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JOURNAL
OF
MORPHOLOGY
FounpEp By C. O. WHITMAN
MANAGING EDITOR
C. E. McCLUNG
ASSOCIATE EDITORS
E. G. CoNnKLIN M. F. GuyEer W. M. WHEELER
Princeton University University of Wisconsin Bussey Institute,
: Harvard University
C. A. Kororp F. R. Lin.ir J. T. PATTERSON
University of California University of Chicago University of Texas
G. A. Drew. H. V. Nea L. L. WoopRurrF ©
Marine Biological Laboratory Tufts College Yale University
Woods Hole, Mass.
VOLUME 36
DECEMBER, 1921,
MARCH, JUNE, SEPTEMBER, 1922
THE WISTAR INSTITUTE OF ANATOMY AND BIOLOGY
PHILADELPHIA
OOEO
Ne yy tye iy Ce AR Te Barns
CONTENTS
No. 1. DECEMBER, 1921
J.T. Parterson. The development of Paracopidosomopsis. Two text figures
andatwelvesplavesu(MIneby MeUTEs)io6 4.4 sasc.ce esa ede Mee Neos oe ene 1
CASWELL GRAvE. Amaroucium constellatum (Verrill). II. The structure
and organization of the tadpole larva. Four text figures and four plates
Gea GHP MCR IS. ORE: Seta P: Lae eee AAP oe 2 REN KOZN SAE ULAR, oA RD ot
FRANK HELVESTINE, JR. Amitosis in the ciliated cells of the gill filaments of
Cyclas) glwo,plates (seven heures). Si20 Maes. il Ades gs P Se A le. 103
D. H. Wenricu. The structure and division of Trichomonas muris (Hart-
mann). One text figure and four plates (thirty-six figures)................. 119
No. 2. MARCH, 1922
ALEXANDER PETRUNKEVITCH. The circulatory system and segmentation in
Arachnida. Two text figures and two plates (seven figures) . ne . 157
W. Haroup LeicH-SHarpe. The comparative morphology of ihe seergi cee
sexual characters of elasmobranch fishes—the claspers, clasper siphons, and
Clisperclandssalviemoirel thas Kivie fi Gunes © ae. mec. cciecn ec. dnese seein ee es 191
W. Haroup LeicH-SHARPE. The comparative morphology of the secondary
sexual characters of Holocephali and elasmobranch fishes—the claspers,
clasper siphons, and clasper glands. MemoirIV. Twenty-two figures..... 199:
W. Harowip Leicu-SHarpe. The comparative morphology of the secondary
sexual characters cf Holocephail and elasmobranch fishes—the claspers,
clasper siphons, and clasper glands. Memoir V. Nineteen figures......... 221
Water N. Hess. Origin and development of the light-organs of Photurus
pennsylvanica De Geer. Five plates (seventeen figures).................. 245,
Sante Naccarati. Contribution to the morphologic study of the thyreoid
gland in Emys europaea. Two plates (five colored figures)................. 279
CHARLES EvuGENE Jonnson. Branchial derivative in turtles. Five plates
Mivierty Our ilo UTES)) ban. mary een eee teem Se tn ay. ett ee, een geen 299
Horace W. StunKArD. Primary neuromeres and head segmentation. Twenty
LOTR CA me at”, oA ei SNe A AUN RE ney Oe Se hot ye 2 es 331
No. 3. JUNE, 1922
Bertram G. Smira. The origin of bilateral symmetry in the embryo of
Cryptobranchus allegheniensis. Thirty-three figures..................... 357
Epira Pinney. The initial block to normal development in cross-fertilized
eggs. I. Crosses with the egg of Fundulus. II. Reciprocal crosses be-
tween Ctenolabrus and Prionotus. Two plates (seventeen figures)......... 401
iil
Pe eet
1V CONTENTS
OuiveErR P. Hay. On the phylogeny of the shell of the Testudinata and the
relationships of Dermochelys. One text figure and two plates............ 421
AupEN B. Dawson. The cloaca and cloacal glands of the male Necturus.
‘Theee plates (sixteen digures) 4.4 W682i «fn O0e eee hy se wie Ha nas 447
Horr Hissarp. Cytoplasmic inclusions in the egg of Echinarachnius parma.
One text figure and four plates (twenty-four figures)..................... 467
No. 4. SEPTEMBER, 1922
CAROLINE Burtinc THompson.: The castesof Termopsis. Nine text figures and
twosplates | odoingh soke IAS IM pee. etek sleet. oes. Reopens A eae eee 495
D. L. Gamsir. The morphology of the ribs and transverse processes in Nec-
furus:macwlatuss y Lirty-one dieuresi’ 2 jc... prsemeides obec eee eee 537
GerorcE H. Bisnor. Cell metabolism in the insect fat-body. I. Cytological
changes accompanying growth and histolysis of the fat-body of Apis mellifica.
Six text figures and three plates (thirty-six figures)................000ee eee 567
AUTHOR’S ABSTRACT OF THIS PAPER ISSUED
BY THE BIBLIOGRAPHIC SERVICE, OCTOBER 17
THE DEVELOPMENT OF PARACOPIDOSOMOPSIS!
J. T. PATTERSON
University of Texas, Department of Zoology
TWO TEXT FIGURES AND TWELVE PLATES (NINETY FIGURES)
CONTENTS
TLS Tira tieR (0 GW OLHTOT Ty BA Aen eae Graphene inane icra ae ts bcs ind ea neh gaat Sars Anita AM eae 1
2. Structure, maturation, and fertilization of the egg...................... 6
BEF MH ECE Hiya LATO L Dicey na. Sy arce oy Sy Sateerhepcatis oferta cakats ce ckaepewehavencin ydvetsrereet 6
7h TEENS ee Res A a Uh BN rc ee a UIE IRA ES 9
Ba LERET EL UUEASY HOD ed Mi icRipc aes eee rl Oe Rue Al Nine pe ar Rs ea TLL 13
ae closwarme Lares h i): 245 tT Oke aa ae athe’ ale gral Wa ae ete ee aterees 14
Gap SHALS HeCVASTON 2 Aes cye ss 5 Cities CG 5.65 EVAR te TA Ee SERS A STE RSE ota 14
Dre eGSE CONG GCI yASLOM eres cei shake essay Mens ope es ae ok Gah ae chee eee dete 15
CMe RG MATOMC IV ASTOMM. ccrssis ccratc fet crave oe eon rao sicker ciclaleee beoasay se ccteborsine nears 18
Gre reerOUnthealvaSlOms seaae west arate ola rae tee ak tees hoa atin oid Maelo e ocala 18
Ce Hiblavs ashipploy olga) kaye A Amn tes eee SretG A At.A CA ras em mae ry eis ger tr 19
TPS BLS: tee TAT UENO Reeeeges Qn on ont eS AAR RENCE. 1 et em 20
ARE GE RON CGI Ie MOLY MENTO i, ios ha ce why vc Re Tee oe awa sae ates ne ae reer 24
MELISCORVA GME Nes POLY Germs tae. weiner ec is s eees Sele mercies este ticle mittee elie ee ere nie Pal
a:) Multiplication of the primary massés:8 2 esi ace MS 27
b. Relation, of: parasitic egg to host: tissues. <. jie.) d ay 4 2) aleve ciate ws eicie 29
Can ISSOCIaclONvOLabNelpOly Germs... secs eee Reece laaitc sole ace 31
d, Origin and history of the sexual embryos... ..... 0.2.5.2... eens 33
eOrigin and history, of the asexualembryassis..22 250. ae ret 5 36
Gise SUUTPATIN TAN TSY 5 eye > sieter sb ols bale tS esas. Sys sagas) SERS cea Spiele, MASE SE atcha orate Reha 39
PARES TERS CSSRTE ATI ERY. Sacer RS as Sy oc cas a RUA Aot lata Shp. aN OT 5b eles tig ese isienaeha s 42
Sov gy 1 LOE CEL GY OA IUGTONG HINT O) C25 12S eg RN Se RESP pe ne eS Rr SS Ae A, 45
1. INTRODUCTION
The object of the present paper is to give a rather complete
account of the development of a polyembryonic insect. We
owe a great deal to Marchal and Silvestri for their pioneer studies
in this field. They have given us a good outline of the general
course of events in the development of some three or four species.
Nevertheless, there are many important points in the embry-
1 Contribution no. 145.
2 J. T. PATTERSON
ology of these insects which are as yet obscure, and there is still
to be written a complete account of a single representative
species. It is highly to be desired that a description be given
of the development of a typical species in each of the three fami-
lies of parasitic hymenoptera in which polyembryony has been
reported. The evidence, although fragmentary in many instances,
clearly indicates that there are significant differences in the type
of development in each of the three families.
This paper is an attempt to give an account of each of the
more important steps in the embryology of the egg up to the
point at which the sexual embryos are formed. For this pur-
pose I have selected a representative of the family Chalcididae.
The species used is Paracopidosomopsis floridanus. Ashmead.
It was chosen primarily because of the ease with which it can
be secured in this vicinity? and because it can be reared readily
under laboratory conditions. In studying the development of
parasitic forms, the latter advantage cannot be over emphasized;
for if the work is to be free from suspicion, one must scrupu-
lously avoid the possibility of the host eggs becoming infected
with foreign parasites. This can only be done when the material
used is reared under laboratory conditions. Another reason for
studying Paracopidosomopsis is the fact that non-viable larvae
appear in the polygerm stages of this species. These are similar
to, if not identical with, the asexual larvae of Litomastix.
I shall first give a brief résumé of the life-history of Paracopi-
dosomopsis, as: it will then be easier to follow the account of
the development of the egg. The parasite lays its egg in the egg
of the common cabbage looper, Autographra brassicae. The two
eggs develop simultaneously, and, as is the case in several other
species, there is one generation of the parasite for each generation
of the moth. The moth reaches its complete development several
days before the parasites, but is not ready to lay its eggs until the
parasites emerge. I have not been able to determine the number
of generations per year for the Austin region. The looper is
2J am deeply indebted to Mr. Thomas H. Jones, of Baton Rouge, La., for
sending me parasitized carcasses at times when they could not be found here
at Austin.
DEVELOPMENT OF PARACOPIDOSOMOPSIS 3
found on various Cruciferae throughout the summer months,
but the main generation appears about the 1st of October, when
the fall crop of cabbage is a month or six weeks old. ‘There is
of course a considerable overlapping of generations. From Octo-
ber Ist until late in December I have been able to rear two com-
plete generations of the parasite in the laboratory. October and
November are therefore the best months in which to collect
material.
The parasite will deposit its eggs at any time during the em-
bryonic period of the host, which lasts from seventy to eighty
hours. After the young caterpillar hatches, the parasite will
no longer parasitize it. There are many interesting points in
the behavior of these insects, especially in connection with egg
laying. They are positively heliotropic and move with great
rapidity toward the light, which fact makes it easy to handle
them in the laboratory.
The individuals in one carcass all emerge at about the same
time, and the females whether fertilized or not are ready to lay.
If males are present, they at once mate with the females. One
male will mate with several females. If a single female be
introduced into a vessel containing a brood of males, which
have become quiet, the entire blood immediately becomes active.
In crawling over the surface of the table or leaf, the parasite
feels its way along by means of the antennae. This is the method
used by the female to find the host egg on the leaves of the cab-
bage. Once an egg is located, the female examines its surface
by the tips of the antennae, which vibrate with great rapidity.
If the egg happens to belong to an insect other than the Auto-
graphra moth, the female leaves it in a little while and continues
her search for the desired egg.
In preparing for oviposition the female mounts the egg and
clasps it with the second and third pairs of legs. The first pair
of legs are either held free or placed on the surface of the leaf.
The tip of the abdomen is then bent down until it comes in
contact with the egg. She then braces herself and forces the
ovipositor into the egg. During the act of laying, the female
remains perfectly motionless, with the head and antennae bent
4 J. T. PATTERSON
down and backward. With the aid of the binocular micro-
scope and strong transmitted light one can easily observe the
egg passing down the lumen of the ovipositor. The egg is forced
out by rhythmic pulsations of the abdomen. As soon as the
ege is deposited, she withdraws the ovipositor, remains motion-
less for a second or two, rubs the pair of hind legs together and
then proceeds to search for another egg. The act of oviposition
varies in time from two to as long as fifteen minutes. If only
a few eggs are present on the leaf, she may in time return and
lay again in the same egg. The number of eggs deposited at
one oviposition is either one or two. My records show that in
about two times out of three two eggs are deposited.
The parasitic egg may be deposited in any part of the host
egg or embryo, but only those which become included in the
tissues of the embryo are able to complete development. The
ege develops whether fertilized or not. Eggs laid by virgin
females always produce male broods, but broods arising from
eges laid by fertilized females are nearly always mixed.
The process of maturation is completed in one and one-half
hours after the egg is deposited. Cleavage then follows, and
the polygerm stage is reached in about seventy-two hours. The
polygerm is composed of a number of primary masses, each of
which consists of a group of embryonic cells surrounded by a
nucleated membrane. The primary masses multiply, by con-
strictions of this membrane, to produce secondary masses, and
these in turn divide to form tertiary masses. Further divisions
follow and the entire polygerm becomes a very complex structure.
The tertiary mass stage is reached in from seven to nine days.
The tertiary divisions produce what I have called compo-
nents. These become scattered throughout the body cavity of
the caterpillar through the dissociation or fragmentation of
the polygerm. The tertiary components form centers for fur-
ther multiplication, or for the formation of groups of sexual
embryos. The sexual embryos begin to form on the fifteenth
day and reach the free larval stage sometime between the twenty-
second and twenty-fourth days. Pupation occurs on the twenty-
eighth day, and the adult insects emerge on the forty-seventh
day.
DEVELOPMENT OF PARACOPIDOSOMOPSIS
5
The time given above is for stages reared in the laboratory
during the months of October to December.
Out in the field
in the sun development occurs much more rapidly. Under such
conditions, the entire life-cycle is completed in about thirty
days.
TABLE 1
STAGE
INES AMR ON UO Baacomneaas Ege ete bios Cod caaE
Second maturation...........
TPCT, be. BOS Ess Geta ECE cee: Ree
REMOTE IONCE ete tits tite See whet acct ata oes as
Est CICA MAGS SPINGIe f°: oScjciis sts fe hs 28 Sate t
PaeriheOrB UME Soe e setts s AOS eT. chal ota
Second. cleavare spindle;,...05 220... ole eyes ee
BEC UCURSENECR TT Sener teks Seer. SoS ke er a
rE SU ete ley sh (Se eR a oo CR Re SD
He celedustacterermetite sors si certian Lae eae
ZEA MRE IO Se Soo SSE De eee ce cree
EME OROURCE Greiner anaes haul a tink an rere tues stern
BYE UAT Cat O VOT Rey crag cee acu aa ooh ase ealie. 2,8 shen
Dempletca MOLY Pera ..c\ cc iete te halos ie ees
Divasiony Of Primary MASSES).2: ae. .% 4. see he bela
Division of secondary masseS.................--
DiVIStonwol enulaly MASSES)... case «eye cinerea
Beginning of sexual embryos...................
Breeton al Scam: Snore esac: ane tbc anee «siete
[PAVE RV 615) Sep eh 2 MRS at A i ae OS ROP, 6-6, CR A
PACH IMBC CES is..caisrse arsine nek uriassk ines ao onal oe Ss
TIME
25 to 30 mins.
60 to 65 mins.
1 hr. 30 mins.
1 hr. 50 mins.
2 hrs. 30 mins.
3 hrs.
3 hrs. 30 mins.
4 to 5 hrs.
7 hrs.
8 hrs.
9 hrs.
19 hrs.
26 hrs.
40 hrs.
45 to 50 hrs.
70 to 72 hrs.
75 to 80 hrs.
77 hrs.
4 to 7 days
7 to 10 days
14 to 15 days
22 to 27 days
28 days
47 days
There is also considerable variation in the rate of devel-
FIGURES
opment in different eggs, irrespective of temperature. In order
to avoid referring repeatedly to the age of different stages, I
have compiled in table 1 the average time for each of the more
important stages.
The data are taken from material reared in
the laboratory and the time is determined from oviposition.
6 J. T. PATTERSON
2. STRUCTURE, MATURATION, AND FERTILIZATION OF THE EGG
a. The freshly laid egg
The freshly laid egg is a pear-shaped cell, surrounded by a
very thin but tough membrane, which is probably a true cho-
rion (fig. 1). The broad or posterior end of the egg corresponds
to the vegetative pole of other eggs. It is from this region
that the true embryonic cells are formed by the process of cleay-
age. The anterior end of the egg gradually narrows down
and finally terminates in a finger-like process, which is char-
acteristic of the eggs of many parasitic hymenoptera. As
development progresses the content of this process is gradually
taken into the egg proper, so that in later stages it is no longer
seen.
As a matter of fact, the different eggs vary greatly both in
shape and in size (figs. 1 to 13). The average unfixed egg meas-
ures about 155 » by 60 u in its major axes. Its content consists
of a very finely granular protoplasm, in which are found a few
yolk or oil spherules (fig. 34). In the fertilized egg there are
found three distinct bodies, the oocytic nucleus, the sperm, and
the so-called nucleolus.
In the freshly deposited egg the Sey is an oval-shaped body
in which the chromatin appears as elongated threads running
more or less parallel with the long axis of the nucleus (figs. 1, 2).
It is always situated well toward the anterior end of the egg,
and usually near the base of the finger-like process. Martin
(14) and Hegner (’14, ’15) have traced out the origin of the
nucleus in two species of polyembryonic hymenoptera (Ageni-
aspis fuscicollis, and Copidosoma gelechiae). According to the
accounts of these writers, it has a rather remarkable history.
In Copidosoma the young oocyte in its nurse chamber has a
very large nucleus, in which is found an irregular, deeply stain-
ing mass of chromatin. The cytoplasm forms a very thin layer
about the nucleus. During the growth period the cytoplasm
increases rapidly, while the nucleus enlarges but very little.
Later the chromatin loosens up and forms a spireme, which
finally breaks up to form thin, irregular-shaped chromosomes.
DEVELOPMENT OF PARACOPIDOSOMOPSIS 7
The chromosomes then become shorter and thicker, and appear
to unite near their ends—a process somewhat resembling synap-
sis. The pairs of chromosomes straighten out and become ar-
ranged in a parallel series, with the points of union lying at the
equator. According to Hegner, the number of rods thus arranged
is eleven or twelve, but in reality there should be only ten, for
the cytological studies of two of my students have demonstrated
clearly that the diploid number of chromosomes in Copidosoma
gelechiae is twenty. At first the parallel pairs of chromosomes
are scattered, but the entire mitotic figure soon undergoes con-
densation, by which the chromosomes become shorter and thicker
and are brought close together. Finally, there is produced a
homogeneous mass of chromatin, and all trace of individual
rods is lost.
Martin’s account of the origin of the nucleus in Ageniaspis,
although differing in details, is in agreement with that just
given for Copidosoma. The entire process is peculiar, especially
in the light of the maturation divisions, which are soon to be
described. It may be, as Hegner (14) suggests, that this pre-
cocious, mitotic-like figure is comparable to the disappearing or
aborting spindle which has been observed in the eggs of tur-
bellarians and polyclads. The important point is that the
chromatin body found at the anterior end of the freshly laid
egg of these parasites is a derivative of the germinal vesicle,
and hence is the true oocytic nucleus.
In fertilization the entire spermatozoon enters the egg. The
head of the sperm is frequently sickle-shaped, with a long taper-
ing tail attached (figs. 1, 3). Apparently, the sperm may enter
the egg at any point on the surface of the posterior region. This
conclusion is based on a study of many fertilized eggs which
had just been deposited. Both Marchal (04) and Martin
(14) believe that they can demonstrate the presence of a micro-
pyle on the surface of the anterior process of the egg of Ageni-
aspis, which would indicate that the entrance of the sperm was
restricted to that point.
The most remarkable body present in the cytoplasm is the so-
called nucleolus. It was first described by Silvestri, who thought
8 J. T. PATTERSON
that it came from the oocytic nucleus, and hence its name. This
structure has given rise to a great deal of discussion, and no
less than five different theories have been advanced to explain
its genesis. It was not observed by Marchal (’04) for the egg
of Ageniaspis, but later Silvestri (’08) and Martin (’14) both
demonstrated its presence in the egg of this species. Martin,
who gives a very clear account of the history of this body, first
~ demonstrated that it arises outside of the germinal vesicle before
the nuclear wall breaks down, and hence could not be regarded
as a true nucleolus. He showed that it first appears in a
young oocyte as a collection of small deeply staining bodies,
among a cloud of very fine granules, situated near the posterior
end of the cell. It gradually increases in size and becomes fully
formed at about the same time the egg attains its full size.
In the meantime, Hegner (’14) also showed that in the egg of
Copidosoma gelechiae the nucleolus was not a plasmosome
coming from the germinal vesicle. However, he reached the
untenable conclusion, based on a study of an incomplete series
of sections, that the matured egg was a composite structure,
produced by the fusion of two oocytes. He thus derived the
nucleus of the egg from the germinal vesicle of one of the oocytes
and the nucleolus from that of the other oocyte.
Silvestri (14) replied almost immediately in an article deal-
ing with the development of Copidosoma buyssoni. In this
paper he admits his error in deriving the nucleolus from the
germinal vesicle of the oocyte, and suggests the possibility that
it may arise from a group of granules lying near the posterior
side of the nucleus. He offers the term oosoma in lieu of nucle-
olus. He also points out that what Hegner regarded as a
composite structure in sections is in reality only the anterior
and posterior ends of the same oocyte—a correction which
Hegner (715) accepts.
The chief interest in the nucleolus lies in the fact that a very
important function has been assigned to it by Silvestri and
Hegner. Silvestri (06) showed that it is distributed to a single
blastomere of the four-celled stage in Litomastix, and suggested
that this cell may be the progenitor of all the germ cells of the
DEVELOPMENT OF PARACOPIDOSOMOPSIS 9
sexual larvae. Hegner (714) has elaborated this idea, and classi-
fies the nucleolus as a germ-line determinant. I have elsewhere
(17 a) pointed out the difficulties which stand in the way of
accepting this interpretation, especially as regards its applica-
tion to the origin of the asexual larvae in Litomastix and Paracopi-
dosomopsis.
I have gone into the subject of the origin and function of the
nucleolus rather fully with the hope of showing how necessary
it is that a reinvestigation of its genesis and fate should be made.
Perhaps this could best be done by the methods employed in the
study of mitochondria.
b. Maturation
I have elewhere (718) described maturation and fertilization,
and the account given here may be confined to a brief statement
of the principal points of interest.
1. The first maturation. The process of maturation is identi-
cal in fertilized and unfertilized eggs. As is the case in many
other hymenoptera, the maturation divisions involve only the
chromatin, and consequently distinct polar bodies are not formed.
The first maturation spindle is formed about fifteen minutes
after the egg is laid, and within the next ten or fifteen minutes
the chromosomes have reached the late anaphase stage (fig. 3).
The long axis of the spindle is not quite parallel with that of the
egg, but it meets the latter at a slightly oblique angle. This
brings the outer end of the spindle near to the surface of the egg
at the base of the anterior process. The chromatin of the first
polar body is therefore found in this region.
The first maturation division results in reducing the number of
chromosomes from sixteen to eight (figs. 26 to 28). In certain
cases one can easily count eight chromosomes in the first polar
body and in the second oocyte (e.g., fig. 27).
2. The second maturation. The second maturation follows
almost immediately after the first is completed, without the
reorganization of a nucleus. Likewise the first polar body chro-
matin forms a spindle and divides without forming a nucleus.
These two divisions may occur simultaneously (fig. 29), or the
10 J. T. PATTERSON
first polar body division may either precede (fig. 28) or follow
(fig. 4) the second maturation division. Consequently, there
is no close correlation between the two divisions. This is exactly
the condition in Ageniaspis as reported by Martin.
Each of the two divisions is equational in character. In
figure 28 is a remarkably clear case of the late anaphase stage
of the first polar body division. At each pole of the spindle
are eight distinct chromosomes (Ai, Az). In figure 29 the ootid
(B.) shows eight chromosomes, and in the second body (B,)
seven are visible. Doubtless one of the chromosomes is hidden
by some of the other seven, for in other figures one can count
eight in the second polar body (fig. 31, B:). The result of these
two divisions is the formation of four groups of chromosomes,
of which three are polar bodies (fig. 29, A:, As, Bi) and one the
ootid (B.). The latter forms the female pronucleus.
3. The formation of the polar nucleus. At this point we shall
describe the formation of the polar nucleus, which is destined to
play an important role in the development of the polygerm.
This body was first described, under the term paranucleus, in
the egg of Ageniaspis by Marchal (’04), who failed to observe
its formation, but who gave a very good account of its later
history.
It was next described by Silvestri for the egg of Litomastix.
The process of maturation in this species is identical with that
of Paracopidosomopsis. Consequently, at the close of matura-
tion the egg of Litomastix contains, in addition to the nucleo-
lus and the female pronucleus, three masses of chromatin lying
close together, but distinct from one another. These are the
three polar bodies. In connection with his account of the first
and second cleavages, he makes the following brief statements
concerning the origin of the nucleolus from the polar bodies:
That the three polar nuclei, ‘‘which during such a period are
close together, fuse together to form a single mass of chromatin,
a nucleus without membrane and with the chromosomes con-
densed”’ (’06, p. 14); and later, ‘‘ During this stage the chroma-
tin mass of the polar bodies is formed into a complete nucleus
with membrane and reticulum very distinct, and is always found
DEVELOPMENT OF PARACOPIDOSOMOPSIS 11
in the anterior part of the egg” (p. 15). Silvestri also gives a
very clear description of the fate of the polar nucleus.
In a paper published two years later, Silvestri (’08) described
the formation of the polar bodies in the egg of Ageniaspis. The
polar bodies are formed exactly as in the egg of Litomastix,
but their subsequent history is somewhat different. The three
masses of chromatin usually become reconstituted, each with a
reticulum and membrane, and all three lying more or less on
top of one another. In some eggs, however, the three polar
nuclei fuse to form a single mass, while in other eggs the second
polar body and the inner nucleus of the first polar body (or only
one of them) divide irregularly into parts, thus producing in
all some four or five nuclei. In the period between the third
and fourth cleavages, the polar nuclei lose their membranes,
and their chromatin becomes scattered in the form of minute
granules. This entire structure is now recognized as the para-
nucleus of Marchal, and the polar protoplasm surrounding the
embryonic cells in which it lies is his trophamnios.
Martin (14) has since reinvestigated the early development of
Ageniaspis, with the express purpose of studying the origin of
the paranucleus. He also finds that three polar bodies are formed,
but his account of their exact origin and position does not seem
to me to be entirely consistent. He states that the two chro-
matin masses which lie toward the center of the egg are both
derived from the first polar body, while the third mass situated at
the extreme anterior end of the polar region is the second polar
body. He bases his conclusion on certain stages in which the
second maturation division precedes that of the first polar body.
It is very difficult to understand how a chromatin mass, such as
that of the second polar body, could reach the position assigned
to it by Martin in his figure 16. Furthermore, he admits that
the time relation may be just the reverse of that seen in this
figure. I believe it is practically certain that the three chroma-
tin masses shown in his figures 17 and 18 are incorrectly labeled.
In each figure I should interpret both the anterior and middle
masses as derivatives of the first polar body, and the posterior
mass, not the anterior, as the second polar body. As to the sub-
12 J. T. PATTERSON
sequent history of the polar bodies, Martin in the main is inagree-
ment with Silvestri. His account of the organization of the
paranucleus and trophamnios is especially clear.
I have given the subject of the formation of the polar nucleus,
which is the homologue of the paranucleus, very careful study,
and the conclusion at which I have arrived is based on an examina-
tion of several hundred eggs, both in sections and in wholemounts.
This conclusion is slightly at variance with that reached by Sil-
vestri in his studies on the egg of Litomastix. So far as the
formation of the polar bodies is concerned, I am in complete
agreement with Silvestri; but according to my observations on
Paracopidosomopsis, the second polar body and the inner
nucleus of the first body fuse to form the polar nucleus, and this
occurs irrespective of the time relations between the second
maturation and the polar body division.
At the close of maturation the female pronucleus moves toward
the sperm, which is situated at the posterior end of the egg,
leaving the three polar bodies at the anterior end. The polar
bodies are almost invariably arranged in a row (figs. 5, Ai, As,
B,). At this stage each polar body consists of a number of
delicate chromatin threads or rods surrounded by a clear space.
In later stages the two posterior polar bodies come to lie close
together, in a single clear space, and somewhat apart from the
third or anterior group of chromatin (figs. 8, 11, 18, 14, 31).
In some eggs one can still recognize the individual chromosomes
(figs. 31, As, B,), but from now on their individuality gradually
disappears, and the single body thus formed consists of a coarse
reticulum of chromatin (figs. 10, 23, P). This body is of course
the formative polar nucleus, and by the time the four-celled
stage is reached it is completely organized and appears as a con-
spicuous figure lying at the base of the anterior process (figs.
TGS: 291.P),
In the meantime the outer nucleus of the first polar body
undergoes certain changes, the most important of which is
the condensation of its chromatin into a single mass (fig. 23,
31, A,). This body very quickly dissolves and disappears. The
changes here recorded have been observed in a large number of
DEVELOPMENT OF PARACOPIDOSOMOPSIS 13
eggs, and there can be no doubt as to the manner in which the
polar nucleus is organized in this species. However, it is only
fair to state that occasionally one finds eggs in which all three
polar bodies would appear to fuse, or at least only a single group
of chromatin threads or rods can be detected (figs. 16, 24).
This appearance is perhaps more apparent than real and is
probably due to one of two causes. Either the polar body,
Ai, has already disintegrated or else it is hidden beneath the
forming polar nucleus.
The real proof that only two polar bodies enter into the pro-
duction of the polar nuclei is seen in certain eggs in which the
disintegration of the third polar body chromatin has been delayed
until the nuclear membrane is completely formed. Such a con-
dition is shown in figures 22 and 36.
Another line of evidence which supports our conclusion is
obtained in studying the mitotic figures of the dividing polar
nuclei. I have shown above that each polar body received
eight chromosomes. ‘Therefore, if three polar bodies enter the
polar nucleus, its subsequent divisions should reveal twenty-
four chromosomes, or the triploid number. I have succeeded in
finding three clear metaphase plates, and in each case the diploid
number of sixteen chromosomes is present (fig. 25).
c. Fertilization
The egg is inseminated by a single sperm, which penetrates
the surface at any point on the posterior half. Polyspermy
never occurs. The entire spermatozoon enters (figs. 1, 3, 5,
S), but the tail disappears and only the head is transformed into
the male pronucleus. After maturation is completed, the ootid
group of chromatin forms the female pronucleus and at the same
time moves toward the sperm, which now lies at the posterior
end. The two pronuclei thus come to lie close together (fig. 6).
Both nuclei then expand, come in contact with each other (fig. 7),
and finally fuse (fig. 8) to form a single large conjugated or
cleavage nucleus (fig. 9, /.N.), which can always be distin-
guished from the smaller cleavage nucleus of the unfertilized egg
eng 10;"'Cu.),
14 J. T. PATTERSON
During the process of fertilization or in the corresponding
period of the unfertilized egg, the nucleolus gradually moves
down from its original position near the middle of the egg
(figs. 1 to 5, No.) to the side of the cleavage nucleus (fig. 10).
3. THE CLEAVAGE STAGES
a. The first division
Tn respect to cleavage, the egg of polyembryonic insects differs
from that of the typical insect egg, in that the cleavage nuclei
are from the first accompanied by cytoplasmic segmentation.
Another point of interest is the fact that the course of develop-
ment is in nowise modified by fertilization. The history of
cleavage, as well as that of the polygerm, is the same in fertilized
and unfertilized eggs. At least, one can detect no difference.
The first segmentation spindle, which is organized about the
cleavage nucleus, is devoid of asters at its poles (fig. 11). It
takes a position at the extreme posterior end of the egg, with its
long axis approximately at right angles to the long axis of the
egg (fig. 12). The chromatin divides in a typical manner. The
chromosomes soon move to the opposite ends of the spindle, and
remain connected for some time by a series of curved interzonal
fibers (fig. 13). The daughter nuclei are then reorganized and
move in opposite directions, finally coming to rest just inside
the cell membrane (fig. 14). The cytoplasmic division follows.
It starts as a furrow extending around the posterior end and in
a plane practically coinciding with the median longitudinal plane
of the egg. Each end of the furrow passes upward for a distance
equivalent to a third or a fourth of the length of the egg proper,
and then curves to the right and to the left, each branch finally
reaching the side of the egg at a point near its middle (figs. 15,
16, 37):
By this manner of division the egg protoplasm is divided into
three parts, of which the two at the posterior end are the true
embryonic cells or blastomeres. The third, or anterior part,
is the polar region or cap, and this contains the polar nucleus.
It includes slightly more than one-third the volume of the entire
DEVELOPMENT OF PARACOPIDOSOMOPSIS 15
egg. The study of many two-celled stages reveals the fact that
the two blastomeres are not always of the same size. Their
disproportion in size may be accentuated by the position the
plastic egg happens to take on the slide.
The nucleolus becomes associated with one of the daughter
nuclei (fig. 14), and is thus included in the blastomere formed
about that nucleus (fig. 15). It passes into the cell unchanged
(text fig. 1, A-#).
b. The second division
In preparation for the second division, each mitotic spindle
is arranged so that the angle formed by its major axis and the
long axis of the egg is less than a right angle, and with this axis
lying more or less parallel with the outside margin of the cell .
(fig. 16 and text-fig. 1, F-H). When the divisions are com-
pleted, the four-celled stage consists of two cells forming the base
of the egg and two lying above these, one on each side (fig. 18
and text fig. 1,7, J, K,O). This is the typical arrangement; but
there are variations from this typical figure in which only one of
the blastomeres forms the base of the egg (text fig. 1, L, M, N,
P). Such variations may be due to one of two causes, either the
blastomeres shift after they are formed or, what is more probable,
the direction of the mitotic spindle in one or both blastomeres
varies from that seen in such figures as 16.
The nucleolus, which, as we have seen, enters one of the first
two blastomeres, again passes unchanged into a single cell (text
fig. 1, J). The nucleolus is thus invariably inherited by one of
the first four blastomeres. Very shortly after the second division
is completed, this peculiar body breaks up and forms a granular
area lying about one side of the nucleus (figs. 17, 18 and text
fig. 1, J—P).
It would be interesting to know whether it is always received
by a definite cell; that is to say, whether in all four-celled stages
the blastomeres inheriting the nucleolus are homologous. While
this point is difficult to determine, nevertheless, after examining
many two- and four-celled stages, I have reached the conclusion
that its distribution is a matter of chance. In the first place, if
J. T. PATTERSON
16
Text fig. 1, A to P A series of camera lucida outline drawings of two- and
four-celled stages.
DEVELOPMENT OF PARACOPIDOSOMOPSIS 17
there is an appreciable difference in the size of the two blasto-
meres, it is seen to pass with about equal frequency into the large
(fig. 15 and text fig. 1, A, H). and smaller cells (fig. 16 and text
fig. 1, D, G). It is found with equal frequency in the right- and
left-hand blastomeres as they lie on the slide; but this would not
disprove homology any more than would size differences, for it
is evident that the position of an egg on the slide is a matter of
accident, so that what appears from above to be the right side
in one egg may correspond to the left side in another egg. The
point raised above cannot, therefore, be decided from a study of
two-celled stages.
In the four-celled stage the typical condition shows the nucleo-
lar cell to be one of the two upper cells, which is invariably smaller
than any of the other three cells (text fig. 1, /—K,O, P). This
condition is found with very great frequency, and were it not
for certain variations, might easily lead one to conclude that the
nucleolus is handed on to a definite blastomere of the four-celled
stage. The most significant of these variations is the one showing
the nucleolus in one of the lower cells (fig. 15). It is impossible
to homologize the nucleolar cell in figure 18 with that in text
figure 1, K.
The reason why the nucleolus is found so often in one of the
upper cells is not to be explained on the basis of homology, but on
entirely different grounds. I have already pointed out that as
the female pronucleus moves to the posterior end of the egg it
is followed by the nucleolus, and by the time the cleavage nucleus
is organized it comes to lie close to this nucleus, usually to one
side and above, rarely below. In the two-celled stage the nucleo-
lus occupies this same relative position with reference to the nu-
cleus of its blastomere (figs. 14, 15). The cytoplasmic division
which produces the four-celled stage will result in placing the
nucleolus in an upper cell. If the nucleolus sinks below the level
of the equatorial plate (fig. 16), the resulting division will produce
a condition like that seen in figure 18. A study of many four-
celled stages shows that any one of the four cells may inherit
the nucleolus, but that it goes into one of the upper cells much
more frequently than into one of the lower cells.
18 J. T. PATTERSON
c. The third division
The third set of divisions produces the eight-celled stage, all
four cells dividing about at the same time. There is nothing con-
stant about the arrangement of the spindles in preparation for
this division, and hence we find a great variety of cleavage figures
in eight-celled stages.
Some time before the division is completed, the polar nucleus
becomes active and undergoes two divisions. It forms a large
spindle which lies at right angles to the major axis of the egg
(text fig. 1, O). The two nuclei produced by this division are
shown in figure 39, P. These two polar nuclei quickly divide
to produce four, which in turn form spindles. A case of this
kind is seen in text figure 1, P, in which two of the spindles are
in side view and two in polar view. The first two polar-nuclear
divisions and the formation of the spindles for the third all occur
before the eight-celled stage is reached. During the latter stage
the eight polar nuclei, produced by the third division, very soon
divide again to form sixteen nuclei (fig. 19).
It is during the eight-celled stage that another remarkable
change also takes place in the polar cap. The protoplasm of
this region gradually moves down along the sides of the eight
embryonic cells (fig. 19), and finally encloses them by a thin layer
(fig. 20). The layer thus surrounding the embryonic cells is
destined to play an important rédle in the formation of the poly-
germ. It is in a way comparable to the trophamnios of Ageni-
aspis,, but we shall refer to it as the polar region or membrane.
In later stages the polar nuclei from the anterior portion of the
polar region also move down alongside the embryonic cells, so
that the polar membrane becomes nucleated.
d. The fourth division
In the fourth division all of the embryonic cells divide, ex-
cept the two which contain the nucleolar substance. The result
is the production of a fourteen- instead of the typical sixteen-
celled stage (fig. 21). In the four-celled stage the nucleolus
breaks down and its content spreads around the nucleus (fig.
DEVELOPMENT OF PARACOPIDOSOMOPSIS 19
39), and gradually becomes scattered throughout the cytoplasm
of the two daughter cells (fig. 40). In the fourteen-celled stage
these two blastomeres are recognized easily, owing to the presence
of the nucleolar granules, which cause the cytoplasm to take a
deeper stain (figs. 41, 42). They lie well toward the top of the
group of enclosed embryonic cells.
It is clear that the presence of the nucleolus or its material
exerts a retarding influence on the divisions of the cells which
happen to inherit it. The inhibitory influence is sometimes
shown in the formation of the eight-celled stage from the four.
In some eggs (text fig. 1, O, P) while the nucleus of the nucleolar
blastomere is still in the prophase condition, the spindles of the
other three cells have reached the metaphase stage.
The fact that the nucleolar substance retards divisions has
been noted by other investigators. Silvestri (’06, ’08) observed
this phenomena in the eggs of Litomastix and Ageniaspis, and
Martin (14) has shown in the egg of the latter species that in
the two-celled stage the nucleolar blastomere does not divide
so quickly as the sister cell. There is thus produced a typical
three-celled stage. At this point of development the nucleolus
breaks down and cannot be traced further.
e. The fifth division
In the fifth division all of the blastomeres, including the two
which inherited the nucleolus material, divide, thus producing
twenty-eight cells. In this stage one can still recognize the four
descendants of the original nucleolar cell by the fact that the
granules in their cytoplasm cause them to take a deeper stain than
the other embryonic cells. In figure 43 three of these cells are
clearly seen; the other lies in an adjacent section. The egg
from which the figure is drawn has twenty-seven cells, but one
of the blastomeres is dividing to produce the typical twenty-
eight-celled stage. The cells do not form a solid mass, for owing
to their rounded condition many interstices are found.
The polar region has formed a definite and complete membrane
around the blastomeres through the gradual movement of its
20 J. T. PATTERSON
protoplasm toward the posterior end. This results in trans-
forming the elongated, pear-shaped egg into a figure more or
less circular in outline. The number of polar nuclei at this
stage is sixteen. In one egg all sixteen polar nuclei are dividing
simultaneously (fig. 44). After this period these divisions become
irregular, as indicated by the fact that one frequently finds single
nuclei in mitosis.
f. The morula stage
After the twenty-eight-celled stage all synchrony in division
is lost, and one may find from one to several blastomeres under-
going division in any egg. Consequently a typical fifty-six- or
112-celled stage is not seen. We may therefore consider together
several eggs which represent steps leading up to the formation
of a solid-ball stage, or what may be called the morula stage.
Figure 45 is a section through a fifty-two-celled stage. There
are present four nucleolar cells, showing that these cells have not
further divided. Figure 46 represents a sixty-celled stage. The
polar membrane is remarkably clear and of almost equal thick-
ness around the entire egg. This stage represents a condition
characteristic of this period of development, viz., a tendency
in certain cells for the cytoplasm to become drawn out into an
elongated process. Such cells often become spindle-shaped.
Figure 47 is a median section through an egg composed of
about seventy cells. It is of peculiar interest in that it represents
the most advanced stage in which one can recognize the de-
scendants of the nucleolar blastomere. In the section drawn
five of these are clearly visible, and a sixth lies in an adjacent
section. It is clear that an irregularity in the division of these
cells has already set in, which is further evidenced in other eggs
showing but one of the four nucleolar cells undergoing division.
The nucleolar cells therefore follow the rule of loss of synchrony in
divisions as do the other cells.
Figure 48 is a median section of a 135-celled stage, which has
to a remarkable degree retained the original pear-like shape of the
egg. At the anterior end there exists a rather interesting con-
dition, which has been noted in some other eggs. A single
DEVELOPMENT OF PARACOPIDOSOMOPSIS 2
large cell (marked X) is embedded in the polar cap. In some
eggs two or even more such cells may be found. The first im-
pression one gains on examining such preparations is that these
have been organized about polar nuclei from the polar protoplasm.
But a detailed study of several eggs showing a similar condition
has convinced me that these cells have separated from the main
mass of embryonic cells and have pressed up into the plastic
polar region. A very clear case, which supports this interpreta-
tion, is shown in figure 49. The two large cells (X) have their
upper portions embedded in the polar cap, while their under
surfaces are still connected with the other embryonic cells by
means of protoplasmic strands.
In stages younger than this one it is not unusual to find several
large blastomeres lying in contact with the under surface of the
polar cap (figs. 45, 47). Silvestri (’06) has noted a similar group
of cells in the egg of Litomastix, and attributes to them an
important significance; but it seems more reasonable to suppose
that they owe their large size to their proximity to the polar cap
which undoubtedly serves as a nutritive organ to the growing
embryonic cells.
Figure 50 is the final morula-like ree that we need consider.
It has 221 cells which form a solid spherical mass. Some of the
cells are spindle-shaped, others are polygonal. The latter class
is frequently grouped together (fig. 50, Y). In one region of
the egg a group of polygonal cells has become transformed into
a nest or cyst, in which the core consists of several cells surrounded
by a layer formed by the fusion of spindle-shaped cells. The
central group is made up of the true or definitive embryonic cells
(fig. 51, D.H#.C.). The outer layer becomes syncytial in charac-
ter (fig. 51, J.M.C.), and finally forms the inner membrane
of the primary mass and their derivatives in the polygerm.
In this stage the polar membrane is of equal thickness about the
entire embryonic mass, and its nuclei are fairly evenly distributed.
At certain points mesenchyme cells, derived from the host
tissue, adhere to its outer surface (figs. 49, 50, M.C.).. In most
eggs these mesenchyme cells are isolated, although in one case
they formed a membrane over about half the circumference.
22 J. T. PATTERSON
However, they never form a complete membrane, as Marchal
(04) observed in the egg of Ageniaspis.
We shall conclude this section by a statement concerning the
fate of the cells which inherit the nucleolar materials, as we
shall have no further occasion to refer in detail to that subject.
In text figure 2 I have outlined in diagram form the history of
the distribution of that body up to and including the twenty-
eight-celled stage. In certain instances the descendants of the
four nucleolar cells of the twenty-eight-celled stage may be
recognized (fig. 47, No. C.)}, but beyond the seventy-celled stage
one can no longer follow their history, at least in preparations
made by the usual methods of technique. There is nothing in
the subsequent history of the egg to show that these cells have
been set aside for special function or that their behavior is
different from that of the descendants of the other three blasto-
meres. It is true Silvestri has formulated the very attractive
hypothesis that the nucleolar cells may become the primordial
germ cells for the sexual embryos which later develop. He has
apparently strengthened this hypothesis by his studies on the
development of the monembryonic egg of certain parasitic
species (Silvestri, ’08) in which he was able to show that a similar
nucleolar-like body is included in the primordial germ cell, and
thus may be regarded as a germ-line determinant.
Aside from the failure to trace these so-called germ cells to the
separate embryos, there are two other objections which are fatal
to his hypothesis. In the first place, it is impossible to conceive
of a mechanism which could operate in such a manner as to
parcel out exactly predestined germ cells to the several hundred
embryos. It would seem that some embryos might receive too
many germ cells, while others might receive none at all. To be
sure, his corollary hypothesis, that the asexual larvae owe their
asexuality to the absence of germ cells, would account for the
latter slip in the mechanism; but I have elsewhere (’17 a) pointed
out that these non-viable larvae are probably the result of an
entirely different cause. In the second place, I hope to show that
in some cases an embryo is derived from a single cell during the
late history of the polygerm. If this can be established as a
DEVELOPMENT OF PARACOPIDOSOMOPSIS 23
fact, then obviously a given embryo does not originate from two
kinds of cells, one of which is derived from predestined germ cells.
The best that can be said for the similarity in the distribution of
the nucleolus in the monembryonic and polyembryonic eggs
QoQaAO*EOQ © CCE
OOOOOOOGO*OOO0OO0O OG
Sh ts lab blases @Q@OOO©
Text fig. 2 Diagram showing the distribution of the nucleolus up to the
twenty-eight-celled stage.
is, that while the latter has inherited this condition from the
former, the nucleolus has ceased to function as a germ-line
determinant, owing to the increase in complexity of development
in the polyembryonic egg.
24 J. T. PATTERSON
4, FORMATION OF THE POLYGERM
The first steps leading to the organization of the polygerm can
be observed as early as the 220- to 225-celled stage (fig. 50). As
we have already noted, the initial step consists in the differen-
tiation of the embryonic cells into two classes. Certain blas-
tomeres become transformed into spindle-shaped cells, while
others, retaining their polygonal shape, become arranged into
groups. The latter constitute the definitive or true embryonic
cells. The spindle-shaped cells become drawn out into long
processes, which assist in dividing up the egg into its primary
divisions. The cells adjacent to the true embryonic cells tend
to fuse together. Their intervening walls soon disappear, and
thus there is formed about the group of embryonic cells a
nucleated membrane (fig. 51, 7.M.C.). The entire structure
thus formed constitutes a primary mass of the polygerm.
These changes occur between forty and fifty hours after the
egg is deposited. During this time, and for the next few hours,
both kinds of cells multiply rapidly. By the time 500 cells are
produced the polygerm is well advanced in its organization.
Such a stage is shown in figure 54. In this preparation the
primary masses are not especially well defined, for many of
the embryonic cells are shrunken and loosely arranged, due in
part to poor fixation. In some places the cells adjacent to the
true embryonic cells have already formed a nucleated membrane
(fig. 54, I.M.C.). Protoplasmic strands from these cells are
seen extending throughout the egg, in between the formative
primary masses (fig. 54, P.S.).
The next change which takes place in the organization of the
polygerm is the lengthening of the egg along its major axis
(fig. 55). There also occurs at the same time a change in the
staining reaction of the true embryonic cells. They take a very
much deeper stain than do the adjacent nuclei and their cyto-
plasm (fig. 55, 1.M.C.).
Figure 52 is a detailed drawing of an oblique section of ayoung
polygerm seventy-two and one-half hours old. The details of
structure are remarkably clear, making the matter of interpre-
DEVELOPMENT OF PARACOPIDOSOMOPSIS 25
tation comparatively easy. The polygerm is surrounded by the
polar membrane, from the inner surface of which processes ex-
tend in toward the center (fig. 52, P.M.). In more advanced
stages these processes are invaded by the polar nuclei, and the
membrane thus formed eventually surrounds each primary mass,
becoming what we shall call the outer envelope or membrane of
the mass.
Another point of interest in this preparation is the condition
of the inner membrane nuclei. These nuclei and their accom-
panying cytoplasm are in the act of forming the inner envel-
ope of the primary masses. Both stain very lightly (fig. 52,
I.M.C.). Various stages in the formation of the inner envelope
are seen in the preparation. In the upper part of the figure
the nuclei lie free in the cytoplasm which surrounds the dark
embryonic cells. On the lower side of the formative primary
mass lying on the right, a portion of the membrane is fairly
well organized.
This account of the development of the inner and outer en-
velopes of the primary embryonic mass differs somewhat from
that given in an earlier paper, from which I may quote: ‘‘ About
seventy hours after oviposition, the nucleated membrane begins
to invade the embryonic cells by the formation of trabeculae,
which divide the embryonic cells into several groups, or primary
masses. During the formation of these masses, or very shortly
thereafter, the young polygerm elongates in the direction of
the long axis of the egg. In addition to the nucleated membrane,
each primary. embryonic mass develops a second envelope, which
lies just inside the nucleated membrane. Apparently this inner
envelope is formed from the peripheral layer of cells of the em-
bryonic mass”’ (Patterson, 718, p. 365). As a matter of fact,
a more extensive study of a completed series of sections shows
that the initial steps in the formation of the inner envelope pre-
cedes the development of the outer membrane.
The true embryonic cells stand out in sharp contrast to all
other structures in the polygerm (fig. 52, D.H.C.). They have
become spherical in shape. In the section four primary masses
are seen. Two of these are practically completed, while two are
26 J. T. PATTERSON
only forming. The number of embryonic cells included in a
primary mass is extremely variable. I have seen cases where not
more than four or five cells were present in a single primary mass;
in other cases I have counted as many as fifty. The number of
cells included in a primary mass is not a matter of any great im-
portance. The embryonic cells are constantly dividing, so that a
primary mass with a few cells would soon have that number
increased. Furthermore, the primary masses themselves soon
divide, especially those which possess a large number of cells.
Figure 56 represents a further advance in the development of
the polygerm. Several of the primary masses are already com-
pleted (Pr.M.). One of these is differentiating into an asexual
embryo (As.L#.). This is the youngest stage in which one can
recognize the asexual embryos. A more advanced stage is
illustrated in figure 57. The primary masses are practically all
completed. There are fifteen of these masses, in addition to a
large conspicuous asexual embryo (As.H#.). The polar membrane
already shows signs of constrictions, which will result eventually
in forming an outer envelope around each mass.
A completed polygerm is one in which all of the primary masses
are found. Such a stage is shown in figure 58. This one has a
large asexual embryo and about twenty primary masses. The
asexual embryo has both the outer and inner envelopes com-
pletely formed, which has resulted in cutting it off from the rest
of the polygerm. Each primary mass has as yet only the inner
envelope completed. It consists of a distinct, rather thick
membrane containing a large number of nuclei (fig. 58, J.M.C.).
The cavity contains a variable number of true embryonic cells,
loosely arranged and spherical in shape.
Figure 59 represents a stage slightly more advanced than
the preceding. The section contains no asexual embryos, but
a very young one is found in one of the lateral sections of the
series. However, asexual embryos are not found in all poly-
germs of this age. The polygerm illustrated in figure 60 has
no asexual embryo. The significance of this will be discussed in
connection with the history of the asexual larvae, given in a
later section of the paper.
