresponding to larva, figure 9, and metamorphic stage I, figure 30,
showing degeneration of basibranchial, ceratohyals, and M. ceratohyoideus
internus, and anterior growth of Mm. abdominohyoideus and ceratohyoideus
internus.

35 Level corresponding to larva, figure 10, showing degeneration of the plate
and of the median portion of M. thoracicohyoideus.

36 Level corresponding to metamorphic stage I, showing further growth of
adult epibranchial, anlage of spiral muscle and degeneration of larval first
epibranchial.

on
~I
for)

PLATE_13

HYOBRANCHIAL APPARATUS OF SPELERPES

LOUISE SMITH

| Wie ie, ema \ .
Che Ch, Ghm ah LL Ch,

34

-s-—

ta
Us — 2a

p
SX

eee

Fh tee So ETE
PCRS Gok | \ L L
Sie By, Sh alee oa
36

577

PLATE 14

EXPLANATION OF FIGURES
Cross-section of metamorphic stage III. X28

37 Level corresponding to adult, figure 22, showing anlage of ‘little horns’
and lingual cartilage.

38 Level corresponding to adult, figure 23, showing changing shape of basi-
branchial and ceratohyals.

39 Level corresponding to levels shown in larva figure 9, in metamorphic
stages II and III, figures 30, 34, and in adult figure 24.

40 Level corresponding to larva, figure 10, and adult, figure 25, showing the
plate and M. thoracohyoideus nearly broken down.

578

HYOBRANCHIAL APPARATUS OF SPELERPES PLATE 14

LOUISE SMITH

/ ne on t i \ None
3 :
need Si, Cl, VE SCh
39

' 1 l

cht ah qhm Bb, ch,
40
579

PLATE 15

EXPLANATION OF FIGURES
Cross-sections of metamorphic stage III. X 28

41 Level corresponding to larva, figure 12, and adult, figure 26, showing the
rapidly ossifying os thyreoideum, the M. thoracicohyoideus degenerating and
M. abdominohyoideus developing, and the M. ceratohyoideus internus beginning
to form the spiral.

42 Level corresponding to larva, figure 14, and metamorphic stage II, fig-
ure 36, showing the further development of the adult epibranchial and spiral
muscle, and the almost complete degeneration of the larval first and second
epibranchials.

43 Level corresponding to larva, figure 15, showing the M. sternohyoideus
completely separated from the thoracicohyoideus and the adult epibranchial and
spiral muscle formed, the posterior ends of the larval epibranchials still present.

580

HYOBRANCHIAL APPARATUS OF SPELERPES
LOUISE SMITH

y I I
\ap aq ah® th Sh

42

ef | Stes
os “2 thea 4 dbo
43
581

JOURNAL OF MORPHOLOGY, VOL. 33, NO. 2

PLATE 15

PLATE 16

EXPLANATION OF FIGURES
Cross-sections of metamorphic stage 1V. X28

44 Level corresponding to metamorphic stage III, figure 38, showing the
infolding of the pharyngeal mucous mmbrane to form the tongue stalk, the fur-
ther flattening out of the basibranchial, and ceratohyals, and the complete loss
of the M. ceratohyoideus externus anteriorly.

45 Level corresponding to adult, figure 24, showing posterior limit of forma-
tion of tongue stalk.

46 Level corresponding to metamorphic stage III, figure 40, showing nearly
complete degeneration of M. ceratohyoideus externus.

47 Level corresponding to metamorphic stage III, figure 42, showing the spiral
muscle beginning to fill out to its adult proportions, and the M. thoracicohyoideus
nearly transformed into Mm. abdominohyoideus and sternohyoideus.

HYOBRANCHIAL APPARATUS OF SPELERPES PLATE 16
LOUISE SMITH

SUBJECT

MBYSTOMA TIGRINUM._ Chromo-
some number and pairs in the somatic

PITTOSES OL. sateny ere ete aes s Ne eee 169
Amphibia caudata and its phylogenetic sig-
nificance. The morphology of the sound-
transmitting apparatus in. 325
Apparatus of Spelerpes bislineatus. The hyo-
PANIC Peo. oss aes oe ne a eie we eeaeetas 2
Asp!anchna ebbesbornii (Rotifer). The de-
Vira) SLT) NG PAB eee Soha awn ter Gc 389

Diao ATUM. Pharynx ‘of Microstoma ... 309

Chromosome number and pairs in the somatic
mitoses of Ambystoma tigrinum.... ..... 169
Clupeoid cranium in its relation to the swim-
bladder diverticulum and the membran-
eusiabyrimth. Vihe.. .::. geese c es ccc 439
Coccids, with especial reference to the forma-
tion of the ovary, origin and differentia-
tion of the germ cells, germ layers, rudi-
ments of the midgut, and the intracellular
symbiotic organism. Embryology of.... 73
Coelenterates. VI. General considerations,
discussions, conclusions. Germ cellsof.. 1
Cranium in its relation to the swimbladder
diverticulum and the membranous laby-
Finthe. heiclapeoid: -o5. 5... +. - ees 439

EVELOPMENT of Asplanchna ebbes-
bornil)(Roetifer):) hess. ....- sys 389

Development of Gelasimus after hatching.