DEVELOPMENT OF PARACOPIDOSOMOPSIS 27
5. HISTORY OF THE POLYGERM
a. Multiplication of the primary masses
In the completed polygerm each primary mass consists of
several embryonic cells surrounded by a relatively thick inner
membrane, and the various primary masses are more or less
separated from one another by ingrowths from the polarmembrane
(fig. 58). Soon after the polygerm is formed, the primary masses
begin to multiply by fission. The division is initiated by a
constriction of the inner membrane, followed by a corresponding
constriction or ingrowth of the polar membrane. In figure
59 some of the primary masses are beginning to divide, and in
figure 60 the one on the left is in the act of dividing.
In the description of these stages we shall refer to the prod-
ucts of division of the primary masses as secondary masses,
and when these in turn divide their products will be referred
to as tertiary masses. In later stages the tertiary masses also
divide a number of times, but such products will be called com-
ponents, whenever they can be distinguished from the ordinary
tertiary masses. During the late history of the polygerm it is
not always easy to determine whether a given mass is secondary
or tertiary. Their general structure is the same and both kinds
are frequently present in the same polygerm, owing to the fact
that the divisions do not occur simultaneously. However, one
can usually distinguish the two kinds of masses by their dif-
ference in size.
Figure 61 is a detailed drawing of a section passing through two
secondary masses that have recently been formed. ‘The mass
on the left has received a single embryonic cell, while the one on
the right has received three such cells. The general rule is for
a secondary mass to have several embryonic cells, but occasion-
ally only a single cell is included.
Figure 62 passes through the middle of a polygerm in which
several of the primary masses are dividing. Some of them have
already completed the division (fig. 62, S.M.). At the points
marked X, Y, Z, are three primary masses in different stages of
division. In all these cases the division has been accomplished
28 J. T. PATTERSON
entirely by the inner membrane or envelope. The constriction
or ingrowth of the polar membrane does not take place until
somewhat later.
The method of division in the formation of tertiary masses
is exactly similar to that just described for the secondaries. No
further details are therefore necessary. The formation of the
tertiaries may begin as early as the end of the fourth day (fig.
73), and continues through the sixth day. From the seventh
to the tenth day the multiplication of the tertiary masses and
their components goes on with great rapidity, and by the eleventh
day they form a very complex structure, which is sometimes
surrounded by adipose tissue developed from the host cells
(fig. 72).
Figures 63 to 68 represent a series of tertiary masses which
have been set free into the body cavity of the caterpillar. They
show the various steps in the multiplication of the com-
ponents of a tertiary mass. Figure 63 is a mass containing a
single embryonic cell. It has just been set free from the main
body of the polygerm. Figure 64 is a slightly later stage in
which the embryonic cells are multiplying. In figure 66 a ter-
tiary has recently divided, and the component on the right has
two cells which have not yet completely separated. Figure
66 is a tertiary component with four cells. Figure 67 is of in-
terest in that it shows how the embryonic cells are being iso-
lated. Ingrowths from the inner membrane have separated
the embryonic cells into groups. In some instances only a
single cell is thus separated, but usually there are two or more
cells in each group. The formation of these groups is then
followed by constrictions of the inner and outer membranes,
which results in producing many new components of the ter-
tiary masses (fig. 68). The components may later completely
separate from each other, becoming scattered throughout the
body cavity of the host and forming new centers of prolifera-
tion. The rate of their distribution to various parts of the body
cavity to a very great extent depends upon their relation to the
host tissues. If the polygerm is embedded in adipose or other
tissue, the scattering of the tertiary masses and their compo-
DEVELOPMENT OF PARACOPIDOSOMOPSIS 29
nents may be greatly delayed (fig. 72). On the other hand, if
the polygerm happens to lie free within the body cavity, the
dispersal of its components may begin very early, even as early
as the primary mass stage.
b. Relation of parasitic egg to host tissues
To understand fully the account of the distribution of the
products of the polygerm, it is necessary to call attention to
the relation of the parasitic egg to the host tissues. The ques-
tion is one of the greatest interest, for it can be demonstrated,
by the means of a very simple experiment, that the development
of the parasitic egg is dependent upon the development of the
host egg. Not only is this true for the late stages, but also for
the initial steps in development.
Marchal (’04), Silvestri (’06), and Martin (’14) have all made
note of certain points on the relationship of the parasitic egg to
the host. In the case of Ageniaspis, Marchal states that in
order for the egg to develop completely it is essential that it be
placed within the embryonic region of the developing caterpil-
lar (Hyponomentus). In all of his preparations showing the egg
of Ageniaspis, he always found it in the body cavity of the em-
bryo, where it normally develops. However, he states that his
preparations are not numerous enough to determine definitely
whether or not some eggs are lost or die if placed in an unfavor-
able position, such as the intestine or yolk. He implies that
some are thus lost, since it does not seem probable that the
parasite could find, by means of its probe, the most favorable
region in which to place the egg. In later stages he discovered
that the epithelial layer which at first forms a cyst about the
developing egg, and then gives rise to the elongated tube of the
chain of embryos, is the product of the host tissue. In the case
of Polygnotus minutus, Marchal discovered the interesting fact
that the egg is lodged in the gastric pouch or stomach of the
host (Cecidomyia), and there, curiously enough, undergoes
its development.
30 J. T. PATTERSON
According to Silvestri’s observations, the egg of Litomastix
may be laid in any part of the host embryo (Plusia), or even in
the yolk outside the embryo. The egg is destroyed if laid either
in the intestine or yolk. In late stages the germ mass may be
found in any part of the young caterpillar, except the intestine
or anterior part of the head. It is most frequently found in the
thorax, either above or below the oesophagus. He also found
the polygerm in the nerve ganglia, especially the brain ganglia.
In Ageniaspis Martin believes that the frequent occurrence
of the egg in the thoracic ganglia of the caterpillar is to be cor-
related with the laying time of the parasite. He finds that the
egg clings to the ganglion in such a manner that the typical
shape of the ganglion is preserved. In late stages of development
he could no longer find the polygerm connected with a ganglion,
which fact leads him to conclude that on account of its growth
- the polygerm is forced out of the ganglion.
My own observationson Paracopidosomopsis very closely paral-
lel those of Silvestri on Litomastix. The egg may be deposited in
any part of the host egg, but disintegrates if it happens to be
placed in the yolk or intestine. In the newly hatched cater-
pillar the egg may be found in any part of the body cavity or
embedded in the tissues adjacent thereto. There are two kinds
of tissues in which it is frequently found, namely, nervous and
adipose.
Both the cephalic and ventral ganglia often contain parasitic
eges. In my preparations I have counted no less than sixty-
three cases of infected ganglia, distributed as follows: one egg
in ventral ganglion, 40 cases; two eggs in ventral ganglion, 3
cases; one egg in supra- or suboesophageal ganglion, 18 cases;
two eggs in brain ganglia, 2 cases. The egg may be deposited
directly in the ganglion. Figure 70 is a portion of a suboeso-
phageal ganglion containing a fertilized egg undergoing matura-
tion. Several similar cases have been observed. Figure 69
is a longitudinal section through the third ventral ganglion con-
taining a well-developed polygerm. Figure 71 is a similar sec-
tion through the fourth ventral ganglion. It contains a large
asexual larvae, several secondary masses, and a small group of
DEVELOPMENT OF PARACOPIDOSOMOPSIS ed:
tertiary masses. Numerous cases similar to these have been
observed. In late stages the embryonic masses break out from
the ganglion and become scattered throughout the body cavity.
I have noticed from my records that most of the cases of
ganglionic infection, especially in the head region, arise in host
eggs that were parasitized during the late embryonic period,
just before the young caterpillar hatches. This is probably to
be explained by the position of the host embryo in the egg. At
the time of hatching the head of the caterpillar is situated at
the apex of the dome-shaped egg, and it is at or near this point
that the female parasite inserts her ovipositor at the time of
laying.
The polygerm is often surrounded or embedded in fat tissue
developed from the host cells. The fat tissue probably starts
to develop from mesenchyme cells such as are shown in figures
49 and 50. The adipose tissue not only serves as a source of
nutriment for the growing polygerm, but it also holds the embry-
onic masses together (figs. 72 to 75), and thus delays their
dispersal.
The relation of the host tissues to the parasitic egg is all impor-
tant in the development of the latter. It can be shown that the
development of the parasitic egg is dependent upon the growth
of the host embryo. This has been demonstrated in the fol-
lowing way. A batch of eggs laid in the laboratory by a virgin
female moth were exposed for an hour to a brood of female para-
sites. Several parasitic eggs were deposited in each host egg.
Two days later these eggs were fixed and sectioned. The moth
eggs of course did not develop, and an examination of the sec-
tions revealed the fact that not a single parasitic egg developed.
Under similar conditions, but with fertilized moth eggs, all of
the parasitic eggs would have been in late cleavage stages.
c. Dissociation of the polygerm
I have already stated that at some period in its history the
polygerm undergoes fragmentation or dissociation. The point
at which dissociation occurs varies greatly in different cases.
JOURNAL OF MORPHOLOGY, VOL. 36, No. 1
ys J. T. PATTERSON
It may take place as early as the fourth day, or it may be delayed
until the eleventh day. Indeed, in some few cases the polygerm
does not completely break up until the larvae are on the point of
being set free. The fragmentation is largely controlled by the
relation of the polygerm to the host tissue. If it lies free in the
body cavity or in loose tissue, dissociation will occur very early;
but if it is embedded in rather dense tissue, such as the ganglion
or fat, the dispersal of the embryonic masses may be greatly
delayed.
The primary masses are organized toward the end of the third
day (figs. 52, 53), and are completed during the early part of
the fourth day. If the young polygerm of this period happens
to be free from host tissue, the separation of the primary masses
may set in. Figure 53 is a polygerm seventy-seven hours old,
and signs of breaking up are apparent. The asexual embryo has
already become completely separated from the other primary
masses. In the same preparation there are several polygerms
from which one or more primary masses have broken away and
lie some distance from the main body of the polygerm.
The usual time for dissociation to occur is during the period
in which secondary and tertiary masses are being formed, that
is, from the end of the fourth to about the tenth day. Figure
69 is a ganglion containing a polygerm ninety-five and one-half
hours old. The secondary masses are beginning to dissociate.
Figure 73 is a polygerm ninety-five hours old and composed of
secondary and tertiary masses. It is undergoing dissociation.
The adipose tissue has nearly all been absorbed and the embry-
onic masses are beginning to scatter.
Figures 74 and 75 are portions of the same polygerm, showing
groups of asexual embryos and tertiary masses, respectively.
The polygerm is nine days and twenty-three hours old. A large
number of tertiary components are found in the body cavity of
the caterpillar, scattered throughout its entire extent.
If bound together by nervous or fat tissue, the embryonic
masses may remain connected until the eleventh day or even
later. Figure 71 is a ganglion containing a seven-day polygerm,
which shows no signs of fragmentation. The polygerm shown
DEVELOPMENT OF PARACOPIDOSOMOPSIS 33
in figure 72 is eleven days old, and only a few masses, on the
left, are beginning to break away. In stages still older one may
find a considerable portion of the polygerm, at the original seat
of infection, still intact.
d. Origin and history of the sexual embryos
The multiplication of the embryoic masses, from the primary
stage to the formation of the tertiary components, is a continuous
process. As already stated, the distribution of the tertiary
masses and their components to various parts of the caterpillar
follows the dissociation of the polygerm. ‘The components thus
distributed became the centers for the formation of groups of
sexual embryos, either directly or after further multiplication,
depending upon the stage at which the scattering occurs. Since
one cannot follow the history of a single tertiary component,
it is not easy to determine at just what point multiplication of
components ceases and embryo formation begins. However,
one can meet this difficulty by studying stages fifteen or sixteen
days old with well-developed sexual embryos, and tracing their
origin back through a series of younger stages.
As the multiplication of embryonic masses progresses the num-
ber of embryonic cells included in each mass naturally becomes
smaller and smaller. This occurs notwithstanding the fact
that the embryonic cells are also multiplying, because the rate
of division of the embryonic cells does not keep pace with the
increase in number of the embryonic masses.
In the tertiary divisions, and more particularly in those of
the components, it is not uncommon for a single embryonic
cell to be separated out into an embryonic mass (figs. 63, 65).
The method of division in the tertiary masses is slightly different
from that found in the case of primary and secondary masses.
Both in the primary and in the secondary masses the division
is effected by a simple constriction of the inner membrane (figs.
60, 62). In the case of tertiary divisions there first grows in
from the inner membrane a number of protoplasmic processes
which divide the embryonic cells into several groups, each con-
34 J. T. PATTERSON
taining one or more of the embryonic cells. The inner membranes
then completely form, and thus separate the groups from one
another (figs. 67, 68). This method of division becomes more
accentuated in the formation of components and the sexual
embryos.
During the thirteenth and fourteenth days the multiplication
of the tertiary masses occurs in the manner just described. At
the end of the fourteenth day tertiary components begin to form
embryonic masses, each of which will produce a-single sexual
embryo. Figure 76 is a section of a tertiary component lying
free in the body cavity, and in which the formation of sexual
embryos is in progress. The section shows six masses, in at
least four of which further divisions will occur. In many places
in the series single embryonic cells are being isolated to form, in
all probability, a sexual embryo (fig. 76, X). Owing to the
fact that a cell may divide immediately after it is isolated, and
usually before the inner membrane is completely organized
about it, it is difficult to establish this point. Nevertheless,
the evidence revealed in an intensive study of this period of
development points to the conclusion that each sexual embryo
arises from a single embryonic cell. Just why components con-
tinue to multiply up to a certain point and then suddenly cease
to divide before producing embryos, is not easy to answer. How-
ever, that this point of departure varies in different cases is
evidenced by the great variation in the number of individual
parasites arising from different eggs.
By the end of the fifteenth day no further divisions of com-
ponents are seen. Each mass represents an individual embryo.
Figure 77 shows the typical condition of this period. Each
embryo consists of several cells, closely pressed together and
surrounded by a well-formed inner membrane. Later, the
embryonic cells form a typical morula stage. From the sixteenth
to the eighteenth day the embryos become well organized. ‘The
inner and outer membranes thin out to form a double-walled,
transparent envelope about each embryo (fig. 78).
On opening up infected caterpillars from the fifteenth to the
eighteenth day, one finds floating in the fluid of the body cavity,
DEVELOPMENT OF PARACOPIDOSOMOPSIS 35
or among the tissues therein, a large number of groups of sex-
ual embryos. Each group results from the fact that the embryos
arising from a single component tend to stick together. The
groups vary in size and shape. Sometimes they form flat or
plate-like structures (figs. 78 to 80). More frequently they are
spherical in shape, which has given rise to the term ‘ball’ stage
inmy notes. The size of the group is determined by the number
of embryos present, and these vary from two to as high as seventy.
In one lot of seventeen groups I counted the following num-
bers: 2 (two), 6, 10 (two), 12 (two), 15, 16, 18 (two) 20 (two),
22 (two), 25, °70.
The embryos develop rapidly from the eighteenth day on,
and sometimes between twenty-second and twenty-fourth days
reach the early larval stage. They then escape from their
capsules into the body cavity of the caterpillar. Once free, the
larvae proceed to devour the contents of the host, first eating
the fatty tissue, and finally devouring the various internal or-
gans. The last of these to disappear are the nervous system
and the intestine. In destroying the internal organs, the larvae
consume such portions as are dissolved by the action of their
salivary secretions. The undissolved parts consist largely of
the chitin of the tracheae. They also destroy all of the body
wall except the superficial layer of chitin.
The larvae pupate on about the twenty-eighth day. During
pupation the non-digested content of the caterpillar hardens
and forms the thin-walled, oval chambers in which the parasi-
tic larvae lie and in which they undergo their transformation
into pupae. According to some observers, a thin cuticular layer
from the larvae forms an inner lining to the chamber and se ves
as a sort of puparium. The layer of chitin of the caterpillar
is perfectly transparent and at first is very flexible. Later,
as drying takes place, it shrinks in on the walls of the chambers
and becomes hard and rigid, the whole thus forming the typi-
cal mummified carcass, characteristic of polyembryonic para-
sites. Under laboratory conditions the parasites emerge from
the pupae on the forty-seventh day.
36 J. T. PATTERSON
e. Origin and history of the asexual embryos
I have already given an account of the history of the asexual
larvae (Patterson, ’18), and shall quote rather freely from that
paper in this section.
The appearance of non-viable, asexual larvae in polyembryonic
hymenoptera was first observed by Silvestri (’06) in Litomastix.
Briefly summarized, his account is as follows. The polygerm of
Litomastix, soon after the polar membrane is established, be-
gins to show differentiation into two distinct regions. ‘The an-
terior part of the egg is made up of large and small cells, while |
the posterior part is composed of small cells only. A constric-
tion develops in the polar membrane, which finally separates
these two regions. Silvestri calls the anterior region the massa
germinigena, and the posterior the massa monembrionale. The
posterior part subsequently differentiates into a single asexual
larva. In the course of further development, the massa germi-
nigena gives rise to a few secondary monembryonal masses,
which develop into asexual larvae, and to a large number of
other masses. This is accomplished by constrictions in the
polygerm. The masses continue to multiply by constrictions,
and from time to time may produce a few asexual embryos, but
a large majority of them develop into sexual embryos. In one
case Silvestri counted 100 asexual larvae arising from one egg;
in a second case he counted about 1700 sexual embryos and 220
asexual larvae. In structure the asexual larva differs from the
sexual larva in that it has no reproductive, respiratory, or cir-
culatory system, and no malpighian tubules.
Silvestri has suggested that the asexual larvae may owe their
asexuality to the absence of germ cells. He bases his suggestion
on the fact that the so-called nucleolus, which in certain monem-
bryonic eggs seems to serve as a ‘keimbahn-determinant,’ is not
inherited by all of the embryonic cells. According to Silvestri’s
suggestion, an embryo arising from cells all of which are deficient
in nucleolar material would be asexual; while one receiving one
or more of these potential germ cells would be sexual. Aside
from the mechanical difficulty (to which I have already referred)
DEVELOPMENT OF PARACOPIDOSOMOPSIS oF
standing in the way of the full acceptance of this hypothesis,
there is the further objection that it does not explain the absence
of organs other than those of reproduction, nor does it take into
account the fact, established by experimentation, that secondary
sexual characters in insects, as well as certain primary organs,
such as those of copulation and oviposition, do not depend upon
the presence of gonads for their development.
In Paracopidosomopsis I have found similar larvae, which
never undergo metamorphosis and are non-viable. In this
species the asexual embryos can be recognized in young polygerms
seventy to seventy-two hours old. Figure 56 shows the youngest
stage that I have found. The young asexual embryo is distin-
guished from the other embryonic masses by two features of
its organization. It has a larger number of cells and the inner
membrane is relatively thicker than in the primary masses.
The embryonic cells multiply very rapidly and soon form a
solid spherical mass (fig. 57, As.H.). At the seventy-two hour
stage the asexual embryo gives evidence of differentiation, and
is surrounded by completed inner and outer membranes (figs.
53, 58, As.#.). It is frequently separated from the rest of
the polygerm. A single asexual embryo may frequently arise
during the primary mass stage of the polygerm, but it is
not the universal rule. In some polygerms of this stage no
asexual embryo is present. Furthermore, it frequently happens
that two or more asexual embryos may arise in a single polygerm
at this early period. Figure 72 shows two young asexual larvae
that must have started their development during the primary
mass stage. The polygerm shown in figure 73 has four asexual
embryos, all in the same stage of development, but situated at
different points in the polygerm. In figure 56 the asexual em-
bryo has arisen at the side of the polygerm; in figure 57, at the
anterior end, and in figure 58, at the posterior end. All of these
facts show that in Paracopidosomopsis an asexual embryo may
arise at any point in the young polygerm, and not habitually
from the posterior region of the egg, as reported for Litomastix
by Silvestri.
38 J. T. PATTERSON
While some polygerms produce asexual embryos at a very
early stage, nevertheless the majority of such embryos do not
appear until after dissociation has taken place. Their produc-
tion in a given polygerm is not confined to a single period of
development, but is a continuous process, extending from the
third to about the fifteenth day. They arise during both the
secondary- and tertiary-mass stages. Sections of practically
every polygerm from twelve to fourteen days old will show
asexual individuals in various stages of development, from
young embryos to fully developed larvae.
During the secondary mass stage one is struck by the frequence
with which they are found in groups. In some groups there
may be as high as ten or twelve individuals. Figure 74 shows
one of these groups embedded in fat. A group of tertiary mass
from this same polygerm is seen in figure 75.
The frequent appearance of asexual embryos or larvae in
groups suggests that, like the sexual embryos, the individuals
of a group have a common origin, probably arising through the
division of a single secondary or tertiary mass.
Single asexual embryos also develop, in conjunction with a
group of sexual embryos. In one case I found an asexual em-
bryo joined to a single sexual embryo, which is still in the morula
stage. In figure 81 is a group of ten sexual embryos and one
asexual embryo all held together by their membranes. In fig-
ure 83 is a fully developed asexual larvae, freed from its capsule,
but still connected by the head to a group of sexual embryos.
In developing into a larvae the asexual embryo becomes bent
upon its long axis, with the ventral surface forming the concave
side (fig. 72, As.H.). Just before escaping from the capsule,
the larva has a characteristic shape, like the letter C (fig. 82).
Once set free, the larvae present various figures, such as are
seen in figures 83 to 86.
The asexual larvae invariably degenerate, apparently they do
not live over three days as free larvae. The first free larvae
appear on the twelfth day, and degenerating specimens are
found on the fifteenth day. The last larvae escape from their
capsules on the sixteenth day and none are found after the
DEVELOPMENT OF PARACOPIDOSOMOPSIS 39
eighteenth day. The beginning of degeneration is marked by a
foreshortening and twisting of the body. The larva becomes
immobile and soon disintegrates (figs. 87 to 90). These larvae
apparently perform no function, for there is no evidence that
they break down the tissues of the host preparatory to assimi-
lation by the sexual larvae. They disappear at least a week
before the sexual larvae are set free from their envelopes.
As one cannot follow the course of development of a single
egg, but must depend upon series of sections and dissections, it
is impossible to determine whether every polygerm eventually
produces asexual larvae. It is possible that some do not. For
the same reason, it is difficult to determine the exact number of
asexual larvae produced by a given egg. This in part is due to
the fact that these larvae are formed continuously from the
third to the fifteenth day, and those first developed degenerate
before the last ones appear. The largest number of larvae found
in a single case is fifteen. The data collected from dissecting
a large number of infected caterpillars, reared in the laboratory,
indicate that no more than twelve or fifteen such larvae are pro-
duced in a single egg.
In conclusion, I should like to point out some of the more
important problems which need further study. These are:
1) The exact origin and the late history of the nucleolus; 2) the
morphology of the sexual larvae, with especial reference to the
origin of germ cells; 3) the morphology of the asexual larvae,
which should be compared with that of the sexual larvae; 4)
the causes underlying the origin of mixed broods and asexual
larvae.
6. SUMMARY
1. There is one generation of Paracopidosomopsis for each
generation of the Autographa moth, at least for the fall months.
2. The parasite will deposit its egg in the host egg at any time,
but does not parasitize the young caterpillar after hatching. It
lays one or two eggs at each oviposition. In about two times
out of three two eggs are deposited.
40 J. T. PATTERSON
3. The egg may be placed in any part of the host egg, but does
not develop unless embedded in the tissues of the host embryo
or larva.
4, The freshly laid egg is pear-shaped, and contains, in addi-
tion to the nucleus, a large nucleolus. The broad end of the egg
is posterior and the narrower end anterior.
5. In fertilization only a single sperm enters. Polyspermy
never occurs.
6. The maturation divisions are typical, and result in reduc-
ing the number of chromosomes from sixteen to eight. The
polar body chromosomes do not form nuclei, and hence are not
accompanied by cytoplasmic segmentation. ‘Two of the three
groups of polar body chromosomes fuse to form a polar nucleus;
the third disintegrates.
7. The egg develops whether fertilized or not. If unfertilized ©
it produces a brood of males. Eggs laid by a fertilized female
produce mixed broods.
8. The cleavage nuclei are from the first accompanied by
cytoplasmic segmentation. . Cleavage is confined to the poste-
rior end of the egg, and eventually results in producing a morula-
like stage.
9. The nucleolus is inherited by only one of the first four blas-
tomeres. Its history can be traced accurately to the twenty-
eight-celled stage, in which its materials are distributed to four
cells. There is no evidence indicating that the descendants of
these four cells become the germ cells of the sexual embryos.
10. The polar nucleus divides, forming several nuclei. These
with the cytoplasm of the anterior third of the egg flow down and
surround the embryonic cells or blastomeres, finally forming a
nucleated membrane or envelope.
11. The morula develops into a polygerm. which consists of
a number (fifteen to twenty) of primary masses. Each primary
mass consists of a group of definitive embryonic cells, surrounded
by an inner membrane. This membrane is formed from certain
blastomeres during the development of the polygerm.
12. The primary masses multiply by constrictions of the inner
membrane, followed by constrictions or ingrowths from the
DEVELOPMENT OF PARACOPIDOSOMOPSIS 41
polar membrane. The products of these divisions are known
as secondary masses, which in turn multiply by similar con-
strictions to form tertiary masses. The tertiary masses later
divide to produce components.
13. At some time during the period of multiplication of the
masses the polygerm undergoes fragmentation or dissociation.
The masses become scattered throughout the body cavity of the
caterpillar, and form new centers either for further divisions or
for the production of sexual embryos.
14. The sexual embryos arise from tertiary components. In
some cases one can trace the origin of an embryo to a single
embryonic cell.
15. Asexual embryos may arise as early as the primary-mass
stage of the polygerm, but the greater number of them develop
during the secondary and tertiary stages. These embryos pro-
duce non-viable larvae, which do not live over three days in the
body cavity of the caterpillar. Not over twelve to fifteen such
larvae arise from one polygerm.
Austin, Texas,
September 17, 1920
42 J. T. PATTERSON
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810-818.
1919 Two new instances of polyembryony among the Encyrtidae.
Science, vol. 49, pp. 43-44.
KoRNHAUSER, SipNEY I. 1919 The sexual characteristics of the Membracid,
Thelia bimaculata. I. External changes induced by Aphelopus
theliae. Jour. Morph., vol. 32, pp. 531-636.
Kuracin, N. 1898 Beitriige zur Kenntnis Entwicklungsgeschichte, von Platy-
gaster. Zeit. f. wiss. Zool., Bd. 63, S. 195-235.
MarcuaL, Paut 1897a Les Cecidomyies des cereales et leurs parasites. Ann.
de la Soc. Ent. de France, T. 66, pp. 1-105. 7
1897 b Contribution A l’etude du developpement embryonnaire des
Hymenopteres parasites, Platygaster. C. R. Soc. Biol., T. 4, pp.
1084-1086.
1898 a La dissociation de l’oeuf en un grand nombre d’individus
distincts et le cycle evolutif de ’Encyrtus fusicollis. C. R. S. Se.,
T. 126, pp. 662-664.
1898 b A new method of asexual reproduction in hymenopterous
insects. Nat. Soc. London, vol. 12, pp. 316-318.
DEVELOPMENT OF PARACOPIDOSOMOPSIS 43
MarcHat, Pavt 1898 c Un exemple de dissociation de l’oeuf de cycle de
l’Encyrtus fuscicollis. C.R. Soe. Biol., T. 10, pp. 238-240.
1898 d_ Le cycle evolutif de 1’Encyrtus fuscicollis. Bull. Soc. Ent.
France, pp. 109-111.
1899 Comparison entre le developpement des Hymenopteres parasites
a developpement monoembryonnaire. C. R. Soc. Biol. T. 1, pp.
711-713.
1902 Observations sur la Biologie des Hyponomeutes. Bull. Soc.
d’études et de vulgarisation di la Zool. Agricole de Bordeaux, T. 1,
fase. 4, pp. 13-26. :
1903 Le cycle evolutif du Polygnotus minutus. Bull. Soc. Ent.
France, pp. 90-93.
1904 a Le déterminisme de la Polyembryonie specifique et la déter-
minisme du sexe dans la polyembryone specifique des Hymenopteres.
C.R. Soc. Biol., T. 56, pp. 468-470.
1904 b Sur la formation de l’intestin moyen chez les Platygasters.
C. R. Soc. Biol., T. 56, p. 1091.
1904 ¢ Recherches sur la biologie et le developpement des hymenop-
teres parasites. I. La polyembryonie specifique ou germinogonie.
- Arch. d. Zool. Exp. et Gen., T. 2, ser. 4, pp. 257-335.
1906 Recherches sur la biologie et le developpement des hymenopters
parasites. II. Les platygasters. Arch. de Zool. Exp. et Gen., T. 4,
ser. 4, pp. 485-640.
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110, S. 419479.
Patterson, J. T. 1915 Observations on the development of Copidosoma
gelechiae. Biol. Bull., vol. 29, pp. 333-373.
1917 a Studies on the biology of Paracopidosomopsis. I. Data on
sexes. Biol. Bull., vol. 32, pp. 291-305.
1917 b Studies on the biology of Paracopidosomopsis. III. Matura-
tion and fertilization. Biol. Bull., vol. 33, pp. 57-67.
1918 Studies on the biology of Paracopidosomopsis. IV. The asexual
larvae. Biol. Bull., vol. 35, pp. 362-377.
1919 Polyembryony and sex. Jour. Heredity, vol. 10, pp. 344-352.
PatTEeRSON, J. T., AND PortEeR, Levia T. 1917 Studies on the biology of Para-
copidosomopsis. II. Spermatogenesis of males reared from unferti-
lized eggs. Biol. Bull., vol. 33, pp. 38-51.
PerRRIER, E., pt GRAvieR C. 1902 La tachygenese ou acceleration embryo-
genique. Ann. Sc. Nat. Zool., 8 ser., T. 16, pp. 133-871.
Ritey,C.V. 1869-70 First annual report of the State Entomologist of Missouri.
1883 Annual Report of the Entomologist, U.S. Dept. of Agriculture,
pp. 99-180.
Ritey, W. A. 1907 Polyembryony and sex-determination. Science, vol. 25,
p. 106.
SACKEN, Baron OsteN 1863 Lasioptera reared from a gall on the goldenrod.
Proc. of Ent. Sco. of Phila., vol. 1, pp. 368-370.
44
J.) T. PATTERSON
SaRRA, RAFFAELLE 1915 Osservazioni biologiche -sull’ Anarsia lineatella Z.
dannosa al frutto del mandorlo. Boll. Lab. Gen. Agr. Zool. Sup.
Seul. Agr., Portica, vol. 10, pp. 51-55.
1918 La variegana (Oelthreutes variegana Hb., Lepidottero Tor-
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della R. Scoula sup. d’Agri. in Portici, vol. 12, pp. 175-187.
Srtvestri, F. 1905 Un nuovo interessantissimo caso di germinogonia (poli-
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1906 Contribuzioni alla conoscenza biologica degli Imenotteri paras-
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8. DESCRIPTION OF PLATES
ABBREVIATIONS
A, first polar body nucleus
A, outer nucleus of first polar body
Ag, inner nucleus of first polar body
As. E., asexual embryo
A. T., adipose or fat tissue
B, second polar body nucleus
B,, second polar body nucleus
Bz, female pronucleus
D.E.C., definitive or true embryonic
cells
E.B., embryonic cell or blastomere
F.N., fertilization or cleavage nucleus
I.M., outer membrane
I.M.C., inner membrane cells or nuclei
M.C., mesenchyme cells of host
No., nucleolus
No.C., blastomere receiving nucleolus
Nu., egg or o6cytic nucleus
O.M., outer membrane
P., Polar nucleus or nuclei
P.M., polar membrane
Pr.M., primary mass
P.S., protoplasmic strand
S., sperm or sperm nucleus
S.M., secondary mass
T.M., tertiary mass
Ist M., first maturation spindle
2nd M., second maturation spindle
All figures in plates 1 to 11 were drawn with aid of the camera lucida.
PLATE 1
EXPLANATION OF FIGURES
1 to 25 are all drawn from whole mounts made by the smear method.
1 Freshly deposited fertilized egg, showing the sperm, odcytic nucleus, and
nucleolus. X 620.
2 Freshly deposited unfertilized egg, showing the oécytic nucleus and nucleo-
lus. X 620.
3 Fertilized egg taken twenty-five minutes after oviposition.
The first maturation spindle is in the late anaphase stage. X 620.
4 to 10 are all drawn from the same preparation.
4 The second maturation spindle is in the late anaphase stage. The first
polar body chromatin is seen at A. The egg is unfertilized. X 620.
5 Fertilized egg in which the second maturation is completed. The female
pronucleus (B:) is migrating toward the sperm. X 620.
6 Fertilized egg with two pronuclei close together. The polar nucleus (P)
is in the process of formation. X 620.
7 Asimilar egg showing the two pronuclei in contact. X 620.
8 Astage showing the two pronuclei conjugating. X 620.
9 An egg showing the fertilized or first cleavage nucleus. X 620.
46
DEVELOPMENT OF PARACOPIDOSOMOPSIS PLATE 1
J. T. PATTERSON
JOURNAL OF MORPHOLOGY, VOL. 36, No. 1
PLATE 2
EXPLANATION OF FIGURES
10 Unfertilized egg showing the female pronucleus and the nucleolus at the
posterior end. This nucleus will form the first cleavage spindle. X 620.
11 Egg showing first cleavage spindle in metaphase. X 620.
12 Egg showing first cleavage spindle in anaphase. X 620.
13 Egg showing first cleavage spindle in late anaphase. XX 620.
14 In this egg the two cleavage nuclei are reorganized and the cytoplasm is
beginning to divide. The nucleolus is closely associated with one of the nuclei.
X 620.
15 The two-celled stage. Note that the nucleolus is included in one blasto-
mere. X 620.
16 A two-celled stage, in which the spindles are formed in preparation for
the four-celled stage. X 620.
17 The four-celled stage. Only one of the four blastomeres receives the
nucleolar material (No.C). The polar nucleus is completely formed. X 620.
18 Another four-celled stage. X 620.
48
PLATE 2
DEVELOPMENT OF PARACOPIDOSOMOPSIS
J. T. PATTERSON
PLATE 3
EXPLANATION OF FIGURES
19 The eight-celled stage. The nucleolar material is distributed to two
blastomeres (No.C). The polar nucleus has undergone rapid division producing
thirteen nuclei. Note that the protoplasm containing the polar nuclei is gradu-
ally flowing around the embryonic cells. X 620.
20 A stage showing thirteen cells, two of which contain nucleolar material.
Usually there are fourteen cells, but in this case one of the blastomeres of the
eight-celled stage has been delayed in its division. The polar nuclei are all
dividing. X 620.
21 Atypical fourteen-celled stage. Itis much flattened on the slide. X 620.
22 to 24 Upper or anterior ends of three eggs showing the polar nucleus.
x 620.
25 Metaphase plate of a polar nucleus, showing the diploid number of chromo-
somes. _ X 1827.
26 First maturation spindle. X 1827.
27 First polar body chromatin (A) and the second oocytic chromatin (B).
There are eight chromosomes in each group. XX 1827.
28 The first polar body dividing (A; and A») and the chromatin of the second
oocyte. Each group has eight chromosomes. XX 1827.
29 First polar body spindle and second maturation spindle. X 1827.
30 First and second polar bodies. X 1827.
31 A similar stage. X 1827.
32 Side view of a cleavage spindle of one of the first four blastomeres. X
1827.
33 Polar view of the first cleavage spindle. ~ X 1827.
DEVELOPMENT OF PARACOPIDOSOMOPSIS PLATE 3
J. T. PATTERSON
-. Gr A
So tae A2
: Jah — By
ip
PLATE 4
EXPLANATION OF FIGURES
It is practically impossible to obtain a median section showing all of the details
of structure. It has therefore been necessary in certain figures to draw in one or
more structures (e.g., nugleolus or polar body nucleus) from adjacent sections.
This was done in figures 34 to 39.
34 A median section showing the first maturation spindle, nucleolus, and
sperm. The latter was taken from the first section on the left. X 1167.
35 <A later stage, showing the three polar bodies and the female pronucleus.
x 1167.
36 This section shows the cleavage nucleus (F.N.), the outer chromatin mass
of the first polar body division (A,), and the polar nucleus (P), which has been
formed by a fusion of the inner chromosome group of the first polar body and the
second polar body. X 1167.
37 A median section of the two-celled stage. XX 1167. .
38 Median section of a four-celled stage. 1167.
39 <A similar section of a slightly later stage. The nucleolus has broken up
and nearly surrounds the nucleus. The polar nucleus has undergone one division.
x LLG
40 The eight-celled stage. > 1167.
41 Median section of a fourteen-celled stage. The two blastomeres which
have received the nucleolar material lie at the top of the group of embryonic
cells. X 1167.
42 A transverse section of a similar stage. X 1167.
52
DEVELOPMENT OF PARACOPIDOSOMOPSIS PLATE 4
J. T. PATTERSON
PLATE 5
EXPLANATION OF FIGURES
43 Slightly oblique section of a twenty-seven-celled stage, showing three of
the four descendants of the original nucleolar blastomere. Nine hours. X 1260.
44 Upper end of a section through a twenty-eight-celled stage, with two of
the sixteen polar nuclei all in mitosis. Nine hours.. X 1260.
45 Median section of a fifty-two-celled stage. The four nucleolar cells are
all shown in the section. Ninteen hours. X 1200.
46 Median section of a sixty-celled stage. Ninteen hours. X 1121.
47 Oblique section of a seventy celled stage. It shows five of the six nucleo-
lar blastomeres present in the egg. Twenty six hours. X 1153.
48 Median section of a 135 celled stage. This egg has retained to a remark-
able degree the original pear shape. Forty hours. X 1035.
PLATE 5
DEVELOPMENT OF PARACOPIDOSOMOPSIS
: : J. T. PATTERSON
PLATE 6
EXPLANATION OF FIGURES
49 Median section of a 169 celled stage. X 1134.
50 Section through a 221 celled stage. Note that some of the cells have
become elongated and spindle shaped. Forty hours. X 1134.
51 Part of a lateral section from the same series, showing certain blastomeres
arranged in the form of anest. > 1167.
52 Oblique section through a young polygerm. The embryonic cells have
already differentiated into two kinds; 1) the definitive or true embryonic cells
(D.E.C.) and2) the inner membrane nuclei (J./.C.). The true embryonic cells,
which take a deeper stain, are in the process of forming primary masses. X 1052.
53 Section through a completed polygerm, showing a single large asexual
embryo (As.£.) and several primary masses (Pr.M.). X 612.
PLATE 6
DEVELOPMENT OF PARACOPIDOSOMOPSIS
J. T. PATTERSON
57
PLATE 7
EXPLANATION OF FIGURES
54 One half of a section through a very young polygerm showing formation
of the primary masses. X 1040.
55 Median section of a young polygerm. X 626.
56 Median section of an almost completed polygerm, showing the youngest
stage at which an asexual embryo can be recognized (As.H.). X 626.
57. A completed polygerm with an asexual embryo at the upper end. X 626.
58 <A later stage with an asexual embryo at the lower end. X 626.
59 Median section of a completed polygerm. X 626.
60 Transverse section of a polygerm showing a primary mass undergoing
division (on the left). X 626.
61 Detailed drawing showing the completed division of a primary mass.
x 1167.
58
PLATE 7
oe
ows
ze
DEVELOPMENT OF PARACOPIDOSOMOPSIS
J. T, PATTERSON
PLATE 8
EXPLANATION OF FIGURES
62 Section of a polygerm showing divisions of the primary masses to form
secondary masses. X, Y, and Z, primary masses dividing. X 767
63 Tertiary mass containing a single embryonic cell. The mass lies free in
the body cavity of the host. X 1267.
64 Tertiary mass showing division of embryonic cell. Some of the nuclei
of the inner membrane are also dividing. 1267.
65 Tertiary mass which has recently divided. The component on the left
has a single embryonic cell; the one on the right has two which have not as yet
separated. X 1267.
66 Tertiary mass showing the multiplication of embryonic cells. X 1267.
67 Tertiary mass preparing for division. The embryonic cells are being
divided up into groups by the activity of the inner membrane. X 1267.
68 Tertiary mass which has divided to produce three components. The
one on the right is preparing for another division. X 1267.
60
PLATE 8
61
—
DEVELOPMENT OF PARACOPIDOSOMOPSIS
J. T. PATTERSON
rms
PLATE 9
EXPLANATION OF FIGURES
69 Longitudinal section through the third ganglion, which contains a number
of secondary masses. X 636.
70 A portion of the brain ganglion youn a fertilized egg in maturation.
X 636.
71 Longitudinal section of the fourth ganglion containing a large asexual
embryo, several secondary masses, and a few tertiary masses. X 636.
72 Longitudinal section of an advanced polygerm removed from a 12 mm.
caterpillar. The polygerm, which is surrounded by adipose tissue of the host,
contains many tertiary masses and also two advanced asexual embryos. Some of
the masses are dividing (at XY). X 260.
_ DEVELOPMENT OF PARACOPIDOSOMOPSIS PLATE 9
s J. T. PATTERSON
PLATE 10
EXPLANATION OF FIGURES
73 Part of a longitudinal section of a polygerm in the body cavity of a 5.5
mm. caterpillar. The polygerm has undergone almost complete dissociation.
One of the four asexual embryos found in the series is seen on the right. X 387.
74 Part of a dissociated polygerm, showing a group of seven asexual embryos
surrounded by fat tissue. The polygerm is nine days and twenty three hours
old. A.T., adipose tissue. XX 208.
75. A portion of the same polygerm, showing teritary masses. X 208.
64
DEVELOPMENT OF PARACOPIDOSOMOPSIS
J. T, PATTERSON
65
PLATE 10
PLATE 11
EXPLANATION OF FIGURES
76 Section of a tertiary mass which has divided up into a number of com-
ponents. In some of the components the embryonic cells are being isolated
through the activity of the inner membrane. Such cells will produce sexual
embryos. X 700.
77 A late stage in the development of a tertiary mass, showing early stages
in the formation of sexualembryos. X 700.
78 An advanced stage of the development of the sexual embryos. X 110.
DEVELOPMENT OF PARACOPIDOSOMOPSIS PLATE 11
J. T. PATTERSON
67
PLATE 12
EXPLANATION OF FIGURES
79, 80, 83, 86 to 90 are from eggs laid by an unfertilized female. The speci-
mens were taken from the body cavity of a half grown caterpillar fifteen days
after the eggs had been deposited. The body cavity contained four normal and
four degenerate asexual larvae, in addition to many masses of sexual embryos.
Figures 81, 82, 84, and 85 are from eggs laid by a fertilized female. The speci-
mens were removed from the body cavity of a 20 mm. caterpillar fourteen days
after the eggs had been deposited. There were found in the body cavity six
free asexual larvae and four larvae still enclosed in their membranes, besides
many masses of sexual embryos.
79 and 80 Two masses of sexual embryos. > 100.
81 Mass of sexual embryos with sexual embryo in capsule. X 100.
82 Asexual larvae in capsule. X 100.
83 Asexual larvae free from capsule, but still adhering to mass of sexual
embryos. X 88.
84 to86 Three asexuallarvae. X 88.
87 to90 Four degeneration asexual larvae. X 88.
DEVELOPMENT OF PARACOPIDOSOMOPSIS PLATE 12
J. T. PATTERSON
69
Resumen por el autor, Caswell Grave.
Amaroucium constellatum (Verrill).
II. La estructura y organizacion de la larva ‘‘renacuajo.”’
Los resultados del presente trabajo que ofrecen novedad son:
1. Una periodicidad en la liberaci6én de las larvas de la colonia
parental. Las colonias numerosas dejan escapar los renacuajos
en bandas durante la aurora o préximamente a esta hora, pero
de vez en cuando una larva escapa durante otras horas del dia.
2. Unas sesenta vesiculas multicelulares, semejantes a blastulas
se invaginan en el manto durante los ultimos estados del desar-
rollo embrionario, permaneciendo aisladas del cuerpo en la
substancia de la ttinica durante todo el periodo de natacion libre
de la larva. 3. Los ecristalinos del ojo no son células retinales
modificadas sino productos que se depositan dentro de las células
ganglidnicas, y son de naturaleza semiliquida o gelatinosa. 4.
Los bastones visuales, diferenciados en las células retinales del
ojo, se proyectan a través de la zona pigmentaria y terminan en
la superficie interna de la copa pigmentaria. 5. Las pruebas
estructurales y fisiol6gicas indican que el ojo funciona en las
respuestas de orientacién del renacuajoalaluz. 6. El estatolito
se forma dentro de una vacuola de la célula estatolitica sensorial
y esta formado de una substancia dura que no se disuelve en los
Acidos fuertes. 7. El autor describe un nervio visceral que se
origina en el ganglio visceral distribuyéndose en la regién del
endostilo. 8. El cord6én nervioso no ocupa posicién dorsal, sino
que a causa de un giro permanente de la cola de noventa grados
hacia la izquierda viene a situarse al lado izquierdo del notocordio.
9. Las dos series de células musculares situadas en los lados
ventral vy dorsal del notocordio, segin indican las pruebas mor-
folégicas y fisiol6gicas, funcionan como unidades de tal modo que
producen un movimiento rotatorio del cuerpo durante la loco-
mocion.
Translation by José F. Nonidez
Cornell Medical College, New York
AUTHOR’S ABSTRACT OF THIS PAPER ISSUED
BY THE BIBLIOGRAPHIC SERVICE, NOVEMBER 7
AMAROUCIUM CONSTELLATUM (VERRILL)!
II. THE STRUCTURE AND ORGANIZATION OF THE TADPOLE
LARVA
CASWELL GRAVE
Washington University, St. Louis, Missouri
FOUR TEXT FIGURES AND FOUR PLATES
This paper, which deals with the structural organization of
the fully developed tadpole larva of Amaroucium constellatum,
is a contribution, in part, to the morphology of ascidians, but it
is especially intended as a further contribution toward the estab-
lishment of a basis for a comparative study of the larval forms
of a number of species of ascidian common to the Woods Hole
region with a view to the correlation, so far as may be possible,
of their specific structural and physiological characters with
observable differences in the distribution and habitat of each
species.
The structures common to ascidian larvae in general have
been so repeatedly described in the many excellent papers pub-
lished during the fifty years that followed the announcement by
Kowalewsky (’66) of his discovery of the chordate affinities of
ascidians, that it seems unnecessary to attempt to cite specific
references to papers except in connection with results or con-
clusions that have not found general acceptance. The points
added to the morphology of the ascidian larva as a result of
this study are enumerated in the concluding paragraphs of the
paper.
1 Since the publication of the first paper of this series (Grave, ’20 b) con-
clusive evidence has been secured that Amaroucium constellatum is not a form
of A. pellucidum, but must be considered a true species, hence the change in
the general title for this, the second paper. The systematic data referred to
will constitute the subject matter of a special paper.
71
JOURNAL OF MORPHOLOGY, VOL. 36, NO. 1
We CASWELL GRAVE
METHODS
Tadpole larvae of Amaroucium may be secured in abundance
at Woods Hole during the months of July, August, and Sep-
tember by placing large colonies of the ascidiozooids in glass
jars before a window in the laboratory. The best results have
been obtained when the colonies were collected the day before
tadpoles were desired and kept in running sea-water during
the night. ‘Tadpoles escape from the colonies in swarms at and
just after sunrise, but they continue to be liberated in small
numbers at any hour of the day. When liberated the tadpoles
swim immediately to the surface of the water and collect at one
side of the jar where they may be easily captured with a pipette.
Immature tadpoles and various earlier developmental stages
may be secured by squeezing a colony in the hand over a culture
dish of sea-water. With the mass of ascidiozooids, tadpoles,
embryos, and eggs thus forced from the colony, a considerable
quantity of gelatinous debris is included, which settles very
slowly, and may therefore be removed by decantation, two or
more changes of water being required. Mature tadpoles may
be had in small numbers in this way also. For a few seconds
after having been squeezed from the colony they lie motionless
upon the bottom of the dish, but soon begin to move sightly,
then to swim about, apparently stimulated by light or by con-
__tact with pure sea-water.
For the study of the general and histological structure of the
tadpole, Bouin’s and Flemming’s mixtures have been found to
give the best fixation. Sections have been made by the paraffin
method and stained with Delafield’s or iron haematoxylin.
For total mounts an excellent transparent stain for tadpoles
fixed with Bouin’s solution is made by adding to 70 per cent
alcohol, slightly acidulated with HCl, an amount of borax carmin
sufficient to give a delicate pink color to the solution. Specimens
should be allowed to remain in the stain twelve hours or more.
STRUCTURE OF THE AMAROUCIUM TADPOLE 73
DEFINITIONS
For the purposes of description, the part of the tadpole which
contains the adhesive papillae and precedes during locomotion
will be referred to as the anterior portion; the tail, as marking
the posterior part of the body, and the sensory-vesicle, conspicu-
ous on account of its pigmented sense organs, will be desig-
nated as dorsal in position. The relation the body of the tad-
pole bears to that of the sessile ascidiozooid has been discussed
by Maurice et Schulgin (’84). A general readjustment of parts
takes place during metamorphosis in which the anterior part
of the tadpole becomes the basal portion of the ascidiozooid.
FORM AND SIZE
The general form of this and other ascidian larvae at once
suggests that of the amphibian tadpole, hence the name ‘tad-
pole’ has been applied to both, but the similarity between ascid-
ian and amphibian larvae, their fundamental chordate charac-
ters excepted, is superficial and disappears with any but the
most casual comparison of either their form, structure, or
activities.
The tadpole larva of Amaroucium, at the period of its com-
plete development, has a total length of approximately 2.25 mm.
The body portion varies in length from 0.74 to 0.78 mm. in
depth from 0.36 to 0.44 mm. and in width from 0.31 to 0.37 mm.
TUNIC
The body and tail proper of the tadpole are enveloped by a
comparatively thick, non-cellular tunic of a glassy, translucent
appearance. In the living tadpole the tunic is flecked at its
outer surface with numerous whitish points, the nuclei of the
flattened test-cells which form a delicate pavement epithelium
over its external surface.
The cytoplasm of test-cells in general is homogeneous in
appearance, but it is not uncommon to find cells which contain
numerous yellowish bodies or are filled with reddish-orange
pigment granules. The quantity of pigment present in test-
74 CASWELL GRAVE
cells, and in cells of other parts of the body also, varies greatly
in tadpoles of different broods, those liberated by highly colored
colonies being more highly pigmented than those produced by
colonies of lighter color. The pigmentation of each tadpole is
approximately the same as that of its parent colony.
The part of the tunic which envelops the tail is greatly com-
pressed dorsoventrally and expanded in the horizontal plane
and entirely constitutes the comparatively wide tail fin of the
tadpole. Seeliger (’85), in his study of Clavellina, noted that
the tunic substance is secreted during the embryonic period when
the tail is folded forward and closely compressed between the
body of the embryo and its chorionic membrane and that the
part which is secreted about the tail is thus caused mechanically
to be spread out on either side and to take on a compressed fin-
likeform. He also noted that the entire tail, the fin included, is
twisted on its axis to the left during the embryonic period, but, in
the Clavellina tadpole, it apparently untwists when the chorion
is ruptured at the time of hatching, for he describes the tail fin of
the free-swimming larva as having a vertical position. Damas
(04) noted the horizontal position of the tail fin of the larva
of Distaplia magnilarva and called attention to its seen in
_ this respect to Appendicularia.
The part of the tunic surrounding the body of the tadpole is
laterally compressed, but the right and left sides are slightly
asymmetrical. Viewed from the dorsal side, a shallow concave
depression is seen on the left near the anterior end, and the
anterior tip end of the tunic which contains the middle adhesive
papilla is found to lie slightly to the right of the medial sagittal
plane of the body (fig. A). These asymmetrical features are
the result of the pressure of the tail during the period of embry-
onic development when it is bent forward beneath the chori-
onic membrane and coiled about the anterior part of the tunic
(fig. B). The imprint of the tail in the tunic takes an oblique
course from below upward across the left side, and therefore
gives to the tunic the form of a screw with a single groove.
STRUCTURE OF THE AMAROUCIUM TADPOLE 75
Fig. A Camera outline drawing of the fully developed tadpole larva as
seen from the dorsal surface, showing the lateral asymmetry of the body, the
horizontal position of the tail fin, and the location of the sense organs in
the sensory vesicle. Abbreviations given on page 93.
76 CASWELL GRAVE
MANTLE
The mantle (ectoderm) varies in thickness in different re-
gions of the body, but it consists at no point of more than a single
layer of cells. The cells of the mantle in general are more or
less cubical in form, but are high and columnar in the parts
forming the rudiments of the oral and atrial siphons, thin and
pavement-like in the region above the sensory vesicle and in
the mantle sheath of the tail (figs. 1, C, 3 and 8).
SG
/ 4 : ‘ - si | :
A Per ye :
PS ht. Pen iat, | EE
Fig. B A drawing of the embryo within its chorionic membrane, showing the
twisted and coiled tail and the four points along the median keel of the body
at which the test-clubs grow out from the mantle to form the test vesicles.
ADHESIVE PAPILLAE
The adhesive papillae, of which there are three arranged in a
vertical series at the anterior end of the body, are tubular out-
growths of the body wall (fig. 1). . Each terminates at the sur-
face of the tunic in an enlarged, goblet-shaped body which opens
outwardly and contains a large lens-shaped mass of elongated,
richly granular cells probably of mesenchyme origin. The cen-
tral canal of each papilla is partially filled with mesenchyme
cells (fig. 7). Toward the end of the free-swimming period
of the tadpole, a contraction of the wall of each papilla takes
place, causing the contents of the terminal, cup-shaped enlarge-
ment to be extruded upon the surface of the tunic. The viscid
STRUCTURE OF THE AMAROUCIUM TADPOLE 77
nature of the extruded material is shown by the fact that the
tadpole adheres to any foreign body against which it chances
to swim, and the most violent movements often fail to release
it from such an attachment.
TEST VESICLES
In the fully developed tadpole a large number of blastula-like
vesicles occupy a considerable part of the space in the anterior
median region of the tunic. They have no organic connection
with the body, but lie midway between the mantle and the ex-
ternal surface of the tunic. They are separated by the stalks
of the adhesive papillae into four unequal groups (fig. 1). The
part of the wall of each test vesicle turned toward the surface
of the tunic is composed of cells much larger than those on
the side facing the body (fig. 9). The test vesicles maintain
this position and orientation during the entire free-swimming
period of the tadpole. The function and history of these bodies
formed the subject of a paper (Grave, ’20b) prepared for the
program of the seventeenth annual meeting of the American
Society of Zoologists, an abstract of which has been published
in the Proceedings of the meeting.
Each test vesicle takes its origin from the mantle wall in the
form of a hollow, club-shaped outgrowth or evagination during
the late embryonic period. Four clusters of these club-shaped
bodies, attached to four median elevations of the body wall,
may be seen in immature tadpoles. The dorsal and ventral
groups project from keel-like ridges, while the anterior groups
are attached to conical papillae situated midway between the
bases of the stalks of the adhesive papillae, and each has the
appearance of a bouquet or rosette (fig. B). Each club-shaped
outgrowth ultimately becomes coverted into a test vesicle,
first by the appearance of a constriction near its point of attach-
ment, then at the point of the constriction it separates from the
body as a pear-shaped structure which gradually assumes a
spherical form and migrates to a position in a zone midway be-
tween the body wall and the external surface of the tunic. The
test vesicles of Amaroucium probably correspond to the ‘bladder
78 CASWELL GRAVE
cells’ which have been described in other ascidian larvae. In
Amaroucium, however, they are not modified cells, but are many-
celled bodies derived from the ectoderm. The number of test-
vesicles is not constant. As accurately as could be determined,
the numbers present in each of eight tadpole larvae are as fol-
lows: 62, 52, 55, 52, 53, 60, 58, and 62.
NERVOUS SYSTEM
In the nervous system of the tadpole the following parts may
be distinguised: a sensory vesicle, visceral ganglion, and nerve
cord which are functional during the brief larval period only;
an hypophysial duct, subneural gland, and definitive ganglion
which persist to function during the life of the sessile ascidiozooid.
The position of these nervous structures in the tadpole and the
relations they bear one to another are shown in figures C, D, and 1.
SENSORY VESICLE
The sensory vesicle is situated between the oral and atrial
siphons to the right of the median sagittal plane of the body
(figs. land 3). Itis oval in form and contains a spacious cavity
or ventricle filled with a clear liquid. ‘Two sense organs are
developed in its wall and project into its central cavity, the
eye occupying a considerable portion of the left side and pos-
terior end, the static organ located on its right and ventral sides.
Except for the parts which form the sense organs and their
ganglia, the wall of the sensory vesicle is thin (figs. D and 5).
THE EYE
The following parts may be distinguished in the eye; a mass
of brownish-black pigment granules arranged in the form of a
cup, the mouth of which is directed obliquely upward and for-
ward; three lenses arranged in a linear series in the axis of the
pigment cup, and a third part which may be called the retina
or retinal ganglion (figs. D, 4, 5, and 6).
A layer of pigment-forming cells in addition to true nerve
cells has been described by Salensky (’93) in the developing retina
of the embryo of Distaplia, but I have been unable to distinguish
STRUCTURE OF THE AMAROUCIUM TADPOLE 79
Figs. C and D Reconstructions of the nervous system from serial sections.
Figure C as viewed from the dorsal surface of the larva; figure D as viewed from
the left side. Outlines of the siphons are included.
80 CASWELL GRAVE
the two types of cells in the eye of either mature or immature
tadpoles of Amaroucium, and my observations are therefore
in agreement with those of Kowalewsky (’71), who found that
the pigment granules lie within the inner ends of the visual
cells of the retina. Cell walls are nowhere definite in the nerve
tissues of the Amaroucium tadpole, however, and it is possible
that the pigment granules are formed in cells distinct from those
in which the visual rods are developed. Studies of the eyes of
the larvae of other ascidians now in progress may clear up this
point.
No migration of pigment granules within the retinal cells was
observed in the living tadpole, and sections of the eyes of tad-
poles fixed in Flemming’s solution after an exposure of thirty min-
utes in the dark showed no observable difference in the distri-
bution of the pigment granules from that of tadpoles exposed to
strong light before similar fixation.
The lenses of the ascidian eye have been described as modified
or transformed cells, but in Amaroucium I find they are not
modified cells, but are deposition products formed within vacuoles
of marginal cells of the retinal ganglion, the nuclei of which,
in the embryo, are similar in size and structure to the nuclei of
the adjacent nerve cells of the retina (fig. 4). The portion of
each lens-forming cell which projects into the cavity of the sen-
sory vesicle is greatly enlarged and contains a large vacuole,
in the center of which a transparent spherical droplet of amber-
colored substance is deposited. In the earliest stage noted the
size of the droplet of lens substance was small. Other stages
showed that the lenses gradually increase in volume until they
entirely fill the vacuoles. Some cases were noted in which more
than one droplet of lens substance were present in the same vacu-
ole, a fact which indicates that the substance is semiliquid in
nature, for the droplets presumably flow together to form a single
lens. The illustrations which show the lens-forming cells and
the lenses (figs. D, 4, 5, 6, and 11) are misleading if they convey
the idea that the lenses are lamellated or have a concentric
structure. They are homogeneous in appearance both in the
living tadpole and in sections. At the center of each lens, how-
STRUCTURE OF THE AMAROUCIUM TADPOLE 81
ever, one or more granules are usually found. The lenses are
easily sectioned and are not crystalline.
The nuclei of the lens-forming cells lose their staining quali-
ties when the lenses have been fully formed and the shrunken
cell bodies persist merely as anchors by which the lenses are
attached to the superior wall of the sensory vesicle (compare
figs. 4, 6, and 11).
The retina consists of a layer of large nerve cells grouped
about the pigment cup. In the axis of some and possibly of
all of the retinal cells a rod-shaped portion is differentiated
-which penetrates the pigment zone and ends at the inner sur-
face of the pigment cup. These portions of the retinal cells
may be termed the visual rods. They are so placed in the zone
of pigment that their long axes coincide with the direction taken
by rays of light focused by the lenses into the pigment cup.
A visual rod is shown in longitudinal section in figure 11 and four
in transverse section are shown in figure 10.
An optic nerve, such as that described by Salensky (’93) in
the larva of Distaplia, connecting the retinal portion of the
sensory vesicle with the visceral ganglion, I have failed to find,
but, as these two parts of the central nervous system of the
Amaroucium tadpole are practically in contact, the visceral
ganglion probably receives the retinal fibers directly.
The eye of the ascidian tadpole is a true direct brain eye and,
as has been pointed out first by Goette (’75) and later by Salensky
(93), Willey (94), and others, it is similar in its structure and
organization to that of the pineal or parietal eye of cyclostomes
and lizards. McBride (’14), on the other hand, possibly with
the observations of Lahille in mind, commits himself to the
view that the eye of the ascidian larva is homologous with one
of the paired lateral eyes of vertebrates. Lahille (90) described
what he interpreted to be the remains of an atrophied eye be-
longing to the right side of the sensory vesicle of the larva of
Distaplia, but Salensky (’93) and others, working with the same
larva, have failed to find any trace of the rudimentary structure
described by Lahille.
82 CASWELL GRAVE
Although the structure and organization of the eye are perhaps
sufficient to support the interpretation that the eye is the organ
by whici the tadpole orients with reference to rays of light, it
may be worth while to state the physiological evidence, secured
since the publication of the paper on the activities and reactions
of the tadpole larva (Grave, ’20), which shows conclusively that
the eye is a functional light-perceiving organ.
During the latter part of their free-swimming period, tadpoles
cease to swim continuously, and intervals of rest, when they
lie quiescent upon one side, become longer and longer. While
examining a tadpole during one of its resting periods with the
microscope, the mirror was so turned as to cut off the transmitted
light. Immediately the light was cut off the tail began to vibrate.
Repeated experiments of the same kind with light reflected from
the mirror, alternately turning it off and on, showed that when
the tadpole was so lying that light from the mirror entered the
pigment cup (on its right side), the tail almost invariably began
to vibrate at the instant the light was turned off and in no case
when the light was turned on. The actual stimulus to muscular
contraction is not transmitted from the eye to the muscle bands
of the tail during the illumination of the visual rods of the retina,
but immediately after the pigment cup is darkened, follow-
ing its illumination. This takes place in the course of normal
locomotion at the moment in each revolution of the body on its
axis when the pigment cup is carried to a position in which rays
of light no longer enter its cavity.
It has been noted from the beginning of the investigation that
the shadow of the hand when passed over resting tadpoles almost
invariably causes immediate renewal of locomotor activity.
The observations just described, which show that the eye is a
functional light-perceiving organ, incidentally explain this
shadow reaction. ©
THE STATIC ORGAN
The static organ consists of a single sensory cell, at the distal
end of which is borne a relatively large, subspherical, black
statolith, and a small number of large nerve cells which form
STRUCTURE OF THE AMAROUCIUM TADPOLE 83
a thickened ganglionic portion in the right lateral and ventral
walls of the sensory vesicle.
The statolith-bearing cell projects for its entire diameter into
the cavity of the sensory vesicle, and is therefore a pendent
structure. In the living tadpole the statolith appears to be con-
tained in a cup-shaped depression at the distal end of the cell,
but sections show that it lies wholly within a vacuole-like cavity
of the statolith cell and is surrounded by a delicate layer of cyto-
plasm (figs. D, 5, and 6). The statolith is composed of a sub-
stance that is not disintegrated by strong acids and is not bleached
by chlorine. It is very hard and, when struck by the edge of
the section razor, is usually torn from its base and dragged through
the tissues.
Tadpoles with two statolith cells are occasionally found, but
they are rare.
The part of the sensory vesicle formed by the ganglionic por-
tion of the static organ is so located that it comes into contact,
at its most ventroposterior end, with the side of the visceral
ganglion, and nerve fibers probably pass from the former to
the latter at this point, but no ‘acustic’ nerve, such as has been
described by Salensky (’93) to connect the ‘gehér organ’ with
the visceral ganglion in the larva of Distaplia, is present in the
tadpole of Amaroucium.
VISCERAL GANGLION
The vertically situated part of the larval nervous system
which connects the sensory vesicle with the nerve cord has been
called the visceral ganglion. A cortex made up of a single layer
of large nerve cells and a longitudinally striated medullary por-
tion may be distinguished in it, but no trace of a neural canal
ean be found either in longitudinal or transverse sections (figs.
C, D, 3, and 10).
From a point on the left side of the ganglion, located just below
the level of the hypophysial duct, a comparatively large bundle
of nerve fibers emerges as a nerve trunk and can be traced ob-
liquely upward and forward to the region above the endostyle a
short distance anterior to the oral siphon where it apparently
84 CASWELL GRAVE
ends, possibly having a distribution to muscle fibers which are
in this region rather richly developed. ‘The origin and course
of this nerve are shown in figures C, D, and 3. Its function
during the free-swimming period of the larva is not evident and, .
on account of the rigidity and immobility of the body, is diffi-
cult to conceive. Non-striated muscle fibers are present in
considerable number in the mesenchyme layer just beneath the
body wall in the region into which the nerve can be traced.
These muscle fibers take a general course from the region of the
endostyle obliquely forward to the ventral side of the body.
Slow writhing contractions of the entire body are very evident
at the close of the free-swimming period when metamorphic
changes have set in, and it is possible the neuromuscular appara-
tus under consideration first comes into function at this time.
Salensky’s conception of the visceral ganglion as the reflex
center or brain of the larva seems to be substantiated by phys-
iological as well as by structural data. My observations on
the reactions of the Amaroucium tadpole (Grave, ’20 b) indicate
that the tadpole orients with reference both to light, by means
of reflexes originating in the eye, and to gravity, by means of
reflexes originating in the static organ. It was found that
the normal response to gravity during the latter part of the
free-swimming period was greatly modified in the presence of
unusual stimulation by light. The visceral ganglion must be
the coordinating center for these diverse reflexes.
THE NERVE CORD
Due to a permanent twist of the tail 90° to the left, the nerve
cord occupies a position on the left side of the notocord in the
space between the projecting edges of the dorsal and ventral
muscle bands (figs. C, 3, 8, and 10). Near the anterior end of
the notocord the nerve cord bends abruptly upward and slightly
to the right to join the ventral end of the visceral ganglion. A
definite neural canal is present throughout its length. Small
nuclei are present here and there in the thin wall of the cord,
but none were found that have the characteristics of nerve cells
(figs. 83, 8, and 10). The cord tapers toward the end of the tail,
STRUCTURE OF THE AMAROUCIUM TADPOLE 85
but it is coextensive with the muscle bands. It no doubt consti-
tutes the pathway for nerve fibers from the visceral ganglion
to the muscle cells, but the endings of fibers in muscle cells could
not be made out.
DEFINITIVE GANGLION, HYPOPHYSIAL DUCT, AND SUBNEURAL
GLAND
The parts of the nervous system described in the foregoing
paragraphs are those which function during the free-swimming
period of the tadpole and degenerate when the larval period is
over. The parts that persist and become the functional nervous
system of the sessile ascidiozooid are the hypophysial duct,
definitive ganglion and subneural gland. As shown in figure
D, they form a vertical series of structures situated immedi-
ately to the left of the sensory vesicle in the median sagittal
plane of the body.
These structures~in their fully differentiated condition in
the adult ascidiozooid have been studied by Metcalf (’00). A
comparison of his figure 47 with figure D of this paper shows
that the entire central nervous sytem of the adult Amaroucium
ascidiozooid is fully formed in the larva, and thus shows clearly
the relation the larval nervous structures bear to those which
persist in the adult.
The hypophysial duct is hollow and its canal is lined with
cilia for about two-thirds of its length (figs. C, D, and 1). At
its anterior end it is continuous with the wall of the oral siphon
and the cavities of these structures are in open communication.
As the ectodermal oral siphon at this stage is in no way connected
with the endodermal pharynx, there can be no doubt, in the case
of Amaroucium, of the primary connection of the hypophysial
duct with the ectodermal, and not with the endodermal part of
the alimentary tract—a fact of considerable significance for
the old controversial question of the homology of the hypophys-
ial duct of ascidians with the hypophysis of vertebrates. The
posterior end of the hypophysial duct terminates blindly in the
region of the atriopore between the lateral horns of the atrium.
The part of the duct which lies posterior to the subneural gland
corresponds to the rapheal duct of the ascidiozooid.
86 CASWELL GRAVE
The definitive ganglion, so called because of its persistence as
the nerve center of the ascidiozooid, lies immediately above the
middle portion of the hypophysial duct (fig. 6). These struc-
tures are in close contact, but are not at this stage connected.
The ganglion is oval in form and is composed of a cortex of cells
and a medulla in which no nuclei are found (figs. 3 and 6). The
nuclei of the cortex are small and do not have the structure
chacteristic of nerve cells, possibly because their functional
activity does not begin during the larval period.
The subneural gland has the appearance of an enlargement
or outgrowth of the middle portion of the hypophysial duct on
the side opposite the definitive ganglion. It is a hollow struc-
ture and its cavity is in open communication with the lumen of
the hypophysial duct (figs. C and D).
THE MIDDLE GERM LAYER
Mesenchyme cells form a discontinuous layer just beneath the
body wall. They are found very infrequently in the posterior
part of the body, especially in the region of the sensory vesicle,
but in the anterior part they are quite numerous and, in the
parts from which the test vesicle and adhesive papillae have
developed, they form a continuous layer more than one cell in
thickness (fig. 9). Their distribution in the middle portion of
the body is shown in figure 3. At no place could they be said
to form an epithelium, and nothing comparable to the mesoderm
or coelom as they are developed in vertebrates is present.
Mesenchyme cells of at least three varieties can be distin-
guished; one in which the cytoplasm is apparently homogeneous
is the most common, but another in which the cell bodies are
loaded with granules is not infrequent. The third variety is asso-
ciated with non-striated muscle fibers, of which some encircle
the body obliquely from the dorsal to the ventral side and are
located just beneath the mantle, some are distributed along
the walls of the pharynx and atrium, and quite numerous sets
of fibers extend in a radial direction from the oral and atrial
siphons as centers.
STRUCTURE OF THE AMAROUCIUM TADPOLE 87
TAIL MUSCLES
Two muscle bandsy each consisting of about eighty very large,
polygonal muscle cells arranged in four longitudinal rows of
twenty cells each, form the dorsal and ventral portions of a
relatively thick envelope for the notocord (figs. 2 and 8). A
single layer of cross-striated contractile fibrillae are differen-
tiated in the cortical layer of each muscle cell. The fibril-
lae take a general longitudinal course, but are inclined about
18° to the right of the longitudinal axis of the tail. The fibril-
lae of adjacent muscle cells join end to end and thus convert the
entire series of muscle cells of each muscle band into a single
muscle. A further indication that the muscle band, rather than
the individual muscle cell, is the morphological as well as the
physiological unit, is afforded by the fact that the alternate light
and dark segments of the fibrillae are so placed that they form
continuous straight transverse rows or lines across the muscle
bands, which are not in any way interrupted or interfered with
by the muscle cell walls. Each muscle band functions as a
unit in a way that indicates that its origin is located at the an-
terior end of the notocord, its insertion at the posterior end.
With each muscular contraction the tail makes a propeller-blade-
like stroke, due to the oblique or spiral course of the contractile
fibrillae in the muscle bands, and the body of the tadpole is thus
made to rotate clockwise during locomotion.
The central portion of each muscle cell contains a nucleus
and cytoplasm in which vacuoles, pigment granules, and larger
spherical inclusions are usually found (figs. 2 and 8).
ALIMENTARY TRACT
The pharynx, which at this stage is not organically connected
with the oral siphon, occupies a large portion of the median
dorsal part of the body (fig. 1). In the part of its dorsal wall
situated immediately in front of the oral siphon, the endostyle
is conspicuously differentiated as a double longitudinal fold.
A deep groove is included between the folds, at the bottom of
which a tract of long cilia is developed (figs. land 2). The ventral
wall of the pharynx is intimately associated, especially at its
JOURNAL OF MORPHOLOGY, VOL. 36, NO. 1
88 CASWELL GRAVE
anterior end, with a comparatively large cone-shaped mass of
yolk material, the yolk granules and masses seeming to be included
within enormously developed endodermal cells (figs. 1 and 2).
The lateral walls of the posterior portion of the pharynx are in
contact with the inner walls of the right and left horns of the
atrium and a series of open communications between the pharyn-
geal and atrial cavities has been formed by the differentiation of
three horizontal rows of ciliated gill openings; the first or dorsal
row consisting of seven small openings, the middle row of six
somewhat larger ones, and the ventral row of five comparatively
large gill openings (figs. 1 and 3).
The part of the alimentary canal in which oesophagus, stomach,
and intestine are differentiated communicates with the pharynx
at a point near the posterior end of the yolk mass and slightly
to the right of the longitudinal axis of the body. The stomach and
intestine have the form of a horizontal loop in the midventral
region of the body beneath the yolk mass. The rectal portion
of the intestine pierces the left horn of the atrium and the anus is
found near the base of the atrial siphon (fig. 1). It is perhaps
needless to state that the alimentary tract is not functional
during the larval period.
NOTOCORD AND ENDODERMAL STRAND
The notocord occupies the proximal two-thirds of the axis of
the tail and penetrates the body to a point directly below the
middle portion of the sensory vesicle, where it ends in contact
with a thickened portion of the pharynx just behind the pointed
posterior end of the yolk mass (figs. A, C, and 1). It retains no
trace of its cellular origin, but at this stage is made up of a thin
cortical sheath of dense material in which granules and larger
spherical bodies are embedded, and a central medullary core
composed of non-staining substance, at the periphery of which
a tracery of delicate strands can be made out (figs. 3, 8, and 10).
A longitudinal linear series of cells, known as the ‘endodermal
strand,’ occupies a portion of the space on the right side of the
notocord between the overhanging edges of the muscle bands.
One or sometimes two ‘strand’ cells can usually be made out in
STRUCTURE OF THE AMAROUCIUM TADPOLE 89
transverse sections of the tail, but none have been observed
along the part of the notocord that lies within the body (figs. 2,
3, and 8).
PERICARDIAL SAC AND HEART
The pericardial sac, a thin-walled oval structure containing
a spacious cavity, is situated in the anterior ventral part of the
body beneath the anterior portion of the yolk mass. <A shallow
but wide invagination of the dorsal wall, which extends the
entire length of the pericardial sac, shows the process by which
the heart is differentiated (figs. 1 and 2). The formation of
the pericardial sac of ascidians has been described as an out-
growth from the embryonic pharynx, but any connection that
may have existed at an earlier stage between these structures
in the Amaroucium embryo has disappeared in the fully formed
tadpole larva.
THE ATRIUM
The atrium is an extensive, U-shaped, thin-walled structure,
the lateral horns of which enfold the posterior portion of the
pharynx (figs. 1 and 3). As has been stated in the section on the
pharynx, the cavities of the atrium and pharynx are connected
by means of three rows of gill openings. The middle portion
of the atrium is joined to the atrial siphon and the atrial cavity
is in open communication with the atrial canal.
SUMMARY
The points that have been added to the morphology and nat-
ural history of ascidian larvae as a result of this study of Amarou- °
cium constellatum may be stated as follows:
1. A periodicity in the escape of larvae from the parent colony
was observed. Large colonies can be depended upon to liber-
ate tadpole larvae in swarms at or near sunrise, but an occasional
larva escapes during other hours of the day.
2. Multicellular, blastula-like test vesicles, about sixty in
number, are evaginated from the mantle during a late embry-
onic stage and lie isolated from the body in the substance of the
tunic during the entire free-swimming period of the larva.
90 CASWELL GRAVE
3. The lenses of the eye are not modified retinal cells, but are
deposition products of a semiliquid or gelatinous nature, formed
within vacuoles of cells located in the margin of the ret-
inal ganglion.
4. Visual rods, differentiated in retinal cells of the eye, pro-
ject through the pigment zone and end at the inner surface of
the pigment cup.
5. The eye is proved, by structural and physiological evidence,
to function in the orienting responses of the tadpole to light.
6. The statolith is formed in a vacuole of the sensory statolith-
cell and is composed of a hard substance that is not disintegrated
by strong acids.
7. A visceral nerve, which originates in the visceral ganglion
and has a distribution in the region of the endostyle, is described.
8. The nerve cord is not dorsal in position, but, due to a perma-
nent twist of the tail 90° to the left, is located on the left side
of the notocord.
9. The two series of muscle cells located on the dorsal and
ventral sides of the notocord are shown by morphological and
physiological evidence to function as units in a way to produce
the rotatory movement of the body during locomotion.
STRUCTURE OF THE AMAROUCIUM TADPOLE 91
BIBLIOGRAPHY
Damas, D. 1904 Contribution 4 l’étude des Tuniciers. Archiv. de Biol.,
AO:
GRAVE CasweLL 1920 a The origin, function and fate of the test vesicles
of Amaroucium constellatum. (Abstract.) Anat. Rec., vol. 17.
1920b Amaroucium pellucidum form constellatum. I. The activities
and reactions of the tadpole larva. Jour. Exp. Zodl., vol. 30.
GortTr, A. 1875 Die Entwickelungsgeschichte der Unke als Grundlage einer
vergleichenden Morphologie der Wirbeltiere. Leipzig.
Kowatevsky, A. 1866 Entwickelung der einfachen Ascidien. Mem. Acad.
Se. Saint-Petersb., ser. 7, vol. 10.
1871 Weitere Studien ueber die Entwickelung der einfachen Ascidien.
Arch. mikr. Anat., Bd. 7.
LAHILLE, F. 1890 Recherchessurles Tuniciersdes cotes de France. Toulouse.
McBripz, E. W. 1914 Text-book of embryology, Invertebrata, vol. 1. Mac-
Millan & Co.
Ritter, W. E. 1909 Halocynthia Johnsoni. A comprehensive inquiry as to
the extent of law and order that prevails in a single animal species.
Univ. Cal. Pub., vol. 6.
SaLensky, W.1893 Morphologische Studien an Tunicaten: ueber das Nerven-
system der Larven und Embryonen von Distaplia magnilarva. Morph.
Jahrb., Bd. 20.
Sreiicer, O. 1885 Dik Entwickelungsgeschichte der socialen Ascidien. Jen.
. Zeitschr. f. Naturw., Bd. 11.
Wititey, ARTHUR 1894 Amphioxus and the ancestry of the vertebrates.
MacMillan & Co.
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EXPLANATIONS OF PLATES
ABBREVIATIONS
ad.p., adhesive papilla
at., atrium
at.s., atrial siphon
ch., chorion
e, eye
en., endostyle
end. sd., endodermal strand
g., definitive ganglion
g.0., gill opening
ht., heart
hyp., hypophysial duct
int., intestine
k., keel
l., lens
L.c., lens cell
m., mantle
mc., muscle cell
m.f., muscle fibrilla
mes., mesenchyme
n, notocord
n.c., neural canal
n.cd., nerve cord
o.s. oral siphon
per, pericardium
ph., pharynx
pg, pigment cup
r., rectum
r.c., retinal cell
ret, retina
sn. g., subneural gland
st., stomach
stat., static organ
$.v., sensory vesicle
t., tail
t.c., test cell
t.f., tail fin
tu., tunic
t.ves., test vesicle
v.g., visceral ganglion
v.n., visceral nerve
vis.rd, visual rod
y., yolk mass
With the exception of figure 1, which was drawn by Miss Besse E. Stocking,
the drawings have been made by the writer of the paper, camera lucida outlines
forming the basis for each. Figures 2 and 4 illustrate structures of the
embryo, all others are of the free-swimming tadpole larva of Amaroucium con-
stellatum. Text figure A taken from a paper by the author published in The
Journal of Experimental Zodlogy, volume 30, number 2, February 20, 1920,
page 243.
ABBREVIATIONS TEXT FIGURE A
R, right side
sc, statolith cell
sv, sensory vesicle
tf, tail fin
tg, groove in test resulting from
pressure of the tail during em-
bryonic development
tv, test vesicles
as, atrial siphon
dap, dorsal adhesive papilla
L, left side
lpo, light perceiving organ
map, middle adhesive papilla
Mc, muscle-cell sheath
n, notocord
os, oral siphon
93
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PLATE 3
EXPLANATION OF FIGURES
4 A transverse section through the sensory vesicle, hypophysial duct,
definitive ganglion, and subneural gland of an embryo. It is reproduced to
show especially the developing lenses within vacuoles of lens cells, the similarity
of lens cells and nerve cells of the retina, and the relation of the pigment granules
of the pigment cup to the bodies of the retinal cells.
5 A drawing of the sensory vesicle and its sense organs as viewed from the
dorsal side of a living tadpole.
6 A slightly oblique transverse section through the nervous system in the
plane of the statolith cell, the latter displaced slightly by the knife. The entire
series of lenses and their position in the axis of the pigment cup are shown.
7 A sagittal section of the middle adhesive papilla, showing its tubular
structure and its content of mesenchyme cells.
S A transverse section of the tail, showing the horizontal fins; the struc-
ture of the notocord; the muscle cells, four on the dorsal and four on the ventral
side of the notocord; the cut ends of muscle fibrillae in the cortical layer of the
muscle cells; the hollow nerve cord on the left side of the notocord in the space
between the edges of the muscle bands; the endodermal strand in the correspond-
ing position on the right, and the mantle sheath closely applied to the muscle
band beneath the tunic.
PLATE 3
STRUCTURE OF THE AMAROUCIUM TADPOLE
CASWELL GRAVE
PLATE 4
EXPLANATION OF FIGURES
9 A median section of a test vesicle showing its structure and its position
in the substance of the tunic. Test cells are shown in various positions, some
located near the surface of the mantle, some above and to the right of the test
vesicle, and others in the tunic epithelium. Some contain pigment granules,
some are without granules.
10 A portion of a transverse section through the posterior part of the eye.
It is reproduced to show especially the four visual rods which are cut trans-
versely in the tangential section of the pigment cup and the extent and struc-
tures of the visceral ganglion.
11 A portion of a transverse section in which a visual rod of one of the retina
cells is cut lengthwise. Two only of the series of lenses are in the plane of the
section.
100
PLATE 4
STRUCTURE OF THE AMAROUCIUM TADPOLE
CASWELL GRAVE
Resumen por el autor, Frank Helvestine.
Amitosis en las células ciliadas de los filamentos branquiales de
Cyclas.
En los filamentos branquiales de Cyclas las células no ciliadas
del epitelio basal producen las células ciliadas del epitelio lateral,
latero-frontal y frontal. La mitosis tiene lugar en las células
del epitelio basal y en las células no ciliadas del eséfago. La
amitosis es el método exclusivo de proliferacioén de las células
ciliadas de los filamentos branquiales. Mediante division ami-
tosica la célula madre del epitelio latero-frontal produce células
transicionales de las cuales nacen células que reemplazan las
células cadueas del epitelio frontal. En las células ciliadas de
Cyclas no puede demostrarse la existencia de un centrosoma, y
entre las mitocondrias y las pestafas vibratiles no puede dis-
cernirse relacion genética alguna. Las pruebas indirectas indican
que los cuerpos basales de las células son derivados del centro-
soma.
Translation by José F. Nonidez
Cornell Medical College, New York
AUTHOR’S ABSTRACT OF THIS PAPER ISSUED
BY THE BIBLIOGRAPHIC SERVICE, NOVEMBER 7
AMITOSIS IN THE CILIATED CELLS OF THE GILL
FILAMENTS OF CYCLAS
FRANK HELVESTINE, JR.
Department of Histology and Embryology, University of Virginia
TWO PLATES (SEVEN FIGURES)
INTRODUCTION
The purpose of this investigation is further to test the validity
of the hypothesis first formulated by v. Lenhossék and by
Henneguy (798) that the basal bodies found in ciliated cells are
derived from the centrosome, and particularly the corollary of
this hypothesis, as expressed by Jordan (713), that ciliated
cells, in consequence of the loss of their centrosome through
partition in the formation of basal bodies, must necessarily
proliferate by amitosis.
Jordan (713) supports the hypothesis as regards especially
the ciliated epithelium of the ductuli efferentes of the white
mouse, and shows for the first time that the cells in this region
multiply exclusively by direct division. In no case was he able
to demonstrate a centrosome or a mitotic figure.
Saguchi (’17) confirms the observation of exclusively amitotic
division in ciliated cells of vertebrates, but he does not agree with
the view held by Jordan that these cells divide amitotically
because the centrosome has been used up in the formation of
the basal granules. He concludes that there is no genetic relation
between basal bodies and the centrosome. He states also that
the basal granules are formed by differentiation from mitochon-
dria, both in vertebrates and in invertebrates. He further
declares that in invertebrates ciliated cells multiply by mitosis
exclusively. It may be stated at once that Saguchi’s descriptions
and illustrations do not adequately support his conclusions.
103
104 FRANK HELVESTINE, JR.
In my investigation the chief interest centers about the ques-
tions: 1, whether ciliated cells in invertebrates divide only by
mitosis; 2, the significance of amitosis in ciliated cells; 3, the
relation of the basal granules to the centrosome, and, 4, the
relation of the basal granules to mitochondria.
The material I have employed in this investigation is the cili-
ated epithelium of the gill filaments of the fresh-water mussel,
Cyclas. I find that in this invertebrate the ciliated cells divide
by amitosis exclusively, and no direct relation between mito-
chondria and ciliogenesis is discernible.
MATERIAL AND METHODS
The material for this study was suggested to me by Prof.
William A. Kepner. The mussels were shelled and then fixed
in various solutions, of which Meves’ was found to be by far the
most satisfactory. Paraffin sections were cut at 5yu, and, after
mounting, were stained with Heidenhain’s iron-hematoxylin.
This technic gave results little short of perfect.
DESCRIPTION
For a histologic description of the epithelium of the gill fila-
ments of Cyclas, I shall divide the epithelium into four regions
according to its position. I distinguish: a, basal epithelium,
or the epithelium lying at the base and between adjacent fila-
ments; b, lateral epithelium; c, laterofrontal epithelium; and, d,
frontal epithelium.
a. Basal epithelium. The epithelium of this region is non-
ciliated (fig. 1, A). The cells on account of their position are
more or less pyramidal. They contain one or two vesicular
nuclei. Among these cells mitotic figures are occasionally
seen (fig. 3, M). The cytoplasm contains many filar and bacil-
lary mitochondria collected for the most part toward the periph-
ery of the cells. From the cells of this region which lie adja-
cent to the gill filaments, ciliated cells are formed which are
pushed up onto the filament, giving rise to the lateral epithelium
(ig. 5, L).
AMITOSIS IN CILIATED CELLS 105
b. Lateral epithelium. Here the cells are for the most part
ciliated. The lateral epithelium of a gill filament at any point
may be composed, on one or both sides, of a single row of tall
cuboidal cells (fig. 4, LZ) or of several rows of columnar cells
(fig. 7, L). (The illustrations are of transverse sections of the
gill filaments. In longitudinal sections of the filament the
several types of cells occur in long rows.)
The cilia of these lateral cells are long and heavy, and are
attached to a single row of basal granules lying under the striated
border (fig. 1, L, 4, Z, 5, £, 7, L). The nuclei are vesicular in
character. Amitosis can occasionally be seen in these cells
(figs. 1, L, 4, L, 5, L, 7, L). This process begins with the inden-
tation of the nuclear membrane (fig. 4, LZ), which indentation
deepens until two nuclei are formed (figs. 1, L, and 5, L). The
nuclei separate, after which there follows a cytoplasmic division
(fig. 7, L). The cytoplasm stains only lightly and contains
filar and bacillary mitochondria, most of which are distal
to the nucleus. No centrosome can be detected in a single in-
stance. Between these cells and the laterofrontal epithelium
several small non-ciliated cells may (fig. 1, J) or may not (fig. 4)
be interposed.
c. Laterofrontal epithelium. The laterofrontal epithelium is
derived from the lateral cells and replaces the worn-out cells of
the frontal region. The parent cell or cells of this region comply
with the same description as the cells of the lateral epithelium;
they are in fact identical with these cells, but in a different posi-
tion. The nuclei, however, soon enlarge and become hyper-
chromatic (fig. 1, P). The first indication of nuclear division
is the indentation of the nuclear membrane (fig. 4, NV). The
nucleus undergoes a multiple division, and from one nucleus,
by amitosis, a number of daughter nuclei are formed (figs. 5,
N, 7, H). Coincident with nuclear division there occurs a dis-
tinct pairing of the cilia and basal bodies (fig. 5, N, 7, H). Cyto-
plasmic division follows, and thus is formed a new type of cell, a
columnar cell with a deeply staining, narrow, elongated nucleus
(fig. 6, T). The cytoplasm contains filamentous and bacillary
mitochondria. Each cell possesses four paired flagella-like cilia
106 FRANK HELVESTINE, JR.
and four relatively large basal granules (fig. 6, 7’). The amitotic
division involves two vertical cell-planes at right angles to each
other. Whether this portion of the epithelium be viewed in
sections cut through the gill filament vertically, as in the illus-
tration, or in a plane at right angles to this, that is, ina plane
that cuts the filament longitudinally, these cells, thus cut lon-
gitudinally, present in either case two basal bodies and two cilia.
This condition demonstrates that these daughter cells of the
amitotically dividing mother cell are of columnar shape and
possess a double pair (that is, four) of basal bodies and cilia.
Amitotic division in the laterofrontal epithelium is not always
in the same plane (fig. 5, N), nor are the daughter nuclei of
uniform size (fig. 7, H). Neither does the development of the
cells of the lateral and laterofrontal epithelium always take
place in the order that I have described. In some cases the
primitive cell of the lateral epithelium undergoes amitosis before
reaching the laterofrontal position (fig. 6, 7’), and the columnar
cells which form a transition®* between the laterofrontal and the
frontal epithelium may be both in the lateral and laterofrontal
position (fig. 6, 7).
d. Frontal epithelium. The frontal epithelium is derived from
the transitional cells of the laterofrontal epithelium. ‘These
transitional cells as they are needed are pushed into the frontal
position. Here a transformation occurs. First, there 1s a par-
tition of the basal granules and a splitting of the cilia, so that
the cell, instead of having two pairs of cilia and two pairs of
basal granules, now has a tuft of cilia and many basal gran-
ules (fig. 5, R). The cilia of these cells are originally long,
but due to their exposed position on the crest of the filament,
where they are constantly in contact with grit and other abrasive
materials, the cilia are broken off and give the appearance of a
brush-like border (figs. 4, 4,5, A,7, A). The nuclei of the fron-
tal cells that are adjacent to the laterofrontal region are of the
same character as the nuclei of the latter region (fig. 5, #). As
the cells are moved crestward the nuclei become for a time more
vesicular (fig. 5, A). When the cells of the frontal epithelium
become injured or worn out the nucleus of the cell becomes
AMITOSIS IN CILIATED CELLS 107
pyknotic (figs. 5, D, 6, C) and finally disintegrates by karyor-
rhexis. Such necrotic material is resorbed basally, and new
cells are pushed over and into place of the degenerated ones.
In the ciliated epithelium of the alimentary tract of Cyclas
I find that cellular proliferation takes place by mitosis (fig. 2).
The epithelium of this region is of the tall columnar variety.
The nuclei of these cells are situated proximally and are pale
staining. Each cell has a tuft of moderately long cilia extending
from its distal border (fig. 2). The cilia are attached to a
double row of basal granules, and from the innermost row of
granules a cone of rootlets extends down into the cytoplasm,
the apex of the cone falling on one side of the nucleus. The
cytoplasm of these cells contains some filar and bacillary mito-
chondria. Centrosomes could not be detected in the cells with
cilia. Mitosis in these cells takes place only before cilia have
appeared, or after they have disappeared.
REVIEW OF LITERATURE
In 1877 Peck*® published a description of the lamellibranch
gill, which has formed the basis for all subsequent text book
accounts. In this comparative investigation Peck devoted
special attention to the histologic details of the gill epithelium in
Anodonta. He distinguishes in this form ‘‘frontal, latero-frontal,
and lateral epithelium.”” He described all cells as being ciliated.
“Those of the latero-frontal rows (a single row on each side of
the frontal epithelium) have the longest cilia, far outreaching
those of the other cells; the frontal epithelium and the more
forward cells of the lateral epithelium come next with finer and
much shorter cilia, and lastly, the inlying lateral epithelium has
but very short cilia.”” He calls especial attention to the cells
of the laterofrontal epithelium, and describes these cells as being
larger than their neighboring cells, and appearing from the
surface like goblet cells with a single coarse flagellum issuing
from them, while if seen in a transverse section of a filament
these cells appear broad and a little flattened, the single flagellum
proving to be an adhering group of long cilia. The nucleus of
these cells he described as large and clear, and as enveloped by
108 FRANK HELVESTINE, JR.
only a narrow layer of cytoplasm. In regard to this epithelium,
Peck gives no further details.
Henneguy? and vy. Lenhossék,‘ working independently, ex-
pressed at about the same time (1898) their opinion regarding
the origin of cilia, and especially the basal granules. The Len-
hossék-Henneguy hypothesis states that the basal bodies of
ciliated cells are identical with the centrosome, that is, derived
from it, and it is based on, 1) a series of histologic analogies
between the basal bodies and the centrosome and, 2) histologic
details that seem significant.
A comparison between the centrosome and the basal corpuscles
brings out the following facts: a) both bodies have the same
form; b) they stain alike and with the same intensity; c) in
unstained preparations the basal bodies refract light to the same
degree as does the centrosome and, d) the position of the centro-
some in certain non-ciliated cells corresponds to the position
of the basal corpuscles in adjacent ciliated cells. The evidence
furnished by certain workers* seems to lend weight to the hypoth-
esis: a) ciliated cells have no centrosome; b) certain ciliated cells
do not divide by mitosis, and, c) ciliated cells resemble the
spermatozoon, the flagellum of which is derived from the cen-
trosome.
In 1913 Jordan? showed that amitosis is the exclusive method
of division in the ciliated cells of the vasa efferentia of the white
mouse. ‘That amitosis is the general mode of division in ciliated
cells was supported by observations on the epididymis of the
rat, horse, bull, mule, rabbit, and dog and the trachea of the cat
and the ciliated cells of the gill of Unio. On the basis of his
results Jordan suggested that the fundamental cause of amitotic
division in the ciliated cells is the destruction of the centrosome
in the formation of basal bodies from which the cilia develop.
Saguchi® more recently (’17) has made an extensive compara-
tive study of ciliated epithelium from various regions in a num-
ber of both invertebrate and vertebrate forms. He claims to be
able to detect centrosomes in ciliated cells of both invertebrates
* The literature pro and con has recently been very fully reviewed by
Saguchi.®
AMITOSIS IN CILIATED CELLS 109
and vertebrates, and he states further that because the centro-
some cannot always be demonstrated is no reason for concluding
that it is lacking. Mitosis, according to this author, occurs
exclusively in the ciliated cells of invertebrates. When these
cells divide by mitosis the basal granules and cilia are said to dis-
appear before division and to be lacking until after division has
taken place. He agrees that in vertebrates the sole method of
division of ciliated cells is by amitosis. In this process the ciliary
apparatus remains unchanged. The difference in the mode of
proliferation in ciliated cells of vertebrates and invertebrates,
he argues, must be due essentially to the degree of differentia-
tion of the cell-plasm. In the development of cilia in cells of
embryonic tissue, Saguchi describes a migration of mitochondria
from the region distal to the nucleus, where they are grouped,
into the cuticle of the cell. Piercing the cuticle, the mitochondria
are described as transforming into cilia. In the efferent tubules
of the mouse and rat ciliated cells are said to be formed from the
cells with brush borders. The mitochondria increase in number
and collect distally to the nucleus. They then proceed to the
distal cell-border and are transformed into rod-like bodies which
sprout short cilia. These cilia pass through the axes of the hairs
of the brush border and gradually lengthen. That the ciliary
apparatus is formed by the differentiation of the mitochondria
and that the centrosome takes no part in the production of cilia
are the chief conclusions of Saguchi.
DISCUSSION
In general I agree with Peck, and can distinguish frontal,
laterofrontal, and lateral epithelium in the gill filament. Peck,
however, did apparently not recognize that such a division must
of necessity be an artificial one, as the type of cells in these
regions varies with their stage of development from the cells of
the basal epithelium, so that one type cannot be said to be
peculiar to one special region. The cell that Peck described as
resembling ‘‘a goblet cell with a single coarse flagellum issuing
from it,” and which he interprets as the large cell of the latero-
110 FRANK HELVESTINE, JR.
frontal epithelium viewed at right angles, I have identified as a
transitional cell arising by amitotic division from the larger
parent cell of the laterofrontal epithelium.
Contrary to the conclusion of Saguchi that mitosis is the ex-
clusive mode of division in ciliated epithelium of invertebrates,
I find that ciliated cells in the gill filaments of the fresh-water
mussel, Cyclas, divide only by amitosis. As to mitosis in ciliated
epithelium, I find that the cells undergoing mitotic division
possess no cilia. Saguchi in his description also states that before
undergoing mitosis the cell loses its cilia. As these cells possess
no cilia during division, it cannot properly be said that ciliated
cells divide by mitosis. Saguchi confirms Jordan’s findings in
vertebrates and concludes with him that amitosis is the exclusive
method of proliferation of ciliated epithelium in these forms.
Since Saguchi admits that the cells in which he saw mitosis in
invertebrates possessed no cilia, and since I have found this to
be the case also in my material, and further that ciliated cells
of invertebrates do divide by amitosis, the conclusion seems
justified that ciliated cells where they proliferate as such do so
exclusively by amitotic division both in vertebrates and in
invertebrates.
From the above it follows that the proximate factor deter-
mining whether a cell of ciliated epithelium is going to prolifer-
ate by mitosis or by amitosis is the absence or presence of cilia.
The question at once arises as to why cells possessing cilia should
always divide by amitosis. Is the cause a structural one or
a functional one? Jordan suggests that amitosis is due to a lack
of a centrosome in these cells, while Saguchi reaches the con-
clusion that amitosis in these cells is ‘“‘due essentially to the
degree of differentiation of the cell-plasm,”’ which latter may be
classed as a functional cause.
Saguchi claims to be able to demonstrate the presence of a
centrosome in ciliated cells. His illustrations and descriptions
do not unequivocally bear out this assertion. The difficulties
attending the identification of such a minute body as the centro-
some from among a large mass of mitochondria render such an
undertaking practically impossible. In my preparations no
AMITOSIS IN CILIATED CELLS 111
undoubted centrosome is discernible. The absence of a centro-
some, or its preemption as basal bodies by the cilia, would seem to
be an adequate structural cause to explain the amitotic division
in ciliated cells. A relation between the formation of the ciliary
apparatus and the centrosome is at once suggested. The Len-
hossék-Henneguy hypothesis states that from the centrosome
by partition the basal granules are formed, and that from these
granules cilia are sprouted. It is very suggestive that the axial
filament of the flagellum of the sperm (comparable to a coarse
ciltum) does grow out from one of the two partition products of
the centrosome of the spermatid.
Saguchi’s description® of the centrosomes in ciliated cells of
the vasa efferentia of the mouse and the rat (pp. 254, 255), and
his illustrations, both indicate the difficulties and uncertainties
involved in an attempt to differentiate centrosomes from mito-
chondria and other cytoplasmic granules, and particularly from
the basal granules. Indeed, his description of the pluricorpus-
cular centrosome in the cells of the rat, ‘curious ring-shaped cor-
puscles,’ which he interprets as ‘derived from the centrosome,’
stating that ‘“‘a centrosome divides repeatedly and forms a ring
by secondary fusion of separated particles” (p. 255), would
seem to accord well with the interpretation of basal bodies as
derivatives of a centrosome. Moreover, both in the case of the
rat and of the mouse, Saguchi describes a diplosome in the non-
ciliated brush-border cells of the efferent tubules, the upper
member of which pair of centrosomes ‘often bears a cilitum’
(p. 255). These observations would seem to support the con-
clusion that basal corpuscles of ciliated cells are derived from
centrosomes; but Saguchi refuses to ascribe to them any such
significance.
I find no relationship, other than spatial, between mitochondria
and the ciliary apparatus. Recent investigations on mitochon-
dria have demonstrated that these cytoplasmic elements have
no direct genetic relationship to structures such as nerve, muscle,
or connective-tissue fibrils, but are fundamental vital elements
of the cytoplasm, probably associated with metabolism. Sa-
guchi, however, concludes and asserts that cilia are formed from
mitochondria. Such a transformation would necessitate not
tt FRANK HELVESTINE, JR.
only a morphological change, but also a chemical change. His
illustrations representing a migration of mitochondria into and
through the cuticle or distal cell-border to form cilia are far
from convincing. The fact that the mitochondria lie between
the nucleus and the distal cell-border in ciliated epithelium holds
some significance for this author. I might suggest that this is
the natural place to look for mitochondria in ciliated cells, or any
other epithelium of the columnar type, as normally these cells
show a marked polarity, and with the nucleus situated well to
the base of the cell, the only position left for the main mass of
mitochondria to occupy is between the nucleus and the distal
border of the cell. Moreover, the analogy between the segrega-
tion of mitochondria about the idiozome of spermatocytes and
the basal bodies of ciliated cells is very suggestive as regards
the homology between centrosomes and basal bodies. These
facts render Saguchi’s claim of a mitochondrial origin of cilia
dubious.
SUMMARY
1. Basal, lateral, laterofrontal, and frontal epithelium can be
distinguished in the gill filaments of Cyclas. The cells of the
lateral, laterofrontal, and frontal epithelium are ciliated and are
derived successively from the non-ciliated cells at the base of the
filaments.
2. Mitosis may occur in the non-ciliated basal epithelium.
The ciliated cells of the lateral, and especially of the latero-
frontal epithelium divide exclusively by amitosis. Mitosis
occurs in the ciliated epithelium of the intestine, but the cells
dividing by this method do not possess cilia and cannot therefore
be classified as ciliated cells.
3. The parent ciliated cell of the laterofrontal epithelium
divides by amitosis, thus producing a group of narrow, cylindric
transitional cells with four basal granules and four cilia each.
These transitional cells, by a partition of the basal granules and
the splitting of the cilia, form cells with tufts of long cilia which
renew worn-out cells of the frontal epithelium. Worn-out cells
of the frontal epithelium disintegrate, passing through a stage of
karyorrhexis, and are resorbed.
AMITOSIS IN CILIATED CELLS its
4. The ciliated cells of the gill filaments of Cyclas reveal no
centrosome.
5. These ciliated cells contain mitochondria in their cyto-
plasm between the nuclei and the distal borders of the cells, but
no genetic relation between mitochondria and cilia is discernible.
6. Indirect evidence points to the conclusion that the basal
bodies of ciliated cells are centrosomal derivatives.
I am indebted to Prof. H. E. Jordan for suggesting this prob-
lem to me and for assistance in the prosecution of this research.
LITERATURE CITED
1 Herwennain, M. 1907 Plasma und Zelle, 8. 284. Fisher, Jena.
Hennecuy, L. F. 1898 Sur le rapports des cils vibratiles avee les centro-
somes. Arch. d’Anat. micr., T 1 (cited from Heidenhain).
3 JorDAN, H. E. 1913 Amitosis in the epididymis of the mouse. Anat. Anz.,
Bd. 438, S. 589.
4 Lenuossix, M. vy. 1898 Uber Flimmerzellen. Verh. d. anat. Ges. zu Kiel
(cited from Heidenhain).
Peck, R.H. 1877 The minute structure of the gill of lamellibranch Mollusca.
Quart. Jour. of Micr. Sci., vol. 17, p. 48.