MBRYOLOGY of coccids, with especial
reference to the formation of the ovary,
origin and differentiation of the germ

cells, germ layers, rudiments of the mid-

gut. and the intracellular symbiotic organ-

RS EAD Fe yas ee er See sia ae oe Ne scl Dis 73

Euplexoptera. The anatomy of the head and

mouth-parts of Orthoptera and......... 251

ISHES. A comparative study of the
bones forming the opercular series of .. 61

ELASIMUS after hatching. The devel-
ODBIENGOL. arise ee ee a er es 485

Germ cells, germ layers, rudiments of the mid-
gut, and the intracellular symbiotic organ-
ism. Embryology of the coccids, with
especial reference to the formation of the
ovary, origin and differentiation of the.. 73

Germ cells of coelenterates. VI. General con-
siderations, discussion, conclusions. ...... 1

Germ layers, rudiments of the midgut, and the
intracellular symbiotic organism. Em-
bryology of coccids, with especial reference
to the formation of the ovary, origin and
differentiation of the germ cells.......... 73

ARGITT, Grorce T. Germ cells of

coelenterates. VI. General considera-
tions, discussion, conclusions. . ; 1

Head and mouth-parts of Orthoptera and
Euplexoptera. The anatomy of the...... 251

HELVESTINE, FRANK, Jr., KEPNER, WM. A.,
AND. Pharynx of Microstoma caudatum_ 309

AND AUTHOR INDEX

Husss, Cart L. A comparative study of the
bones forming the opercular series of fishes 61

Hyman, O. W. The development of Gelasi-
Mus after batching). se eee ss ee 485

Hyobranchial pu of Spelerpes bislinea-
Gus? The ee n n e e 527

EPNER, Wm. A., AND HELVESTINE,
Frank, JR. Pharynx of Microstoma
caudatum .

ee The clupeoid cranium in
its relation to the swimbladder divertic-
ulum and the membranous............. 439
Layers, rudiments of the midgut, and the in-
tracellular symbiotic organism. Embry-
ology of coccids, with especial reference to
the formation of the ovary, origin and dif-
ferentiation of the germ cells, germ...... 73

EMBRANOUS labyrinth. The clupe-

oid cranium in its relation to the swim-
bladder diverticulum and the. ........ 439
Microstoma caudatum. Pharynx of. ........ 309

Midgut, and the intracellular symbiotic organ-

ism. Embryology of coccids, with espe-

cial reference to the formation of the ovary,

origin and differentiation of the germ cells,

germ layers, rudiments of the............ 73
Mitoses of Ambystoma tigrinum. Chromo-
some number and pairs in the somatic... 169
Mouth-parts of Orthoptera and Euplexoptera.
The anatomy of the head and............ 251
PERCULAR series of fishes. A compara-
tive study of the bones forming the.... 61
Orthoptera and Euplexoptera. The anatomy
of the head and mouth-parts of.......... 251

Ovary, origin and differentiation of the germ
cells, germ layers, rudiments of the mid-
gut, and the intracellular symbiotic organ-
ism. Embryology of coccids, with espe-
cial reference to the formation of the..... 73

AIRS in the somatic mitoses of Amby-
stoma tigrinum. Chromosome number
BOT es sts ee eae era ee ee eae eras 169
PARMENTER, CHARLES L. Chromosome num-
ber and pairs in the somatic mitoses of
Ambystoma tigrinum: =. ..............-- 169
Pharynx of Microstoma caudatum........... 309
Phylogenetic significance. The morphology
of the sound-transmitting apparatus in
Amphibia caudata and its................ 325

EED, H. D. The morphology of the
sound-transmitting apparatus in Am-
phibia caudata and its phylogenetic sig-
MIICHICE me: [cas Banc ae hee eke oe anne we 325
(Rotifer). The development of Asplanchna
EDDESHOEDI A. anus cleans casei aan dee 389

HINJI, Grorce OrtHay. Embryology of
coccids, with especial reference to the for-
mation of the ovary, origin and differenti-

ation of the germ cells, germ layers, rudi-
ments of the midgut, and the intracellular
SYM DIOviG OFEANISIO. =~. due vce oe: < ais crew ae 73

585

586

Significance. The morphology of the sound-
transmitting apparatus 1n Amphibia cau-
data and its phylogenetic....-..------- 55

INDEX

325

i. re
Smrru, Louisr. The hyobranchial apparatus

of Spelerpes bislineatus..... ..----+-:-->
Sound-transmitting apparatus 1 Amphibia
caudata and its phylogenetic significance.
The morphology of the.......-------+----
Spelerpes bislineatus. The hyobranchial ap-
paratus Ol Ae steerer eer ee ae
Swimbladder diverticulum and the membran-
ous labyrinth. The clupeoid cranium in
its relation to the......-..----------+:>

325

527

. 439

Symbiotic organism. Embryology of coccids,
with especial reference to the formation of
the ovary, origin and differentiation of the
germ cells, germ layers, rudiments of the
midgut, and the intracellular: ee. teres

pe]
A gan coe ae Guorce W. The de-

velopment of Asplanchna ebbesbornii

(Riotifer)oascsc mecca sees ebayer 389

TRACY, Henry C. The clupeoid eranium in
its relation to the swimbladder diverticu-
lum and the membranous labyrinth.....

ee Hacutro. The anatomy of the
head and mouth-parts of Orthoptera
and Buplexoptera...---.-----+++++++°°: 2

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