6 Sacucui, S. 1917 Studies on ciliated cells. Jour. Morph., vol. 29, p. 217.
bo
or
1d By. Ma
EXPLANATION OF FIGURES
All figures except figure 2 were drawn from transverse sections of the gill fila-
ments of Cyclas. The tissue was fixed with Meves’ fluid, cut at 5u, and stained
with iron hematoxylin. The magnification is 1300 diameters, except in figure 5
where is it 1500 diameters. In order not to obscure the basal bodies and their
cilia, cytoplasmic details, including mitochondria and the cytoreticulum, are
added only in figure 5. A 7; Leitz oil-immersion lens was employed in this study.
1 Transverse section showing basal epithelium and portions of two adjacent
gill filaments. The cell in the laterofrontal position is of the primitive type with
a tuft of long cilia (P). Interposed between the laterofrontal and lateral epithe-
lium is a small non-ciliated cell (J). The cell of the lateral epithelium which is
also ciliated (Z) contains two nuclei and is evidently in a phase of amitotic
division. The cells of the basal epithelium are roughly pyramidal in shape and
are non-ciliated (A).
2 Area from the ciliated epithelium of the intestine. The cells are tall
columnar and have a tuft of cilia, a double row of basal granules, and a cone of
rootlets extending into the cytoplasm. One of the cells is undergoing mitosis,
but this cell has no cilia.
3 Transverse section showing basal epithelium and a small portion of
the filament. A mitotic figure (7) is seen in a cell of the non-ciliated basal
epithelium.
4 Section of a complete filament. The frontal epithelium (A) has short,
broken-off cilia. Two pyknotiec nuclei of degenerating cells are seen close to the
basement membrane (D). The cell of the laterofrontal epithelium shows a
nuclear indentation, the initial step in amitosis. The cell of the lateral epithe-
lium (Z) lies next below the laterofrontal epithelium.
114
AMITOSIS IN CILIATED CELLS PLATE }
FRANK HELVESTINE, JR.
PLATE 2
EXPLANATION OF FIGURES
5 Right half of complete filament. A binulceated cell of the lateral epithe-
lium (L), differentiated from the non-ciliated cells of the basal epithelium, is
still almost in a basal position. Amitotiec nuclear division has occurred in the
cell (NV), in the laterofrontal position, with the pairing of the cilia and the basal
granules. A degenerating cell (D) of the frontal epithelium, with a pyknotic
nucleus surrounded by a vacuole, is situated toward the interior of the filament.
The cells of the frontal epithelium (A) have short worn-off cilia and vesicular
nuclei. The mitochondria, predominantly of bacillary and filar form, are aggre-
gated in the distal border of the cells.
6 Transverse section of half of a filament. The primitive cell of the lateral
epithelium has suffered amitotie division, forming transitional cells (7). Each
daughter cell is of the tall columnar variety and shows in vertical sections a pair
of cilia and a pair of basal bodies. A remnant (J/) of the primitive cell (VM and T)
is undergoing belated nuclear division. The pairing of the basal granules and
the cilia is conspicuous next the uppermost of the group of daughter cells (7).
Two of the frontal cells (C) are in stages of degeneration.
7 Transverse section of gill filament, showing frontal epithelium (A), mul-
tiple direct nuclear division with pairing of cilia and basal bodies in the latero-
frontal epithelium (H), and the lateral epithelium of one side composed of two
cells (L).
116
PLATE 2
AMITOSIS IN CILIATED CELLS
FRANK HELVESTINE, JR.
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Resumen por el autor, D. H. Wenrich.
La estructura y division de Trichomonas muris (Hartmann).
Este flagelado que habita en el ciego del rat6n mide 12 a 16
micras de longitud por 5 a 10 micras de espesor, y posee los
siguientes orgdinulos: Ntcleo, citostoma y blefaroplasto con las
estructuras que en él se insertan—los tres flagelos anteriores, el
flagelo posterior que corre a lo largo del margen de la membrana
ondulante, el bast6n basal cromdtico en la base de dicha mem-
brana, el axostilo, las filas externa e interna de granulos croma-
ticos y el cuerpo parabasal. Esta tiltima estructura es el cuerpo
parabasal de Janicki (’11) pero aparece solamente con ciertos
métodos téenicos. En la divisién pueden reconocerse estados
comparables a la profase, metafase, anafase y telofase de las
células de los metazoarios. Durante la profase se forman seis
cromosomas dobles (hendidos longitudinalmente), mientras que
el cariosoma desaparece gradualmente como en el caso del nuc-
leolo de los metazoarios.
El nuevo bast6n basilar cromitico se origina como una hilera
de pequefios grdénulos que se inserta por uno de sus extremos en el
blefaroplasto. La nueva membrana ondulante y el flagelo pos-
terior se desarrollan al mismo tiempo que el bast6n cromatico
basal. Un pequefio blefaroplasto nace por gemacion del primi-
tivo, y ambos permanecen reunidos por una paradesmosis durante
la division. La membrana nuclear persiste durante la mitosis.
El comportamiento de los cromosomas durante la metafase y
anafase es semejante a los dellas células de los metazoarios. El
axostilo primitivo degenera, formdndose uno nuevo a expensas de
cada blefaroplasto. El borde interno del bast6n cromatico basal
produce por gemacién una nueva fila de grinulos cromaticos.
La divisién de la célula se retrasa hasta que las dos series de
orgdnulos est’in completas. El nucleo y el cuerpo celular son
las Unicas partes que se dividen ecuacionalmente, mientras que
todas las demas partes necesarias aparecen como eccrine
de las estructuras primitivas correspondientes.
Translation by José F. Nonidez
Cornell Medical College, New York
AUTHOR’S ABSTRACT OF THIS PAPER ISSUED BY
THE BIBLIOGRAPHIC SERVICE, NOVEMBER 7
THE STRUCTURE AND DIVISION OF TRICHOMONAS
MURIS (HARTMANN)
D. H. WENRICH
Department of Zoology, University of Pennsylvania
ONE TEXT FIGURE AND FOUR PLATES (THIRTY-SIX FIGURES)
CONTENTS
LILO NCTE Msn da bts 6 bo OME GO SIGH tt SORES Maa sc DIE Oe Se AMES ore ee 119
Misirene GEANOEMe LIU S omer ai iat-acEois ae ae oeinid asi aac tere ee eas 120
AL Misterials, 0.3.00 6.6 ox. RE I Ee Sr Eee eS 120
12> WMIQUNOT Bisles Beas tert toe ee Son ote ceil o Rea ere on ce eae 121
MWSEVC He nAulye TN ClyICUal siesta eo che or sco, eee achie oeac Diats bicera Date 122
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Paw Sent Me eed ney Stes Aes: eee eR MES es Soon Se Py SEE, POA 123
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Ds itterences dwe:to difterent fixablves.. ...- os ecese oe he oe ve tee aleve 130
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Aamiheeblephanoplast Veracca c sie. «co oe nee a tee eed 138
5. Other structures: 4.) =... <- Po Gl enbelias Di Maes! vere seas d cce 139
LED SUIS RD PES aS oT Se SO Be Om a SER ee Eg Oe at CUM RAS ce de ee a SAE eA 140
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15 PGE. 05 SL {sR Fh be oot A PEE ISERIES cock Ge cas ORE 141
pumMimary ol, the more Important. TEsULtst.)...ic8. <5) decd Sh di aloes we slew eae o 145
nee Ren ERE TRC reer ova he, cin Wei Aa Sev toe Gh SOR oie say Sd oe Meise his, ohn ae 147
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INTRODUCTION
The structure and division processes of various species of
Trichomonas have received the attention of several investigators,
but there is much disagreement among them regarding details
of structure in the vegetative condition and events during division
even in the same species. Such differences are very noticeable,
for example, in the most extensive accounts in recent years, one
119
JOURNAL OF MORPHOLOGY, VOL. 36, No. 1
120 D. H. WENRICH
by Kofoid and Swezy (’15) and two by Kuczynski (14, 718).
The structure and division of Trichomonas muris were described
in these papers as well as in the earlier one of Wenyon (’07).
Since I have been able to secure some material which seems to
be especially favorable for the study of cell structure and division
in this species, and since my findings are not in entire agree-
ment with any of the authors mentioned above, it would seem
to be worth while to place on record my observations. I have
begun an investigation of the various intestinal protozoa of
rats and mice, but the present account will be limited to the one
species.
MATERIALS AND METHODS
A. Materials
Trichomonas muris (Hartmann) is found chiefly in the coecum
of mice and to a less extent in the large intestine. Only rarely
has it been found in the small intestine and then only at the
lower end.
The first material from the coecum of a mouse (Mus musculus)
in which I found the division stages numerous was secured in
December, 1916. Slides made from this material have proved
to be the most valuable in the collection, and many of my figures
have been made from them. Since that time 102 additional mice
have been examined, of which fifty-one were wild and fifty-one
were albinos. Of the wild mice, nine were Peromyscus leucopus
and the others were the house mouse, Mus musculus. Only
two of the Peromyscus and only five of the forty-two house
mice showed infection with Trichomonas muris, while fifteen
of the fifty-one white mice were found to harbor this species.
Young mice showed less tendency to infection than adults and
the degree of infection was extremely variable. It ranged from
occasional specimens to cases when the entire contents of the
coecum appeared to consist of Trichomonas and a few bacteria.
In these latter cases division stages were common in the mass
of coecal contents as well as near the mucous membrane.
STRUCTURE AND DIVISION OF TRICHOMONAS 121
B. Methods
Aside from preparations of living flagellates in fresh coecal
contents mixed with salt solution, cover-glass preparations fixed
and stained in various ways and mounted in balsam were em-
ployed. Coecal material, usually from the region adjoining the
mucous lining, was mixed with a little salt solution and smeared
out thin on clean cover-glasses. With few exceptions these
smears were fixed without allowing them to dry, although
occasionally some were dried and subsequently stained with
some modification of the Romanowsky method. These dried
smears do not give satisfactory preparations and have not been
used as the basis of the observations here recorded.
For the wet smears the following fixatives, usually heated to
about 40°C., have been tried: Schaudinn’s sublimate and alcohol,
with and without the addition of acetic acid; Worcester’s formol-
sublimate-acetic; Flemming’s stronger and weaker solutions;
Perenyi’s chrom-nitric acid; Carnoy’s alcohol-chloroform-acetic ;
Bouin’s picro-formol-acetic; Allen’s (16) modification of Bouin’s
(B 15); sublimate-acetic, and picro-mercuric. The most satis-
factory of these have proved to be Schaudinn’s, Bouin’s, Allen’s,
and Flemming’s, in about the order named. Some other fixa-
tives were used in special experiments which will be described
elsewhere.
For staining, Delafield’s, Heidenhain’s iron alum-haematoxylin,
and safranin (after Flemming’s) have been tried, but most
of the smears have been stained with Heidenhain’s haema-
toxylin, which has always given the most satisfactory results.
Alcoholic solutions of haematin, haematoxylin, and iron-alum,
according to the methods described by Dobell (’14) and Kofoid
and Swezy (15), were tried, but did not give results as satis-
factory as the twenty-four-hour staining in iron alum-haema-
toxylin, so their use was not continued. Various counterstains
were tried, but none of them appeared to add to the value of
the preparations, and were not generally employed.
122 D. H. WENRICH
THE VEGETATIVE INDIVIDUALS
A. Form
Both in the living and the fixed condition the body of this
species of Trichomonas is rather fusiform, with a length of from
one and a half to two times the greatest width. There is some
tendency for the so-called dorsal side to be more convex than
the opposite, somewhat flattened, ventral side. In the free-
living condition the region of greatest width is usually near the
middle, but the flexibility of the pellicle permits a variety of
shapes, especially when the animals are creeping or forcing their
way through the coecal debris. Then the body may change
shape rapidly and some of the variations are to be seen in the
fixed material. For example, figure 7 shows an animal with
the anterior end much more pointed than the one in figure 8.
Figures 8 and 17 show animals with the greatest width at the
posterior end instead of in the middle, as is more common.
Adverse conditions, such as lowered temperature, changes in
the constitution of the surrounding fluid, or desiccation, often
lead to considerable changes in form, the most common modifi-
cation being the rounded-up condition (figs. 11, 18, and 15).
The rounded form also seems to be characteristically assumed
during the process of division (figs. 20 to 30). When confined
in cramped quarters the form changes are exceedingly various.
In free-swimming animals the undulating membrane is spirally
arranged on the surface of the body, and they rotate on the long
axis, without any appreciable changes in diameter. On the
other hand, fixed and stained individuals often give the impression
of being flattened and of lying on one side with the undulating
membrane at one edge, as seen, for example, in figures 1 to 4.
In those specimens showing the spiral arrangement of the un-
dulating membrane and accompanying structures, it is seen that
the direction of the spiral is from the left over to the right, as
shown in figures 5 and 17 and in text figure A.
STRUCTURE AND DIVISION OF TRICHOMONAS 123
B. Size
Wenyon (’07) called attention to the great variation in size
in the Trichomonas of mice giving the length as from 3 to 20 un.
Kofoid and Swezy (’15) emphasized a similar variation in size
for T. augusta. In both cases the authors raise the point that
differences in size alone do not furnish sufficient criteria for the
separation of species. I have found two species in mice which
do differ as to size, and it may be that the range of sizes observed
by Wenyon had a greater significance than he supposed. Care-
ful study and measurement of the flagellates found in mouse
no. 1, for example, revealed a larger species which I take to be
T. muris, ranging in length from 8 to 20u, with an average of
TABLE 1
Table showing results of measurements, from certain host mice and for certain
fixatives
UL AVERAGE LENGTH,
MOUSE NUMBER ; FIXATIVE csp ace MICRONS RANGE, MICRONS
1 Schaudinn’s 100 13.1 10-16
19 Schaudinn’s 50 12.7 10-16
24 Schaudinn’s 100 12.8 10-16
24 Allen’s 100 15.7 11-22
12.9, and a smaller species ranging in length from 6 to 9u, with
an average of 7.2u. The smaller species may be T. parva
Alexeieff, and can be differentiated on morphological grounds
other than size, and shows only three chromosomes in division.
In other mice pure infections of each species*were found as well
as other mixed infections.
Measurements have been made from several series of slides,
and the results indicate slight racial differences for the different
hosts as well as differences due to various methods of fixation.
Table 1 indicates some of these differences.
The results for different fixatives is strikingly illustrated by
mouse no. 24, where the average length for animals fixed with
Allen’s fluid (15.7) is 22.6 per cent more than the average
length for those fixed in Schaudinn’s fluid (12.8). It is prob-
124 D. H. WENRICH
able that Schaudinn’s fluid causes shrinkage, and possibly Allen’s
fluid may cause swelling.
In making these measurements great care has been taken to
secure an unbiased selection of individuals for measurement.
Only individuals which appeared normal and had the axostyle
approximately straight were measured. All such individuals in
Text figure A Vegetative individual of T. muris, partly diagrammatic; a,
blepharoplast; b, undulating membrane: c, caryosome; d, nucleus; e, inner row
of chromatic granules; f, parabasal body; g, outer row of chromatic granules; h,
chromatic basal rod; 7, posterior flagellum as chromatic margin of undulating
membrane; 7, axostyle; k, chromatic ring at point of emergence of axostyle; l,
cytostome; m, anterior free flagella; n, posterior free flagellum.
any one field of the microscope were measured. Successive
fields were treated the same way, duplication of fields being
prevented by the use of a mechanical stage. All measurements
were made by the aid of an eye-piece micrometer which had been
calibrated for the set of lenses used. The measurements obtained
agree very well in a general way with those of Kuczynski (14)
and Kofoid and Swezy (’15).
STRUCTURE AND DIVISION OF TRICHOMONAS 125
C. Organization of the cell
The organelles of this species of Trichomonas are those typical
of the genus and are indicated in text figure A. They include
besides the nucleus (d) and cytostome (1) the series of structures
attached to the blepharoplast (a), consisting of the three anterior
free flagella (m), the long posterior flagellum running as the
chromatic margin (7) of the undulating membrane (b) and con-
tinuing posteriorly as a free flagellum (n), the chromatic basal
rod (h) at the base of the undulating membrane, the axostyle
(7), the parabasal body (f)-and the inner (e) and outer (g) rows
of chromatic granules.
The protoplasm is enclosed in a cell membrane or pellicle,
which, as previously noted, is flexible enough to permit vari-
ations inform. ‘These form variations may be classed as ‘eugle-
noid’ in type. Pseudopodia formation has been described by
several authors, for example, by Kuczynski (’14) and by Kofoid
and Swezy (15), but I have seen such apparent pseudopodia
only under conditions which appeared to be either degenerative
or precystic, and therefore I do not regard this phenomenon as
normal for the active individual. The protoplasmic projection
shown in figure 16 is probably the result of mechanical injury
in making the smear, and is not a pseudopodium.
The protoplasm itself is rather fluid in nature, as is indicated
by the rapidity with which form changes occur. It appears to
be somewhat vacuolated, although not to the extent seen in
some other species, such as T. augusta, as figured by Kofoid
and Swezy (15), or T. mirabilis, as figured by Kuczynski (’18).
The appearance or non-appearance of vacuoles seems to vary
somewhat from host to host and from cell to cell. Variations —
from one fixative to another are discussed further on.
The nucleus (text fig. A, d) lies in the anterior third of the
body dorsal to and usually a little to the left of the axostyle
which occupies the position of the principal axis. It is usually
oval or broadly elliptical in shape, being approximately 4 to 5y
long and 2.5 to 3u wide. At the periphery is a delicate nuclear
membrane or caryotheca, which is sometimes difficult to see.
126 D. H. WENRICH
Within the membrane the chromatin occurs as small granules
scattered upon a fibrous network, and as a caryosome (c) of
relatively large size which is usually surrounded by a clear area.
The exact number of the small chromatin granules had not been
determined, but in the early prophases they are reduced to six,
which are paired or split.
The clear area about the caryosome is sometimes large with
a diameter as much as one-half to two-thirds that of the nucleus
(figs. 1, 2, 7, 8, etc.). In other instances it is much smaller
(fig. 13). In figure 1 the caryosome appears to be double, but
this condition is rare. Careful focusing usually discloses fine
fibrous connections between the caryosome and the network at
the periphery of the clear area (figs. 7, 8, 10, 12). There is no
apparent constancy in the position of the caryosome, since it is -
found at either extremity of the nucleus or in any intermediate
position. |
Occasionally there is seen a nucleus like that shown in figure 9,
but such nuclei often are accompanied by signs of degeneration,
and the condition is regarded as abnormal.
I have not been able to make out a rhizoplast connecting the
nucleus with the blepharoplast as described by Kofoid and
Swezy (15).
The cytostome is an opening at the anterior margin of the
body on the side of the major axis opposite the nucleus. This
side is usually considered as ventral. The cytostome is not so
large as that described and figured for T. augusta. There appears
to be a short cavity leading into the interior along the ventral
side of the axostyle.
~The blepharoplast is a deeply staining granule, or possibly a
pair of granules at the anterior end of the major axis of the body.
To it a series of other organelles are attached, as already men-
tioned. The nature of this focus of organization is difficult to
determine. By some authors it is regarded as homologous with
the similarly named structure in some of the simpler flagellates,
such as the haemoflagellates, and by others it is assumed that
in the Trichonomads it is composite, being composed of a number
of granules equal to the number of flagella attached. Martin
STRUCTURE AND DIVISION OF TRICHOMONAS 127
and Robertson (’11) thus describe it for Trichomonas (Tetratric-
chomonas?) gallinarum. Kofoid and Swezy (715) believe it is
composed of two parts, one of which is a centrosome and the
other the basal granule for the flagella. In the material that I
have studied this structure frequently appears to be double,
that is, composed of two approximately equal parts, and the
posterior flagellum is attached to the anterior moiety, while the
chromatic basal rod is connected with the posterior one. Such
a condition was also described by Wenyon (’07). Since the
three anterior flagella take the stain so slightly, it is difficult
to determine what their relation is to the blepharoplast com-
ponents.
Because the three anterior flagella do not stain very deeply,
they are difficult to make out. This difficulty is often increased
by the presence of spirochaetes of similar caliber and staining
power and by the flagella taking a position in contact with, or
under, the body. In the drawings they have been omitted when
not plainly seen. In all cases in which they could be clearly
discerned, they appeared to be of equal length, wavy, and about
one-half the length of the body, although sometimes shorter.
Figures 2 to 5 and 7 to 15 show the flagella in their typical
condition.
Hartmann (10), Wenyon (’07), and Kuczynski (14, ’18)
figure these flagella just as I have found them, but the figures
for this species given by Kofoid and Swezy (’15) have the anterior
flagella as long as or longer than the body of the animal. On
account of this and other differences, one may be led to suppose
that the latter authors were dealing with a different species.
The posteriorly directed flagellum running as the chromatic
margin of the undulating membrane is very much longer than
the others, extending the length of the body, making six to eight
undulations in its course and projecting posteriorly as a free
flagellum as long as the anterior flagella. This posterior part
is similar to the anterior flagella in caliber and staining power,
but the intracytoplasmic portion appears to be much thicker
and takes the stain intensely. There is some variation in stain-
ability depending on the fixative employed, as will be noted
elsewhere.
128 D. H. WENRICH
As has been observed by other authors, the undulating mem-
brane seems occasionally to be broken, allowing the entire
flagellum to become free. Individuals with the posterior flagel-
lum free are not rare in fixed and stained preparations (fig. 15).
The chromatic basal rod takes origin in the blepharoplast,
or possibly the posterior portion of it, and extends along the
surface of the body at the base of the undulating membrane.
It, together with the undulating membrane, takes a spiral course
on the living animals, as in figures 5 and 17, as previously noted,
passing posteriorly from the left over to the right. It ends free
in the cytoplasm. It appears to be a body of some rigidity
because changes in its position are usually accompanied by corre-
sponding changes in the form of the body. As described by
Wenyon (’07), it may project from the body as a stiff thread.
It is broadest near the middle, tapering to a slender distal ter-
minus, and to a less slender proximal or anterior end attached
to the blepharoplast. Near the anterior end it often exhibits
a bend, which may even be S-shaped, which suggests a high
degree of flexibility of that region (fig. 14, e.g.).
Most observers have represented this structure as a homo-
geneous rod. In this species I have been considerably puzzled
about its organization, for frequently it appears to have em-
bedded within it a row of granules on the inner side, similar to
the row which lies close to it, but deeper in the cytoplasm (figs.
11 and 14). At other times the additional row seems to be just
in contact with the rod (figs. 2, 10, 18, 21), and again the row
may be adjacent to but not in contact with the rod (fig. 8).
These observations indicate that new rows of granules take their
_origin from the basal rod and migrate inward, possibly replacing,
during division, the one that is always found close to and parallel
with the rod.
This outer row of chromatic granules close to the chromatic
basal rod is very characteristic of this species and extends from
60 per cent to 90 per cent of the length of the rod out from the
blepharoplast. It is figured by Hartmann (’10), Wenyon (’07),
Kuezynski (14, 718), and Kofoid and Swezy (’15). Another
row of similar granules is found deeper in the cytoplasm, and
STRUCTURE AND DIVISION OF TRICHOMONAS 129
close to the axostyle on its dorsal side. It is easily seen in the
region posterior to the nucleus, but its anterior extension is
frequently obscured (figs. 1, 2, 4, 7, 9, ete.). In some cases it
is traceable forward outside the nucleus up to the blepharoplast.
Posterior to the nucleus this row is nearly parallel to the longer,
more peripheral one. The inner row of granules is mentioned
and figured by Wenyon (’07) and by Kuezynski (’14), but seems
to be absent from the form described by Kofoid and Swezy (715)
under the name of T. muris.
In the region between the nucleus and the blepharoplast
there are often additional granules similar to those in the two
rows (figs. 7, 10, 14). The presence of these extra granules
often makes it difficult to determine the anterior limit of the
nucleus, on account of their resemblence to the granules of
chromatin within the nucleus and the faintness of the nuclear
membrane.
The axostyle is a hyaline cylindrical rod attached to the
blepharoplast and it traverses the major axis to project slightly
at the posterior end, where it tapers rapidly to a sharp point.
At the point of emergence there is the ring of deeply staining
substance (text fig. A, k) mentioned by Kofoid and Swezy.
In the region of the nucleus the axostyle is frequently somewhat
curved around that body which appears to lie slightly to the
left of it. The axostyle seems narrower in the region near the
blepharoplast than elsewhere. J have never seen any cases of a
capitulum in this species such as Kuczynski (’18) mentions.
The flexibility of the axostyle is indicated by the frequent
occurrence in fixed material of a decided bend at the most
flexible region just posterior to the nucleus (figs. 2, 3, 11, 13,
15), but I have never seen this structure used as an organ of
locomotion, as maintained by Kofoid and Swezy (’15) for T.
augusta.
There are no chromatic granules in the axostyle except in
new ones growing out from the blepharoplast in the telophase of
division. However, the deeper row of granules often appears
to be in contact with the axostyle in the region immediately
anterior to the nucleus (figs. 1, 2, 3, 7, 8, etc.).
130 D. H. WENRICH
The parabasal body is a cylindrical curved rod, of a diameter
comparable to that of the axostyle, connected by a narrow attach-
ment to the blepharoplast and lying dorsal to and to the right
of the nucleus. Its texture is apparently different from that of
any other structure in the cell and its staining reaction with
haematoxylin is different from the other structures. While it
appears to be homogeneous, its texture is of a looser, more
spongy nature than that of the structures so far mentioned. Its
appearance compares well with the figures of it given by Janicki
(11). It is quite variable in length, as indicated by figures
3, 4, 5, 6, and 16, but when it is longer it often has a constriction
(figs. 4, 5), or a thinner place (fig. 16), marking off two regions.
One wonders if the distal portion may not become detached and
serve some function in metabolism.
I have never seen any indication of a central core or thread as
described by Cutler (’19) for the parabasal of Ditrichomonas
termitis. On the contrary, in an animal which was either round-
ing up for encystment or else had started to degenerate (fig. 6),
the parabasal appeared as a granular peripheral case enclosing
a non-staining area.
Since Kofoid and Swezy (’15) employed mainly Schaudinn’s
fluid which seems to dissolve out the parabasal, this elusive
organelle was apparently overlooked by them, and they applied
the term ‘parabasal’ to the chromatic basal rod. ‘The homology
of the above-described parabasal in Trichomonas muris with the
similar structures figured by Janicki (’11) for Devescovina,
Parajoenia, Stephanonympha, and Trichomonas and by Cutler
(19) for Ditrichomonas termitis seems to me to be justifiable,
but a homology between the chromatic basal rod and these
parabasals of Janicki, as claimed by Swezy (716), would, in my
opinion, be open to some question.
D. Differences due to different fixatwes
It will be profitable, I think, to consider at some length some
differences of appearance in the organization of Trichomonas
muris which are correlated with the use of different fixatives.
The conditions found in the series of slides from mouse no. 24
STRUCTURE AND DIVISION OF TRICHOMONAS MSE
illustrates this point. In this case the entire set of instruments,
reagents, glassware, microscope, etc., were placed in a warm
room at 37°C. a number of hours before the mouse was killed.
The mouse was taken into the same warm room, killed, opened,
and the coecal contents examined. The coecum was found to
be swarming with Trichomonas, so fixations were made with
Allen’s, Bouin’s, Carnoy’s, Schaudinn’s, sublimate-acetic and
weak F'lemming’s fluids. After fixing for half an hour at 37°C.,
the subsequent washing and further treatment were carried out
at room temperature, and all the slides were stained at the same
time and in the same way with the same stock solutions of iron
alum and haematoxylin. The chemical differences in the differ-
ent fixatives would therefore appear to be the variable factors
in this experiment, so that differences in appearance can, I think,
be attributed to different effects of the fixatives on the organisms.
In avy smear of this kind, of course, there are always thicker
and thinner areas, and the intensity of the stain varies with the
thickness of the film on the cover-glass. It is therefore possible
to compare for a wide range of intensities of the stain.
The general cytoplasm may first be considered. Figure 1
indicates the results from fixation with Carnoy’s fluid. Little
vacuolization is indicated, and such vacuoles as there are do not
show any stainable contents. Figure 2 is from a smear fixed
in sublimate-acetic, and here not only are the vacuoles well
defined, but the contents have taken the stain. Some few
individuals on this smear did not show the vacuole contents
stained, but the great majority did. The smears of this series
fixed in Schaudinn’s fluid showed an occasional individual with
vacuole contents stained. In the other series which were fixed
with Schaudinn’s fluid vacuole contents did not usually take the
stain. Figure 3 is from a smear fixed in weak Flemming’s fluid,
and the structure of the protoplasm is much like that in figure 1.
The various organelles may next be considered. Schaudinn’s
fluid and sublimate-acetic gave somewhat similar results except
for the protoplasmic vacuoles already mentioned. The nucleus,
blepharoplast, posterior flagellum, chromatic basal rod, and
specific granules are all sharply differentiated, although in the
132 D. H. WENRICH
sublimate-acetic slides the chromatic basal rod was not so in-
tensely stained as in those fixed in Schaudinn’s. Similar results
were obtained by the use of Allen’s and Bouin’s fluids, except all
structures appeared swollen in comparison with those prepared
with other fixatives. Also the free flagella were better stained
after the last two fixatives named than after the first two. In
the case of Carnoy’s fluid (fig. 1) the results varied considerably
with the stain. In the animals showing an average intensity
of the stain, the nucleus was very black, often failing to show
any structure, while the chromatic basal rod and the chromatic
margin of the membrane failed to stain. In contrast, the two
rows of chromatic granules were stained very deeply. In the
specimen drawn (fig. 1) the chromatic margin was not so strongly
stained as is indicated and the nucleus was lighter than in the
majority of individuals. The blepharoplast was also faintly
stained on these slides, while the free flagella and the axostyle
were farely well defined in most cases. After weak Flemming’s
fluid all the structures were rather indistinctly differentiated by
the stain, and yet these slides were the only ones in which the
parabasal body appeared.
I did not find the parabasal body until after reading the paper
by Cutler (19), who describes its occurrence in Ditrichomonas
termitis. According to Cutler, this structure was not constant
in material prepared with the usual fixatives, but by employing
Flemming’s without acetic acid and other fixatives which con-
tained neither acetic acid nor corrosive sublimate, he was able
to demonstrate it consistently. Following his suggestion, I
employed on the same lot of material from mouse no. 29 Allen’s,
Bouin’s, and Flemming’s fluids each without acetic; also 1 per
cent chromic acid containing 1 per cent urea and several strengths
of formalin, together with unmodified Schaudinn’s and Allen’s
fluids as controls. The latter two fluids gave the best general
fixation, but the Flemming’s without acetic and the 1 per cent
chromic acid both brought out the parabasal in some individuals
when subsequently stained with iron-alum haematoxylin. Since
Janicki (’11) found the parabasal in T. bactrachorum which
had been fixed with an ‘osmic acid mixture,’ I was led to scrutin-
STRUCTURE AND DIVISION OF TRICHOMONAS 133
ize all of my slides which had been fixed with Flemming’s fluid,
with the result that I detected this structure in T. batrachorum
and T. augusta from the leopard frog and in some slides of T.
muris fixed with weak Flemming’s. Later I found the same
structure in T. caviae in material fixed with weak* Flemming’s
and Flemming’s without actic. The parabasal was most clearly
differentiated in the slides of T. muris fixed in weak Flemming.
Since in the weak Flemming the amount of osmic is reduced and
since, further, the parabasal appeared in slides fixed with 1 per
cent chromic acid, it would seem that the chromic acid is as
much if not more responsible for bringing out this structure than
is the osmic acid. Also, my experience does not parallel that of
Cutler (’18) in the case of formol, since none of my formol-
fixed preparations showed the structure.
In the slides fixed with weak Flemming from mouse no. 24
a great majority of the flagellates showed the parabasal plainly,
while in a few it was difficult or impossible to make it out. In
the slides from mouse no. 29 fixed with 1 per cent chromic and
with Flemming’s without acetic only a small percentage of the
flagellates exhibited the parabasal. There thus appear to be
individual variations with the same technique as well as differ-
ences due to differences in technique. Kuczynski (’14) found
the parabasal in only four out of more than fifty guinea-pigs
and in none of the mice, although over a hundred were examined.
The above results point to the necessity of employing a variety
of methods of technique, since reliance upon a single method
might readily lead to erroneous conclusions.
E. Encystment
Encystment in Trichomonas has been much disputed, there
being few observations of a conclusive nature showing the exist-
ence of cysts. Wenyon (’07) called attention to the existence in
the faeces of the mouse of large numbers of rounded-up indi-
viduals which he stated could live for a week or more outside the
host if kept moist. Some others, which were much contracted
and rounded up, he thought were encysted, and he figures such
a specimen in his figure 35, plate 11. I have seen many of the
134 D. H. WENRICH
rounded-up kind, especially in material from hosts which had
been dead several hours. I have also seen in some hosts con-
siderable numbers of the contracted forms in the coecal contents.
In figure 36 I have represented one of these, and it is very similar
to the one figured by Wenyon. In figure 35 there is shown one
which is apparently in the process of changing to the rounded
and contracted condition. Iam inclined to the belief that these
animals are preparing to encyst, since there is no sign of degenera-
tion except the apparent disappearance of the free flagella.
DIVISION
All authors who have studied carefully the division of any
of the species of Trichomonas agree that the process is compli-
cated and appears to take a relatively long time for its accom-
plishment. Kuczynski (’14) gives eight hours as the time for T.
augusta. It is also generally agreed that the flagellates remain
active during the entire process, the flagella and undulating
membrane continuing to vibrate even in the rounded-up con-
dition which is characteristically assumed during part of the
time. The extensive activities of the post mitotic phase have
been well described and illustrated for T. augusta by Kofoid
and Swezy (’15).
Since it is possible to recognize in the division of the nucleus
stages comparable to those of mitosis in metazoan cells, it will
be convenient to refer to these stages under the conventional
terms, prophase, metaphase, anaphase, and telophase.
A. Prophase
1. The nucleus. The first changes in the nucleus which indi-
cate the approach of mitosis result in the formation of the pro-
phase chromosomes out of the scattered chromatin granules of
the ‘resting’ nucleus. There are always six of these chromo-
somes, and each one consists of a pair of closely associated
moieties. The parts are often somewhat elongated and the two
components lie side by side. These prophase elements remain
connected with each other and with the caryosome, until the
end of the prophase stage, by the fine strands of non-chromatin
ee a a
STRUCTURE AND DIVISION OF TRICHOMONAS 135
reticulum of the nucleus (figs. 10 to 17 and 21). Occasionally
the six elements become arranged in the form of a chain, recalling
the chains of split chromomeres sometimes seen in metazoan
prophases (fig. 15). In cases where the fixation has not been
good, the two parts of each element appear to be fused together,
so that the nucleus seems to have six single granules in it in
addition to the caryosome. This condition seems to be more
prevalent in the later than in the earlier prophases (fig. 19).
Since in the earliest stage in which the prophase chromosomes
can be distinguished they are already double, it has been im-
possible to determine whether or not the doubling is the result
of antecedent splitting.
In the earlier stages the six chromosomes are always outside
the clear area surrounding the caryosome, but later the boundary
of the clear area disappears, and the caryosome then seems to
be more directly connected with adjoining chromosomes by the
non-chromatic reticulum (fig. 15). In cases where the chromo-
somes appear to be single, due to fusion, and where the peri-
caryosomal space can no longer be defined there seem to be
seven chromosomes instead of six, since the caryosome is not
always easily distinguishable from the chromosomes. In all
such cases, however, careful study has resolved the group of
Seven into six chromosomes and one caryosome. During the
progress of the prophase changes the caryosome gradually loses
its staining power just as do nucleoli of metazoan cells, and at
the metaphase no trace of it is visible. Figure 22 shows a very
late prophase or early metaphase with the spindle partly formed
and a faintly defined vestige of the caryosome.
Number of prophase chromosomes. Wenyon (’07) reports the
number of prophase chromosomes as six and says that they early
divide into two, giving six pairs of granules. In his figure 2,
plate 11, for example, he shows six pairs of granules besides a
caryosome. My results are thus in agreement with his. Kuc-
zynski (’14) describes eight prophase and four metaphase chromo-
somes and again insists on these numbers in his later paper
(18). In this later paper, however, he admits (p. 128) that
“Over 70 per cent of the observed prophase nuclei of the Tri-
JOURNAL OF MORPHOLOGY, VOL. 36, NO. 1
136 D. H. WENRICH
chomonads named (T. muris, T. augusta, T. Caviae, and T.
batrachorum) contain seven sharply outlined chromosomes
although in many cases, of which a number have been pictured
(e.g., plate I, figs, 16, 17; plate II, fig. 20; plate IV, fig. 57;
plate VII, fig. 96), the probability is great that the position of
the chromosomes interferes with the certain recognition of an
eighth. Chromosome-groups of only six, of uncertain separa-
tion, occur much more seldom.”’ In all the figures mentioned
in the quotation (except in fig. 96), and in some others not
mentioned, the groups can be resolved into six split prophase
chromosomes and one caryosome. In the few cases where
Kuezynski thinks he finds eight, I am inclined to the belief
that he may have counted as separate chromosomes the two
parts of one which had become rather widely separated; then,
with the caryosome, the number eight is obtained.
Kofoid and Swezy (715) give five as the chromosome number
both for the prophases and the metaphase for T. muris and T.
augusta. If the form which they called T. muris is the same
species as the one I have been studying, the difference in chromo-
some number needs to be accounted for. I will merely refer
to the great difficulty in elucidating these small details in such
minute organisms, even when the technique has been good, and
to the further possibility that the form studied by them was of a
different species.
As for other species, since Kuczynski finds and figures con-
ditions in T. caviae so similar to those in T. muris, I am inclined
to believe that there are six chromosomes in T. caviae. Dobell
(09) found six chromatin bodies in T. batrachorum, but hesitated _
to call them chromosomes. Martin and Robertson (11), on
the other hand, described for 'T. eberthi eight prophase and four
metaphase chromatin units, although they prefer not to call
them chromosomes. It can hardly be argued that all species
of Trichomonas should have the same number of chromosomes,
but since Dobell and Wenyon have both found six and since the
numbers in the species studied by Kuczynski are probably six
instead of eight, the situation in T. eberthi, might bear rein-
vestigation. yy
STRUCTURE AND DIVISION OF TRICHOMONAS 137
I have not found stages with the so-called ‘nuclear cloud’
as described by Kofoid and Swezy (’15) as shown in their figure
49, nor have I seen the spirene stage shown in their figure 50.
My figure 9 shows a condition somewhat similar to their figures
46 and 47, but I think such nuclei are abnormal, particularly
since they are so much larger than usual and often accompany
other evidences of degeneration.
2, Chromatic basal rod. Coincident with the intranuclear
changes of the early prophase, the new chromatic basal rod
makes its appearance. Usually it appears some time before
the blepharoplast has divided and is very difficult to recognize
in its earliest stages. Figure 11 shows the earliest stage in
which I have been able to find this structure, and here it will
be seen to consist of a row of very fine granules closely connected
together and joined to the blepharoplast. Figure 10 shows a
stage which seems to be a little later, judging by the nuclear
changes, and here also the new chromatic basal rod is a row of
granules, but much longer than the one in figure 11. I was
unable to trace it past the nucleus and up to the blepharoplast.
The new rod is always in a characteristic position, dorsal to,
and to the right of, the nucleus (figs. 10 to 17). Although rela-
tively slender at first, it gradually increases in size until by the
time the blepharoplast divides it is easily recognizable. After
the division of the blepharoplast the new rod does not always
maintain its position near the surface of the body. In figure 19,
for example, the new blepharoplast is at the upper surface, while
the new rod extends from it around the nucleus, deep into the
protoplasm to the lower surface.
3. The new undulating membrane and chromatic margin. As the
new chromatic basal rod grows, irregular thickenings appear
along its length, as indicated in figures 12 and 15. A little
later one can see the new chromatic margin of the new undulating
membrane closely applied to the new rod (figs. 19 to 21). In
its first recognizable condition this chromatic margin is of much
smaller caliber than the old one, its undulations are low and
in length it cannot be traced beyond the distal end of the new
rod (figs. 20 to 26). In figure 19 it was possible to trace the new
138 D. H. WENRICH
chromatic margin along only apart of the course of the new rod,
although presumably it extended the whole distance. In the
part which could be made out, however, it remained close to
the rod, and hence transversed the deeper protoplasm along
with the latter organelle. This deeper position would hardly
be expected if the new chromatic margin, or posterior flagellum,
had been split off from the peripherally placed old one.
I have not been able to see evidence of a splitting of the
undulating membrane and the chromatic margin, as described by
Kofoid and Swezy (715), although I have searched long and
diligently for such evidence. My evidence indicates that the
new chromatic margin grows out along the new chromatic basal
rod as a new structure just as the other flagella grow out as new
structures. In figure 18 I have drawn an individual which
appeared to have the old chromatic margin double for the
anterior half of its length. The two portions appear to be of
equal caliber. The nucleus could not be made out distinctly
and there are other indications of degeneration, so that I regard
this individual as abnormal, especially since I have carefully
examined such large numbers in all stages of division without
ever finding any other specimen that indicated a splitting of
the membrane.
Wenyon (’07), Martin and Robertson (’11), and Kuczynski
(714, ’18) also find the new posterior flagellum growing out as a
new structure, although Dobell (’09) describes the splitting of
the undulating membrane in T. batrachorum. I am inclined to
agree with Kuczynski that Dobell, and Kofoid and Swezy have
been misled by the secondary filament in the undulating mem-
brane of T. augusta and T. batrachorum, and I am quite con-
vinced that splitting of the undulating membrane does not
normally occur in T. muris.
4. The blepharoplast. After the new chromatic basal rod
has been formed, the new blepharoplast appears, connected to
the old one by the paradesmose (Kofoid and Swezy, 715). Figure
17 shows a relatively early prophase with the new rod attached
to a small granule, which in turn is connected with the old
blepharoplast. In my opinion, this small granule is the new
STRUCTURE AND DIVISION OF TRICHOMONAS 139
blepharoplast. In nearly all cases the new blepharoplast is
smaller than the one attached to the old chromatic margin, and
it would not be unexpected if it should begin as a small bud
from the main or mother blepharoplast. The daughter blepharo-
plast continues to separate from the old, until the two are on
opposite sides of the nucleus. ‘The paradesmose connecting them
remains on the outside of the nuclear membrane which appears
to persist during division (fig. 23). Figure 22 shows the two
blepharoplasts in place and the spindle forming in the nucleus,
while the chromosomes are not quite completely aligned in the
equatorial plate.
5. Other structures. On account of the poor stainability of the
anterior flagella and on account of their frequent position on
or close to the cell to which they belong, and on account of the
presence ofttimes of large numbers of slender bacilli and wavy
spirochaetes, the behavior of these structures in division has
been difficult to follow. I am convinced, however, that the
accounts of other authors are correct to the effect that one or
two of these flagella accompany the new blepharoplast, while
the other two or one remain with the old or parent blepharoplast
(figs. 25 and 26). New flagella to make the full number appear
to be formed as new outgrowths from the blepharoplasts (fig. 32).
Late in the prophase the axostyle becomes separated from the
blepharoplast and begins to degenerate (figs. 20 to 22). New
axostyles grow out from the blepharoplasts in the telophase,
as will be described later. -
I have not been able to detect any peculiarities in the behavior
of the parabasal body during the prophases. I have drawn
figure 16 to show that there cannot possibly be any confusion
between the parabasal and the outgrowing new chromatic basal
rod. The parabasal is unusually long in this specimen and there
is a thin region over the nucleus which suggests that the distal
end may possibly become detached. ‘This idea is also suggested
by figures 4 and 5, where there is a constriction; but in these
latter cases there is no evidence of approaching division.
I have already suggested that new long rows of chromatic
eranules grow out from the chromatic basal rod. On the other
140 D. H. WENRICH
hand, there is some evidence of division of these granules, as
seen, for instance, in figure 8. Here the long row seems to be
double in the distal part and the two rows appear to lie close
together. The distance between them is foreshortened, how-
ever, in this position. The duplication in connection with the
short row behind the nucleus is difficult to interpret, and I am
not sure that division of the granules is indicated.
B. Metaphase
Figures 22 to 26 show a series which includes a very late
prophase or early metaphase (fig. 22), metaphases, and early
anaphases, which indicate very well the behavior of the chromo-
somes in these stages. In figure 22 the chromosomes are still
similar to those of the earlier prophases, the two parts of each
being closely approximated with their long axes parallel. Al-
though the fibers of the forming spindle have already become
attached to the chromosomes, the latter have not as yet lined
up into a definite plate. It appears from these figures that
whatever directive influence the spindle fibers may have in the
separation of the chromosomes, it is exercised for some of them
before the plate has become established. All the figures with an
equatorial plate show the two parts of some of the chromosomes
already drawn out so that they are in contact only at their ends,
while others are just in the process of being separated. Since
I have seen a great many animals in the stage indicated by figures
23, 24, and 25 and none showing stages between them and figure
22, I judge that some of the chromosomes are separated during
ae formation of the metaphase plate.
As seen in the figures mentioned, the number of chromosomes
in the metaphase is definitely six, the number found in the
prophases. Martin and Robertson (’11) and Kuezynski (14,
718), as previously noted, believe that eight(?) prophase chromo-
somes are reduced to four multiple elements in the metaphase.
I think I have demonstrated the probability that the prophase
number in Kueczynski’s figures is six, and the tendency for the
metaphase chromosomes to clump probably accounts for the
apparent number, four. Kofoid and Swezy (15) do not show
STRUCTURE AND DIVISION OF TRICHOMONAS 141
any metaphase figures for T. muris, and even in their extensive
figures for Tl’. augusta they have nothing corresponding with my
figure 22. ‘They therefore missed the evidence showing that the
process of separation at the metaphase and anaphase corresponds
to the details as seen in the corresponding stages in metazoan
mitoses, except for the precocious separation toward the two
poles before the equatorial plate is completely formed. Their
figures 20 and 21 for T’. augusta in which the metaphase chromo-
somes are seen as single elements elongated in the direction of the
spindle axis possibly show conditions in which the constriction
between the separating chromosomes has been eliminated by
contraction of the chromatin in the process of fixation.
C. Anaphase
Figures 26 and 27 illustrate anaphases. I have not seen so
many anaphases as I have metaphases, and presume that this
phase is of shorter duration. During this stage the chromosomes
appear to become elongated (fig. 26) and constricted (fig. 27).
Figure 27 shows the smallest chromosome as having divided
precociously and the daughter elements are much nearer the
poles than those of the other chromosomes.
D. Telophase
After the chromosomes have been completely separated and
the two daughter groups have arrived at positions some distance
apart, the nucleus which has been elongating during the anaphase
(fig. 27) becomes constricted in the middle (figs. 28 and 29),
thus forming the two daughter nuclei. The nuclear membrane
persists throughout this process. In the early telophases the
chromosomes begin to change their appearance, becoming less
dense and more granular. The constriction which first appeared
in the anaphase becomes more pronounced and each of the former
chromosomes appears to be made up of two rounded or slightly
elongated parts in contact at the ends (fig. 31). These eventually
give rise to the scattered granules seen in the resting nucleus
and the new caryosome becomes established surrounded by its
142 D. H. WENRICH
characteristic clear area. I have not been able to make out
the precise method of origin for the caryosome.
The entire number of six chromosomes can usually be seen
when a polar view of the telophase group can be had, such as is
shown for the lower nucleus in figure 30. The complete number
is also seen in the side views of figure 31. Kuczynski (14)
likewise shows six in a similar stage in his figure 66 of T. muris.
His figures 64 to 67 and 69 also show well the constriction of the
anaphase and early telophase chromosomes that I have mentioned.
Since the telophase chromosomes appear to resolve themselves
each into two chromomeres, and since the earliest prophase
chromosomes which can be recognized as such are already double,
one naturally wonders if the two parts of a prophase chromo-
some may not be represented by the two telophase chromomeres.
Since the two telophase chromomeres are arranged end to end,
while the two parts of a prophase chromosome are arranged side
by side, and since the number of chromatin granules in the
resting nucleus is rather large and indefinite, the direct relation-
ship suggested is improbable.
While the two daughter nuclei are becoming reorganized into
typical resting nuclei, complete sets of other organelles are being
established for the two new individuals. The origins of most
of these organelles have been discussed in connection with the
prophase. The chromatic basal rod and the flagella merely
complete a development initiated at the earlier phase. The
new axostyles, however, apparently do not begin to grow out
until the telophase. There is a suggestion of a new axostyle
growing out from the old blepharoplast in the early stage shown
in figure 29, but in figures 32, 33, and 34 the new axostyles are
distinctly seen. In figure 34 it will be noted that the new
axostyle growing out from the older, larger blepharoplast is
longer than the other one, as might be expected. It will also
be seen from these figures that there is a row of chromatic
granules along the new axostyles. These appear to be imbedded
in the axostyles and are probably intimately concerned in the
formation of these organelles. These granules must go to the
surface later or disappear, for they do not occur within the adult
STRUCTURE AND DIVISION OF TRICHOMONAS 143
axostyle. It is possible that the chromatic granules seen in the
adult along the axostyle from the blepharoplast to behind the
nucleus (figs. 3, 7, and 8) are the same as the ones which appear
to be concerned in the formation of the new axostyles.
It is probable that the degeneration of the axostyle in the
late prophases accounts for the rounding up of these animals
at about that stage in the division process.
Kuczynski (’14, 718) saw and figured the degeneration of the
old axostyle and the growing out of the new ones from the
blepharoplasts, and Martin and Robertson (’11) report the same
thing for T. eberthi. Wenyon states that the axostyle (‘pointed
organ’) divides by longitudinal division, but offers no evidence
in support of this statement. Kofoid and Swezy (715) show one
figure (fig. 60) which they interpret as showing division of the
axostyle in T. muris. But the figure is also open to the interpre-
tation as a partial superposition of two independently formed
elements, and since it is the only one they could find after pro-
longed search, the evidence is not very conclusive. Since the
evidence of the degeneration of the old axostyle and the origin
of new ones as outgrowths from the blepharoplasts is so con-
clusive in my material and in the results reported by Kuezynski,
the origin of this structure by splitting may be regarded as
extremely doubtful, at least for T. muris. Dobell (09) and
others were undoubtedly in error in believing that the new
axostyles developed from the paradesmose. This structure
retains its connection with the two blepharoplasts for some
time after the division of the nucleus (figs. 31, 34), but eventually
disappears.
Kofoid and Swezy state for T. muris that the long row ot
granules disappears during metaphase and reappears in the
telophase. I have been able to find them at practically all
stages of division but, as previously noted (p. 128), there is
evidence that the old row may be replaced by a new one which
is budded off from the ventral (inner) side of the chromatic
basal rod. Just how the new chromatic basal comes to have an
associated row of granules has not been determined.
144 D. H. WENRICH
T have not been able to find stages showing any division of
the parabasal body. In all the anaphases and later stages
(figs. 26 and 28) there appears to be a parabasal for each blepharo-
plast. Whether the old one disappears and two new ones grow
out, or whether the old one remains and one new one grows
out, or whether some other mode of origin may prevail has not
been determined. One point should be noted, however, namely,
- that in these anaphases and telophases the parabasal attached
to the daughter blepharoplast is always smaller than the one
connected with the old blepharoplast. Considering a possible
analogy with the chromatic basal rod, this fact might be inter-
preted as indicating that the old parabasal persists and a new
one grows out from the new blepharoplast. The one attached
to the old blepharoplast is not so long as the longest ones seen
in the non-dividing and earlier prophase stages (figs. 3, 4, 5, 16),
but is comparable in length to the portion proximal to the con-
striction as seen in figures 4 and 5 or proximal to the fainter
area in figure 16. The suggestion already made that the portion
distal to the constriction may become detached will be recalled.
The origin of all the new structures has been discussed, except
the cytostome. This structure is not much in evidence during
the metaphases and anaphase, but two cytostomes appear in
the telophase. It is possible that the old one, like the axostyle,
disappears, and two new ones are formed. Before division of the
cell body, all the organelles in the two sets apparently become
developed to a condition corresponding to that of the original
set.
No cases have been found showing the constriction of the cell
body in my fixed and stained slides, but I have frequently
observed this process in the living animals. It takes place
rapidly and the two separating individuals always appear to be
of equal size and completely developed. The long interval
between the division of the nucleus and the division of the cell
body doubtless serves to allow the new organelles to attain com-
plete development before the daughter cells separate.
I think it is worth while to point out that according to the
evidence which I have presented there appear to be only two
STRUCTURE AND DIVISION OF TRICHOMONAS 145
parts of this complicated flagellate that divide equationally.
They are, 1) the nucleus, including the chromosomes, and, 2)
the cell body. The blepharoplast, chromatic basal rod, posterior
flagellum, and possibly also the parabasal body and one or two
of the anterior flagella of the parent appear to be retained by
one of the new daughter individuals, while the other daughter
is supplied by new outgrowths, including a new small blepharo-
plast budded off from the parent one. The old axostyle, and
possibly also the old cytostome, disappear and a new one is
formed for each new cell. New chromatic granules appear to
have a different origin, as previously described. This behavior
is paralleled by that of the Infusoria, exemplified by Paramecium,
which remains active during the process of division. Some of
the cilia and one of the contractile vacuoles are taken by each
daughter cell, and new ones are formed to make the complete
set of organelles. Part of this development in Paramecium
takes place after the separation of the daughter cells, whereas
in Trichomonas development of the new organelles appears to
- be completed before the daughters separate. Since in both cases
the daughter cells come to resemble each other completely, their
hereditary potentialities must be equally descended from the
parent. An equational division of the nuclear material would
therefore be sufficient to insure equality between the daughter
cells, granting that the nuclear material constitutes the physical
basis of heredity.
SUMMARY OF THE MORE IMPORTANT RESULTS
1. Trichomonas muris (Hartmann) from the coecum of the
mouse measures 10 to 16u long by 5 to 10 wide, but varies in
size slightly from host to host and to a larger extent as a result
of the use of different fixatives.
2. Different fixatives also give rise to different staining reac-
tions of the protoplasmic vacuoles, nuclei, and other organelles.
3. The anterior free flagella are short, not more than half
the length of the body, and stain faintly with iron-alum haema-
toxylin stain. The posterior flagellum stains intensely as the
chromatic margin of the undulating membrane, but its posterior
146 D. H. WENRICH
free extension is similar to the anterior flagella in length and
staining capacity.
4. The chromatic basal rod is thicker in the middle and tapers
toward both ends. It appears to give origin to the outer row
of chromatic granules by a kind of budding process.
5. There is a deeper row of chromatic granules near the
axostyle extending from behind the nucleus up to the blepharo-
plast.
6. There is a parabasal body similar to the one described by
Janicki and Kuczynski. It has a position dorsal and to the
right of the nucleus. It varies in appearance and occurrence
from host to host, from flagellate to flagellate, and from one
fixative to another. It has appeared after the use of weak
Flemming’s, Flemming’s without acetic, and 1 per cent chromic
acid solutions.
7. In the prophase of division the chromatin becomes organized
into six double (split ?) prophase chromosomes and the caryo-
some gradually disappears. A new chromatic basal rod grows
out from the blepharoplast and appears first as a row of fine
granules. It is connected with the small new blepharoplast
which a little later becomes budded off from the main, or parent,
one. The two blepharoplasts are connected by a paradesmose.
8. Before the metaphase has been reached the axostyle becomes
detached from the blepharoplast and begins to disintegrate.
9. In the metaphase six definite chromosomes are found, but
the two parts of each tend to separate in the late prophase |
while the equatorial plate is forming.
10. In the anaphase the chromosomes become granular and
each divided into two equal parts by a transverse constriction.
The body of the nucleus divides by simple constriction, the
nuclear membrane persisting through the process.
11. In the telophase six chromosomes, each doubled by the
transverse constriction, can be seen. These become organized
into the ‘resting’ nucleus. A new axostyle grows out from each
blepharoplast. The origins of the new parabasal bodies and
cytostomes were not definitely made out.
STRUCTURE AND DIVISION OF TRICHOMONAS 147
12. The two sets of organelles retain the common proto-
plasmic body until development is complete, and then the cell
body divides rapidly.
13. The only parts of the cell to divide equationally are,
a) the nucleus, including the chromosomes, and, b) the cell body.
LITERATURE CITED
ALLEN, Ezra 1916 Studies on cell division in the albino rat (Mus norvegicus,
var.alba). IL. Experiments on technique, ete. Anat. Rec., vol. 10.
Curiter, D. Warp 1919 Observations on the protozoa parasitic in the hind
gut of Archotermopsis wroughtoni Desm. Part I. Ditrichomonas
(Trichomonas) termitis Imms. Quart. Jour. Mic. Sci., vol. 63.
DoseLt, Ciuirrorp C. 1909 Researches on the intestinal protozoa of frogs and
toads. Quart. Jour. Mic. Sci., vol. 58.
1914 Cytological studies on three species of amoeba, ete. Arch. f.
Protist., Bd. 34.
HARTMANN UND KisskaLr 1910 Practikum der bakteriologie und protozoologie.
Il Teil. Protozoologie, von M. Hartmann. 2te aufl. Jena.
JANickI, C. 1911 Zur Kenntniss der Parabasalapparat bei parasitischen
Flagellaten. Biol. Cent., Bd. 31.
Kororp, C. A., AND Swezy, Ottve 1915 Mitosis and multiple fission in tri-
chomonad flagellates. Proc. Amer. Acad. of A. and 8., vol. 51.
Kuczynski, M. 1914 Untersuchungen an Trichomonaden. Arch. f. Protist.,
Bd. 33.
1918 Ueber die Teilungs vorgiinge verschiedener Trichomonaden und
ihre Organisation im allgemeinen. Arch. f. Protist., Bd. 39
Martin, C. H., anp Ropertson, Muriet 1911 Further observations on the
coecal parasites of fowls with some references to the rectal fauna of
other vertebrates. Part I. Quart Jour. Mic. Sci., vol. 57.
Swezy, Outve 1916 The kinetonucleus of flagellates and the binuclear theory
of Hartmann. Univ. of Cal. Publ. in Zool., vol. 16.
Wenyon, C. M. 1907 Observations on the protozoa in the intestine of mice.
Arch. f. Protist., Suppl., Bd. 1.
JOURNAL OF MORPHOLOGY, VOL. 36, No. 1
EXPLANATION OF PLATES
The drawings have all been outlined with the aid of a camera lucida, using a
Spencer 1.8 mm, oil-immersion objective and a Zeiss no. 12 compensating ocular.
The draw-tube was set to make a magnification of 4000 at the level of the table
where the tracing wasdone. In reproduction the magnification has been reduced
to 3000. Details of structure were completed with ink while the object remained
under observation, then each drawing has been checked two or three times by
subsequent comparison with the object. All figures from material stained with
iron-alum haematoxylin. Fixation will be indicated for each figure. For these
fixing fluids the following abbreviations will be used: Allen’s for Allen’s ‘B-15,’
Carn. for Carnoy’s fluid, Schaud. for Schaudinn’s fluid, sub.-acet. for sublimate-
acetic, and wk. Flem. for the weaker fluid of Flemming.
PLATE 1
EXPLANATION OF FIGURES
1to3 Vegetative individuals showing some differences due to use of different
fixatives; fig. 1, Carn., chromatic basal rod not stained; fig. 2, sub.-acet., vacuole
contents stained; fig.3, wk. Flem., parabasal body stained.
4to6 Wk. Flem., parabasal body stained, constricted in figs. 4 and 5, under-
going change in fig. 6.
7to9 Schaud., vegetative individuals; fig. 7, view from left side; fig. 8, possi-
ble division of rows of chromatic granules; fig. 9, hypertrophied (abnormal ?)
nueleus.
148
PLATE 1
VISION OF TRICHOMONAS
Dae
STRUCTURE AND DI
ENRICH
Ww
149
PLATE 2
EXPLANATION OF FIGURES
Figs. 10 to 15, 17, 18, Schaud.; fig. 16, wk. Flem.
10 to 17 Prophases; fig. 18, apparent division of chromatic margin of undu-
lating membrane. Figs. 10 and 11, early granular stage in the formation of the
new chromatic basal rod. Figs. 12 to 15 and 17, six double split chromosomes
besides the caryosome which gradually loses its staining capacity. Fig. 16, new
chromatic basal rod together with the parabasal body. Fig. 17, budding of a
small new blepharoplast to which the new chromatic basal rod is attached.
PLATE 2
CTURE AND DIVISION OF TRICHOMONAS
STRU
WENRICH
D. H.
PLATE 3
EXPLANATION OF FIGURES
Figs. 19 to 21, 25, Schaud; figs. 22 to 24, 27, Allen’s; fig. 26, wk. Flem. Figs.
19 to 21, late prophases: figs. 22 to 25, metaphases; figs. 26 arid 27, anaphases.
19 Paradesmose between blepharoplasts; new chromatic basal rod and new
chromatic margin penetrating deep into the cytoplasm; chromosome moieties
fused, giving the appearance of six single elements.
20 Blepharoplasts 180 apart; axostyle detached and beginning to degenerate.
22 Begininng of the spindle and beginning of separation of daughter chromo-
somes; remnant of caryosome seem.
26
97
New parabasal body attached to the daughter blepharoplast.
Constriction in the anaphase chromosomes; a small chromosomes has
divided precociously.
152
ATE 3
PL
STRUCTURE AND DIVISION OF TRICHOMON
H. WENRICH
D
PLATE 4
EXPLANATION OF FIGURES
Fig. 28, wk. Flem.; figs. 29 to 32 and 36, Schaud.; figs. 33 to 35, Allen’s.
28 Early telophase: delayed separation of chromosomes; two parabasal bodies.
29 Constriction of nuclear membrane; degenerating axostyle.
30 to 34 Telophases. Fig. 30, side view of one, and polar view of the other
daughter nucleus. Fig. 31, constriction in daughter chromosomes; paradesmose
intact. Figs. 32 to 34, formation of ‘resting’ nuclei and outgrowth of new axos-
tyles.
35 and 36 Precystic changes.
154
pi
STRUCTURE AND DIVISION OF TRICHOMONAS PLATE 4
D. H. WENRICH
155
AUTHOR’S ABSTRACT OF THIS PAPER ISSUED
BY THE BIBLIOGRAPHIC SERVICE, DECEMBER 27
THE CIRCULATORY SYSTEM AND SEGMENTATION
IN ARACHNIDA
ALEXANDER PETRUNKEVITCH
Yale University
TWO TEXT FIGURES AND TWO PLATES (SEVEN FIGURES)
The circulatory system in Arachnida has been made repeat-
edly the subject of studies and is fairly well known. Never-
theless, several points have escaped observation, partly on
account of technical difficulties, partly because the attention
of the investigators was directed toward other aspects of their
study. Thus it came to pass that the relation of the circulatory
system to the problem of segmentation in arthropods received
less attention than it deserves. Indeed, in this respect the cir-
culatory system may be more valuable than the nervous system
and may, with a certain portion of the alimentary canal, of
which I shall speak in a later contribution, help to establish
definite homologies within the phylum of arthropods, and thus
not only clear the relationships between the various classes
belonging to that phylum, but also throw light on their phylogeny.
Many years ago, in the spring of 1905, while on a trip to
Jamaica, West Indies, I collected scorpions and spiders, preserv-
ing them in the only fluid then obtainable in Jamaica, a mixture
of alcohol with ether. The specimens were simply thrown into
a jar and left in the fluid, as I did not intend to use them for any
anatomical or microscopical study. Quite recently I wanted a
few sections through scorpion embryos for class demonstration,
so I imbedded and sectioned some quite young scorpions which
had been carried by their mother on her back and of which I
had many specimens representing stages before and after the
first moult. I also took embryos out of the uterus. My sur-
prise was great when I found that the fixation of the tissues was
157
158 ALEXANDER PETRUNKEVITCH
remarkably good and that prolonged sojourn in the preserving
fluid made the material considerably less brittle than it usually
is on account of the voluminous yolk. A cursory examination of
the sections has revealed so many interesting deviations from the
usually accepted descriptions of the anatomy and embryology
of scorpions, that a number of carefully oriented and sectioned
series of various stages were made. All sections were purposely
made 20u thick. Exact orientation for sagittal, frontal, and -
transverse series-was comparatively simple on account of the
size, shape, and coloration of the material. A few of the series
are absolutely symmetrical. The most satisfactory staining
proved to be haematoxylin followed by orange G.
When the study of the circulatory system revealed the remark-
able similarity in this respect between scorpions and spiders,
I prepared corresponding series through very young spiders
fixed for the purpose in my sublimate mixture. Two species
of scorpion (Centrurus insulanus and C. ecarolinianus) and three
species of spiders (Agelena naevia, Lycosa carolinensis, and
Pholcus phalangioides), belonging to three different families,
form the basis of the present study. While it would be very
interesting to extend it over other groups of Arachnida, the
diversity of the material leaves no doubt that similarity here is
not accidental, but is the expression of true homology, and
that generalization is therefore warranted and helpful.
To avoid possible misinterpretation of results, obtained only
from the study of sections however perfect, a few scorpions
were injected through the heart. These scorpions, Centrurus
carolinianus of Texas, were obtained alive through the courtesy
of Professor Painter, of the University of Texas, who kindly
took the trouble to collect and mail to me a dozen specimens.
Injection was made in a manner similar to that which I used for
Lycosa several years ago, but the technique in the case of the
scorpion is somewhat more complicated, owing to the fact that
the heart is only imperfectly visible through the chitin. For
this reason it becomes necessary to open the chloroformed speci-
men in a saline solution to expose the heart. The tergites of
the second to sixth abdominal segments are carefully removed,
CIRCULATORY SYSTEM IN ARACHNIDA 159
beginning with the posterior edge of the sixth tergite. The
ligaments of the heart are cut close to the hypodermis with
a sharp scalpel, as otherwise the heart would sustain injury.
Freshly precipitated carmine as injection fluid proved to be
quite satisfactory. Not only the large vessels become injected
to their end near the base of the.claws in the legs, but many
ramifications of pedal arteries appear dark red. The injected
specimen is next fixed in 95 per cent alcohol, dehydrated in ab-
solute alcohol, and cleared in cedar oil, in which it becomes
sufficiently transparent for further preparation. All organs ob-
structing the view are now carefully removed with the aid of
two needles under a binocular dissecting microscope and the
entire circulatory system exposed to view.
Usually the circulatory system in scorpions is described as
consisting of a dorsally situated heart which gives rise anteriorly
to the cephalic aorta and posteriorly to the posterior aorta. The
heart itself is said to consist of eight chambers with a pair of
ostia each, or eight pairs of ostia altogether, typically one pair
for each segment of the body. The cephalic aorta is described
as giving rise to a pair of arteries near its base, and a little further
to another pair. The latter assume a downward course, pass
on each side of the oesophagus, forming a ring from which six
pairs of arteries are said to be given off to the appendages, while
a single median supraneural artery runs from the ring backward
above the nervous system. The usual description of the finer
ramifications, as well as of the arteries given off by the heart,
is irrelevant to our purposes and may be entirely omitted.
The microscopical structure of the heart seems in all Arthro-
poda to be more or less the same. Its wall is composed of three
layers. The outer layer, the adventitia, consists of connective
tissue. The heavy media or muscularis is formed either by a
spiral muscle or by symmetrically arranged semicircular mus-
cle fibers which meet in the middorsal and midventral line, as
has been described by Bergh for insects. The inner layer or
intima is, whenever present, nothing but a very thin transparent
membrane which, according. to Verson, may be nothing but the
sarcolemma of the muscle fibers of the media.
160 ALEXANDER PETRUNKEVITCH
a
Text figure A. Centrurus insulanus, late embryo. Sagittal section in the
plane of symmetry, showing all organs developed at that age. The black spot
above is one of the median eyes. In front of the eye is the dorsal dilator of the
pharynx. Abdominal tergites marked with Roman numerals J to XII without
reference to their embryological history. 1, the first abdominal neuromere;
12, the last neuromere; Cbp, basal plate of comb; GO, genital opening; PG, poison
gland; SG, sting; ST, sternum; the dotted lines showing its anterior and posterior
margins; UP, upper lip (rostrum). Further explanation in the text.
CIRCULATORY SYSTEM IN ARACHNIDA 161
This description of the gross anatomy of the circulatory system
is, as we shall see, not quite correct, but served to show the simi-
larity between the scorpion and Limulus. On the other hand,
it created differences between scorpions and spiders especially,
since the circulatory system of the latter had only recently been
described correctly. Moreover, the exact position of the heart
in both groups remained unnoticed, and yet therein lies its value
from the morphological point of view.
The best material for the study of the circulatory system
in the scorpion is furnished by that stage of embryos represented
in our text figure A, in which all organs are already fully de-
veloped, but the nervous system has not yet reached its final
state of contraction. The postabdomen is still short, the length
of each segment being smaller than the diameter. The last
neuromere has already begun to fuse with the preceding one and
is almost entirely withdrawn into the fourth postabdominal
segment. The first and second abdominal neuromeres have
moved forward, passed the diaphragm, are completely within the
cephalothorax, and are fused with the thoracic ganglia into
one mass in which the separate neuromeres remain however
clearly defined as they do, even in adult scorpions. The third
abdominal neuromere is just on the verge of passing the dia-
phragm, while the fourth, which in the adult scorpion forms the
last portion of the thoracic ganglionic mass, is still in the abdomen
just behind the genital opening and on a level with the basal
plate of the comb. In this connection we may state that Buxton
had recently shown that the comb receives its nerves from the
eighth postoral ganglion of the suboesophageal mass, i.e., from
the third abdominal neuromere—an observation which I am
able fully to confirm.
In median sagittal sections the diaphragm appears as a
thin line. Its ventral portion runs from the ventral body wall
just in front of the genital opening to the endosternite which
lies above the nervous system. Its dorsal portion arises from a
vertical transverse crest of the endosternite and proceeds up-
ward to the dorsal body wall, where it is attached between the
carapace and the first abdominal tergite.
162 ALEXANDER PETRUNKEVITCH
The midgut may easily be recognized because of the presence ~
of a cardiac valve, because of its thin walls and, further, because
it is still filled with embryonic yolk. It has the appearance of
a wide tube with larger lateral branches or diverticula usually
known under the name of ‘liver.’ The anterior portion of the
midgut proper runs at almost a right angle to the longitudinal
axis of the body and belongs to the cephalothorax because it
lies in front of the diaphragm. Its posterior end extends only
to the end of the fifth abdominal segment. The diverticula
of the midgut extend forward almost to the anterior end of the
cephalothorax and backward through the entire: preabdomen,
the last dorsal diverticulum reaching even into the first post-
abdominal segment. The gross anatomy and segmental arrange-
ment of the diverticula are not easy to understand. Sagittal
sections show a clear separation of the diverticula into ten
metamerically arranged groups, two of which are in the cephalo-
thorax and eight in the preabdomen. Their metameric nature
is especially emphasized by the dorsoventral muscles and the
hypocardiac ligaments. The ligaments shown in text figure
A as ventral projections of the heart exist only in the preabdomen.
Of the muscles, one pair is in the cephalothorax where they are
attached by one end to the carapace between the two cephalo-
thoracic diverticula and by the other to the endosternite above
the nervous system. There are eight pairs of dorsoventral
muscles in the preabdomen passing between the diverticula,
right and left of the midgut, from the back to the ventral sur-
face of the body. The first pair is easily overlooked as it lies
closely applied to the posterior surface of the diaphragm. Dor-
sally, these muscles are attached to the anterior end of the first
abdominal tergite on the outside of the epicardiac ligaments.
Ventrally, they are attached, like the cephalothoracic pair, to
the endosternite above the nervous system. All other dorso-
ventral muscles are attached at both ends to the chitin of the body
wall. The dorsal attachments are to the outside of the epicardiac
ligaments. The ventral attachments are slightly farther apart
than the dorsal, one pair for each abdominal sternite, the second
pair lying at the sides of the genital opening, and the third
at the sides of the basal plate of the comb.
CIRCULATORY SYSTEM IN ARACHNIDA 163
Although the diverticula are divided by the dorsoventral
muscles into ten groups, there are only six pairs of ducts con-
necting them with the midgut proper. The first pair of these
transverse ducts is in the cephalothorax and connects the two
cephalothoracic diverticula with the anterior portion of the mid-
gut. The first, second, third, and fourth abdominal diverticula
have a pair of transverse ducts each in the corresponding seg-
ments. The last pair of connecting ducts opens into the midgut
in the fifth abdominal segment. These ducts are long, extend-
ing backward over three segments and establishing a connection
between the midgut and all four posterior metameric groups of
diverticula.
Transverse sections through the second to fourth abdominal
segments show that each pair of diverticula is composed of
two dorsal, two ventral, and one lateral lobes. . All lobes are
filled with embryonic yolk and all those on the same side are
naturally connected with each other near their base. The dor-
sal right and left lobes are separated by the pair of dorsoventral
muscles. Below the midgut proper, which occupies approxi-
mately the center of a transverse section, the dorsoventral mus-
cles pass on the outside of the inner pair of ventral lobes. In
the cephalothorax only dorsal lobes are present. The last pair
of transverse ducts gives off side branches to the fifth and then
to the sixth abdominal diverticula, while the ends of the ducts
lead into the seventh pair into which the eighth pair also opens.
The hindgut begins in the terminal region of the fifth abdominal
segment. It is considerably smaller than the midgut, but has
both relatively and absolutely much thicker walls. It is devoid
of embryonic yolk and in the last postabdominal segment forms
a considerable widening beyond which it is again suddenly con-
stricted and opens with a small anus in the midventral line at
the end of the segment.
The heart lies entirely in the preabdomen, extending from
the diaphragm which separates the latter from the cephalothorax,
almost to the end of the seventh abdominal tergite. Its an-
terior and posterior limits are clearly defined by the valves
of which I shall speak later.. The heart has seven pairs of ostia
164 ALEXANDER PETRUNKEVITCH
(not eight as usually erroneously stated). The presence of the
ostia gives the heart the appearance of an eight-chambered organ.
In reality there are neither valves nor any constriction or im-
pediment in the spaces between the successive ostia. There is,
therefore, nothing that would have the morphological value of
chambers. In transverse sections the ostia occupy a position
approximately half-way between the dorsal pole and the equator
of the heart. The first pair lies exactly at the posterior edge of
the first abdominal tergite. The position of all seven pairs may
be best understood from text figure A. It is the same in all
specimens and does not change with maturity. The structure
of an ostium with its valve is represented in figure 3 on a large
scale. This is a frontal section, and the media or muscularis
is therefore sectioned at right angles to its muscle fibers. Each
fiber has the appearance of a rectangle. The adventitia of con-
nective tissue is well defined and shows long, darkly stained
nuclei. Multinuclear fibers of connective tissue run from the
edges of the ostium laterally, converging and forming a ligament,
the so-called pteripyle.
The position of the ligaments by which the heart is suspended
is well known in spiders, owing to several researches, especially
those of Causard. There is scarcely any difference in this respect
between spiders and scorpions. Text figure B, drawn from a
complete series of transverse sections through a late embryo,
represents the heart of the scorpion with all its ligaments of a
single group projected into the same plane. All told, there are
eight metamerically arranged groups of ligaments, each group,
except the first, composed of four pairs. The shortest of these
are the epicardiac ligaments which pass on each side of the dorsal
sinus and are attached to the basal membrane of the hypodermis,
thus clearly demonstrating their connective nature, since all
muscular fibres, as for example those of the dorsoventral muscles
represented in the figure, pass between the hypodermal cells
and are inserted in the base of the cuticle. ‘The second pair are
the pteripyles. Their distal end merges with the somatic con-
nective-tissue layer which separates the dorsal longitudinal
muscles of the abdomen from the hypodermis. The third pair,
CIRCULATORY SYSTEM IN ARACHNIDA 165
CN Ds LEP
\ ! 4
EY P-- S ia ae
StS » v2 <\ \ e Cah tien,
"2? (Ze
Sal sean obit
d Hayle,
Lf63 xb
te
LM_--?
Text figure B. Centrurusinsulanus, lateembryo. Transverse section through
the heart in the region of the third abdominal segment, showing the ligaments.
The drawing was made from two sections, as not all ligaments are in the same
plane. The epi- and hypocardiac ligaments are in one plane, while the pteripyles
and alar are ligaments inanother. AL, alar ligaments; CN, cardiac nerve; DS,
dorsal pericardial sinus; DV M, dorsoventral muscle; H, heart; HYP, hypodermis;
LEP, epicardiac ligaments; LHY, hypocardiac ligaments; LM, dorsal longitudinal
muscles; LS, lateral pericardial sinus; PE, pericardium; PTP, pteripyle; VS,
ventral pericardial sinus.
166 ALEXANDER PETRUNKEVITCH
often called alary muscles, are the alary ligaments. Their liga-
mentary nature has been elucidated by Causard. They are
directed at almost right angles to the longitudinal axis of the
heart. Distally they are not attached to the body wall, as
usually stated, but merge with a layer of connective tissue, evi-
dently representing the splanchnic coelom covering and separat-
ing the diverticula of the midgut from other organs situated above
the latter. The fourth pair are the hypocardiac ligaments.
They are by far the strongest and longest, and are easily mis-
taken for muscles, especially where they intercross with the dorso-
ventral muscles. From here on they continue diverging and
unmistakably and finally merge with the splanchnic layer of
connective tissue which covers the diverticula of the midgut
from below. The first group of ligaments consists of two pairs
only. The epicardiac ligaments are attached to the anterior
edge of the first tergite. The hypocardiac ligaments are more or
less normally developed, but the pteripyles and alary ligaments
are wanting.
Since there are no muscles for the dilatation of the heart, diastole
is accomplished through the elasticity of the heart ligaments.
This explains why the muscularis of the heart is so powerfully
developed. During systole the heart has to overcome the re-
sistance of the ligaments, while the contraction of the latter dur-
ing diastole is not impeded by the relaxed muscles. There is
nothing unusual in such arrangement, as a similar condition exists
in almost all joints of the appendages in Arachnida, where flexing
is accomplished by muscular contraction and extension by the
elasticity of the interarticular chitinous membrane. I have
counted 120 pulsations of the heart in one minute.
The pericardium appears as a thin membrane, and the space
between it and the heart is, in sections, invariably filled with
coagulated blood plasm, and consequently is clearly discernible.
Owing to the presence of epicardial and hypocardial ligaments,
this space is subdivided into four regions which may be termed
sinuses, though they communicate with each other in those re-
gions of the heart where there are no ligaments. The lateral
sinuses are the largest, next in size is the ventral sinus, while the
dorsal sinus, almost round in shape, is the smallest of the four.
CIRCULATORY SYSTEM IN ARACHNIDA 167
In the dorsal midline of the heart, partly imbedded in a groove
in the wall of the heart, the cardiac nerve extends from one end
of the heart to the other (text figure B, CN). The nerve is
clearly visible in all transverse sections and unquestionably
corresponds to the cardiac nerve described in Chilopoda
(Duboseq), Protracheata, and other Arthropoda. As my mate-
rial is not specially prepared for the study of nerves, I am unable
to find a connection of the nerve with the brain, but such
connection has been described by Police in Euscorpius.
The structure of the anterior aortic valve is best understood
from median sagittal sections and sections which traverse the
valve more or less at right angles. In the first (fig. 1, AV)
the valve appears as a line attached to the dorsal wall of the heart
exactly under the epicardiac ligaments, inclined downward, and
about two and a half times as long as the diameter of the heart at
the place of the attachment of the valve. In reality the valve is
a muscular membrane arising from the dorsal half of the wall of
of the heart and attached to the sides of the vessel throughout its
length. The anterior edge of the valve is longer than the diameter
of the vessel. The valve has, therefore, a peculiar shape, being
concave or troughlike at its free edge and convex or arched at
its base. About half-way between its base and end the valve
is drawn tight in the equator of the transverse section of the
vessel. Such a section is represented in figure 4, which also
shows that the valve is not a fold, but consists of a single layer
of transverse muscular fibers with elongated nuclei. There is
always a greater accumulation of blood-cells above the valve than
below it, showing that the action of the valve is perfect.
The structure of the posterior aortic valve is more difficult
to ascertain, and is somewhat different from the anterior one.
Text figure A represents the position of the posterior valve as
being not far from the posterior edge of the seventh abdominal
tergite. This position is constant in specimens of all ages. The
valve seems to have the shape of a cone, the open free apex of
which is directed posteriorly, while the broad base is attached
to the wall of the heart along its entire circumference. This
valve, too, has a single layer of circular muscle fibers composing it,
168 ALEXANDER PETRUNKEVITCH
but the length of the posterior valve is many times smaller than
that of the anterior valve. Indeed, the posterior aorta which
begins at this place is a thin vessel gradually becoming smaller
as it traverses all the segments of the postabdomen. It may
be traced through the poison gland into the sting, where it ends
apparently without any ramifications.
I have stated that the valves are muscular in structure. It may
be objected that I have adduced no evidence in support of this
assertion and that one may just as well claim that the valves are
internal projections of the same connective tissue which as its
adventitia surrounds the heart. Indeed, I have no sections
through either the anterior or posterior aortic valves to prove
or disprove either of the contentions. But I have already men-
tioned the fact that spiders possess the same types of valves.
A comparison of figure 6 with figure 1 will show that the position
and appearance of the anterior valve in both scorpions and spiders
is the same. Similarly, a comparison of figures 4 and 7 will
disclose the identity in structure of the anterior aortic valve in
these two orders of Arachnida. Now I happen to have a great
many sections through young spiders, and these show the intima
lining both surfaces of the valve and continuing directly as
intima of the heart itself. In many cases there is a slight loosen-
ing of the intima from the muscularis, with the consequence
that it appears as an uninterrupted line. The intima of the
heart being the sarcolemma of the muscle fibers, it is not con-
ceivable that it could line any but muscular tissue.
Let us now turn our attention to the anterior aorta and the
arterial blood vessels of the cephalothorax (fig. 1). The anterior
aortic valve, having the exact direction of and lying immediately
in front of the diaphragm represents the exact demarkation line
between the heart and the aorta. Otherwise, the transition from
the heart to the aorta would be scarcely perceptible. Shortly
beyond the valve the aorta gives rise to a pair of small arteries
supplying with blood the pair of dorsoventral muscles which
separate the first cephalothoracic diverticula of the midgut from
the second.
CIRCULATORY SYSTEM IN ARACHNIDA 169
The aorta itself continues as a considerable vessel under the
brain until it reaches the third neuromere of the suboesophageal
ganglionic mass. Here the aortic arch around the oesophagus
is formed. The arch is very short and connects the aorta with
the right and left thoracic sinus opposite the base of the second
pedal artery as shown in figures 1 and 2. Two pairs of vessels
issue from the aortic arch. The first pair are the large cephalic
arteries shown in figure 1. The second pair cannot be shown
to advantage in the planes chosen for our drawings and is there-
fore not represented there. But these vessels are nevertheless
constant in their position and easily found. Their roots are
in the right and left anterior inner edge of the aortic arch, near
its junction with the sinus and almost directly above the third
vessel connecting the sinus with the supraneural artery. The
two vessels run convergingly upward, feeding the wall of the
oesophagus.
Each thoracic sinus gives rise to small and thin vessels con-
necting the sinus with the supraneural artery, and to four large
vessels for the appendages. Of these vessels the first is the
largest and splits almost immediately into two branches. The
outer branch (fig. 1 and 2, 1, AP) is the first pedal artery, while
the inner, longer, and stouter branch is the pedipalpal artery
(APP). The latter gives off a thin branch directed inwardly,
following in its course the curvature of the ganglionic mass,
and connecting with the supraneural artery just behind the
pharynx. A branch of this supplies the tissues in front of the
pharynx.
Posteriorly, the right and left sinus merge with each other and
form a connection with the supraneural artery. This artery
is single and runs in the median line above the ventral nervous
system and closely applied to it. Anteriorly it runs to the very
end of the ganglionic mass, turning downward in its course and
now continuing backward in the midventral line below the gan-
glionic mass as subneural artery. Posterior to the thoracic sinus
the supraneural artery continues as a straight vessel in the median
line above the nervous system and closely applied to it through
the entire abdomen. I have not followed its course in the post-
abdomen.
170 ALEXANDER PETRUNKEVITCH
Nine single interneural vertical arteries connect the supra-
neural with the subneural artery. These vertical arteries pass
exactly between adjoining neuromeres, the first artery separa-
ting the pedipalpal from the first pedal neuromere, the ninth
lying immediately behind the fourth abdominal neuromere of
the suboesophageal ganglionic mass. Median longitudinal con-
necting vessels seem to exist between all vertical arteries, but
only between the fifth and sixth, and between the sixth and
seventh vertical arteries the connecting vessels are invariably
well discernible, as shown in figure 1.
The subneural artery does not extend beyond the suboeso-
phageal ganglionic mass, but ends behind the fourth abdominal
neuromere, where it receives the ninth vertical artery, which
may be, therefore, in a way regarded as a direct continuation of
the subneural artery. At the place of their junction a single
blood vessel is given off ventrally. This is the comb-artery
shown in figures 1 and 5. It gives off a pair of branches, one for
each comb, and another pair of smaller branches for the genital
opercula.
Each cephalic artery gives off several branches, the most
important of which is the ophthalmic artery shown in figure 1.
Beyond the ophthalmic artery the main vessel may be termed
cheliceral artery. Inside the chelicera the cheliceral artery forms
two ramified branches, one for the flexor and the other for the
extensor of the movable finger.
COMPARISON WITH SPIDERS
Having thus described the most important features of the cir-
culatory system in scorpions, we now. may proceed to its com-
parison with the corresponding system in spiders. A glance
at plate 3 of my paper published in the Zoologische Jahrbiicher
for 1920, vol. 31, will reveal both the similarities and diversities
of structure. What I described there as ‘Kopfarterie’ corre-
sponds with the cephalic artery of the scorpion almost to the
minutest details, and shows the same ramifications. On the other
hand, in the spider the cephalic arteries represent the upper
branches of the thoracic arteries, the lower branches of which
CIRCULATORY SYSTEM IN ARACHNIDA t7t
lead to the thoracic sinuses, whereas in the scorpion the cephalic
arteries arise from the aortic arch. This means simply a further
extension of the arch in the spider, so that the aortic arch of the
scorpion corresponds with the thoracic and connecting arteries
of the spider. The homology is unmistakable, and it may be
wiser to speak in the spider also of an aortic arch instead of a
thoracic and a connecting artery.
The thoracic arch, then, of the spider opens into the thoracic
sinus at the base of the second pedal artery as in the scorpion.
As in the scorpion, the pedipalpal and the first pedal arteries
are branches of the first arterial stem given off by the thoracic
sinus. The aorta recurrens of the spider, shown in my drawings,
is the supraneural artery. But for the comparison of the supra-
neural and subneural arteries of the scorpion and spider we have
to consult the description of these arteries given by Causard,
and his figures on plate IV. Instead of quoting passages in the
original, I translate them with such omissions as have no rela-
tion to our subject.
“We will now consider the arteries which issue from the aortic
arches. For a long time two roots were described issuing from
the posterior end of each goose-foot [my thoracic sinus,—A. P.],
forming by their junction a sort of supraganglionic anastomosis |
which gives rise to a longitudinal artery directed backward and
‘running along the dorsal surface of the ganglionic mass. Schnei-
der gave this artery the name supraneural. He has also shown
that in front of this anastomosis there are five others. There
are therefore altogether six anastomoses which this author de-
scribes as thin and delicate. This is true of the five anterior ones,
but cannot be accepted as characteristic of the last one, which
has a considerable diameter. Moreover, the supraneural artery
is rather stout; how could it be fed by two such fine roots? These,
as he shows, are often incomplete, the supraneural artery aris-
ing from a single root which may be either the right or the left
one. When the root is complete this anastomosis has the shape
of a V.
“The anterior anastomosis has the shape of a V open poste-
riorly [ie., of an angle with a vertex directed forward—A. ape
re2 ALEXANDER PETRUNKEVITCH
It is situated immediately under the oesophagus and gives rise
anteriorly to a thin artery which is closely applied to the inferior
surface of the oesophagus. This is the swboesophageal artery.
The four following anastomoses are rectilinear and each gives
rise to a vessel which issues from the middle of their ventral
surface and traverses the ganglionic mass from end to end to
its ventral surface. Schneider gave to these arteries the name
of median cerebellar arteries, as he did in the case of the scorpion.
I prefer to call them ganglionic median arteries [my vertical
or interneural arteries—A. P.]. The supraneural artery gives
also rise to a certain number of more or less short arteries of this
kind, the first being omitted at the beginning of the supraneural
artery and corresponding therefore with the sixth supraganglionic
anastomosis. I was able to find seven or eight such arteries,
thus bringing their total number to 12 or 138 . . . . What
Schneider does not mention is the fact that all these arteries
connect on the ventral surface of the ganglionic mass with a
median longitudinal lacune” . . . . (pp. 61-62).
Although I have no conclusive evidence at this moment,
either to confirm or to disprove some of the statements con-
tained in the above quotation, it seems to me that Causard
has been misled by imperfect injections. We easily recognize
in the suboesophageal artery of Causard that portion of the
supraneural artery, which is shown in the scorpion in our figure
2 as SOA. But I think that both Causard and Schneider have
overlooked the connection of the ‘anastomoses’ with the supra-
neural artery. Causard, indeed, has seen their connection with
the subneural artery by means of the vertical arteries (gan-
glionic median arteries). On the other hand, the number of
these vertical arteries given by Causard as 12 or 13 seems to be
decidedly too great. A careful examination of sagittal series
of sections through young spiders shows invariably the presence
of eleven distinct neuromeres in the thoracic ganglionic mass.
The first belongs to the pedipalpi, the second to fifth to the
legs; the tenth corresponds to the same neuromere in the scorpion,
which in the latter is already in the abdomen. The eleventh
neuromere is imperfectly divided into two. The anterior portion
CIRCULATORY SYSTEM IN ARACHNIDA 173
is the eleventh neuromere proper, while the posterior portion
represents the remnant of the abdominal neuromeres, whether
contracted and fused or lost altogether makes no difference as
regarding our proposition. We thus have at the most eleven
interganglionic surfaces, if we count the partition of the eleventh
neuromere as complete. Therefore, there cannot be more than
eleven vertical arteries, since arteries passing through instead
of between ganglia are not known.
The heart of the spider has four pairs of ostia in the Theraph-
osidae and only three pairs in the true spiders, as against seven
pairs in the scorpions. From the position of the aortic valve,
it is safe, however, to accept that the reduction in the number of
ostia took place in a progressive direction from the rear end of
the heart forward. What has happened to the rear portion of the
heart, which has lost the ostia? I think it must have shrunk
in size, become considerably thinner and changed into what
became the proximal end of the posterior aorta. We have
seen that the posterior aortic valve has a structure distinctly
different from that of the anterior valve. It would be scarcely
necessary to assume a progression of the posterior aortic valve,
a shifting of its position with the loss of ostia. Is it not more
likely that the posterior aortic valve is a modified remnant of
the last pair of ostia valves which have become functionless as
such, when the ostia themselves closed? With other words,
that the posterior aortic valve of a Theraphosid is the remnant
of the fifth pair of ostia valves, while in true spiders it is the rem-
nant of the fourth pair?
From the above comparison of the circulatory system of the
scorpion with that of the spider we may now draw the following
important conclusions: the scorpion represents the more genera-
lized and therefore more primitive circulatory system among
Atachnida, the spider the more modified and therefore the more
advanced. The most permanent structure in the circulatory
system of Arachnida is the anterior aortic valve which is at-
tached at the anterior edge of the first abdominal tergite and
therefore marks the limit between cephalothorax and abdomen.
The reduction in the number of ostia stands in direct, relation-
174 ALEXANDER PETRUNKEVITCH
ship with the loss of segmentation in the abdomen and proceeds
in the same direction, that is, from the posterior end forward.
The changes in the neural.portion of the circulatory system do
not extend over the thoracic haemomeres because of the per-
manency of the thoracic appendages, but follow the changes in
the position of abdominal neuromeres. As the contraction of
the longitudinal connectives between neuromeres brings ab-
dominal neuromeres into the thorax, abdominal vertical arteries
are also shifted in position, while the complete disappearance of
the last abdominal neuromeres brought about a corresponding
complete disappearance of the last vertical arteries.
COMPARISON WITH LIMULUS
The circulatory system of Limulus has been excellently de-
scribed by Milne-Edwards, and such errors as he has admitted
in his description have been later corrected by Patten and Reden-
baugh. J have made injections of adult large specimens to
verify the results, and can only confirm their correctness. It
is different, however, with the interpretation of the structures, and
here I disagree both with the older and later investigators.
Alphonse Milne-Edwards worked eight years before Lankester,
and although the idea that Limulus is an Arachnid had been
already advanced by Latreille and later by Owen, yet the knowl-
edge was not sufficient to admit of incontrovertible homologies.
Consequently, notwithstanding the great similarity in the struc-
ture of the nervous and circulatory systems, Milne-Edwards
felt justified in pointing out the differences and in refusing to
place Limulus either among Crustacea or among arachnids.
For reasons which it is not worth while reviewing at present,
Milne-Edwards considered the first pair of appendages in Limulus
homologous, not with the chelicera, but of the pedipalpi in
scorpions. :
Lankester’s interpretation of Limulus was colored by his
theory of tagmata into which (according to him) the body of
an arthropod is divided. He finds that the body of Arachnida
is composed of three tagmata of six somites each and that the
genital openings are placed on the first somite of the second
CIRCULATORY SYSTEM IN ARACHNIDA 5
tagma or mesosoma. Following this idea, he finds the same
tagmata in Limulus, the mesosoma being represented by the
genital opercula and the five gill-plates, while the metasoma is
reduced to a very small area around the anus, including the last
pair of lateral spines.
Patten and Redenbaugh do not attempt to change the inter-
pretation of Lankester, but correct it in regard to the chilaria.
The presence of a distinct neuromere for this pair of appendages
having been established by Kingsley, our authors naturally
ascribe to them the value of a distinct metamere and consider
the chilarial somite as belonging to the cephalothorax. For
them, as for all previous investigators, the articulation between
the carapace and the abdomen is the segmentation line separat-
ing the cephalothorax from the abdomen.
It would be useless to describe here in detail the entire cir-
culatory system of Limulus, but certain features of it must be
considered. The heart occupies the same position as in other
arthropods and extends from about the middle of the line passing
through the side eyes back to about the middle of the abdomen.
The heart has no opening posteriorly and the superior abdominal
artery is connected with the heart only indirectly through the
collateral arteries. Therefore, though occupying the same posi-
tion as the posterior aorta of Arachnida, the superior abdominal
artery of Limulus cannot be regarded as homologous with the
latter. The number of ostia is greater than in the scorpion,
inasmuch as Limulus has eight pairs. Patten and Redenbaugh
describe and figure a pair of rudimentary ostia in front of the
aortic valve. These may be the last remnants reminiscent of
a still older time when the ancestor had a heart extending farther
forward. The aortic valve has almost the same structure as in
Arachnida. In front of the valve ‘‘a pair of tendinous bands,
comparable to a pair of alary muscles, run forward and upward a
short distance beyond the limits of the pericardium, and attach
themselves to the carapace close to the insertions of the tergo-
proplastral muscles” (p. 127). I may add that this connection
is so strong that in removing the carapace the heart is easily
injured, unless particular care is given to sever the connection of
176° ALEXANDER PETRUNKEVITCH
these tendinous bands, which is certainly not the case with the
heart ligaments of the subsequent metameres.
The aorta is exceedingly short and forms almost at once two
vessels which are rightly regarded as the aortic arch. These
vessels are large and long, run at first forward, then curve down-
ward, pass the oesophagus on each side and open into the ‘vascu-
lar’ ring a little to the inside of and above the base of the first
pedal artery. The entire ventral circulatory system of Limulus
is perineural; i.e., it sheaths completely the nervous system. Not
only the postoral neuromeres of the suboesophageal ganglionic
mass, but the supraoesophageal forebrain as well is enclosed in
this perineural circulatory system. The haemal sheath extends
through the entire length of the ventral nervous cord in the
abdomen. Accordingly, neither supraneural, nor subneural, nor
interneural or vertical arteries are present.’ The cheliceral ar-
teries issue from the ventral surface (actual, not morphological)
of the vascular ring. In all this Limulus is very different from
the scorpion and other Arachnida. Yet the similarity is never-
theless quite striking. If the forebrain portion of the vascular
ring were removed, the rest of it would present an identical
appearance with the two thoracic sinuses of the Arachnida. The
similarity is increased by the existence of five nervous bridges
connecting the right and left ganglia of the five pedal neuromeres.
These nervous commissures are naturally ensheathed by the
corresponding perineural vessels which, therefore, represent the
five arteries in the scorpion connecting the thoracic sinuses with
the supraneural artery. But in what way could we explain
the origin of the scorpion type of neural circulatory system from
the Limulus type or vice versa? Has the perineural system
broken up into two sinuses and neural blood vessels, or have the
latter altogether a separate origin?
The relatively great size of the oesophagus and the position
of the forebrain in front of and not above the suboesophageal
ganglionic mass in Limulus may have something to do with the
differences between this animal and Arachnida. But this posi-
tion itself is by no means original. Notice the position of the
mouth in the middle of the ventral surface of the cephalothorax
CIRCULATORY SYSTEM IN ARACHNIDA si
and the position of organs in front of the mouth, which morpho-
logically have to be considered as postoral. Notice the plastro-
buccal muscles going “from the anterior neural side of the plastron
to the oesophagus” and the strands of muscles attaching the
proventriculus to the carapace in the region of the median eyes.
The former undoubtedly represent the pharyngeal dilators of
Archnida, the latter the dilators of the sucking-stomach in spiders
and the corresponding pair of dorsoventral muscles in the scor-
pion. Although considerably in front of the posterior edge of
the carapace in Limulus, these muscles are not far in front of
the aortic valve. Notice that in severing the carapace from
the abdomen with a knife, the opercular plate remains with the
carapace. Notice, further, that the suboesophageal ganglionic
mass in Limulus consists of seven neuromeres, the sixth be-
longing to the chilaria and the seventh to the opercula; that,
owing to the perihaemal type of blood system, the vessels for
the chilaria and opercula issue from the vascular ring; notice
all this and you get the idea of what happened to Limulus in the
course of its phylogenetic development. On the ventral surface
two somites, corresponding to the first and second abdominal
somites in Arachnida and characterized by the chilaria and oper-
cula, became fused with the thoracic somites, while at the same
time the corresponding neuromeres moved forward and fused
with the suboesophageal ganglionic mass. On the dorsal surface
a general displacement forward took place. In this displacement
two things remained unchanged: the position of the mouth and
the attachment points of the foregut and of the heart in the re-
gion of the aortic valve. What was above and behind the mouth,
with the forward bending of the back came to lie in front and
above the mouth. Part of the heart followed the displacement
because of the permanent attachment at the aortic valve. Of
the tergites, those of the chilarial and opercular somites had to
follow the forward motion of the original carapace and weredrawn
into the hollow of the horseshoe-shaped carapace as it was formed
through the forward displacement. ‘These tergites fused with the
carapace along their front and sides, but are still visible even in
the adult and especially in the so-called trilobite stage of the
178 ; ALEXANDER PETRUNKEVITCH
young. The cephalothorax of Limulus is therefore the result
of fusion of the original cephalothorax with the chilarial and
opercular somites, and the articulation between the carapace
and abdomen is in reality an articulation between the opercular
and first gill somite, or what corresponds to the division line
between the second and third abdominal somites in Arachnida,
as exemplified by the genital and comb somites in the scorpion.
The division line between the last thoracic and first abdominal
(chilarial) tergites lies immediately in front of the attachment of
the heart, i.e., somewhat in front of the line passing through the
two lateral eyes.
The history of this forward displacement and fusion of origin-
ally abdominal somites cannot be gleaned from a study of the
external segmentation of Limulus embryos. On the ventral
surface segmentation is clear, but on the dorsal the first visible
segment is already the first gill segment. Something similar
may be seen in the scorpion. Here, in the adult, the third
abdominal tergite corresponds to the first lung sternite and there-
fore in reality representing the fourth abdominal somite. The
second tergite, corresponding to the comb, represents the third
abdominal somite. But the first abdominal tergite is the result
of a fusion of the first and second tergites of the corresponding
embryonic somites. The external segmentation is clear in young
embryos on the ventral surface, and in quite young embryos
is at least indicated by the even segmentation of the nervous
system as seen in longitudinal sections through these stages.
But when segmentation appears in the shape of transverse de-
pressions on the dorsal surface, the first visible abdominal ter-
gite corresponds already with the same tergite of the adult and
is therefore already the result of fusion. It may be argued that
if in Limulus abdominal tergites fused with the carapace, the
same may have happened in the case of the missing first tergite
of the scorpion. But this interpretation meets with too many
objections. Of these perhaps the clearest is presented in the
case of solpugids in which the thorax is still externally segmented.
In my monograph of Palaeozoic Arachnida (713) I have pointed
out that the Xiphosura must have developed independently
CIRCULATORY SYSTEM IN ARACHNIDA 179
of the scorpions. The idea that Limulus is an arachnid as it
is usually expressed, or more correctly that the Arachnida have
a common ancestor with Xiphosura, must now be completely
abandoned. Geologically, Limulus is older than the scorpion
and already the oldest limuloid shows the same type of segmenta-
tion as the recent. Neither has the idea of Versluys on the origin
of gills from lung books any bearing upon the question of origin
of Limulus or Arachnida. With a stress onimaginationone may
derive Limulus from a eurypteroid ancestor, but to derive the
latter from originally air-breathing Arachnida on the basis of no
other evidence than conjectures which rest on,a comparison of
gill-plates with lung books and in the absence of any remains
of air-breathing Arachnida antedating eurypteroids, seems to be
a rather hazardous undertaking.
It may be interesting to mention in this connection that early
stages in the embryonic development of scorpions show clearly
eighteen postoral neuromeres, the first of which soon passes in
front of the mouth and represents the cheliceral somite. The
study of preoral neuromeres in the same stages is too compli-
cated to admit of impartial judgment. In later stages, after
the passage of the mouth behind the cheliceral neuromere, one
may clearly count three pairs of nerves issuing from what appears
to be three corresponding neuromeres. ‘The first pair are the
optic nerves of the median eyes, the second the nerves of the
lateral eyes, and the third the nerves of the upper lip. These
nerves are much finer than both optic nerves and can be traced
with certainty only in sections parallel to the plane of symmetry
(sagittal). The adult scorpion has therefore four preoral and
seventeen postoral neuromeres. Five of the latter belong to the
thorax and twelve to the abdomen. It happens that the abdomen
of the scorpion shows twelve tergites. Yet one should not
conclude from this coincidence of figures that each of the neuro-
meres mentioned belongs to a corresponding tergite. Nothing
of the kind. [I have already mentioned that the comb receives
its nerves from the third abdominal neuromere, as may be easily
demonstrated on sagittal and frontal sections. In early stages
the neuromeres do not possess longitudinal connectives and
180 ALEXANDER PETRUNKEVITCH
are recognizable without difficulty only because of the constric-
tion between adjoining neuromeres. The last neuromere is
clearly situated in the last abdominal segment, and only later
moves forward and fuses partially with the penultimate neuro-
mere. There is, therefore, in the adult scorpion an abdominal
segment in excess of neuromeres. From an examination of nu-
merous series I have no doubt that it is the first postabdominal
or caudal segment and which therefore may have the value not
of a true somite, but of an anterior subdivision or ‘segment of the
same somite to which the second postcaudal segment also belongs.
Here, then, something happened the reverse of the fusion of
sclerites in the first two abdominal somites, namely, the sub-
division of the sclerite ring of a single somite into two distinct
sclerite rings or segments, without a corresponding subdivision
of other structures in the same somite.
It may be objected that such formation of pseudo-segments
has not as yet been described, either for Arachnida or other
Arthropoda, and that it were simpler to accept that the neu-
romeres really correspond to the visible segments, but in moving
forward lost connection with them and began to furnish nerves
to the next following. In other words, that the first abdominal
neuromere originally furnished the nerves for the genital opercula,
lost connection with the latter, and ceded this morphological and
physiological function to the second neuromere; that the same
happened to the second neuromere in relation to the comb, which
now received its nerves from the third neuromere. But this
explanation, besides being more complicated, suffers from another
weakness. ‘The roots of nerves follow the displacements of their
neuromeres, but the nerves themselves obtain their connection
with the original appendages, even if some branch of the nerve
passes to another somite. This may be seen in Limulus and in
many other arthropods. But in the case of the first abdominal
neuromere of the scorpion there is not even a considerable or
appreciable displacement forward, so that there would be no
morphological reason of any kind for a loss of connection with the
genital opercula if these belonged to the first neuromere.
CIRCULATORY SYSTEM IN ARACHNIDA 181
We may therefore form the following conclusions regarding
segmentation in scorpions and in Arachnida in general. The
body of an Arachnid is composed of twenty one somites, to wit:
1 the first ocular (median eyes in the scorpion, anterior median
eyes in the spider); 2 second ocular (lateral eyes in the scorpion,
eyes with inverted retina in spiders, i.e., anterior lateral, and the
four posterior ones) ; 3 rostral (upper lip); 4 cheliceral; 5 pedipal-
pal; 6 to 9 thoracic pedal; 10 to 21 abdominal. ‘The first three
are originally preoral in position. The fourth or cheliceral
becomes preoral during development. The attachment of the
heart to the anterior edge of the dorsal wall of the first abdominal
somite and the formation at this place of the aortic valve
indicate the division line between the thorax and the abdomen.
The tenth somite is always rudimentary, having lost its identity
in all but its neuromere. The genital opening is on the eleventh
somite (second abdominal). A further fusion and ultimate loss
of the identity of somites in Arachnida involves the posterior end
of their body, beginning with the twenty-first somite and pro-
ceeding forward. In some eases, as in the eighteenth somite
in the scorpion, secondary or spurious segmentation may take
place, which has no relation to the original metamerism. If
there be more than three originally preoral somites, these would
have to be sought for in front of the first ocular somite.
Turning once more our attention to Limulus, we may first
of all consider the homology of the thoracic and abdominal
somites established in a way excluding all doubt. The six pairs
of appendages belong to the same somites as in Arachnida,
the chilaria represent the tenth, the opercula the eleventh somite,
the five branchial neuromeres correspond to the twelfth to six-
teenth somites, and of the three postbranchial ganglia the last
is the result of fusion of the nineteenth to twenty-first neuromeres,
if the ancestor of Limulus possessed that many postbranchial
somites.
The homology of the preoral somites is more troublesome.
Patten and Redenbaugh describe three preoral neuromeres, the
olfactory, median ocular, and lateral ocular. Shipley following
Carpenter recognizes only two somites, the median ocular and
182 ALEXANDER PETRUNKEVITCH
the rostral. I think we may consider it as fairly conclusive
that the median ocular and lateral ocular somites are homologous
in Limulus and the scorpion. Whether the rostral somite of the
scorpion corresponds with the somite designated as rostral in
Limulus by Carpenter, is not so sure, but if it does not, then
Limulus must possess just the same some evanescent somite be-
tween the lateral ocular and cheliceral. As for the olfactory
somite, its homologue in Arachnida would have to be sought in
one of those two pairs of obscure parietal ganglia described
by Schimkewitsch for tetraneumonous spiders.
In the presence of a perineural circulatory system, in the ex-
istence of eight pairs of ostia in the heart, and of a pair of chila-
rial nerves, Limulus shows evidence of its origin from an arthro-
pod ancestor lower and more primitive than the Arachnida. But
in every other respect Limulus shows advanced development
different from that in Arachnida and most likely standing in direct
relationship to its particular mode of life. It seems as if the
older interpretation of Limulus as a descendant of Trilobites
must be revived. The shape of the trilobite carapace, the posi-
tion of the mouth, the probable similarity in the position of the
foregut as suggested in figure 24 of Raymond’s beautiful mono-
graph, the larval stages showing segments which were inter-
preted as cephalothoracic, but some of which probably are ab-
dominal tergites drawn into and fused with the thoracic ones,
point to a similarity more than casual. At any rate, the problem
should be reinvestigated from the new point of veiw.
COMPARISON WITH OTHER ARTHROPODA
We have seen that the aortic valve has a uniform structure
and a permanent position in Arachnida, permitting of strict
homologies within that class. We have also seen to what con-
clusions we arrive through the assumption that the rule holds
good in the case of Limulus also. One would expect that a struc-
ture so permanent in one or perhaps two classes would prove to
be the same in the case of all other Arthropoda, if the diverse
forms united under this immense phylum are of monophyletic
origin. Unfortunately, this is not the case.
CIRCULATORY SYSTEM IN ARACHNIDA 183
In his work on the organs of circulation in Schizopoda, pub-
lished in 1883, Delage writes (I translate the original): ‘To
determine exactly the length of the heart one should first of all
well define its limits. It happens that these limits are not easily
traceable because the diameter of the heart is not greatly differ-
ent from that of the aortae at their points of origin. (Delage has
in mind both the anterior and posterior aorta.) They are marked
by the presence of cardioaortic valvules which have not yet been
described by anyone. Moreover, these valvules are identical
with those which are found in the same place in Amphipoda.”’
“Within these limits the heart extends from the level of the last
maxillary segment to the superior portion of the last thoracic
segment.’”’ The anterior aorta, the median stem of which ends
in the upper lip, gives off four branches in its course: the common
trunk of the ophthalmic arteries, the cerebral artery, and the two
antennal arteries. It may be of interest to notice that the
sternal artery in Schizopoda arises from the heart.
The structure of the aortic valves themselves is different from
those of Arachnida. They are paired lateral structures, as in
all other Crustacea. The position indicated by Delage, taking
into account evidence derived from the study of all other organs,
is two somites nearer the head than in Arachnida. In such
Decapoda as the crayfish and the lobster the heart is distinctly
limited and considerably modified. Instead of arising from the
aorta, the antennal arteries arise directly from the heart and
have their own valves. Yet the aorta has also valves at its
base and these are of the same type as in Schizopoda and
Amphipoda. The position of these valves coincides exactly with
the semilunar sulcus of the carapace, the two ends of which open
into the so-called cervical groove. There are therefore differences
in regard to structure of the circulatory system in closely related
orders of the same sub-class—differences which cannot be
understood without special study directed to their elucidation.
We know still less of the Protracheata, Pycnogonida, and
the four classes formerly comprised under the general name of
Myriapoda. Although I have some investigations under way,
I am not prepared as yet to make any definite statement.
184 ALEXANDER PETRUNKEVITCH
The circulatory system of insects is somewhat better known
in this respect, yet here also the data are quite inadequate to
form a clear judgement. Popovici-Baznosanu has described the
heart in the Chironomus larva and states that the aortic valves
are situated close to the anterior end of the fifth segment. In
other larvae the heart had been described by other authors as
situated near the rear end of the body. In some larvae the heart
is not even situated directly under the dorsal body wall, but les
considerably deeper in the body cavity. The structure of the
cardio-aortic valves, too, seems to be not only different from that
of the aortic valve in Arachnida, but not always of the same type
in allinsects. Moreover, according to Zawarzin there are modified
ostia in the aorta itself. It is evident that the first step must be
in finding the true limits of the heart itself in insects. Mean-
while all conjectures would be entirely out of place.
SUMMARY AND CONCLUSIONS
1. In comparing the segmentation in arthropods the uncer-
tain method of counting somites beginning with the anterior
end should be abandoned. Instead, some structure should be
chosen which has permanent value for a number of forms within a
class and used as a starting-point of comparison.
2. Such structure in the case of Arachnida is furnished by
the cardio-aortic valve which marks the division line between
the last thoracic and first abdominal segments.
3. The method applied to Limulus leads to the conclusion
that the carapace of Limulus is more complicated than in Arach-
nida, having two abdominal tergites drawn into the horseshoe-
shaped thoracic tergite with which they have fused anteriorly
and laterally.
4. A further conclusion is that the midcorporal articulation in
Limulus is not between thorax and abdomen, but between the
second and third abdominal somites.
5. The structure of the circulatory system in Arachnida fol-
lows a general plan given in the text.
6. The number of postoral somites in adult Archnida is seven-
teen. Five of these are thoracic and twelve abdominal.
CIRCULATORY SYSTEM IN ARACHNIDA
185
7. The genital opening is on the second abdominal somite.
8. The first caudal segment in scorpions is not a true somite,
but merely the anterior division of the fourteenth postoral somite.
9. If the number of preoral somites in Arachnida is not more
than four, as represented by the median ocular, lateral ocular,
rostral, and cheliceral somites, then the total number of somites
in Arachnida is twenty-one.
EXPLANATION OF PLATES
ABBREVIATIONS
AC, Cheliceral artery
AO, Aorta
AP, Pedal artery
APP, Pedipalpal artery
1. AT, First abdominal tergite
AV, Anterior aortic valve
BR, Brain
CA, Comb-artery (ninth vertical ar-
tery)
Cop, Basal plate of comb
CO, Aortic arch connection with
thoracic sinus
D, Diaphragm
DP, Dilator muscle of the pharynx
DS, Dorsoventral muscle separating
the first cephalothoracic diverticle
of the midgut from the second
GB. 2, Gnathobase of the second leg
GP, Genital plate (operculum)
H, Heart
LC, Longitudinal connective between
the fourth and fifth abdominal
ganglia
M, Mouth
MA, Anterior edge of carapace
ME, Median eyes
MG, Midgut
MP, Posterior edge of carapace
PH, Pharynx
SAA, Supraneural (epineural) artery
SBA, Subneural (hyponeural) artery
ST, Sternum
UP, Upper lip (rostrum)
VA, Vertical or interneural artery
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Resumen por el autor, William H. Leigh-Sharpe.
Morfologia comparada de los caracteres sexuales secundarios
de los peces elasmobranquios—los 6rganos copuladores,
sus sifones y gl4andulas. Memoria III.
Los elasmobranquios mas antiguos carecen'de 6rganos copula-
dores; también puede afirmarse con certeza que carecen de
sifones y glindulas de dichos érganos. Los fésiles mds préximos
a estos presentan un tipo directo de 6rgano copulador; probable-
mente no existen en ellos sifones 0 solamente aparecen bajo una
forma rudimentaria. Mas tarde el tipo de érganos copuladores
en forma de rollo aparecié, prediciendo 4 los Scyllidae; probable-
mente estos 6rganos iban acompafiados de un sif6n. Los Lam-
nidae son geol6gicamente mas recientes que los Scyllidae, y
poseen una glandula en el 6rgano copulador, por lo menos en las
formas recientes. Mas tarde se produjeron las rayas, las cuales
se asemejan a las de los tiempos recientes.
Translation by José F. Nonidez
Cornell Medical College, New York
AUTHOR’S ABSTRACT OF THIS PAPER ISSUED
BY THE BIBLIOGRAPHIC SERVICE, FEBRUARY 27
THE COMPARATIVE MORPHOLOGY OF THE SECOND-
ARY SEXUAL CHARACTERS OF ELASMOBRANCH
FISHES
THE CLASPERS, CLASPER SIPHONS, AND CLASPER GLANDS
MEMOIR III
W. HAROLD LEIGH-SHARPE
London, England
FIVE FIGURES
The previous memoirs appeared in the Journal of Morphology
as follows: Memoir I, volume 34, page 245, 1920; Memoir II,
volume 35, page 359, 1921. The first contained a general intro-
duction to the subject and an account of Scyllium catulus, 8.
canicula, Acanthias vulgaris, and Raia circularis. The second
dealt with Galeus vulgaris, Mustelus vulgaris, Lamna cornubica,
and Rhina squatina.
The present memoir describes the following species:
Mederetic he Hee plerinsss5 eee is crete doy wee oe da ee eed wha ded 192
Pleuracanthus paralleluss. ppm. « se ose oie mtntobscat: 193
SAPD USPS Tene oe] Ais] OPO) 1c bE Yepake pF Cg a nn 193
EDMEITROM SOUS EL LCETICGIUS: 545% Poficicetde << o cinc.d oc vis conc s COM ee vee 197
Pe MClaMIA GHEOMACE VMI, So mee sete Sate tigt cobs csegodcs eet 197
The fossil aspect of this subject is unsatisfactory. Soft parts,
the clasper siphons and clasper glands, as is only natural, are not
preserved. The claspers, mainly the skeleton, alone are in-
dicated. After an examination of the fossil collection in the
Natural History branch of the British Museum, South Kensing-
ton, London, England, by the courtesy and under the personal
superintendence of Dr. A. 8. Woodward, I have selected five
examples which have some bearing on the matter.
191
192 W. HAROLD LEIGH-SHARPE
The figures have been executed from them especially for me
by Miss Edith C. Humphreys, and the catalogue number is
appended. A summary of the conclusions drawn from these
observations follows.
a7
S
cni.
Fig. 1 Cladoselache kepleri. K., kidney; *, see text.
CLADOSELACHE KEPLERI
This primitive Palaeozoic fish occupies the unique position of
the oldest known elasmobranch. It possesses no claspers; in
consequence, the bases of the pelvic fins appear wider apart than
isusual. Figure 1 represents the specimen P. 9269, the Newberry
sp. from the Upper Devonian (Cleveland Shale), Berea, Ohio,
U.S. A. (William Clark collection).
SEXUAL CHARACTERS—ELASMOBRANCH FISHES 193
The streak marked K in the figure has been misinterpreted as
a clasper, an error which, owing to the influence of E. Stromer
von Reichenbach, has found its way into the text-books. How-
ever, this is the only specimen in which this streak occurs, and,
further, as recently as about 1914 at the points marked * in the
figure, Doctor Woodward has caused excavations to be made
and microscope slides prepared of the abstracted fragments.
These sections reveal the same structure as that of the kidney of
recent forms, the organ having become calcified.
There being no claspers, it is a safe assumption that there are
also neither clasper siphons nor clasper glands.
PLEURACANTHUS PARALLELUS
Subsequently, in late Palaeozoic times, there appeared elasmo-
branchs with claspers—the Pleuracanthei. Figure 2 represents
the specimen from the Carboniferous ‘gaskoéhle’ at Tremosna
near Pilsen. I have not seen this fossil. Dermal denticles
appear to be preserved, and, if the restoration of Xenacanthus
decheni is to be relied on, the spoon-shaped conformation at the
tip of the claspers had already been evolved.
The point of interest is that the clasper, from its skeleton,
appears to be of the ‘straight’ or ‘direct’ type, by which is meant,
not that it is without a gentle curve longitudinally, but that it is
- not rolled up in a scroll-like manner; on the contrary, the groove
is very wide open. Such a condition I interpret as being primi-
tive here, and secondary in the skates. The type of clasper
suggests that possibly clasper siphons were not yet evolved, or
were present only in a rudimentary form.
Acanthodes wardi is devoid of claspers, and therefore of no
use in this investigation.
SQUALORAJA POLYSPONDYLA
This early Mesozoic chimaeroid fish, to be compared subse-
quently with Chimaera, is beautifully preserved in the specimen
P 2276 found in the lower lias at Lyme Regis, Dorsetshire, Eng-
land, a jurassic formation (fig. 3).
194 W. HAROLD LEIGH-SHARPE
“Arising immediately within the point of union of the pubic
and iliac regions is the basal cartilage which . . . . is
prolonged backwards into a powerful clasper . . . . the
cartilage becomes more calcified and . . . . broader. The
Fig.2 Pleuracanthus parallelus (after Fritsch.') B., basipterygium; D.D.;
dermal denticles; cl., claspers. A., restoration of Xenacanthus decheni.?
inner edge is straight, but the outer edge exhibits a gentle sigmoid
curve which results in the widening of the rounded terminal
extremity; and at the end of each clasper (especially the left)
1 Fritsch, Ant., Fauna der Gaskéhle und der Kalksteine der Permformation
Bohmens—Prag. Bd. 3, Heft I, Taf. 93.
2 Op. cit., p. dh.
SEXUAL CHARACTERS—ELASMOBRANCH FISHES 195
a small tuft of dermal hooklets is preserved. Thefinrays ..
. . completely shown on the right are altogether twelve in
number, and the length of the supporting cartilage is scarcely
more than one-half of the appended clasper.’’$
a]
6
+
A
A
is
w
en ily cm.
Fig. 3 Squaloraja polyspondyla, dorsal aspect. p.pb., prepubic process;
pub., pubic bar; il., iliac process; b., basipterygium;7., cartilaginous fin rays;
d., edge of skin; cl., claspers; d.h., dermal hooklets;d.¢. dermal tubercles; V.c.,
vertebral column.
The prepubic processes no doubt formed the base of attach-
ment of the anterior claspers, which Parker has given reasons for
believing a third pair of limbs, metameric with the pelvic clas-
pers, making the Holocephali the sole exemplars of hexapodous
vertebrates. This suggestion has not met with support.‘
3 Quoted from Woodward, A. S., Squaloraja polyspondyla, Proe. Zool. Socy.,
1886, p. 527.
4 Parker, T. J., Nature, 1886, vol. 34, p. 635.
196 W. HAROLD LEIGH-SHARPE
The analogy and possible homology between the basipterygium
and a femur probably led to the introduction of the term of
pterygopodia applied to the claspers.
Dermal denticles and tubercles are preserved in places, and the
tips of the claspers are provided with recurved dermal hooklets.
These appear to be seven close together with one more remote,
15
cm.
Fig.4 Rhinobatus intermedius. cl., clasper; Rh., rhipidion; H., possible
position of hypopyle.
and are stout, affording an early example of an apparatus for the
prevention of elision of the claspers from the oviduct of the
female, since they curve forwards; backwards, when the clasper
is bent forward in copula.
The chief point of interest is that the clasper suggests from its
skeleton that it is rolled up in a scroll-like manner, recalling that
of Scyllium. It is probable, therefore, that each clasper was
accompanied by a clasper siphon.
SEXUAL CHARACTERS—-ELASMOBRANCH FISHES 197
RHINOBATUS INTERMEDIUS
It was not until a much later date that the skates arrived.
These appear to resemble recent forms, and some are even as-
signed to existing genera. Figure 4, to be compared subse-
quently with a modern species of Rhinobatus, is drawn from the
specimen 49516, from the upper cretaceous at Sahel Alma, Mount
Lebanon (Lewis collection). It does not follow that the primi-
A
iis) ig Wigiy &
om TAD) APA PATA ES OAT) OES PS BaD
)
Oo
cM.
Fig.5 Cyclobatis oligodactylus. cl., clasper; B., basipterygium
tive forms possessed a clasper gland as do their modern congeners,
but at least such a corollary is probable. The specimen gives
indications of a well-developed rhipidion.
CYCLOBATIS OLIGODACTYLUS
Also from the upper cretaceous, Hakel, Mount Lebanon, is the
small and well-preserved specimen P 601 (Egerton collection).
Originally described as resembling the Torpedinidae, but now
known to be related to the Trygonidae, figure 5 does no more
198 W. HAROLD LEIGH-SHARPE
than indicate a likeness to the skates in general ‘as regards the
claspers, which are well developed, suggesting a male of mature
age.
SUMMARY
The conclusions drawn from a chronological survey of fossil
forms are:
1. The oldest elasmobranchs (Cladoselache) are without
claspers. It is almost certain they are without clasper siphons
and clasper glands also.
2. The next fossils have a direct type of clasper. Possibly
clasper siphons were not yet evolved or were present only in a
rudimentary form.
3. Subsequently the scroll type of claspers appeared, sug-
gestive of the Scylliidae. Probably these were accompanied by
a clasper siphon.
4. The Lamnidae are geologically more recent than the
Scylliidae, and these have progressed a stage further and evolved
a clasper gland, at any rate in recent forms.
5. Later the skates arrived and resemble those of recent times.
Resumen por el autor, William H. Leigh-Sharpe.
Morfologia comparada de los caracteres sexuales secundarios
de los Holocéfalos y peces elasmobranquios—los 6rganos
copuladores, sus sifones y glandulas. Memoria IV.
Chiloseyllium y Pristiurus poseen sifones semejantes a los de
Scyllium. Los Holocéfalos poseen rasgos peculiares y carac-
teristicos de su clase, entre los cuales pueden mencionarse un par
de 6rganos copuladores anteriores, en adicién del par encontrado
comunmente, los cuales pueden retraerse dentro de bolsas; la
hembra posee una bolsa en Callorhynchus; en los machos existen
6rganos copuladores frontales; en Chimaera existen Organos
copuladores bifurecados y un cuerpo longitudinal. Los cuatro
Batoideos son semejantes entre si y a Raia en lo referente a la
glindula del 6rgano copulador, y difieren de Raia circularis en
la ausencia de un tubo sifonal extendido hasta la extremidad
posterior del 6rgano copulador. Rhinobatus se diferencia de
los restantes por ser en algunos aspectos mas primitivo, con una
pequena glandula pero con una sentina y una garra.
Translation by José I. Nonidez
Cornell Medical College, New York
AUTHOR’S ABSTRACT OF THIS PAPER ISSUED
BY THE BIBLIOGRAPHIC SERVICE, FEBRUARY 27
THE COMPARATIVE MORPHOLOGY OF THE SECOND-
ARY SEXUAL CHARACTERS OF HOLOCEPHALI
AND ELASMOBRANCH FISHES
THE CLASPERS, CLASPER SIPHONS, AND CLASPER GLANDS
MEMOIR IV
W. HAROLD LEIGH-SHARPE
London, England
TWENTY-TWO TEXT FIGURES!
The preceding memoirs appeared in the Journal of Mor-
phology as follows: Memoir I, volume 34, page 245, 1920;
Memoir II, volume 35, page 359, 1921; Memoir III, volume 36,
page 191, 1922. The first two contained an account of the
commoner British species, Seyllium catulus, S. canicula, Acan-
thias, Raia, and Galeus, Mustelus, Lamna, Rhina, respectively.
The third dealt exclusively with the fossil aspect of the subject.
The present paper describes certain species to which I have
kindly been allowed access at the Natural History Branch of the
British Museum, South Kensington, London, viz.:
Glniloseys unm POMC cAd Um rat ecam ey ote cee hoes tee aloe os oe eee 200
EIS UMTS ie AMO SGOEMIESE Coes a2? Ce 22 oe oe citer bares an kis Siete ee Oe 201
SG HeLa TAOS LOSE Wer oe copys yee yale 8 Seeds Sc ee hse we aR 201
Wallon live btisesm tunel Cua. 25%. gene cei Gate ALA eed aoe a aw oie 208
POE EU LEMMON Abe! ae eon-a-4. e Dae oor acta eo as ees Coat eee eed
eG OnMPASbInaeate saree. wt Meee ce om sixcdarla ce or Meiiavina csi eo
ML STAN] DA 220 [5011 ie, OR, gener ere cre ae ee OO in ee 217
PUM reads! ST OMLOUUS: eke seta n os is. aeeenedeecare Seto tise Steels wie 218
1The figures are specially drawn from the author’s dissections and’ preparations
by Miss Edith C. Humphreys, to whom best thanks are tendered.
199
200 W. HAROLD LEIGH-SHARPE
CHILOSCYLLIUM (HEMISCYLLIUM) PUNCTATUM
The barbelled dogfish
The following description is based on the specimen numbered
55, exhibited at the Great International Fisheries Exhibition.
JO
oO
cm.
Fig. 1 Chiloscyllium punctatum. <A., apopyle; H., hypopyle; S., siphon;
S1., slit.
It was taken at Singapore, was 3 feet in length and weighed
6 pounds at capture (fig. 1).
This animal possesses a siphon of the type found in Scyllium,
long and narrow, about 4 cm. in length. The claspers are
denticled as in Seyllium, but the closed portion of the scroll-like
SEXUAL CHARACTERS—ELASMOBRANCH FISHES 201
clasper formed by the overlapping edges is very short. On the
inner side of the dorsal surface of the clasper is a slit whose
cavity leads not forwards, but slightly backwards, and hence
cannot be truly regarded as a pseudosiphon.
PRISTIURUS MELANOSTOMUS
The black-mouthed dogfish
This near relative of Scyllium differs from that genus more
than might be supposed. The specimen examined, one of two
from an unnamed locality, measures 62 cm. in length. The
siphons are long and narrow, in this case 12.5 em., poorly de-
veloped, with but feebly muscular walls. They are propor-
tionately longer than in Scyllium, but lie immediately under the
skin as in Acanthias. The claspers terminate in a gimlet-like
coil (fig. 2), which, when unrolled, as in the inset, shows slight
indications of a rhipidion. The actually closed portion of the
scroll-like clasper is moderately long as in Scyllium.
CHIMAERA MONSTROSA
The king of the herrings, or rabbit fish
Although the Holocephali are not strictly elasmobranchs, it is
both important and essential that they should be included, since
they exhibit the features which form the subject of this investi-
gation. They possess the peculiarity of an anterior pair of
claspers, or grapplers, in front of the pelvic fins in addition to
the usual posterior pair. The anterior claspers are capable of
being retracted into a glandular pouch, where they are usually
held in retreat. They are absent in the female, but the pouch
may in some cases be present in a rudimentary form. There
is also a frontal clasper in the male. In Harriotta the claspers
are said to be poorly developed, and the frontal clasper absent.
The specimens here described are from a batch of about nine
received from Rockall, September, 1920.
In the females, the largest of which is 79 em. long, I failed to
find any vestige or rudiment of either anterior claspers or their
pouch: there appears to be no trace of them, at any rate in the
present species, Chimaera monstrosa.
JOURNAL OF MORPHOLOGY, VOL. 35, NO. 2
202 W. HAROLD LEIGH-SHARPE
The male examined is 68 cm. in length, the fish in each sex
being measured to the extremity of the whip-like tail. The
claspers are not obvious, being covered in the natural position
by the pelvic fins, as seen on the (observer’s) right in figure 3.
Fig. 2 Pristiurus melanostomus. A., apopyle; H., hypopyle; S., siphon;
FRh., rhipidion.
They are of a type wholly different from Callorhynchus and the
elasmobranchs, since each bifurcates into an external and an
internal radius. At the bifurcation of the radii is the apopyle.
Whilst the fish is very smooth, the clasper radii are fiercely
denticled, though lacking at the tip the dermal hooklets so
characteristic of the fossil Squaloraja (Memoir III, p. 198).
SEXUAL CHARACTERS—ELASMOBRANCH FISHES 203
The external radius, which is capable of flexion outwards to
prevent elision from the oviduct, is denticled at the tip on the
outer border, and all along the inner border, while the more
stationary internal radius is denticled all along its outer margin
which is grooved, the denticles being represented by stippling
in the figure. The two radii, when approximated, together form
a clasper groove or tube suggestive of the elasmobranchs. The
whole external radius is therefore analogous in function to the
Ant. CL. Wes Peli. z
Bn =
ip QD), \ 8
Spi) lV Mos
Vy! | ni
Pp \\\
; ‘ict 4.
\
Ex.R
& S ra)
IntR. aes
Fig. 3 Chimaera monstrosa. Ant. cl., anterior clasper partly protruded ;
Pch., pouch with anterior clasper retracted; Ex. R. and Int. R., the external and
internal radii of the posterior clasper; O. C., opening of cavity; A., apopyle.
spur in Acanthias, the radii being long and slender and apparently
not erectile.
Leading into the apopyle is a cavity which I cannot consider
the homologue of the siphon for the following reasons: 1) It
does not appear to be a smooth sac with muscular walls; 2) it is
situated in the swollen proximal end of the clasper, and not on the
ventral surface of the abdomen; 3) part of the skeleton of the
clasper (the basal portion from which the two radii originate) has
to be cut through to reveal its interior, which is not the case in
204 W. HAROLD LEIGH-SHARPE
any siphon I have as yet investigated; 4) it appears to have a
homologue in Callorhynchus which is merely a folded portion of
the clasper-tube.
Lying within this cavity is an elongated longitudinal body
which is neither solid nor tubular, but rolled upon itself in a
scroll-like manner (fig. 4, L. B.). A microscopic examination
of this body in transverse section reveals (fig. 5) that it is com-
Gr.
oO
cm.
Fig.4 Chimaera monstrosa. Gr., grappler, the pouch having been dissected
away; M., muscle; Cav., cavity; L. B., longitudinal body; Hx. R. and Int. R., the
external and internal radii of the posterior clasper.
posed of compact bands of striped muscle, some spongy tissue, a
supporting base of cartilage, and covered by a most curious
epithelium. This epithelium consists of a single layer of ex-
tremely long, narrow, rod-like cells, with a nucleus in the centre
where there is a slight dilatation to receive it. Such cells are
always associated with a sensory epithelium, (e.g., olfactory) in
higher types, and are probably not glandular.
SEXUAL CHARACTERS—ELASMOBRANCH FISHES 205
The anterior claspers can each be retracted into a slit-like
pouch which is placed transversely to the animal’s axis (con-
trast Callorhynchus, where it is longitudinal); thus, in figure 3,
the clasper on the (observer’s) left is partly protruded, while that
on the right is completely withdrawn. A reference to figure 6
Ep. |
\
Le
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Uf
_INN
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SS =
SH
uf
=
| Fig. 5 Chimaera monstrosa, a transverse section of the longitudinal body
(haemalum-eosin). Hp., epithelium of the longitudinal body, a single cell
of which is shown highly magnified in the inset A; Cart., cartilage; M., muscle;
Ep. Cav., epithelium of the cavity.
shows that the pouch is lined with stratified epithelium, im-
mediately below which are fibrous connective tissue and the
striped muscle bands of the derma, and that a few goblet-cells
are present.
In the channel, however, formed by the continuity of the base
of the pouch and the anterior clasper, numerous goblet-cells are
found as indicated in figure 7,G. C. Goblet-cells are also present
206 W. HAROLD LEIGH-SHARPE |
in the epithelium of the clasper, more numerous on its concave
surface. These, no doubt, secrete mucus, which fact has given
rise to the expression ‘glandular pouch,’ although the walls of
the pouch are in the main but slightly glandular.
As regards the clasper, only that portion of it which I have
called the grappler in Callorhynchus, is represented here. It is
composed of practically nothing but cartilage, over which is drawn
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Fig. 6 Chimaera monstrosa, a transverse section of the wali of the pouch
of the anterior clasper (haemalum-eosin). Fp., stratified epithelium; M., striped
muscle.
Fig. 7 Chimaera monstrosa, a longitudinal section through the anterior
clasper (haemalum-eosin). Cart., cartilage; Hp., stratified epithelium. M.,
muscle; G.C., goblet cells.
SEXUAL CHARACTERS—ELASMOBRANCH FISHES 207
a normal type of stratified epithelium(fig. 7). The grappler is
spoon-shaped, slightly concave, deeply serrated on its morpho-
logical inner border, but without denticles. Its function would
appear to be truly a clasping or grappling one. There is no
compact gland in connection with the anterior clasper as there
is in Callorhynchus. ;
The frontal clasper (fig. 8, A) is a knocker-like structure on
the forefront of the head, which is used for clasping, and helping
to overtake, catch, and turn the female. It is limited to the
male, and, from indications of scars on the females, its place of
; \
Fig. 8 Frontal claspers. A., Chimaera monstrosa. B., Callorhynchus
antarcticus.
application appears to be near the pectoral fins. It is strongly
denticled on its under surface, and, when not in action, is kept
bent in a little socket-like depression in the skin, into which it
just fits. In the female the position of the frontal clasper is
indicated by a flat area on the skin of the forehead.
208 W. HAROLD LEIGH-SHARPE
CALLORHYNCHUS ANTARCTICUS
The southern beauty
This curious member of the Holocephali agrees with Chimaera
in the essential characters mentioned in the introduction to
* that genus, but is widely different as to details. I have examined
16
oO.
cm.
Fig. 9 Callorhynchus antarcticus. Ant. Cl., anterior clasper partly pro-
truded; Po., pouch with anterior clasper retracted; A., apopyle; f., fused portion
of clasper tube; H1., true hypopyle; H2, false hypopyle.
an adult male from Hobart Town 66 em. long and an adult
female from Table Bay 73 em. in length. While the posterior
claspers are here again covered by the pelvic fins, they are of the
ordinary scroll type with nothing specially remarkable. The
closed portion of the clasper tube formed by the overlapping
edges is very long, and a peculiarity is that, subsequent to the
unrolling of the scroll, at the extreme tip of the clasper, is a
short closed portion formed by actual fusion of the clasper edges
SEXUAL CHARACTERS—ELASMOBRANCH FISHES 209
(figs. 9 and 10, f), or possibly by a fused rhipidion. This is the
first recorded case in which a closed tubular passage is formed
in the claspers by actual fusion. No denticles nor accessory
structures are present on these claspers; neither, as far as I can at
present ascertain, is there any siphon or gland to be found in
connection with them.
As in Chimaera, the anterior claspers can each be retracted
into a slit-like pouch which is longitudinal, or parallel to the
animal’s axis, not transverse as in Chimaera; thus in figure 9
10
oO
Cm.,
Fig. 10 Callorhynchus antarcticus, the right posterior clasper unrolled.
Cav., position of cavity at proximal end of clasper tube; f., fused portion of clas-
per tube.
the clasper on the (observer’s) left is partly protruded, while
that on the right is completely withdrawn. The curious anterior
claspers are extraordinarly complicated, and, the pouch having
been dissected away, the relations between the various parts
are shown in figure 11 in two positions. Fundamentally there
is the grappler not unlike that of Chimaera, a spoon-shaped
cartilaginous structure, slightly concave, without a serrate
border, but covered on its anterior convex face with complicated
dental tubercles, a type with five cusps, the coronillae (fig. 12).
On the outer side of the grappler are two soft, slightly fimbriated
expansions, apparently outgrowths of the walls of the pouch,
210 W. HAROLD LEIGH-SHARPE
11
12
man.
Fig. 11 Callorhynchus antarcticus, the right anterior clasper in two posi-
tions, the pouch having been dissected away. Gr., grappler; Gl., gland; D.,
gland duct; O., orifice of gland duct; Fr., frills; Cor., coronillae.
Fig. 12 Callorhynchus antarcticus, a single coronilla from the grappler.
C., cusps; P. C., pulp cavity.
SEXUAL CHARACTERS—ELASMOBRANCH FISHES 211
the frills, whose function is problematical. Associated with the
grappler on its inner side is a globular, compact gland, with a
completely closed duct, opening by a pronounced and well-
defined orifice at the end of a short tube. As the whole clasper
organization protrudes from the pouch, the gland orifice emerges
after the grappler.
Since the gland here is associated with the anterior clasper, and
none with the posterior, it is conceivable that this is morphologi-
cally the same gland shifted forwards, having taken its duct
with it. Taking into consideration its absence in Chimaera and
its conjunction with surrounding parts, I do not think this is
likely, but that it is of independent origin. ‘The gland does
not at all resemble those of the Batoidei, as indeed there is no
reason why it should, being globular instead of elongated, and
completely surrounding its containing sac leaving a lumen which
is very small, instead of being confined to one side of it.
Histologically (fig. 13), the gland resembles that of Lamna in
being composed of compact masses of tissue, not penetrated by
ducts, and separated from each other by partitions of connective
tissue. The individual cells, however, are different. Instead
of being elongated, they are spherical, as shown in the inset,
with spherical central nuclei.
In the female anterior claspers are not present, but there is a
similar and similarly situated pouch to that of the male. The
pouch, the limits of whose large cavity are indicated by a dotted
line in figure 14, is entered by a small sharply defined orifice,
posterior to which is a directive or guiding groove, sloping down
to the orifice. The material is not in a good enough state of
preservation to determine the presence and nature of a gland or
body at the bottom, or dorsal aspect, of the female pouch, but
_I am inclined to think one is present as a spongy mass. Con-
sideration of the organs in both sexes leads to the belief that the
narrow tubular duct of the gland of the male, with its strikingly
penis-like extremity, is introduced into the pouch of the female,
otherwise we should find the pouch of the female wide open,
revealing the extremities of the large cavity, instead of guarded
by a narrow orifice, just large enough to admit and hold the penis
22 W. HAROLD LEIGH-SHARPE
12
Ie
ee
14
cm
Fig. 13 Callorhynchus antarcticus, a transverse section of the gland of the
anterior clasper (haemalum-eosin). Gl., gland; Z., lumen; M., muscle.
Fig. 14 Callorhynchus antarcticus, female. Po., pouch, the limits of whose
internal boundaries are indicated by a dotted line; O., orifice of pouch; d.g.,
directive groove.
SEXUAL CHARACTERS—ELASMOBRANCH FISHES 213
of the male, with the directive groove to guide the latter into
it. Possibly the spongy mass at the base of the female pouch,
will, in the future, be revealed not as a gland for secretion, but
as a body for the absorption of the secretion of the gland of the
male whose function might be hedonistic.
The whole of the closed portion of the posterior clasper tube
in the male and the cavity of its proximal end is filled with
decomposed débris. Possibly this is what Schneider pronounced
to be spermatozoa (Memoir I, p. 265) as there is no reason why
it should not be, but it is not stored in what I am terming a
siphon in these papers, since Callorhynchus does not possess one.
Save that it is stouter and curved in a different manner, the
frontal clasper closely resembles that of Chimaera (fig. 8, B).
It is limited to the male sex.
The material, which is nearly fifty years old, is not in a good
enough state of preservation to make out the following points,
which will have to be supplemented in a future memoir from
fresh material. 1).The nature of the cavity and its walls in the
proximal end of the posterior claspers in the male. 2) The
nature of the walls of the pouch of the female. 3) The presence
of a body and its nature in the pouch of the female.
TORPEDO MARMORATA
The electric ray
Notes. In none of the Batoidei that follow in this memoir is the siphon tube
carried down the clasper to open by an aperture posterior to the hypopyle as it
does in Raia circularis (Memoir I, p. 260).
The largest specimen, from which figure 15 is drawn, was
captured at Algoa Bay in 1891, and measures 43 cm. in length.
This study has also been supplemented from other animals from
Madras.
The claspers are singularly blunt and clumsy, and terminate in
a double spoon-shaped conformation, suggestive of a burrowing
bivalve mollusc, e.g., Solen. On their outer edge is a long slit
whose cavity does not lead forward and only slightly backward.
This is neither a pseudosiphon, which has been defined for Galeus
214 W. HAROLD LEIGH-SHARPE
and Mustelus, nor a pera, which has been defined for Mustelus in
Memoir II. On their inner edge, near the tip, is a slot whose
cavity leads neither forwards nor backwards.
The glands of the Batoidei are, as far as this investigation has
gone, of the same type. This has already been described in
S11.
7 . Sle. ‘cm.
Fig. 15 Torpedo marmorata. Cl. Gl., clasper gland; A., apopyle; H., hypo-
pyle; SI. 1., slot; SI. 2., slit.
detail for Raia circularis, q. v., Memoir I, pp. 260 to 262. Briefly,
the gland is an elongated bilobed body, with a longitudinal
groove, containing a single row of papillae; superficially it
resembles a date-stone and is confined to the dorsal side of the
siphon sac. The duct of the siphon is thus also the duct of the
gland, and debouches in this and the following genera at th,
SEXUAL CHARACTERS—ELASMOBRANCH FISHES 215
apopyle. The gland becomes more and more twisted out of
one plane in each succeeding genus. A transverse section
(fig. 16) reveals, in each case, that its histological structure is the
same as in Raia. Owing to their larger size, the glands only are
shown in section in this memoir; the relation of the gland to the
siphon sac is seen in Memoir I, figures 10 and 11.
5
aD,
W
se
3
i
Fy OX
poo
BOD
PES o
we,
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— St y
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mm. S35
Fig. 16 Torpedo marmorata, a transverse section of the clasper gland (hae-
malum-eosin).
TRYGON PASTINACA
The sting-ray
The specimen considered (fig. 17) was taken in the Bay of
Biscay in March, 1892. It exemplifies the two-spined stage,
and measures 69 cm. in length, and 40 cm. at the greatest breadth
across the pectoral fins. The flesh of the inner edge of the clasper
is drawn up into a pronounced ridge-like fold, the pent. The
hypopyle is very close to the apopyle, and there the clasper-gland
duct debouches. ‘The rhipidion is pronounced, and is hard and
shell-like with an edge like a knife-blade, so that, in spite of an
epidermis over it, it is easy to cut the finger on it. The clasper
gland is as in Torpedo (fig. 18).
216 W. HAROLD LEIGH-SHARPE
Oo
mn}.
Fig. 17 Trygon pastinaca. Cl. Gl., clasper gland; H., hypopyle; Pe., pent;
Rh., rhipidion.
Fig. 18 Trygon pastinaca, a transverse section of the clasper gland (hae-
malum-eosin). P., papilla.
SEXUAL CHARACTERS—ELASMOBRANCH FISHES 217
MYLIOBATIS AQUILA
The eagle-ray
The specimen investigated (fig. 19), captured at Madeira,
measures 75 cm. in length and 46 cm. at the greatest breadth
across the pectoral fins. ‘The flesh of the inner edge of the clasper
Fig. 19 Myliobatis aquila. Cl. Gl., clasper gland; H., hypopyle; Pe., pent;
Rh., rhipidion.
is drawn up into a ridge-like fold, the pent, not so pronounced as
in Trygon. The clasper is stouter, but otherwise resembles
Trygon more closely than any other two genera approximate to
each other. The rhipidion is situated more anteriorly, but
resembles that of Trygon in being hard and shell-like with a
knife-blade edge. The clasper gland is as in Torpedo (fig. 20).
218 W. HAROLD LEIGH-SHARPE
RHINOBATUS PRODUCTUS
The long-nosed skate
This specimen (fig. 21), taken at San Diego, California, in
May, 1891, measures 85 cm. in length. The first point to strike
the observer is the extreme similitude of this species to its fossil
forerunner, Rhinobatus intermedius (Memoir II, p. 197).
The long, thin, delicate, tapering claspers with a spathe-like
expansion at the extremity are unmistakable, easily distinguish-
ing them from those of the foregoing genera. The closed portion
4
Fig. 20 Myliobatis aquila, a transverse section of the clasper gland (hae-
malum-eosin).
of the clasper tube is long, and towards the outer edge of the
dorsal aspect of the clasper is a slit, asin Torpedo. The claspers
are denticled all over, which is unusual in a skate-like form. The
rhipidion is narrow and elongated, and not in the form of a
fan; it is entirely concealed, unless the edge of the clasper tube
is rolled back, as in the lower inset. At the place where the
siphon tube debouches in Raia circularis is a well-marked sen-
tina, into which there is no opening from the interior. The
sentina is covered by an expansion resembling the web between
a frog’s toes, and on its outer side is a non-articulate immovable
claw which does not resemble that of Acanthias. When the web
SEXUAL CHARACTERS—ELASMOBRANCH FISHES 219
is contracted this is not seen, but by a muscular movement the
web can be expanded as in the lower inset, in which case the claw
protrudes, and serves to prevent elision of the clasper from the
oviduct.
16
cm.
Fig. 21 Rhinobatus productus. Cl. Gl., clasper gland; S., siphon; S. Ap.,
aperture of siphon tube; A., apopyle; H., hypopyle; P. F., pelvic fin; Cl. T.
clasper tube; SJ., slit; RA., rhipidion; Sn., sentina; Cl., claw.
The clasper gland, while conforming to the general type as in
Raia, Torpedo, etc., is the one which presents the most minor
differences. It is elongated and extremely narrow, and more
twisted out of one plane than the others. So small is its diameter
and so small the gland components that it is possible to cut
through two papillae in a transverse section (fig. 22). The origin
of the groove, which ultimately becomes the siphon tube, is
unusually far forward in position.
220 W. HAROLD LEIGH-SHARPE
Rhinobatus appears to be a more primitive skate-like form for
the following reasons: 1) The claspers are denticled all over.
2) The rhipidion is not well developed, and is not in the form of
afan. 3) The clasper gland is very small in comparison with the
size of the animal. 4) A similar fossil species dates back to
cretaceous times.
On the other hand, it is specialized in the following points:
1) The web expansion over the sentina with, 2) a claw.
Fig. 22. Rhinobatus productus, a transverse section of the clasper gland,
(haemalum-eosin). Ci., one component of the gland; C2, another component;
P1., papilla of the first component; P2, papilla of the other component; M.,
stripe muscle; S. S., siphon sac.
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Resumen por el autor, William H. Leigh-Sharpe.
Morfologia comparada de los caracteres sexuales secundarios
de los Holocéfalos y peces elasmobranquios—los 6rganos
copuladores, sus sifones y gl4ndulas. Memoria V.
Notidanus posee 6rganos copuladores primitivos en forma de
hoja, contenidos en una vaina formada por la aleta pélvica.
Posee una cavidad peculiar, la cual lo mismo que la de los Holocé-
falos, puede representar un estado en el proceso de formacién de
un sif6n. Los Spinacidae poseen espinas en los 6rganos copula-
dores (las cuales tal vez falten en los jévenes), mientras que el
sifon con paredes musculares de Spinax es tal vez el mas tfipico
hallado hasta el presente. Cestracion carece de sif6n; Pristio-
phorus posee un sif6n grande y sacular. Ademas de estos carac-
teres hay notables semejanzas en los 6érganos copuladores de
Cestracion y Pristiophorus, pero el primero posee un gancho
semejante a una aguja de crochet, donde el ultimo presenta a
modo de una espuela. Rhinochimaera difiere de Chimaera por
carecer de organos copuladores bifureados y por poseer ganchitos
dérmicos semejantes a los del fésil Squaloraja. Las dos especies ©
de Chimaera descritas en el presente trabajo difieren considerable-
mente por sus 6rganos copuladores de la Chimaera monstrosa
descrita en la Memoria IV. Las dos especies de Raia descritas
son de tipo muy diferente al de R. circularis, y sus 6rganos
copuladores son en extremo complicados. La inervacién del
érgano copulador y su saco y glandula son objeto de descripcién
en el presente trabajo, con los resultados de una serie de expe-
rimentos sobre la estimulaci6n nerviosa.
Translation by José F. Nonidez
Cornell Medical College, New York
AUTHOR’S ABSTRACT OF THIS PAPER ISSUED
BY THE BIBLIOGRAPHIC SERVICE, FEBRUARY 27
THE COMPARATIVE MORPHOLOGY OF THE SECOND-
ARY SEXUAL CHARACTERS OF HOLOCEPHALI
AND ELASMOBRANCH FISHES
THE CLASPERS, CLASPER SIPHONS, AND CLASPER GLANDS
MEMOIR V
W. HAROLD LEIGH-SHARPE
London, England
NINETEEN FIGURES!
The preceding memoirs appeared in the Journal of Morphology
as follows: Memoir I, volume 34, page 245, 1920, Scyllium catu-
lus, 8. canicula, Acanthias, Raia circularis; Memoir II, volume 35
page 359, 1921, Galeus, Mustelus, Lamna, Rhina; Memoir III,
volume 36, page 191, 1922; Fossil forms; Memoir IV, volume
36, page 199, 1922, Chiloscyllium, Pristiurus, Chimaera, Callo-
rhynchus, Torpedo, Trygon, Myliobatis, Rhinobatus. The present
paper deals with certain species to which I have kindly been
allowed access at the Natural History branch of the British
Museum, South Kensington, London, viz.:
PMR MMTAINIS | PEISCUIS 3 2 Se Sees ahs re cee ches ae ere winsas Btls Se eae ES 222
BeUNRIPCE Ce Ot ieee cas soe a pale urdanad ota < 4 eee rae) eR 225
Mer LTOMUOLUS ssi bAMICMS 02s 2is cele assis anion ken ca wee oad sell ack 227
eat SELNIS APIO BUN orate es eid Bo ae ee ewe clee ne vend 228
Wes racroneplilippines: se Aes okie eee ee eRe Ae nee 229
ASG TMS AGIED AUS oe 08 Poke aue Woy er apres ak Foie Suid ekcih ood teat 230
Pa OCRIMITON eb ACD: fon cca e ac he Cente ates ee ee Nite 232
(Lee 2 Wace) aA Ea Ree SES Se eo Oe aa 234
SeANITSRPEO ESD SEMI C AT EGK corse para 9 atayacias SeRamesaps/ ee SSA =. step ag eee as eS 236
Lies CLD Eee ie RR SR ee) 1 tenes ee ae Was ee 236
PPE MUL ANG ae te tare eet eho te Aan ee. Ostet oe 243
1 The figures are specially drawn from the author’s dissections and prepara-
tions by Miss Edith C. Humphreys, to whom best thanks are tendered.
221
Zo2 W. HAROLD LEIGH-SHARPE
NOTIDANUS (HEXANCHUS) GRISEUS
The six-gilled shark
This primitive Protoselachian fish exhibits, as may be supposed,
characters quite different from those of the species previously
considered. I was fortunate enough to be able to investigate a
Oo
cm.
Fig. 1 Notidanus griseus. Sh., prolongation of the pelvic fin forming a
sheath for the clasper; Sh. S., sheath sac; Ph. Cl, phyllaceous clasper.
large specimen measuring 77 inches, or nearly 2 meters, in length,
taken in Japan, in 1905, and from this the figures are drawn. I
have also examined small specimens from Nice, and in these
the claspers are of a bright lemon-yellow color.
The pelvic fins are prolonged posteriorly in such a way as to
form a sheath in which the claspers are effectually concealed,
so that, at first sight, the small specimens may be mistaken for
SEXUAL CHARACTERS—ELASMOBRANCH FISHES 223
females, unless the fins are parted, and, in the large specimen,
the fins may be mistaken for the claspers. The claspers are
obviously in a very primitive condition, and look like a duplica-
tion of the fins, resembling a rolled-up leaf, so that the closed
cm.
Fig. 2 Notidanus griseus. Sh., sheath formed by pelvic fin; Cl., clasper;
A., apopyle; H., hypopyle; S. Ap., aperture of ‘siphon’ tube; E£z., position of the
external sheath sac indicated in dotted outline; Su., sulci; a-a’, b-b’, cartilages
cut through in dissection.
tubular portion is brief. They are not covered with denticles,
and have little or no skeletal support.
Between the outer border of each clasper and its sheathing
fin is an external sac which is not merely a groove formed in the
union of clasper and fin, but a space of considerable length (fig.1).
In figure 2 the position of this sac is indicated in dotted outline.
224 W. HAROLD LEIGH-SHARPE
Though pointing anteriorly, the sheath sac cannot be considered
as homologous with a pseudosiphon.
Leading into the apopyle is a shallow cavity which appears to
be homologous neither with a siphon, nor with the cavity of
Fig. 3 Notidanus griseus. A transverse section through the party wall
between the cavity and the sheath sac, in the position indicated by the dotted
outline in figure 2 (haemalum-eosin). Fp., stratified epithelium; M., muscle.
Fig. 4 Spinax niger. 1, 2, 3, 4, spines; H., hypopyle; S., siphon; Sm., sen-
ticetum; a., its apex.
Chimaera. It offers a homology with the former as to situation,
confirmed by its being a sac with muscular walls capable of
fulfilling the normal functions of a siphon.
A portion of the thin partition between this cavity and the
external sheath sac, from the area bounded by the dotted outline
in figure 2, was removed and sectioned, with the result that it
SEXUAL CHARACTERS——ELASMOBRANCH FISHES 225
reveals (fig. 3) that both cavities are lined with stratified
epithelium.
The apex of the cavity cannot beattained without cutting through
the cartilages a - a’, b - b’ (fig. 2); in this it differs from a siphon,
but resembles the cavity of Chimaera. On either side of the
cavity is a deep sulcus leading down to the aperture of the cavity
at the apopyle.
SPINAX (ETMOPTERUS) NIGER
The black dogfish
This smallest of the elasmobranchs affects deep water. I
have examined specimens from 200 fathoms, from Christiansund;
from 200 fathoms, from the coast of Portugal, and the specimen
from which the figures are drawn was taken in 1904 at Faro,
Algarve, Portugal, from 365 fathoms, and is 26 em. long.
This memoir deals largely with types like Acanthias which
depend upon movable spines rather than upon a rhipidion for
fixative purposes during impregnation. These, in Spinax, are
four in number, set within a senticetum which can be erected,
in which case they take up the positions indicated in figure 4.
One, number 3, points forward, while number 4 differs from the
rest in being flat and blade-like, with a keen edge. The apex (a)
of the senticetum is a tapering flap which projects over the
spines, which, in a position of rest, are, according to Jungersen
(b, Memoir I, p. 265), hidden by a pair of cartilaginous plates
covered by skin.
The claspers are stout, denticled all over, and adnate with the
pelvic fins. There is no rhipidion.
A series of embryos and young forms, taken at Bergen in 1901,
and specially drawn for me by Michael G. L. Perkins at the
University Museum, Cambridge, England, exhibits very clearly
the development of the claspers and their state of coalescence
with the pelvic fins (fig. 5).
The siphon is peculiarly stout, and may be mistaken at first
sight fora gland. Sectioning, however, reveals that its solidarity
is due to the extraordinary thickness of its muscular walls,
which are here developed to an unusual extent (fig. 6). The
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Ep., stratified epithelium
eosin).
226
SEXUAL CHARACTERS—ELASMOBRANCH FISHES PAT
epithelium lining the sac is the best example at present seen of
the stratified variety, comprising four or five rows of cells, with
an occasional mucus-secreting cell, as in Galeus. I should
consider that in this type the siphons are the best adapted to
perform the normal functions assigned to those organs.
6
Oo
cm.
Fig.7 Centrophorus lusitanicus. Cl., clasper; A., apopyle; S., siphon
CENTROPHORUS. LUSITANICUS
The Portuguese arreghonda
This sexually immature specimen, measuring 72 cm., taken on
the coast of Portugal, shows a strong resemblance to Spinax in
the following points (fig. 7):
The claspers are short, adnate with the pelvic fins; but there
are no spines present in this specimen. The apopyle is wide with
a tumid border.
228 W. HAROLD LEIGH-SHARPE
There is no rhipidion. The siphon is short, and similarly
situated to that of Spinax, but, instead of possessing thick walls,
its walls are remarkably thin.
Fig.8 Echinorhinus spinosus. A., apopyle; S., siphon
ECHINORHINUS SPINOSUS
The spiny or bramble shark
This species can only be, at present, described from a specimen
taken at Nice, which though 85 cm. in length is immature.
Figure 8 does no more than indicate a general resemblance to
Centrophorus, the absence of spines and the presence of a similar
siphon.
SEXUAL CHARACTERS—ELASMOBRANCH FISHES 229
CESTRACION (HETERODONTUS) PHILIPPI
The Port Jackson or bull-head shark
An old and badly preserved specimen, taken at New Zealand,
89 cm. in length, was examined.
The claspers are long and stout, with a flexure recalling that of
Mustelus (fig. 9). The apopyle, which is far removed from the
cloaca, leads into a deep pouch, which is continued posteriorly in
Fig. 9 Cestracion philippi. A., position of apopyle; Ha., hamus; Hy.,
hypopyle.
a nearly closed tube with soft edges. A small rhipidion is
present, posterior to which is a blunt, hard, movable hamus,
with a recurved, crotchet-like head.
On the opposite side of the rhipidion to the hamus is a deep
cavity, the crumena, not similar to any pouch previously de-
scribed. There is a small thick-walled siphon present.
Notwithstanding the specialization of the claspers, as regards
the development towards siphon formation, this genus appears
to be one of the most primitive of recent forms, as might perhaps
230 W. HAROLD LEIGH-SHARPE
be expected of a genus surviving from so geologically ancient
times. *
Situated in the clasper, anterior to the apopyle and leading
posteriorly into it, is a blindly ending cavity whose boundaries
are indicated by a dotted line in figure 10, and which is much
more simple than that of Notidanus, and on about the same
developmental level as that of Callorhynchus.
Fig. 10 Cestracion philippi. A., apopyle; Hy., position of hypopyle; Ha.,
hamus; Cr., crumena; Rh., rhipidion; Cav., boundary of cavity of the clasper
indicated in dotted outline; S., siphon.
PRISTIOPHORUS CIRRATUS
The saw-fish shark
A mature specimen of this species, taken at Tasmania in 1885,
was examined, 109 cm. in length, a portion of the anterior end
of the saw having been broken off and not included in this
measurement.
As regard the claspers and their accessory structures, this
animal bears a surprising likeness to Cestracion, although their
SEXUAL CHARACTERS—ELASMOBRANCH FISHES 231
relationship is probably far apart. ‘The apopyle, which is some
distance from the cloaca, leads into a deep pouch, which is con-
tinued posteriorly in a closed tube with soft edges, all as in
Cestracion (figs. 11 and 12). The small rhipidion in a similar
position is so reduced as to be negligible. The hamus is replaced
by a long, straight, thorn-like spur in a similar position. This
Sp.
Sk.
Fig. 11 Pristiophorus cirratus. Sp., spur, enclosed in a sheath of skin
(Sk.); S., siphon.
Fig.12 Pristiophorus cirratus. A., apopyle; H., hypopyle; Sp., spur shown
erected in the upper sketch; S., siphon.
spur can be erected as in figure 12 upper diagram, and is sugges-
tive of that of Acanthias save that it is perfectly straight, and
when at rest lies in a sheath of skin of its own. There is, how-
ever, no crumena. The clasper tube is more perfectly closed,
though still a scroll, and the hypopyle more distinctly defined.
The claspers are stout and have a flexure. .
The greatest difference between Cestracion and Pristiophorus
is that, whereas the former has a small siphon, in the latter it is
JOURNAL OF MORPHOLOGY, VOL. 36, NO. 2
Zao W. HAROLD LEIGH-SHARPE
an enormous bag. The deep pouch into which the apopyle
leads is actually the wide mouth of the posterior end of this bag,
so that no siphon tube is developed.
RHINOCHIMAERA ATLANTICA
It is stated that the males of Harriotta, a deep-sea member
of the Holocephali, which this species closely resembles, have
hitherto only been described from immature specimens. The
following account of Rhinochimaera is made from a mature
specimen, 84 cm. in length, taken off the southwest of Ireland in
1910.
In all its details Rhinochimaera shows a close similarity to
Chimaera monstrosa (Memoir IV, p. 201), except that the clasp-
ers, instead of being bifurcated into external and internal radii
as in Chimaera, are single, resembling the more slender of the
radii of the other genus.
Rhinochimaera agrees with Chimaera in the following points
(fig. 13): The pelvic fins are of the same conformation and cover
over the claspers so as to conceal them. Leading into the apo-
pyle is a cavity similar and similarly situated to that of Chimaera,
which has already been described at considerable length. - An-
terior claspers are present, which can be retracted into a slit-like
pouch, thus the clasper on the (observer’s) left is almost fully
protruded, while that on the right is almost withdrawn. The
clasper is almost entirely composed of cartilage, but instead of
being serrated on its morphological inner border it is beset with
hooklets, like those of the pelvic claspers, which serve the same
function as aserration. The histological details are the same, as
far as I have been able to examine them. The clasper pouch,
however, is set obliquely to the animal’s axis (transverse in
Chimaera, longitudinal in Callorhynchus), and the clasper is
not spoon-shaped.
On account of its prolonged rostrum, inter alia, Harriotta has
already been classified as showing affinities with the fossil Squa-
loraja polyspondyla, rather than with Chimaera; so also should
Rhinochimaera. And, in the organs we are considering, there
is another close resemblance, for the delicate, very flexible,
posterior claspers, instead of being freely denticled, terminate in
SEXUAL CHARACTERS—ELASMOBRANCH FISHES 233
a raspberry-shaped protuberance, grooved on the outer side of
the middle line, owing to the presence of a raised external fleshy
pad, and bearing attenuated, movable, dermal hooklets, as
shown in the inset, strikingly like those of the fossil Squaloraja
16
Pch.
: \
/ \\
l lf " OQ
}] YY a\ M
cm.
D.H.
R.
Fig. 13 Rhinochimaera atlantica. Cl., posterior clasper; Cav., position of
cavity of clasper; Ant. Cl., anterior clasper; Pch., pouch of anterior clasper;
,D. H., dermal hooklets; &., enlarged view of the posterior (distal) end of the
right clasper.
(Memoir III, fig. 3). The hooklets, which point anteriorly, and,
when not erected, lie flat against the protuberance, appear to
be more numerous than in Squaloraja, there being at least six
principal ones on each side of the groove, besides other minute
subsidiary ones which grade into scale-like forms; doubtless,
some are not preserved in the fossil. There is no frontal clasper.
234 W. HAROLD LEIGH-SHARPE
CHIMAERA (HYDROLAGUS) COLLIEI
This small Pacific species, taken near Monterey, California,
measuring 49 cm. in length, differs from Chimaera monstrosa
(Memoir IV, p. 201) in the following particulars: The anterior
5
(A: Ant. Cl.
Fig. 14 Chimaera coiliei. Ezt. R. and Int. R., the external and internal
radii of the posterior clasper. Cl. Gr., clasper groove; A., apopyle; Ant. Cl.,
anterior clasper; Pch., pouch of anterior clasper; Sp., spike; Pg., peg; Cor.,°
corolla of skin, an enlarged view from the posterior aspect of the distal end of the
left external radius.
claspers bear but 3 spines on their morphological inner border
and their pouch is more widely open. The pelvic fins, of similar
shape, do not so completely cover the posterior claspers, which
are markedly different from those of the other species (fig. 14).
Both the external and the internal radii are grooved, so that,
SEXUAL CHARACTERS—ELASMOBRANCH FISHES 255
when the radii are apposed, as indicated on the (observer’s) left,
a temporarily closed passage is formed. The denticles are
larger and differently situated, surrounding the posterior half of
each radius. Posterior to, and unconnected with the hypopyle,
Ant. Cl:
6
3
|
vy
Ext RZ ; O
Int.R. ClLGr cm.
Fig. 15 Chimaera mirabilis. Hat. R. and Int. R., the external and internal
radii of the posterior clasper; Cl. Gr., clasper groove; A., apopyle; Ant. Cl., ante-
rior clasper; Pch., pouch of anterior clasper.
the internal radius ends in a cartilaginous spike, and the external
radius in a peg, having a hole in its center from which all tissue,
if any, has disappeared, and surrounded by a foxglove-like
corolla of skin, as shown enlarged in the inset. The proximal
curvature of the external radius is very striking. Leading into
the apopyle is a very minute cavity, entirely embedded in carti-
lage, whose nature could only be investigated by sectioning. The
frontal clasper is huge in proportion to the size of the animal
and the spikes on it larger.
236 W. HAROLD LEIGH-SHARPE
CHIMAERA MIRABILIS
A male and a female of this small species, each measuring 77
cm., were examined from the west coast of Ireland. They rival
Chimaera monstrosa in length on account of their extremely
long tails. They differ from that species in the following par-
ticulars: The anterior claspers have five spines on their mor-
phological inner border; their pouch is more widely open and set
at a slightly oblique inclination to the animal’s axis. The
posterior claspers are small and their division into an external
and an internal radius does not take place until half way down
their length. They are slightly grooved, and approximation of
the radii forms a narrow tube as in Chimaera colliei, but the
denticles are but small. The apopyle is half way along the in-
ternal radius and no cavity leads into it (fig. 15). There are
neither anterior claspers nor their pouch present in the female.
The frontal clasper of the male is small.
RAIA
TYPE: RAIA CLAVATA
The thornback
How far various species of the same genus differ from one
another must form the subject of a future memoir; meanwhile,
no two genera could present more differences than the two types
of Raia here discussed: R. circularis (Memoir I, p. 260) and R.
clavata. Unfortunately, erection is not the same in the two
cases, for, while in the former it consists in the diametrical expan-
sion of a soft clasper by a suffusion of blood, here it is effected
by the unfolding of the clasper edges and the protrusion of
complicated structures by muscular contraction, from the
position of rest shown in figure 19A to the condition shown in
figure 16. The clasper gland, though similar in structure, is
situated more posteriorly in the lobe of the pelvic fin, and its
duct, really that of the containing sac, is not carried down the
clasper as a closed tube to open posterior to the hypopyle, as in
R. circularis, but debouches at the apopyle, as in Torpedo, Try-
gon, etc. (Memoir IV). A deep sentina is present as in Rhino-
SEXUAL CHARACTERS—ELASMOBRANCH FISHES 237
batus (Memoir IV, p. 218), while its inner border is raised into a
prolonged spike having a strong cartilaginous support to prop
the oviduct open. The rhipidion is elongated and not in: the
CUGE
cm.
Fig. 16 Raia clavata. Cl. Gl., clasper gland; A., apopyle; H.. hypopyle;
Rh., rhipidion; Sqg., signal; Sn., sentinel; Ps., pseudosiphon; Sp., spike; St.,
sentina,
form of a fan; owing to its being twisted out of one plane, its
function is very effectively performed. Posterior to the hypo-
pyle, at the level of the commencement of the rhipidion, is a
soft, fleshy pad, the signal, resembling the ‘foot’ of a bivalve
mollusc, which, during erection, turns laterally through an
238 W. HAROLD LEIGH-SHARPE
angle of 180°, and whose apex, from pointing inwards, becomes
directed outwards, also propping the oviduct open, and dis-
tending it. Posterior to the signal, but remaining on the oppo-
site face, is a knife-blade-like structure, the sentinel, so keen is
whose edge, that, in spite of an epidermis over it, I repeatedly
cut my finger on it. The oviduct is thus distended and stimu-
lated in three directions. A pseudosiphon is present on the
inner border of the concavity, serving partly as a sheath for the
sentinel, when not in the erect condition. A shield is present,
not so well developed as in R. blanda, and covered by the signal
in figure 16.
The greater part of the year 1920 was spent in the electrical
stimulation of pithed skate. Various species were used, in-
cluding both R. circularis and R. clavata, but the results in the
latter case were more conclusive owing to less difficulty in iso-
lating the nerve twigs.
Previous practice in dissection revealed (fig. 17) that the
siphon (S) and its contained gland are innervated by a posterior
branch of the 52nd? spinal nerve, and that the clasper is inner-
vated by a posterior branch of the 54th spinal nerve.
The animal figured is 84 cm. long. The spinal cord was severed
just behind the medulla and the brain destroyed; it was also
necessary to divide the cord again just anterior to the 50th
spinal nerve to prevent any electrical reflux agitating the pec-
toral fins. The results point to the conclusion that the sugges-
tions tentatively put forward in Memoir II, page 371, are not
tenable for this species. This does not directly prove any
point, since erection is of a different type in the two species, and
the circumstance that the claspers of R. circularis taken im-
mediately after copulation were both in a state of erection may
well point to the fact that that phenomenon is there due to a
chemical stimulus.
The results of electrical stimulation were as follows:
1. Stimulation of the posterior branch of the 52nd _ spinal
nerve causes, first, secretion from some or nearly all of the
papillae of the gland followed by a slow undulating contraction of
2 The basis of this count is that of fish having thirteen spinal nerves in the
anterior group of the brachial plexus.
SEXUAL CHARACTERS—ELASMOBRANCH FISHES 239
RAIA CLAVATA.
Figure 17
240 W. HAROLD LEIGH-SHARPE
the containing siphon sac (s) in-an anteroposterior direction, but
no erection of the clasper.
2. A series of successive stimuli to the posterior branch of the
52nd spinal nerve causes prolonged spasmodic secretion, and the
sudden activation of secretion from hitherto dormant papillae,
but no contraction. During sustained stimulation contraction
appears to be inhibited. On the cessation of stimulation a
similar undulating contraction of the siphon sac follows. After
prolonged stimulation there may be from two to as many as six
such contractions following one another at gradually lengthen-
ing periods.
3. Stimulation of the posterior branch of the 54th spinal nerve
causes erection of the clasper which continues for fifty seconds,
the clasper continuing to remain erect during, and for some
time after, a series of successive stimuli, but no secretion or sac
contraction.
4. If the posterior branch of the 52nd spinal nerve be severed,
and the common portion of this spinal nerve be stimulated,
movements of the pelvic fin follow, but no secretion or sac
contraction.
5. If the spinal cord be stimulated between the origin of the
52nd and 54th spinal nerves (and, indeed, at other places, in which
cases other responses occur), secretion, followed by sae contrac-
tion, and clasper erection supervene simultaneously; wherefore,
in order to obtain perfect results in cases 1 and 3, it is better to
sever the spinal cord between the origin of the 52nd and 54th
spinal nerves.
6. Besides being distributed to the accessory structures of the
clasper, such as the signal, etc., the posterior branch of the 54th
spinal nerve also gives off prominent branches to the muscles
that work the whole clasper, such as the one indicated at, N
(fig. 17). If these branches are not cut, stimulation of the whole
posterior branch causes not only erection, in the sense of that
word as previously used, but also flexion of the clasper in an
anterior direction, exactly as would be required in copulation.
If these secondary branches be severed, stimulation of the main
branch still causes erection, but in not so complete and perfect a
SEXUAL CHARACTERS—ELASMOBRANCH FISHES 241
manner as when the other branches are left intact, so that antero-
flexion of the clasper is brought about. In fine, the antero-
flexion helps to originate the erection and is normally part of it,
though, on nerve severance, erection alone can still. be induced
by stimulation.
Secretion of the clasper gland takes the form of the protrusion
of long, glairy, transparent strings of substance from the papillae,
suggestive of the squeezing out of an artist’s oil colors from their
Fig. 18 Raia clavata. A diagrammatic representation of the secretion of a
single string of albumin from a papilla of the clasper gland.
containing tubes. For the sake of simplicity, only one such string
is Shown diagrammatically in figure 18. This substance exhibits
all the chemical and physical properties of albumin; hence the
clasper gland must in the future be considered an albuminous
gland. Long strings of albumin in an undisintegrated state were
found lying in the siphon sacs of fresh skates dissected for
practice which had died a ‘natural’ death, without resort to
electrical or other stimulation. The secretion of many papillae
at once is very striking to observe. Since all do not secrete
242 W. HAROLD LEIGH-SHARPE
together, but here and there two or three papillae are passive, I
gather that all the gland components are not in a state of
activity simultaneously. I regret I have not obtained such
results as yet in R. circularis.
cim.
Fig. 19 Raia blanda, the right clasper in, A., the normal position, B., the
erect position. A., apopyle; H., hypopyle; Rh., rhipidion; Ps., pseudosiphon;
Sg., signal; Sh., shield; Sn., sentinel; Sp., spike; St., sentina.
I have not yet met with skates in copula, but a reliable au-
thority informs me that in these larger species only one clasper
is inserted at a time.
The specimens of R. clavata were from Weymouth, Plymouth,
and other parts of the English Channel.
SEXUAL CHARACTERS—ELASMOBRANCH FISHES 243
RAIA BLANDA
The blonde ray
This skate is of the same type as R. clavata, and all the
accessory structures of the clasper are similar, save that the
spike is smaller and the shield parallel with and covering and
protecting the signal, and of the same hard constituency as the
sentinel, is larger, as is also the signal (fig. 19). The clasper
gland is as in R. clavata.
Resumen por el autor, Walter N. Hess.
‘Origen y desarrollo de los 6rganos luminosos de Photurus
pennsylvanica De Geer.
El primer indicio de la formacién de los 6rganos luminosos de
la larva en el embrién se manifiesta a la edad de quince dias.
En este momento un grupo de células adiposas, con sus grandes
glébulos de grasa que se tifien en negro por el acido ésmico,
emigran ventralmente en el segmento octavo y vienen a situarse
en la regién ocupada por los futuros érganos luminosos de la
larva. Los 6érganos luminosos del adulto comienzan a desarro-
llarse unas pocas horas antes de la formacién de la ninfa, en cuyo
momento un gran nimero de esferas grasosas, en el sexto y
séptimo segmento abdominal, se desintegran y las células adiposas
asi liberadas, con sus glébulos grasosos tefiidos en negro, emigran
a la regién de los futuros 6rganos luminosos del adulto.
Durante unos dias, tanto en el embrién como en la ninfa, no
existe diferencia perceptible alguna en ninguna de las células del
érgano luminoso, pero finalmente tiene lugar una diferenciacién
en ellas, transformandose en las capas reflectora y fotogénica.
Aun después de ser perceptibles ambas capas, los glébulos de
grasa coloreados en oscuro pueden a menudo observarse en
sus células. La presencia de glébulos grasos oscuros en todas
las células de los 6rganos luminosos en vias de desarrollo, lo mismo
en la larva que en la ninfa, junto con el método de formarse
estas células, conduce al autor a la conclusién de que los organos
luminosos de Photurus pennsylvanica son de origen enteramente
mesodérmico. Los 6rganos luminosos larvarios cesan de
funcionar durante el segundo dia de la vida adulta, a cuyo tiempo
son fagocitados.
Translation by José F. Nonidez
Cornell Medical College, New York
AUTHOR’S ABSTRACT OF THIS PAPER ISSUED
BY THE BIBLIOGRAPHIC SERVICE, FEBRUARY 20
ORIGIN AND DEVELOPMENT OF THE LIGHT-ORGANS
OF PHOTURUS PENNSYLVANICA DE GEER
WALTER N. HESS
Department of Entomology, Cornell University
FIVE PLATES (SEVENTEEN FIGURES)
CONTENTS
MNMALT RE LUIRIE reece A crate Sma. te ae Pe seen ik Fae Ns = AE NERS OHSS area es Sym Deniers 245
EAN TRCLIOUS. SEN Stet: uN, Sa Bee «a ale th OP ene ere Peek eee vet 246
History of embryological work on the light-organs of insects............... 247
Location and structure of the light-organs..................ccneccceecceces 250
Origin and development of the-larval light-organs...................-.000- 251
Origin and development of the adult light-organs............... 2... eee eee 254
Phagocytosis of the larval light-organs: 3: 2.0. /sc06 0 des ba leak nek 262
SUTTER Wy REN Reo Arama cite toy) 5 tries Acted hn ci icptuc co aiaionay ever hra sire lacsysayeahi es 263
Smee ER er ere ee ats cS aes ce Aas chccais pass nis SiSES Sie Cian d «ap eee ads 265
INTRODUCTION
In spite of the fact that the light-organs of fireflies have been
the object of much study during the past century, comparatively
little has been done on their development. During the past
few years, however, this phase of the subject has received more
attention, though as yet the question of their origin is not
settled.
Shortly after the publication of the two brief conflicting
reports, regarding the origin and development of the light-
organs in insects, by Vogel (713) and Dubois (’13), the author
undertook a study of the development of these organs. Dur-
ing the winter of 1916 to 1917 there appeared two more articles
on the origin of these organs (Williams, ’16, and Dahlgren,
17), but as these contradicted one another, the question was
considered still unsettled. At that time the present study,
which had been largely devoted to the development of the
light-organs in the embryo, was interrupted by the war, so a
brief preliminary report was published. Upon resuming this
245
246 WALTER N. HESS
work during the past year, a careful study was made of the
development of the light-organs in both the embryo and the
pupa. This has resulted in the modification of the tentative
views expressed in the preliminary report.
The author is indebted, especially, to Dr. William A. Riley,
under whose supervision the greater part of this study was
made, also to Dr. O. A. Johannsen for his helpful suggestions and
criticisms.
MATERIALS AND METHODS
Eggs were obtained, for a study of the embryonic develop-
ment of the light-organs, by confining ripe females in jars that
had been partly filled with earth and moss. Since oviposition
occurred very readily in captivity, it was easy to obtain a com-
plete series of eggs by removing the insects to different jars
each day.
The different larval and pupal stages were obtained by col-
lecting second-year larvae in April and confining them in Jars
with earth and moss. By observing them each day, desired
stages could be selected.
Two complete sets of eggs, late larvae, and pupae were ob-
tained as described above. The eggs were collected at intervals
of twenty-four hours, from the time of oviposition until hatching.
The pupae were collected at twenty-four-hour intervals from the
time of pupation until emergence. Several stages of late larvae
were obtained previous to pupation. One set of this material
for histological purposes was fixed by heating in water at a
temperature of 80°C. for four minutes, after which it was trans-
ferred to alcohol. These eggs were punctured with a fine needle
immediately after heating, in order to insure good preservation.
The chorion of each egg was later dissected off to expose the
embryos. The other set of material was fixed in Flemming’s
fluid (strong formula). These eggs were punctured and allowed
to remain for twelve hours in the fixer, after which the chorions
were removed and the eggs were returned to the fixer for another
twelve hours. The first twelve hours of fixation was sufficient
to harden the embryos so that their chorions could be dissected
off, while the last twelve hours insured. good fixation. In the
DEVELOPMENT OF LIGHT-ORGANS 247
case of the larvae and pupae, the posterior four segments were
removed by scissors and placed in the fixers.
The material was all imbedded in paraffin. In the case of
the embryos, a binocular microscope was used to insure proper
orientation for sectioning. Sections of the larvae and pupae
were cut from 6 to 10y in thickness, while those of the embryos
were cut 3 and 4y thick. MHeidenhain’s iron hematoxylin was
used for staining all sections.
HISTORY OF EMBRYOLOGICAL WORK ON THE LIGHT-ORGANS OF
INSECTS
There are, in general, three conflicting views regarding the
origin of the light-organs in insects. One view is that they are
derived by a proliferation of the hypodermis and hence are ecto-
dermal; another, that they are formed from both ectoderm and
mesoderm and, lastly, that they are formed from fat-cells and
hence are of mesodermal origin.
Two views were suggested by early workers concerning the
possible ectodermal origin of these organs. The one of these
that is least generally accepted was advanced by von KoOlliker
(57) and Lindemann (’63), who maintained that the structures
are nervous in origin. Von Ko6lliker compared these organs
with the electric organs in fishes, which, together with the fact
that the light-organs of insects are under the control of the
nervous system, led him to conclude that they are of nervous
origin. Lindemann considered the organ a definite part of the
nervous system. The other of these views was supported by
Owsjannikow (’68), Heinemann (’86), Dubois (98, 713), and
Marchal (711), who upheld the idea that these organs arise from
the hypodermis by a proliferation of its cells. Owsjannikow
considered the organ in the nature of a gland and hence ecto-
dermal in origin. Heinemann, who worked on the light-organs
of the elaterid beetles, considered that those organs, as well as
the light-organs in the Lampyridae, were derived from the hy-
podermis. Marchal was also of the opinion that they are formed
from ectoderm. The observations of all these workers, unless
it is Dubois, are of little value, as they studied only the adult
organs.
JOURNAL OF MORPHOLOGY, VOL. 36, NO. 2
248 WALTER N. HESS
The question of the origin of these organs can be settled only
by a study of the development of the photogenic tissues. With
the exception of three recent papers, Vogel (’13), Williams (716),
and Dahlgren (’17), this has not been done by any one except
Dubois. Dubois (’98) studied the embryology of both Lampyris
noctiluea and Pyrophorus noctiluca. He believed that he was
able to follow the development of the photogenic organs through
the different stages, from the beginning of segmentation to the
adult insect. He discovered a close histological resemblance
between the blastoderm cells and the photogenic cells of the larva,
pupa, and adult. Furthermore, he concluded that the cells of
the hypodermis multiplied, and by proliferation formed directly
the photogenic organs of the larva. For some reason Dubois’
work has attracted little attention, and is practically ignored
in general discussions of the subject. Moreover, he misinter-
preted the normal structure of the body wall in Lampyris, and
for this reason his conclusions have been severely criticised by
the few who have discussed them.
Wheeler and Williams (’15), in their study of a mycetophilid
fly of the New Zealand caves, found that the light-organs were
a modified portion of the malpighian tubules. These structures,
as is well known, are of ectodermal origin, and this furnishes a
clear instance of light-organs from the ectoderm in insects.
Dahlgren (’17) studied the development of the adult light-
organs in the pupa of Photurus pennsylvanica. He states that
the light-organs are formed by a proliferation of cells from the
ventral abdominal hypodermis, which multiply and later differ-
entiate into the two layers of the light-organs. He does, however,
express some doubt as to the possible origin of the reflector layer.
Two different workers upheld the view that the light-organs
arise from both ectoderm and mesoderm. Gegenbauer (’74)
advanced the idea that the non-luminous, or reflector, layer of
the light-organs is derived from the fat-body, while the photo-
genic layer is formed from the hypodermis. Wielowiejski (90),
in opposition to Dubois, states that the so-called reflective, or
urate, layer of the photogenic organs is derived from fat-cells,
while the light-giving, or luminous layer, on the contrary, is
DEVELOPMENT OF LIGHT-ORGANS 249
composed of cells derived from oenocytes, and is, therefore,
ectodermal. Here again the conclusions were based on a study of
the mature organs, and hence are not conclusive.
With the exception of the two previously mentioned papers
by Vogel and Williams, all authors who favor the theory of fat-
cell origin have based their conclusions only on a study of the
adult organs. Many of these, including Peters (’41), Leydig
(57), Schultz (65), Emery (84), Seaman (’91), Wheeler (’92),
Bongardt (’03), and Berlese (’09), seem to be of this opinion,
largely because the structure of the mature organ resembles
somewhat that of fat-cells, and also because certain cells of the
photogenic organs secrete urates and other products, comparable
with fat-cells.
Recently two important papers have appeared, which were
based upon a definite study of the developmental tissues; one
by Vogel (713) and the other by Williams (’16). Vogel studied
the embryonic development of this organ in Lampyris noctiluca,
beginning with a stage in which the organ was already clearly
differentiated. He made a comparable histological study of
its cells with those of the neighboring tissues, from which he
concluded that the cells of the photogenic organ, in this stage,
agree with the neighboring fat-cells in shape, size, and relations
of their nuclei. On this he based his conclusions that the light-
organs arise from fat-cells.
Williams (716) worked upon our native species, Photurus
pennsylvanica and Photinus consanguineus, and apparently
confirmed the work of Vogel. Although he studied the develop-
ment of both the larval light-organs and the light-organs of the
adult, his observations were devoted almost entirely to the adult
light-organs. In the case of the adult he found that the fat-
spheres broke down, liberating their cells, and that these became
distributed along the hypodermis in the region where the light-
organs were to form. He also found that the cells of the early
organs formed a gradual gradation from the rather dark pig-
mented cells lying against the hypodermis, to those on the side
towards the fat-cells and continuous with them.
250 WALTER N. HESS
Buchner (’14) and Pierantoni (’14) both consider the light-
organs a symbiotic structure in which there are found luminous
bacteria, or fungi, that cause the light. Buchner showed a very
close similarity between the granules in the photogenic layer of
the light-organs and the symbiotic bacteria of the homopteron,
Aphrophora spumaria.
LOCATION AND STRUCTURE OF THE LIGHT-ORGANS
The light-organs of all the luminous Lampyridae were found
lying next to the sternal side, in the eighth abdominal segments
of larvae, and in one or both of the sixth and seventh abdominal
segments of the adults. In the larvae the organs appear as two
small elliptical discs (fig. 1). In an adult male of Photurus
pennsylvanica the light-organs cover the entire sternites of
the sixth and seventh abdominal segments (fig. 2), while in the
female of this species the organs occupy only about two-thirds
of the corresponding sternites (fig. 3). During the first one or
two days of adult life, the larval light-organs can be seen to emit
light from the eighth abdominal segment (fig. 3). The phagocy-
tosis of these organs however, is usually so far advanced by the
end of the second day of adult life that they no longer emit >
light.
In the species studied, the arrangement of these organs, in
the larva, is shown diagramatically by figure 4. The photo-
genic layer (P) lies next to the ventrolateral sternite of the eighth
abdominal segment. In the adult male (fig. 5) the photogenic
layer (P) extends entirely across the sternite next to the hypo-
dermis, while the reflector layer (R) completely covers this layer
on its dorsal side in both the larva and the adult.
The mature light-organs whether in the larva (fig. 9) or in
the adult (fig. 17), are of the same general structure. Both
are composed of two layers of cells, the inner reflector layer
(R) and the outer photogenic, or luminous layer (P). The former
is composed of fairly regular polygonal cells in which are located
a large quantity of crystals of urate salts. This layer in life is
opaque and chalky in appearance. The ventral layer is com-
posed of two parts: the tracheal structures (7’) and the photo-
DEVELOPMENT OF LIGHT-ORGANS 25
genic cells (P). The photogenic cells, whose walls are often
indistinct, contain numerous granules of a non-urate composi-
tion, called photogenic granules. Tracheae penetrate both
layers of the light-organs, and in the region of the photogenic
layer they are profusely branched. ‘These branches each end
in a tracheal end-cell, which sends its tracheal capillaries, or
tracheoles (7 C), in among the photogenic cells. The photogenic
layer is overlaid on the ventral side by a thin layer of hypodermal
cells, which in the region of the light-organs secretes a non-pig-
mented cuticula.
ORIGIN AND DEVELOPMENT OF THE LARVAL LIGHT-ORGANS
The eggs of Photurus pennsylvanica, in this climate, require
an average of about twenty-six days to complete their embryonic
development. This period, however, is influenced largely by
weather conditions.
Since the embryonic light-organs are of such minute size,
it was often difficult to locate the structure in cross-sections,
hence, for most of this work only sagittal sections were used.
Up to and including the thirteenth day of incubation, the em-
bryos were found to be bent backwards so that they nearly
formed a circle. On the fourteenth day the embryos began to
turn, and at the head end there was a slight indication of their
coiling up. ‘The posterior end, however, was still turned slightly
backward. The embryos on the fifteenth day showed more
evidence of coiling up than on the fourteenth day, for now the
posterior end was also turned slightly forward. Sagittal sections
of the lateral portions of the eighth abdominal segment (fig. 6)
show the presence of many fat-cells, as well asa clearly defined
hypodermis surrounding the body. Those embryos which were
killed in Flemming’s fluid and stained in Heidenhain’s iron
hematoxylin show avery clear differentiation between the fat-cells
and those of the hypodermis, since the fat-cells contain many
fat-globules which are colored dark by osmic acid. No such
globules were found in any of the hypodermal cells. As the
embryos begin to coil up, the abdominal segments become larger,
forming a space of considerable size in the region of the future
DAS WALTER N. HESS
body cavity. This space, in the fifteen-day embryos, is largely
filled with fat-cells.
The earliest indication of the formation of the light-organs
was found in the fifteen-day embryos. At this time some of the
large fat-cells with their dark colored globules, located in the
eighth abdominal segment, were collecting together and _ be-
coming closely applied to the hypodermis in the region of the —
future light-organs (fig. 6). This section, which was prepared
as described in the preceding paragraph, shows a very clear differ-
entiation between these cells and those of the hypodermis. In
fact, the cells of the early light-organs are larger than those of the
hypodermis, and in addition they contain the dark colored
globules which are so characteristic of the fat-cells. The nuclei
of the fat-cells are also larger than those of the hypodermis.
At this time the cells of the light-organs were found to be continu-
ous with the fat-cells of the body proper. The dark colored
globules, in the cells of the light-organ that were located nearest
to the hypodermis, were smaller and fewer in number than those
of the fat-cells in the body dorsal to the light-organ. In fact,
there appeared to be a gradual gradation in the size and amount
of these globules from those cells that were found nearest to the
hypodermis, where they were smaller and fewer in number, to
the cells dorsal to the light-organ where the globules were larger
and greater in number. There was no indication, at this time,
of the two layers which are so characteristic of the mature light-
organs.
A large eylindrical group of tracheal epithelial cells (7) was
found just dorsal to the light-organ, but as yet it had not secreted
any chitin.
In the sixteen to seventeen-day embryos the lght-organs
are entirely laid down so far as the contribution of fat-cells
is concerned (fig. 7). In fact the organs at this time are regular
in outline and their cells do not appear to be so closely applied
to the hypodermis. The individual cells of the light-organs,
at this stage, appear similar to those of the fifteen-day embryo,
except that they are now closer together; their cell boundaries are
less distinct, the dark colored fat-globules are smaller in size and
DEVELOPMENT OF LIGHT-ORGANS | 253
fewer in number. So far as could be determined, the cells of the
entire mass are alike in size and structure and give no indication
of a differentiation into two layers. The similarity between the
light-organ cells and the neighboring fat-cells is very evident
even at this stage. The only difference in structure that could
be determined between these two groups of cells is in respect
to the size and abundance of the fat-globules. These globules
appear slightly larger and a little more abundant in the fat- _
cells than in the cells of the light-organ.
The group of tracheal cells, dorsal to the light-organ, have now
secreted a lining of chitin and have become connected to the light-
organ.
At the age of about twenty days there occurs a differentiation
of the cells of the light-organs into the two layers: the photogenic
layer (P), which lies next to the hypodermis, and the reflector
layer (R), which surrounds the cells of the photogenic. layer,
except in the region of the hypodermis (fig. 8). At this time the
cell walls of all the cells that compose the light-organs are rather
indistinct. Those, however, of the reflector layer appear slightly
more distinct than those of the photogenic area. The cells of
the two layers resemble one another in shape and size, but in
structure they appear much different. The cells of the reflector
layer seem to be considerably vacuolated and less granular,
while those of the photogenic layer give a much denser appear-
ance due to denser granulation. ‘The fat-cells at this time are
much vacuolated. They no longer appear similar to the
cells of the photogenic layer, but they do resemble very much
those of the reflector area. The dark osmic acid colored globules,
so characteristic of the early light-organs and the fat-cells were
not noticeable in the preparations of this period. Theirabsence,
however, may be due to fixation, since these globules appear in
the preparations of a corresponding period in the development of
the adult light-organs in the pupa.
At the end of twenty-two days the embryos begin to emit light
from the light-organs. At this time they are capable of moving
about within the chorion, through which membrane the light-
organs appear as two minute spots of light. The two layers of
254 WALTER N. HESS
the light-organs are now well differentiated, although considera-
ble change takes place subsequently.
The entire light-organ appears to be attached to the hypoder-
mis by a delicate, non-cellular membrane,
The mature light-organ, in a larva one year old (fig. 9), shows
very distinctly the arrangement of the two layers, namely, the
reflector layer (R) and the photogenic layer (P). The cells of
the photogenic layer contain many rather small granules, yet
in the cells of this layer of the adult light-organ, except along
their margins, these granules are very large. The cells of the
reflector layer are much less granular. With the fixers and
stains used the cell walls of the photogenic cells are rather
indistinct, while those of the reflector cells are noticeable.
As will be observed by comparing the larval light organ with
the mature adult light-organ, the cells of the two structures —
are similar in appearance, yet the reflector layer, in the larva,
is thinner than in the adult organ in proportion to the thick-
ness of the photogenic layer in the two stages. One large
trachea sends its branches to the cells of the larval light-organ.
The hypodermal cells in the body wali are now much reduced
in size in comparison with those of the embryo. ‘These cells are
especially small on the ventrolateral side of the light-organ.
Here they secrete a rather thick cuticula, but, due to its trans-
parency, the light from the organ easily penetrates it.
ORIGIN AND DEVELOPMENT OF THE ADULT LIGHT-ORGANS
Since the adult light-organs of insects are developed during
the pupal stage, a large number of larvae of Photurus pennsyl-
vanica were collected during April, for the purpose of obtaining
material for the study of the development of this organ. About
the 25th of May certain of these larvae built their pupal chambers,
and in about five days they transformed to pupae. )
Several of the active larvae were taken about a week or ten
days before their normal pupation period, and sections were made
of the sixth and seventh abdominal segments, to determine the
nature of the various histological structures. A transverse sec-
tion (fig. 11) shows a normal layer of hypodermal cells (4),
DEVELOPMENT OF LIGHT-ORGANS 255
just dorsal to which are large fat-spheres (/) containing many
large fat-cells. Certain sections show these spheres closely
appressed together and lying upon the hypodermis. ‘There
were often two layers of these fat-bodies lying near the hypo-
dermis, although some sections showed only one. The cells
of these fat-spheres near the hypodermis are destined to form
the light-organs. They contain many dark colored fat-globules
which are characteristically colored by the osmic acid. Williams
called them the photogenic fat-spheres. At this stage several
large and small leucocytes are scattered about among the fat-
masses.
Williams, in his discussion of the early stage in the develop-
ment of the light-organs, places considerable emphasis upon the
small leucocytes which he terms fat ‘haemocytes.’ According
to this author, there occurs at this time a partial investment of
the photogenic fat-spheres by a band of yellowish-brown material.
He does not think that it is a secretion of the fat-spheres, but
that it is formed from material in the blood, which, together
with certain haemocytes, have been attracted to one side of the
fat-body. He finds the small leucocytes almost always in con-
tact with this investment and not applied elsewhere to the fat-
spheres. He then concludes that the investing cap, as well as
the small leucocytes, seems to be instrumental in breaking up
the fat-body. This is described as taking place by the inflec-
tion of the cap in which the fat-sphere is squeezed, or constricted,
until the thin membrane opposite the envelope can no longer
stand the strain and ruptures. This pressure often serves to
distort the nuclei. He finds the investment only on the side
of the fat-spheres next to the alimentary canal, which make it
possible for the fat-cells, as soon as they are liberated to migrate
immediately to the body wall and there form the photogenic
layers.
Although considerable attention was given to the nature of the
fat-spheres just previous to the formation of the light-organs,
no such investment membrane could be made out with any degree
of certainty. In certain cases there appeared what seemed like
a little denser mass of insect blood about these fat-bodies, but
256 WALTER N. HESS
in no case was there definitely evident a semicircular band-like
structure on the intestinal side of the fat-spheres that appeared
to be functioning in breaking down these globular masses of
cells. It is true that there were a few small leucocytes present
at this stage and as the light-organs began to develop, they
seemed to increase slightly in numbers. They were not, however,
found attached to the fat-spheres as Williams described them.
Whether they may function in connection with the early forma-
tion of the light-organs was not determined. Because of the
apparent increase in numbers, it is possible that they help in
breaking down the fat-bodies.
Since there is a considerable variation in the stage of develop-
ment of the light-organs in different larvae and pupae, at the
same age with respect to the time of pupation, it is difficult
exactly to correlate stages in the development of the light-
organs with definite periods preceding and following pupation.
Among the different series of the developmental stages studied,
of the same age, a difference of fully one day is frequently
noted in the development of these organs.
A typical larva, taken about one-half day before pupation,
appears sluggish and distended with blood. On sectioning, the
light-organ cells usually appear as is shown in figure 12. Certain
of the large fat-spheres have ruptured and their cells are being
distributed along the hypodermis next to the basement membrane.
In certain instances, where the fat-spheres are found lying near
and closely appressed against the basement membrane, they
are flattened and their cells distributed along the hypodermis,
without apparently leaving the fat-spheres. A little later, how-
ever, these fat-spheres liberated their cells. A regular gradation
in the cells of the fat-spheres in such instances can be seen;
those next to the basement membrane are rather flattened,
their nuclei larger, their fat globules smaller and fewer in number,
and their cell walls more distinct than the cells farther from the
hypodermis. As these cells spread out in this manner, the old
coverings of the fat spheres disappear, due undoubtedly to the
action of the leucocytes.
er.
DEVELOPMENT OF LIGHT-ORGANS Bae
The fat cells, when they first leave the fat-spheres, are large
and nearly circular, though somewhat irregular in outline. ‘Their
nuclei are large and fairly distinct, though they are often more
or less concealed by the fat-giobules which are colored dark
by osmic acid. Their nuclei are not elongated and distorted,
as Williams described. The cytoplasm of the cells show a
more or less vacuolated condition. Soon after the fat-cells
are liberated from the fat-spheres and become distributed along
the hypodermis, their cells divide rapidly, and it is not uncommon,
in sections of this stage, to find them in mitosis. Occasionally
the fat-cells can be observed dividing before’ leaving the fat-
spheres, but in all such cases, the spheres have ruptured and
are lying against the basement membrane.
It is true that there are numerous leucocytes present about
the fat-spheres and the newly liberated fat-cells, though the
enveloping membrane of the fat-spheres described by Williams
does not appear to be present. The large leucocytes are espe-
cially abundant, and it is possible that they function in break-
ing down the fat-bodies. The small leucocytes are also present
in considerable numbers, and it seems possible that they may also
have a similar function. Some of these leucocytes are un-
doubtedly functioning in breaking up some of the fat-masses
for the actively developing tissues. Neither the small nor
the large leucocytes are observed among the cells of the
light-organ, though they are often found lying near, or in the
region of, these cells.
The hypodermis at this stage also shows evidence of considera-
ble activity, for its cells are much elongated (fig. 12) and some
show evidence of division. Sections of some of the larvae at
this stage do not show these elongated hypodermal cells, though
as arule they appear to be attenuated to a considerable extent.
It seems possible that the stage of their elongation may not
correspond with the early origin of the light-organs, so the two
structures may not appear the same at the same stage in different
specimens.
Branches from the larger neighboring tracheae make their
appearance very early, and at this stage these smaller tracheae
258 WALTER N. HESS
(7) may be seen extending down among the fat-spheres in
the region of the undifferentiated cells of the light-organ (U).
They do not extend to the hypodermis at this stage. Their
cells often show evidence of active mitosis. It is not until late in
the development of the light-organs that these tracheal cells form
the mature trachea and tracheoles.
A section of the light-organs of a typical pupa taken one-
half day after pupation (fig. 13) shows a considerable modifica-
tion of that of the previous figure. The fat-spheres are no
longer observed in a ruptured condition, and it seems very
probable that all of those destined to function in the devel-
opment of the light-organs have liberated their cells. The
undifferentiated cells of the early developing light-organ (U)
at this stage are about three cells deep and they are entirely
undifferentiated, so far as any evidence of a differentiation into
two layers is concerned. These cells now appear considerably
different from those of the early liberated fat-cells shown in
figure 12. Their nuclei are larger and they contain a denser
chromatin mass. Their cytoplasm appears to contain a fine net-
work of granular protoplasm. ‘The large dark colored fat-globules
are much less numerous, although a few of them can be seen inthe
cytoplasm of all these cells. It seems very probable that they
function as a reserve supply of food, and are used up during the
increased activity of these cells while they are forming into the
new light-organs. There is still evidence of mitotic division
among these undifferentiated light-organ cells.
The hypodermis presents some complicated, yet interesting,
conditions at this time. Its cells no longer show the attenuated
condition of figure 12, but, instead, they lie along the cuticula.
They present avery irregular appearance, for many of them appear
as if they might be wandering up among the undifferentiated
cells of the light-organ. Their cytoplasm is of a fine granular
nature and resembles rather closely that of the light-organ cells
at this period. Their nuclei are rather large and also resemble
very closely the nuclei of the light-organ cells. The size of the
two groups of cells varies very little, although as a rule those of
the hypodermis are smaller. To add to the difficulty of inter-
DEVELOPMENT OF LIGHT-ORGANS 259
pretation at this stage, the basement membrane has largely
broken down, so that no very definite line of separation between
the two groups of cells could be found. Since modifications of
a similar character were observed in the hypodermis remote from
the light-organ, it was concluded that these peculiarities are
associated with the normal metamorphosis of this tissue.
The dark colored globules which were present in the fat-
cells are still present, in a small number in all the undifferentiated
cells of the light-organ, but none are present in the cells that
are definitely known to be hypodermal. The presence of these
fat-globules in the fat-cells and the cells of the developing light-
organ, but not in the hypodermal cells, leads one to conclude that
these undifferentiated light-organ cells are derived entirely from
fat-cells.
The tracheae by this time have penetrated the light-organ
cells at frequent intervals, and at many places they have ex-
tended their cells to the hypodermis. Frequent mitotic divisions
are still observed among these cells. Some of the tracheal
cells, which appear to be grouped in masses, are frequently
observed lying just dorsad of the light-organ cells. As a rule,
several of the cells from these larger masses, extend down be-
tween the cells of the light-organ. These tracheae, while in an
immature stage of development, grow down among the light-
organ cells at more or less regular intervals and they resemble
in location those of the mature light-organs.
A slightly later stage than figure 13, which was taken of a pupa
one day after pupation, is represented by figure 14. Even at
this stage the cells of the light-organ show no evidence of a
separation into the two layers, but, on the other hand, they all
appear to have the same general characteristics and resemble
very closely those of the undifferentiated light-organ cells shown
in figure 13. It seems evident, however, that their cytoplasm
is slightly more granular and the fat-globules are slightly less
abundant, but, aside from the fact that they are now four or
five cells in depth, there is little real difference. Emphasis here
should be placed upon the fact that all the undifferentiated cells
of the light-organ (U), at this stage, appear to be alike histologi-
260 WALTER N. HESS
cally and that they all contain the dark colored fat-globules.
If certain of these cells had been proliferated from the hypoder-
mis, it seems very probable that there would be two different
types of cells present. The hypodermis, which was so irregular
and indefinite in outline in the previous stage, now shows
its cells all arranged in a regular manner along the cuticula,
except for an occasional cell.
Ina pupa four days old, the cells of the future light-organs show
a decided advance in development (fig. 15). The cells of the
two layers can be fairly clearly distinguished, though they still
appear to intergrade to a certain extent. Those of the photogenic
layer (P) are larger, nearly spherical, and more regularly arranged
than those of the reflector layer (RP). Their cytoplasm is of a
nearly uniform dense granular nature, except for an occasional
dark colored fat-globule, around which there appears to be a
lighter area. Their nuclei are larger, but their chromatin con-
tent does not appear as dense as formerly. The cells of the re-
flector layer (R) are smaller, rather irregular in outline, and
their cytoplasm is made up of a fine granular network. The
fat-globules are still present in the cells of both layers.
The hypodermis with its basement membrane is now repre-
sented by a narrow border of cells lying next to the cuticula.
The developing tracheae (7') show little advance over those
of the previous stage, except that they have enlarged, and their
cells now rest firmly upon the hypodermis. Their cells do not
appear to be dividing at this stage.
A little later stage, represented by a pupa five days old (fig.
16),*shows the two layers of the light-organ clearly differentiated.
The photogenic layer (P) is composed of much enlarged cells,
which, except for their larger size and semirectangular nature,
appear much the same as the photogenic cells of figure 15. The
cells of the reflector layer are of the same general appearance as
they were in the previous stage. The cells of both layers still
retain some of the dark colored fat-globules.
The development of the light-organs from the stage represented
by figure 16 to the mature organ requires a period of about one
week. The most noticeable change takes place in the tracheal
DEVELOPMENT OF LIGHT-ORGANS 261
cells, from which mature tracheae with their tracheal end-cells
develop. These tracheae (fig. 17, 7’), like the tracheal end-cells,
are formed from the tracheal epithelium. The boundaries of
these end-cells are not distinctly seen, yet their nuclei (1 C N)
appear much the same as the nuclei of ordinary tracheal epithe-
lium. The large tracheal branches in the region of the photogenic
layer gives off many smaller branches which divide and often
redivide, each branch finally ending in a tracheal end-cell. In ~
the region of the photogenic layer the tracheal epithelium is
much thicker, and it is here that the tracheal end-cells are formed.
Since these cells are very abundant, they form a contiguous mass,
arranged in the form of a cylinder about the large tracheal
branches, and applied closely to the neighboring photogenic cells.
The tracheal branches bear taenidia, but the capillary tubules,
or tracheoles, which arise from the tracheal end-cells and extend
among the cells of the photogenic layer, do not, although they
are chitinous. Where these tracheoles enter the photogenic mass
there are little depressions, which probably are located in the
divisions between the cells of this structure.
The cells of the photogenic layer are found to contain, ex-
cept along their peripheral boundaries, much larger granules
than those represented in the previous figure. These are called
photogenic granules. The cells of the reflector layer, even in
the mature organ, closely resemble in general outline and ap-
pearance fat-cells. Those of the photogenic layer, on the other
hand, show little similarity. The large fat-globules, which are
present in the cells of both layers, during the early stages in
the development of the light-organs, disappear shortly before the
organs reach maturity.
The pupae continue to emit light from the larval et -organs
throughout the pupal period, but the adult light-organs do not
begin to function until one or two days before the end of the
pupal period. It requires from sixteen to eighteen days for the
completion of the pupal period.
Thus, in the development of the light-organs, groups of fat-
cells become localized in the regions of the future light-organs,
which for a considerable period after they become so localized
262 WALTER N. HESS
all show the same general characteristics. These cells later be-
come differentiated into the photogenic and reflector layers,
but both before and after they become so differentiated all these
cells contain the dark fat-globules which are so characteristic of
fat-cells after treatment with osmic acid. .These observations
lead me to conclude that all the cells of the light-organs are derived
from fat-cells, and hence are mesodermal in origin.
PHAGOCYTOSIS OF THE LARVAL LIGHT-ORGANS
The larval light-organs of Photurus pennsylvanica begin to
show evidence of breaking down soon after the pupa changes
to an adult, and from this time on their light becomes fainter,
until it finally disappears about forty-eight hours after the emer-
gence of the adult. At the end of the second day of adult life,
just before the luminosity disappears, a cross-section of the organ
has the appearance of that shown in figure 10. At this stage the
cells of the reflector layer (FR) are still intact and their structure
appears normal. Those of the photogenic layer, on the other
hand, show definite evidence of breaking down. They are no
longer together in a mass, but are separated into differ-
ent groups. The structure of their cell walls is very indis-
tinct and their granules are less prominent. Surrounding the
cells of the photogenic layer, and to a certain extent intermingled
among them, are many large leucocytes (LL). Whether they
have a phagocytic action was not determined, yet their presence
in such numbers suggests very probably that they are function-
ing in the destruction of the light-organ. No other blood cells
are found in the neighborhood of the light-organ in sufficient
numbers to make it seem possible that they are functioning
in the destruction of this structure. Anglas (’00) apparently
found similar cells during metamorphosis in Vespa. He did
not attribute to them a phagocytic function. A section of this
organ taken at the end of the third day of adult life shows very
little evidence of the light-organ cells. Numerous large leu-
cocytes are present at this time in the region of the old larval
organ, but the cells of this structure are indistinct and most of
their walls have broken down. ‘This indicates that the destruc-
DEVELOPMENT OF LIGHT-ORGANS 263
tion of the light-organ is very rapid as soon as luminescence
ceases and that the leucocytes are probably the chief agents in
destroying it.
SUMMARY
1. The first indication of the formation of the light-organs,
in the embryo, is noticeable at the age of fifteen days, just as the
embryo revolves from its backward-turned position and starts
to coil up.
2. At this time groups of fat-cells, with their large globules
which are colored dark by osmic acid, migrate ventrally in
segment eight and come to lie in the region of the future light-
organs. These undifferentiated light-organ cells are now con-
tinuous with the groups of fat-cells dorsal to them.
3. As soon as the fat-cells become localized in the region of the
future light-organs, their dark colored globules become smaller
in size and fewer in number. In fact, in the fifteen-day embryos
there appears to be a gradual gradation from the cells lying next
to the hypodermis, which contain smaller and fewer of these
globules, to the fat-cells near the central part of the body, which
contain more and larger globules.
4. In the sixteen- and seventeen-day embryos the light-organs
are regular in outline, and they have become separated from the
other fat-cells. The fat-globules are now smaller and fewer in
number than on the fifteenth day. All celis that compose the
light-organ are apparently now of the same histological structure.
5. At the age of twenty days there begins to take place a
differentiation of the cells of the light-organs into the photogenic
and reflector areas.
6. At the age of twenty-two days the light-organs become
functional and appear as two minute spots of light.
7. The larvae emerge on about the twenty-sixth day of incu-
bation.
8. These larvae require nearly two years (about twenty-two
months) to reach maturity, at which time they pupate.
9. In mature larvae, about one-half day before pupation,
the cells of the fat-spheres, which lie near the hypodermis in
JOURNAL OF MORPHOLOGY, VOI. 36, NO. 2
264 WALTER N. HESS
the ventral part of the sixth and seventh abdominal segments,
are liberated and become distributed along the hypodermis.
These cells contain numerous fat-globules, which appear dark
after treatment with osmic acid. .
10. The fat-cells, which are liberated from the fat-spheres
during the last day of larval life and the first one or two days
following pupation, compose a layer about three cells deep above
the hypodermis. Sections of the light-organs at this stage show
some of these cells in mitosis.
11. The undifferentiated cells of the light-organs, at this
stage, are all of the same general histological appearance, which
suggests a common origin.
12. The cells of the photogenic and reflector layers, in the
five-day pupae, are clearly differentiated. At this time the
cells of both layers still contain some of the’ dark colored fat-
globules.
13. Tracheal epithelium, by the rapid division of its cells,
now extends from the region of the body cavity down between
the cells of the light-organs at regular intervals. It later gives
rise to the trachea of the light-organs, together with their tra-
cheal end-cells and tracheoles.
14. Shortly before the light-organs become mature, in both
the embryo and the pupa, the fat-globules disappear and the
organ takes on its characteristic adult structure.
15. The light-organs of both the larva and the adult are
formed from fat-cells which become differentiated into the
photogenic and reflector layers of the mature light-organs.
Hence the light-organs are entirely mesodermal in origin.
16. In the breaking down of the larval light-organs, which
occurs about forty-eight hours after the emergence of the adults,
the cells of the photogenic layer become separated into small
groups, soon after which their cell walls and cytoplasmic contents
become indistinct. Soon after the cells of the photogenic layer
break down the cells of the reflector layer meet the same fate.
Numerous .large leucocytes are found surrounding the cells of
the breaking down light-organs at this period. It seems probable
that they are the chief agents in the destruction of these organs.
DEVELOPMENT OF LIGHT-ORGANS 265
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is
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267
w\
PLATE 1
EXPLANATION OF FIGURES
1 Larva, ventral view of abdomen. LO, larval light-organ, located on the
eighth abdominal segment.
2 Adult male, ventral view of abdomen. AO, adult light-organ, located
on the sixth and seventh abdominal segments.
3 Adult female taken immediately after emergence, ventral view of abdomen.
AO, adult light-organ; LO, larval light-organ.
4 Larva, cross-section to show position of the light-organs. R, reflector
layer; P, photogenic layer.
5 Adult male, cross-section to show position of the light-organs. MR, reflector
layer; P, photogenic layer.
268
PLATE 1
ALTER N. HESS
PLATE 2
EXPLANATION OF FIGURES
6 Sagittal section of the light-organ of a fifteen-day embryo, which shows the
light-organ in a very early stage of development. F, fat-cells; FG, fat-globules;
H, hypodermis; 7’, trachea; U, undifferentiated cells of the light-organ.
7 Sagittal section of the light-organ of a seventeen-day embryo. C, cuticula,
F, fat-cell; H, hypodermis; M, muscle; 7, trachea; U, undifferentiated cells of the
light-organ.
8 Sagittal section of the light-organ of a twenty-day embryo, which shows a
beginning of the differentiation of the cells into the two layers of the mature light-
organ. C, cuticula; F, fat-cell; H, hypodermis; P, photogenic layer; R, reflector
layer.
Note. All the material that was used for the histological preparations that are
illustrated on this plate and the three following plates was fixed in Fleming’s
fluid and stained in Heidenhain’s iron hematoxylin.
270
DEVELOPMENT OF LIGHT-ORGANS PLATE 2
WALTER N. HESS
i)
~I
>
PLATE 3
EXPLANATION OF FIGURES
9 Cross-section of the light-organ of a one-year-old larva. C, cuticula;
F, fat-sphere; H, hypodermis; LL, large leucocyte; p, photogenic layer of light-
organ, R, reflector layer of light-organ; SL, small leucocyte; 7, trachea.
10 Cross-section of the larval light-organ, forty-eight hours after the emer-
gence of the adult insect. C, cuticula; F, fat-spheres; H, hypodermis; LL, large
leucocyte; M, muscle; P, photogenic layer of larval light-organ; R, reflector
layer of larval hight-organ; SZ, small leucocyte; 7’, trachea.
11 Portion of a cross-section of the seventh abdominal segment of a larva,
taken about one week before pupation, to show the arrangement of the fat-
spheres and hypodermis. £8, basement membrane; /, fat-sphere; FG, fat-globules
H, hypodermis; LL, large leucocyte; WM, muscle; O, oenocyte; PC, primary cuti-
cula; SC, secondary cuticula; SZ, small leucocyte; 7’, trachea. .
272
DEVELOPMENT OF LIGHT-ORGANS PLATE 3
WALTER N. HESS
PLATE 4
EXPLANATION OF FIGURES
12 Portion of a cross-section of the seventh abdominal segment of a larva,
taken one-half day before pupation. B, basement membrane; F, fat-sphere;
H, hypodermis; LL, large leucocyte; PC, primary cuticula; SC, secondary cuti-
cula; SL, small leucocyte; 7, trachea; U, undifferentiated cells of the light-organ.
13 Portion of a cross-section of the seventh abdominal segment of a pupa,
taken one-half day after pupation, to show a little later stage than figure 12 in
the formation of the light-organ. C, cuticula; F, fat-sphere; H, hypodermis;
LL, large leucocyte; M, muscle; SL, small leucocyte; 7, trachea; U, undifferen-
tiated cells of the light-organ.
14 Portion of a cross-section of the seventh abdominal segment of a pupa,
taken one day after pupation. It represents a slightly later stage than figure 13.
For labels see figure 18.
274
PLATE 4
DEVELOPMENT OF LIGHT-ORGANS
WALTER N. HESS
275
PLATE 5
EXPLANATION OF FIGURES
15 Cross-section of the fourth stage in the development of the adult light-
organ, taken four days after pupation. It illustrates an early stage in the differ-
entiation of the light-organ into two layers. C, cuticula; H, hypodermis; P,
photogenic layer of light-organ; R, reflector layer of light-organ; 7’, trachea.
16 Cross-section of the fifth stage in the development of the adult light-organ,
taken five days after pupation. This represents a stage in which the cells of the
two layers of the light-organ are definitely differentiated. The dark fat-globules,
so characteristic of fat-cells after osmic-acid fixation, are distinctly visible in all
the cells of the light-organ at this stage, as well as in the four previous stages.
For labels see figure 15.
17. Cross-section of the adult light-organ fully developed. C, cuticula; HCN,
nucleus of tracheal end-cell; H, hypodermis; N, nucleus of photogenic cell; P,
photogenic layer; R, reflector layer; T, trachea; TC, tracheole.
276
PLATE 5
DEVELOPMENT OF LIGHT-ORGANS
WALTER N. HESS
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Resumen por el autor, Sante Naccarati.
Contribucién al estudio morfologico de la glindula tiroides de
Emys europea.
Kn Emys europea la tiroides es un 6rgano medial impar, de
forma esferoidea y color rojizo, situado encima del coraz6n en la
cavidad del arco formado por el tronco innominado. El volumen
y peso de este 6rgano presentan considerable variaciones, que
dependen en su mayor parte de la edad y tamano del animal;
el peso medio es 0.025 gramos y su longitud media 5mm. La
irrigaciOn sanguinea de la glindula se lleva a cabo mediante dos
arterias tiroideas superiores y otras dos inferiores y el mismo
numero de venas. Variaciones y anomalias en el numero y
distribucién de los vasos tiroideos son bastante frecuentes. La
inervacion tiene lugar mediante el vago y el simpatico.
La tiroides de Emys no difiere esencialmente en estructura
histol6gica de la de los demas vertebrados, incluso el hombre.
En la capsula de tejido conectivo fibroso existen cromatdforos
esparcidos Las células del epitelio son generalmente cuboideas,
menos frecuentemente cilindricas o aplanadas y estan en contacto
directo con el coloide. El nticleo ocupa siempre la parte basal
de la célula; es distintamente vesicular, bastante grande, provisto
de grdanulos cromdticos y sin nucleolo. Entre los alveolos
adyacentes existen escasas fibras eldsticas delicadas derivadas
de las ramificaciones de la red elastica mis grosera que cubre la
superficie de la glandula. Los gradnulos de secrecién son mds
grandes y menos numerosos que los grdanulos de grasa y las
mitocondrias. Son claramente fuchsin6filos con el método de
Galeotti. El coloide intravesicular no difiere del que se halla
en la tiroides humana.
Translation by José F. Nonidez
Cornell Medical College, New York
AUTHOR’S ABSTRACT OF THIS PAPER ISSUED
BY THE BIBLIOGRAPHIC SERVICE, DECEMBER 27
CONTRIBUTION -TO THE MORPHOLOGIC STUDY OF
THE THYREOID GLAND IN EMYS EUROPAEA
SANTE NACCARATI
Department of Histology, Royal University, Rome
FIVE COLORED FIGURES
NOTES ON THE EMBRYOLOGY OF THE THYREOID
The thyreoid, together with the thymus, the postbranchial or
suprapericardial bodies, the carotid gland, the thymous lobules,
and the parathyreoids (also called epithelial corpuscles), belongs
to the group of organs called branchial derivatives.
The first embryogenetic researches on the thyreoid go back to
Huschke (26), Rathke, Remak (’55), and Gétte (’67) and, with
many others which followed not long after, were conducted
almost exclusively on mammals. They served to establish
that the thyreoid is derived from a thickening and hollowing of
the ventral wall of the pharynx at the level of the second pair of
branchial arches.
His (’68) established that the thyreoid is derived from two
equal lateral rudiments in the pharyngeal wall.
Miiller (71), Sessel (’77), and Kolliker (79) found that in
mammals the thyreoid is derived from a single medial rudiment
in the form of a hollow diverticulum (Miiller and Seessel) or of a
solid bud (K6lliker) from the ventral wall of the pharynx, with
which it remains in temporary connection.
Born (’83) and Fischelis (’85) found that in the pig embryo
the thyreoid comes from three rudiments, originally independent,
a middle thyreoid rudiment arising from the ventral wall of the
pharynx at the level of the second pair of branchial arches, and
two lateral thyreoid rudiments, supposed to be formed from
the epithelium of the fourth entodermic branchial pouches.
279
JOURNAL OF MORPHOLOGY. VOL. 35, No. 2
280 SANTE NACCARATI
Other researches were conducted by Wolfer (’80), Stieda (’81),
Dohrn (’87), Gétte (75), DeMeuron (’86), Maurer (’85), Balfour
(78), Kastschenko (’87), Piersol (’88), Prénant (’94~-99),
Van Bemmelen (’85-’93), Platt (96), Simon, Soulié (97),
Verdun, Jacoby (’94, ’96, ’97), Verson (’07), and others more
recently, too many to be quoted here, with resulting confirmation
of the origin of the thyreoid from three distinct and originally
independent rudiments, i.e., one middle and two lateral thyreoid
rudiments, in mammals.
According to Maurer (’99) Echidna, and according to Syming-
ton (’97, ’98) the Edentata and Marsupialia are exceptions to
this rule, in that they retain the independence, of the three
rudiments, having, in addition to a two-lobed thyreoid, two
organs developed from ‘the lateral rudiments, homodynamic
with the postbranchial or suprapericardial bodies, by which
name they are called.
Livini (’02) made a careful study of the embryology of the
organs of the thymus-thyreoid system in Amphibia urodela, and
found the thyreoid arises as a single medial solid epithelial
bud from the caudal wall of an entodermic spur of the pharyn-
geal floor which enters in intimate contact with the ecto-
derm. He considers this spur as a rudiment of the hypobranchial
groove of the Tunicata. However, the level at which the thyreoid
arises cannot be established because when its bud is already
recognizable, neither the branchial pouches nor the cartilages
of the branchial arches have yet been differentiated.
NOTES ON THE COMPARATIVE ANATOMY OF THE THYREOID
Cyclostomata. In the lamprey the thyreoid gland is but
little developed in the adult. In the larvae (Ammocoetes)
it remains in open communication with the buccal cavity, at a
level between the third and the fourth branchial slit, in such a
manner that it may be considered as a diverticulum of the
ventral wall of the pharynx. With the coming of the meta-
morphosis it becomes a glandular organ of vesicular structure
and is isolated from the pharynx. The arrangement in the
larvae recalls a homologous relation between the thyreoid in the
THYREOID IN TURTLES 281
Cyclostomata and the hypobranchial groove of Amphioxus, on
the one hand, and between the latter and the ventral furrow of the
branchial basket in the Tunicata on the other. This homology is
based on the following considerations:
I. The thyreoid of Petromyzon in the Ammocoetes stage is a
sort of muciparous gland, which, if it in some respects differs
from the endostyle of the Tunicata, nevertheless presents such
points of resemblance to it that the two formations may be
regarded as homogeneous (Dohrn, ’80).
II. During the development this gland is transformed into
an organ corresponding to the thyreoid of other vertebrates.
In the Elasmobranchii the thyreoid is a single medial organ
sometimes spherical, sometimes cylindrical, sometimes triangular
or pear-shaped; its volume and weight differ in different animals;
sometimes (as in Scyllium catulus) it is cranially located at the
angle of bifurcation of the branchial artery; sometimes it is
located in the vicinity of the tongue, between the coracohyoideal
and coracomandibular muscles (as in Acanthias vulgaris and in
Mustelus laevis); sometimes it is immediately under the skin
(as in Squatina angelus).
Within the thyreoid of the Elasmobranchii there exist, accord-
ing to Thompson (’10), masses of small solid cells, partly epithe-
lial, partly adenoid, which have been thought to indicate a
homology with the parathyreoids and the thymus.
In the Teleostei, according to Maurer (’85), the thyreoid is a
single organ until, at a certain age, it divides into an accumula-
tion of follicles which surround the branches of the branchial
artery on every side. In Amiurus, according to Thomson, the
thyreoid consists of a number of vesicles scattered here and
there, enclosed in the matrix of the connective tissue; the cells
which line the vesicles are cylindrical, very low, and in some cases
almost flat.
In the Urodela the thyreoid is a double organ, yellowish in
color, spherical or egg-shaped; its largest diameter is less than
1 mm.; it is slightly flattened, and very superficially located,
between the mylohyoid and sternothyreoid muscles, in the
immediate vicinity of the jugular vein.
282 SANTE NACCARATI
In the Anura, also, the thyreoid is a double organ (single in
the embryo), lateral, egg-shaped, pinkish in color; each half is
4 mm. in diameter; both are located in the ventral side of the
animal, at the posterior horns of the hyoid bone, in front of the
jugular veins, to which they are closely adherent.
In the Ophidia the thyreoid is a single discoidal organ located in
the median line at the base of the heart, between the two carotids
in the young animal it is at the lower extremity of the thymus.
Its diameter is about 3 mm. and its weight about 2 mgm.; by
its grayish-white color it is easily distinguished from the two
thymous lobes, noticeable for their brighter color, whose medial
margins cover it.
In the Lacertilia also (aside from a few genera like Monitor,
in which it consists of two lobes located at the side of the neck
in front of the carotids) the thyreoid is a single medial organ
just under the skin on the ventral side of the trachea, along which
it lies with its greatest diameter transverse to the diameter of the
trachea. It is dark gray; fusiform, largest in the middle, the
two extremities narrowing until, as they reach the lateral ex-
tremities of the neck, they terminate in a fine filament, often
bifurcated. In the Lacerta viridis it reaches a length of 1 em.
My research on the thyreoid in the Squamata, on the species
Zamenis viridiflavus, Tropidonotus natrix, Lacerta viridis, and
Lacerta agilis, confirming this statement, will form the subject
of another paper.
In the Aves the thyreoid consists of two rounded or oval lobes,
pink in color, varying in size with the animal, and located at the
sides of trachea near the syrinx, attached to the ventral side
of the carotid, generally at the level of the vertebral artery. In
the pigeon, for example, the thyreoids occur in the anterior wall
of the thorax, near the junction of the thorax and neck. They
are ovular in form, with their longest diameter lengthwise of the
body; their lowest point is slightly above the point where the
main branches of the carotid artery divide. In front of them are
the jugular veins on the outside and the oesophageal arteries on
the inside.
THYREOID IN TURTLES 283
Mammalia. Aside from the human thyreoid, the detailed de-
scription of which may be found in any treatise on anatomy, it
may be remarked that, in general, this organ in mammals con-
sists of two lateral lobes at the sides of the trachea, between the
first and the ninth tracheal rings—the exact position depending
on the animal; the weight and volume also vary with the animal.
In general the two lobes are joined to each other by an isthmus,
usually thin, as in the dog, cat, rabbit, rat, and guinea-pig; in
old animals it may atrophy or disappear. Thus in the donkey
the isthmus is easily seen in the young animal, while in the old it
is reduced to a slight atrophic filament without glandular struc-
ture. In the horse and sheep, on the other hand, the isthmus is
so thin that often it cannot be distinguished; this has led some
to believe that in these animals it occurs only exceptionally.
Aberrant nodes of thyreoid tissue have been described in non-
human mammals; in these, however, it has not been possible
to recognize the pyramid ‘of Lalouette or the appendix of
Morgagni.
PERSONAL INVESTIGATIONS
My researches on the thyreoid of Chelonia were carried out
on the two Italian species, Emys europaea and Testudo graeca.
There is very little difference in the macroscopic aspect and no
difference at all in the microscopic appearance of the thyreoid in
these two species. I will give the description of the thyreoid of
Emys europaea and refer to Testudo graeca for the main differ-
ential points. ;
Macroscopic anatomy. In Emys europaea the thyreoid is a
single medial organ of spheroid form and pinkish color, located
in the cavity of the arch formed by the truncus innominatus.
From the anatomic-topographiec point of view, in order to reach
the thyreoid by trepanning it is necessary to apply the point of the
instrument half a centimeter above the point of union of the
hyoplastral with the hypoplastral plates. Removing the bone,
and taking pains to hold the animal’s forepaws well apart so as to
withdraw from the operative field the two scapuloclavicular
ligaments (with the animal’s neck extended), one finds a small
284 SANTE NACCARATI
rounded body, easily recognized by its pink color, across the
adipose tissue and suprapericardial connective tissue, larger or
smaller than a pea according to the size of the animal.
In the classic treatise of Bojanus (1819-21) in which are
reported with clearness and precision all the characteristics
of the macroscopic anatomy of Emys europea, the thyreoid is
taken for the thymus. The latter, when it exists, is a long double
organ of a light gray color, located in front of the carotids, with
which it is in close contact, at the point of conjunction of the
neck with the thorax. It is strange that so able an anatomist
should have fallen into such an error. The pink color of the
thyreoid is due to the blood which it contains, the amount of
which is very considerable (according to Tschuovsky, 560 ce. of
blood pass each minute through 100 grams of human thyreoid
tissue). When the excess of blood in the thyreoid of the tortoise
is eliminated, it acquires the appearance of an opalescent lens.
Volume and weight. The volume and weight of the thyreoids
of Emys europaea are very variable. The most noteworthy
variations are due to the size and age of the animal. With the
purpose of establishing as exactly as possible the average weight,
I have weighed the thyreoid of thirty Emys and found that in
adults weighing about 275 gm. the thyreoid has an average
weight of 0.025 gm. In general, 100 gm. of body weight corre-
sponds to about 10 mgm. of thyreoid. For man this proportion is
about five times as big. If the weight of the Emys is taken with-
out its carapace and plastron, which averages about 40 per cent
of the total, according to my measurements of thirty animals,
the proportion is 16 mgm. of thyreoid to 100 gm. of the animal’s
weight. There are great individual variations from the average.
In another paper I have prepared, in tabulated form, the weights
of the thyreoid and other glands in groups of several species of
reptiles, including Emys europaea.
As regards the volume of the thyreoid in Emys europaea, what
I have said regarding the weight holds good, namely, that it
varies within very wide limits, according to the size of the animal.
In general it may be stated that in an animal of 300 gm. weight,
the maximum diameter of the gland is about 5 mm.
THYREOID IN TURTLES 285
Topographical relations. As I have said above, the thyreoid in
Emys europaea lies within the large upward-curving arch formed
by the truncus innominatus, just above the heart. As _ this
arch leans slightly toward the right, the gland is not absolutely
in the middle, but is a little to the right. In front (on the ani-
mal’s ventral side), the thyreoid is separated from the thoracic
wall by a lamellar connective, transparent and fairly tough, con-
sisting of several layers, continuous below with the pericardium
and surrounded above by the large vessels of the neck. The
vascular arch along which the thyreoid lies is closely connected
with it, both by means of the vessels and by means of the connec-
tive tissue. The rear wall of the gland (toward the animal’s
dorsal side) is in front of the trachea, with which it is not in con-
tact. It must be noted that in Emys europaea the trachea
divides into the two bronchi a little above the thyreoid, while in
Testudo graeca the division occurs much higher, near the base
of the tongue.
Circulation and innervation. As in a man, the thyreoid in
these Chelonia is highly vascularized. ‘The blood flows to it
through the two superior and the two inferior thyreoid arteries.
The inferior pair are short, but very capacious; they issue from
the truncus innominatus, and penetrate the gland at right angles,
passing through its outer inferior margin. Regarding the
behavior of the large vessels as they leave the heart, it should be
remembered that, whereas the left aorta reaches the left bronchus
without branching, the right aorta on the contrary, before
curving, sends off a large but very short trunk (truncus innomi-
natus) which forms a superior concavity and then divides into
the right and left carotid and subclavian arteries, after sending
off the inferior thyreoid arteries and the oesophageal arteries.
The superior thyreoid arteries are longer but thinner; they
branch from the carotids and turn downward and inward, issuing
in the outer superior ‘margin of the thyreoid gland. ‘These
arteries (unlike the inferior thyreoid arteries which are always
present) are sometimes missing.
It must be noted that the division of the truncus innominatus
into subclavian and carotid sometimes occurs a little higher on
286 SANTE NACCARATI
the right than on the left; when the animal’s neck is extended, the
point of bifurcation of the two carotid arteries and the right
subclavian is in a line with the right forepaw.
In connection with the thyreoid arteries it must be noted that
there are many variations, especially of the superior pair, which
often, instead of penetrating the gland directly, join the inferior
pair, thus entering the gland as a single trunk. When this occurs,
the superior thyreoid artery turns downward immediately after
leaving the carotid and follows a course,of about 1 cm., while
the inferior artery, turning slightly upward, follows a very short
course. The trunk which results from their union is so short
and thick that it resembles an arterial sinus.
At other times the superior thyreoid artery is missing, and is
replaced by three or four small arteries forming a network around
the upper tip of the gland. At still other times there may be
a median artery which arises from. one of the two carotids near
the hyoid bone and turns downward along the median line of the
neck, reaching the upper tip of the gland.
The ramifications of these arteries, finely divided, form a
plexus around the fibrous capsule which surrounds the gland,
and penetrate the parenchyma, where they form a very fine
capillary network interwoven with the thyreoid vesicles, which
they enclose, passing through the intervesicular septa. The
musculature of these vessels is very distinct.
The veins which originate in the form of fine branchlets
traversing the vesicles compose on the surface of the gland a
thick venous network, a large plexus from which issue the princi-
pal veins (inferior thyreoid); the latter unite with the accessory
pectoral veins and empty into the subclavian vein formed by the
confluence of the jugular and axillary veins.
The fine perivesicular veins are without musculature and ap-
pear as little tubes with endothelium alone, traversing the inter-
lobular connective tissue. Fine elastic fibers passing through
this tissue seem to provide a kind of support for the larger
vessels. The lymphatics are also very numerous; as in human
thyreoid, they arise as small vacuoles between the cells lining the
vesicles; these unite to form intervesicular canals, and those in
THYREOID IN TURTLES 287
turn join to form larger trunks (the interlobular canals). These
last follow the course of the arteries, veins, and nerves till they
reach the external surface of the gland, where they form a dense
network, from which emerge the larger branches through which
the lymph is emptied into the lymphatic ganglia of the neck.
The innervation of the thyreoid is by the sympathetic. The
fine non-medullated fibers accompany the arterial ramifica-
tions in the gland. The vagus also sends two fine branchlets
into the gland through the laryngeal nerves, but their distribu-
tion is not constant.
HISTOLOGY
For the microscopic study of the thyroid of Emys europaea I
have made use of specimens preserved in—
. Formalin, from 5 per cent to 10 per cent aqueous solution.
. Mercurie chloride
. Zenker’s fluid
. Flemming’s fluid
. 96 per cent alcohol
. Miiller’s fluid
The sections were stained in different ways. For the general
study of the thyreoid tissue, preservation in 10 per cent formalin
and staining with Ehrlich’s acid-haematoxylin and the aqueous
solution of eosin gave good results. Fixation in Flemming and
staining with ferric haematoxylin (Heidenhain) and eosin per-
mitted greater accuracy in studying the delicate structure of the
cellular elements. Safranin and carmine have been very useful
in delicate cytological study. For studying the elastic fibers
fucselin and Weigert’s fluid were used combined as follows:
a. Fueselin.
b. Fueselin-Van Gieson: Weigert’s fluid.
c. Weigert’s fluid; borax carmine, alcoholic solution of the
Naples Zoological Station.
d. Weigert’s fluid safranin.
e. Safranin, picric acid, Weigert’s fluid.
There is no substantial difference in structure between the
thyreoid of Emys europaea and Testudo graeca and that of the
oor wWhN re
e
288 SANTE NACCARATI
other vertebrates, including man. It presents externally a
fibrous connective-tissue capsule in which, here and there, are
scattered pigmented cells (chromatophores). From this capsule
issue numerous connective-tissue septa, which, gathering on the
inside of the gland, form a network enclosing the vesicles. . These
vesicles, called also follicles or alveoli, are irregularly rounded, from
50 » to 300 » in size, and are lined with simple epithelium, the
cells of which are mainly cubical, less often cylindrical or flat,
and are in direct contact with the interior of the vesicular cavity,
in which is contained the colloidal fluid, an amorphous, homo-
geneous substance presenting under the microscope transverse
streaks or fissures and staining with acid stains; for example,
it stains pink with haematoxylin-eosin and yellow with Van
Gieson or with safranin and picric acid. The interior surface of
the epithelial cells, namely, the surface looking in the lumen of
the vesicles, is not clearly defined, but it has a broken appearance,
recalling that of the colloidal substance, and probably, since it is
not constant, due to the latter’s remaining adherent to the cells.
The protoplasm is homogeneous and contains fine grains. The
nucleus always occupies the basal part of the cell, is well
marked, vesicular, rather large, and provided with chromatin
granules and does not have a nucleolus. The limits between the
cells are quite clear, and in the cellular walls, which are in con-
tact with the connective tissue limiting the alveoli, there is a
basal membrane, not always, however, well differentiated. In
specimens colored with safranin and picrie acid there are cells
having a nucleus which contains granules colored red (chromatin)
noticeable against the brighter background of the rest of the
nucleus, and cells whose nucleus is entirely colored red (fig. 3).
These two kinds of cells correspond to the two types, principal
and colloidal, described by Langendorff, who interpreted them
as different aspects assumed by the same cell at different func-
tional periods. This interpretation seems very probable because
the aspect and the disposition of the cells in the different alveoli
is so variable that they suggest many functional phases from
the beginning elaboration to the complete secretion of the colloid.
THYREOID IN TURTLES 289
The gland is subdivided into lobules by larger connective-
tissue septa derived from the external fibrous-connective capsule,
and the lobules in their turn are subdivided into alveoli by
thinner septa of the same nature. The blood vessels, the lym-
phaties, and the nerves run into the intervesicular and inter-
lobular septa, where they form a highly complicated network.
In the small thyreoid arteries I have not found it possible to
demonstrate those thickenings or buds (Schmidt’s ‘Zellknospen’)
of which Ko6lliker (02) speaks. The intervesicular substance is
rather scanty, and is formed of areolar connective tissue, ex-
tremely rich in blood vessels, which constitute a capillary net
surrounding the alveoli and extending its finest branches into
the epithelium. Between each alveolus and the next are scanty
delicate elastic fibers which accompany the blood vessels and
are derived from the ramifications of the coarser elastic network
covering the surface of the gland. The elastic fibers are numerous
and well demonstrable only in the external connective capsule.
Toward the interior of the gland they grow thinner and scarcer
till they disappear entirely in the walls of the most central of the
alveoli. Elastic fibers are more frequent in the thyreoid of
young animals.
Under the microscope the intravesicular colloid does not
differ essentially from that of the human thyreoid. In the
interior of the alveoli there are, at times, free epithelial cells,
detached from the alveolar walls, as if some cellular desquama-
tion had occurred (fig. 3). This condition noted in the thyreoid
of individuals suffering from Basedow disease was at first
given a pathological significance; later it was seen that it was
a normal phenomenon, a form of holocrine desquamation of cer-
tain thyreoid cells (Pende, 718).
The granules of secretion, as in the cells of the human thyreoid,
appear larger and less numerous than the granules of fat and the
mitochondria. They stain distinctly red (fuchsinophile) with the
method of Galeotti. This method, proposed by Galeotti for the
study of the granules of secretion, is of the utmost importance for
finer cytologic researches and should never be omitted. The
fixative for the employment of this method is either Flemming’s
290 SANTE NACCARATI
or Hermann’s fluid. The sections must be very thin, about
4or6u.
The technique is as follows:
1. The section is stained from five to ten minutes at the tem-
perature of 50°C. with a freshly prepared saturated solution of
fuchsin in aniline water.
2. Wash in water for about thirty seconds.
3. Transfer to a semisaturated solution of picric acid in
50 per cent alcohol for twenty or thirty seconds.
4. Prolonged wash in water until the section does not yield
any more picric acid.
5. Staining for four or five minutes with a4 per cent solution
of methyl] green in 90 per cent alcohol.
6. Rapid transfer to grades of alcohol, during which the
sections yield much stain.
7. Transfer to xylol and mount.
Sections thus prepared show the following characteristics:
The nuclear chromatin, the centrosomes, and the granules of
secretion are bright red, the protoplasm and the connective
green. The strongly basophile substances, such as mucin and
chondrin, take also a green, but more intense stain. The picric
acid, acting as a mordant on the methyl green, renders it a
plasma dye. In good sections the plasma takes an emerald
green stain. If it takes a yellowish-greenish stain, the section
can be utilized, provided that the fuchsinophile granules take a
distinctly bright red stain. Sometimes (either because of a
much prolonged action of the picric acid or for other reasons) the
section does not stain at all with methyl green. It is advisable
to repeat this method several times until a good section is
obtained.
Figure 4, showing only a part of the epithelium of the vesicle,
gives the appearance of a few cells stained with the method
of Galeotti.
THYREOID IN TURTLES 291
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1886-7 Studien, usw. XI. Thyreoidea und MHypobranchialrinne.
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FiscHetis, Pu. 1885 Beitrige zur Kenntnis der Entwickelungsgeschichte der
Glandula thyreoidea und Gl. Thymus. Arch. mik. Anat., Bd. 25.
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1891 Der Tractus thyreoglossus und seine Beziehung zur Zungenbein.
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und der Carotiddriisen. Anat. Anz., Bd. 12.
1897 Zur Entwickelung der Nebendriisen. Anat. Anz., Bd. 13.
KastscHENKo, N.- 1887 Das Schicksal der embryonal Schlundspalten bei
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1888 Schilddriise, Thymus und Kiemenreste der Amphibien. Morph.
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1898 Der Derivate der Schlundspalten bei der Eidechsen. Verh.
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Bd. 18.
1899 Die Schilddriise, Thymus und anderen Schlundspalten Derivate
bei der Eidechse. Morph. Jahrb., Bd. 27.
292 SANTE NACCARATI
pE Meuron, P. 1886 Développement du Thymus et de la glande thyreoide.
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THYREOID IN TURTLES 293
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PLATE 1
EXPLANATION OF FIGURES
1 Thyreoid of Emys europaea. Fixation: alcohol 96 per cent. Staining:
alcoholic carmine solution of the Zoological Station of Naples, Weigert’s fluid
picric acid. 8* objective, 4 ocular. The colloid is stained yellow, the cellular
nuclei red, the elastic fibers blue. A portion of the external capsule has been
left in place. Note how numerous are the elastic fibers in the capsule and how
extremely scarce are they in the intervesicular connective.
2 Same. Fixation: Fiemming’s fluid. Staining: Heidenhain’s haematoxy-
lin. 7* objective, 4 ocular. In some places the intervesicular connective has
given way and the alveoli appear detached.
1
PLATE
dis. , , eye? om j TORE 3 * Yeu)
i Soigs' is Sow ggg “VW a 8
-THYREOID IN TURTLES
SANTE NACCAKATI
PLATE 2
EXPLANATION OF FIGURES
3 Same. Fixation: Flemming’s fluid. Staining: Safranin-picrie acid. 1/15
imm., ocular 4. The two different aspects of the thyreoid cell, viz., the prin-
cipal and the colloidai celis, are distinctly shown. Some cells detached from
the epithelium are shown in the colloid.
4 Same. Fixation: Flemming’s fluid. Staining: Galeotti’s method. 1/15
imm., ocular4. Only a part of the section has been drawn, in order to demon-
strate the granules of secretion within the cell bodies.
5 Same. Fixation: Formalin. Staining: Haematoxyiin and eosin. Objec-
tive, Zeiss BB, ocuiar 4. An island of thymus substance was found in the
thyreoid of this animal and is shown in this section.
296
PLATE 2
THYEROID {N TURTLES
SANTE NACCARATI
297
Resumen por el autor, Charles E. Johnson.
Derivados branguiales en las tortugas.
El tema de este trabajo es el desarrollo de los derivados bran-
quiales de las tortugas, representadas por las formas Chelydra
serpentina, Chrysemys marginata y Trionix sp. El timo per-
sistente se origina en la porcién dorsal de la tercera bolsa visce-
ral, mientras que un brote transitorio aparece en conexién con
la porcion dorsal de la segunda bolsa visceral. Una paratiroides
se desarrolla en la porcién ventral de la tercera bolsa. La cuarta
bolsa no produce ningtin 6rgano persistente y las puebas de la
existencia de estructuras transitorias son dudosas. La quinta
bolsa origina una paratiroides persistente y, aunque faltan pruebas
directas, es posible que produzca algunas veces un timo rudi-
mentario.
En estados jévenes hall6é el autor con gran constancia un
diverticulo ultimobranquial bien desarrollado, situado a cada
lado del cuerpo, pero el del lado derecho est& generalmente
destinado a formar una estructura sumamente pequena, cuando
se compara con la del lado izquierdo, y en algunos casos dicha
estructura parece faltar por completo en estados mas avanzados.
Las bolsas cuarta y quinta y el diverticulo ultimobranquial se
diferencian a expensas de lo que al principio es una sola evagina-
cién de la pared faringea lateral. La quinta bolsa es de natura-
leza rudimentaria y durante un periodo considerable mantiene
conexiones celulares con la vesicula ultimobranquial. Esta se
caracteriza por la forma vesicular voluminosa que a menudo
exhibe. Al llegar la época de salir el embrién del heuvo ha
adquirido la estructura de un organo linfoide.
Translation by José F. Nonidez
Cornell Medical College, New York
AUTHOR’S ABSTRACT OF THIS PAPER ISSUED
BY THE BIBLIOGRAPHIC SERVICE, DECEMBER 27
BRANCHIAL DERIVATIVES IN TURTLES!
CHARLES EUGENE JOHNSON
Department of Zoology, University of Kansas
FIVE PLATES (TWENTY-FOUR FIGURES)
INTRODUCTION
Studies on the branchial derivatives of reptiles have been
confined chiefly to lizards and snakes. The most recent work
on these groups is that of St. Remy et Prenant (’03-’04). In
the former group, represented by the genera Anguis and Lacerta,
these authors found that a thymus body is formed by the second
and third gill pouches only. The derivative of the second pouch
is of variable size; whether it persists into the adult stage or
not they were unable to determine. The third pouch gives
rise also to a persisting epithelial body or parathyreoid. The
fourth pouch gives origin only to a transitory epithelial body.
The fifth pouch is of very rudimentary nature; it attains the
form of a small blind pocket which soon disappears without giv-
‘ing rise to derivatives of any kind. A right and a left ultimobran-
chial evagination is present in the early stages, but the left one
alone is destined to develop into a glandular organ; the right, as a
rule, very soon disappears entirely, but in one instance a rudi-
mentary ultimobranchial body was found on this side in an
embryo Anguis of 6 cm. length.
In snakes, represented by the genera Coluber and Tropido-
notus, a somewhat different condition was found. In this
group the first and second gill pouches give rise to rudimentary,
transitory thymus bodies, that of the second being the larger.
The third pouch likewise produces a transitory thymus bud very
similar to that of the first or the second pouch, but in addition
1 Technical assistance for a part of the present work was made possible through
the research fund of the University of Kansas.
299
JOURNAL OF MORPHOLOGY, VOL. 36, NO. 2
300 CHARLES EUGENE JOHNSON
there is formed a persisting parathyreoid body. The persisting
thymus is developed from the fourth and fifth pouches. In
Coluber, moreover, the fourth pouch gives origin to a persisting
parathyreoid, while a very rudimentary transitory body of this
kind, as a rule, is formed by the fifth pouch. In Tropidonotus,
on the other hand, the fifth pouch only exceptionally gives rise
to a parathyreoid, which likewise is of transitory nature. The
right ultimobranchial evagination does not disappear, as in the
lizards, but, like the left, undergoes progressive development
into a glandular organ. The two are symmetrically situated in
Coluber, but in Tropidonotus the position of the right one is
somewhat variable.
For the turtle group very little work appears to have been done
in connection with the branchial derivatives. A brief account
by van Bemmelen (’93) has reference to Chelonia viridis. Ac-
cording to this account, the earlier phases of the development of
the gill pouches correspond with the conditions in lizards and
in snakes, but in later stages there is greater similarity with the
processes in birds than with those of reptiles. Five pairs of
visceral pouches are recognized, of which the first three become
perforate, as does probably the fourth pair also. From the
second pouch arises an epithelial bud which develops into the
anterior lobe of the thymus; but the pouch itself becomes, as in
snakes, an isolated vesicle which is destined to disappear, as in
the case of the corresponding pouch in birds. The third pouch
becomes an expanded epithelial vesicle provided with numerous
secondary evaginations. It separates from the epidermal and
the pharyngeal epithelium, and the secondary evaginations
give rise to the thymous tissue, in the midst of which the central
epithelial cyst persists as a homologue of the ‘carotid body’
of lizards. The fourth and fifth visceral pouches arise simul-
taneously with the ‘suprapericardial’ (ultimobranchial) evagina-
tion, from a lateral ‘blinddarmférmigen Falte’ at the caudal end
of the pharynx (recessus praecervicalis), in the same way as in
snakes. These outpouchings soon become separated from the
pharynx and form a complex of three connected vesicles. If
these vesicles, says van Bemmelen, in their further development
BRANCHIAL DERIVATIVES IN TURTLES 301
were to proceed in the same manner as in the case of the serpents,
then the first two, which represent the fourth and fifth pouches,
must develop into thymous tissue, while the third and hindmost
remains epithelial. But this does not occur; all three maintain
an epithelial character, and even in much later stages are
found in this condition, situated between the aorta and the
pulmonary arch.
MATERIAL AND METHODS
The present study is based upon a series of sectioned embryos
varying in size from 4 mm., greatest length, to newly hatched
specimens. The stains employed were borax carmine and Lyon’s
blue. Wax-plate reconstructions of the structures involved
were made from specimens of Chrysemys of 10 mm., greatest
length, and of 8 mm. carapace length (ce. 1.); and from embryos
of Chelydra of lengths of 5 mm., 9 mm., and 9.5 mm.
I am indebted to Dr. B. M. Allen for material to supplement
certain stages in my own series.
The earlier part of the work unfortunately was undertaken
with very inadequate material and resulted in the erroneous
conclusion that the body closely associated during its develop-
ment with the ultimobranchial body was a derivative of the
fifth visceral pouch instead of the fourth. In the meantime there
appeared the work of Shaner? whose excellent models, especially
of a 9.5-mm. Chrysemys picta, leave no doubt as to the origin of
the body in question. While a corresponding stage is lacking
in my own material, I have a somewhat older specimen of Chely-
dra which proves the correctness of Shaner’s results.
THE VISCERAL POUCHES
The account of the earlier stages in the development of the
visceral pouches is based upon embryos of Chelydra and of
Chrysemys. The conditions in the two genera are essentially
similar, and selection is made from one or the other accordingly
2 R.F. Shaner, The development of the pharynx and aortic arches of the turtle,
with a note on the fifth pulmonary arches of mammals. Am. Jour. Anat., Nov.,
1921.
302 CHARLES EUGENE JOHNSON
as the more favorable stages are at hand to illustrate the suc-
cessive steps in the developmental processes. For the later stages
a number of specimens of Trionyx also are available. As in
lizards, the first gill pouch does not give rise to any parts of the
organs under consideration and may therefore be omitted from
further reference in this connection.
In an embryo Chrysemys of 4 mm. the first three visceral
pouches are clearly differentiated. Just behind the third pouch
is a fourth conspicuous evagination from the lateral pharyngeal
wall. In form this diverticulum is more rounded than the pre-
ceding pouches. Its lateral wall is flattened and has rather
broad contact with the ectodermal epithelium; and on its dorsal,
ventral, and posterior walls constrictions occur in the sections
by which the limits between the diverticulum and the pharyn-
geal wall proper appear clearly defined, but anteriorly these
limits may be recognized only in a general way. The size and
form of this evagination readily distinguish it from the typical
visceral pouch and in its walls appear no differentiations that
might indicate, as such, the developing fourth and fifth visceral
pouches or the ultimobranchial diverticulum. In this specimen
the first pair of the associated aortic arches is complete, but the
second and third pairs are visible in their dorsal portions only.
A somewhat more advanced condition is shown in a 5-mm.
Chelydra (fig. 1). The first three pouches have increased in
depth. The first and second are already perforate and the third
is nearly so. The second aortic arches are conspicuous, but
reach only about half way to the ventral aorta. The third arches
are now complete, as are also a very slender pair of fourth arches.
The diverticulum behind the third pouch has grown very con-
siderably and two distinct areas or divisions in its wall are
now discernible: an anterior larger part which is elongated in the
dorsoventral direction, parallel with the third visceral pouch,
and a second smaller part which appears as a diverticulum from
the first, pushing out from its posterior wall. The former is in
close contact with the ectoderm nearly throughout its length;
and on its inner surface, along this line of contact, it presents
a conspicuous furrow. In the light of subsequent stages, the
BRANCHIAL DERIVATIVES IN TURTLES 303
larger of these two secondary diverticula represents the develop-
ing fourth visceral pouch; the smaller one represents an early
stage in the differentiation of the diverticulum destined
to give rise to the ultimobranchial body. A fifth pouch cannot as
yet be positively identified (fig. 1, V, p. 5?), though potentially
present.
At this stage there is on each side, between the pharynx
proper and the diverticulum just mentioned, a slender blood
vessel which inosculates with both the fourth aortic arch and
the aortic root; it ends at the boundary line between the fourth
visceral pouch and the ultimobranchial diverticulum. ‘This
vessel is shown by later stages to be the fifth aortic arch.
The next available specimen is an embryo Chrysemys of 6.5
mm. (figs. 2, 3, and 13). In this embryo the fourth gill pouch
also is perforate. ‘The first three pouches, except for an increase
in size, reveal no new features requiring comment. The pharyn-
geal outpouching, which in the preceding developmental stages
embodied in one the fourth and fifth pouches and the ultimo-
branchial diverticulum, is now for the first time distinctly
differentiated into its three component parts. Furthermore, the
fifth visceral arch has arisen, interposing itself so as to separate
the fourth pouch anteriorly from the other two components
of the original vesicle. At its pharyngeal end the fourth pouch
is broadly continuous with the remaining portion of the vesicle.
The latter now consists largely of the ultimobranchial diver-
ticulum, the fifth pouch appearing to be merely an anterior, some-
what laterally projecting, secondary outgrowth. The complex
of diverticula as a whole has been constricted from the pharynx
so as to open into it through a short but still relatively wide
passage formed by the confluence of the mouth of the fourth
pouch with a very short common opening of the fifth pouch
and the ultimobranchial diverticulum. The ultimobranchial
pocket has a depth equal to about two-thirds the dorsoventral ex-
tent of the fourth pouch. It is somewhat elliptical, flattened
lateromedially, and its walls are thick. The fifth pouch is small
and that of the left side is developmentally more advanced
than its fellow. While the fifth pouch, as before remarked,
304 CHARLES EUGENE JOHNSON
appears to be a secondary diverticulum from the ultimo-
branchial pocket, this is evidently the result of the early
differentiation of the ultimobranchial vesicle, involving as it
does a relatively large area on the primary pharyngeal outpouch-
ing, one in which the diminutive fifth pouch is unable, as it
were, to express itself until at a somewhat later stage. The short
common passage or stalk previously referred to, by which the
fifth pouch and the ultimobranchial diverticulum join the fourth
pouch in opening into the pharyngeal cavity, evidently represents
originally a portion of the pharyngeal wall proper, and the
relations existing are consequently of secondary nature. The
rudimentary fifth pouch had been carried bodily out from the
pharynx by the ultimobranchial evagination.
In the fifth visceral arch at this stage there is a complete
aortic arch, which, about one-fifth of its distance from the
dorsal aorta, gives off a more slender posterior branch, the sixth
aortic arch; this, after making a loop about the fifth visceral
pouch, rejoins the fifth arch. The sixth aortic arch of the right
side is incomplete ventrally. The sixth aortic arch lies in the
angle formed by the ultimobranchial diverticulum and the fifth
pouch, the former being wholly medial to the vessel. »
The fifth visceral pouch is in intimate contact with the ecto-
derm along its lateral edge. The ultimobranchial outgrowth
nowhere touches the outer germ layer; its basal or proximal end
is opposite the origin of the trachea, and the distal end is directed
ventrally, parallel to the long axis of the fourth pouch.
In an embryo Chelydra of 7.5 mm. and one of 9 mm., the second
and third pouches are still perforate, and in the latter specimen
the fourth also is open. The pouches are much flattened antero-
posteriorly and their dorsoventral axes have increased consid-
erably in length. Because of the greatly narrowed ectodermal
and entodermal connections, the dorsal and ventral portions of
the second and third pouches appear in the sections as closed
vesicles and the posterior wall of the dorsal extensions of these
two pouches is now much thicker than the anterior wall. A
more pronounced advance, however, is apparent in connection
with the posterior complex of diverticula (figs. 4 and 5) where the
BRANCHIAL DERIVATIVES IN TURTLES 305
fifth visceral arch, by its increase in depth, has separated the
fourth visceral pouch more widely from the associated fifth
pouch and ultimobranchial body. Also a second process of
separation, proceeding simultaneously, is well under way, namely,
the pinching off of the complex as a whole from the pharynx by
the constriction of the common connecting stalk.
_ The fifth visceral pouches in these stages appear to attain
their full development as such. In the larger specimens the
right pouch is distinctly larger than the left, but in the smaller
the two are of about equal size. Contact with the ectoderm is
still maintained, but is more restricted than in the preceding
stage. On each side of the body a neck-like stalk connects the
fifth pouch with the ultimobranchial diverticulum. While,
as remarked, the right pouch in the larger specimen of Chelydra
is larger than the left, other specimens of this genus as well as
of Chrysemys indicate that there is considerable variation in the
comparative size of right and left pouches in different embryos.
In the 9-mm. Chelydra the long, or dorsoventral axis, of the larger
right pouch is about one-fourth that of the fourth visceral pouch.
The ultimobranchial body, beyond an increase in length
and the clearer demarcation noted above, exhibits no important
changes.
A notable feature in connection with the aortic arches at this
stage is that the middle segment of the fifth arch, or that which
forms the anterior limb of the loop, is exceeded in caliber, although
slightly, by the posterior limb or that which represents the s xth
aortic arch. In another embryo Chelydra of 7.5 mm., which in
other respects is in a corresponding stage of development, the
sixth aortic arch is already much larger than the fifth. In both
specimens the pulmonary artery is now present as a branch of the
sixth arch immediately above its junction with the fifth.
In a 9.5-mm. Chelydra, the second visceral pouch has lost
its connection with the eetoderm; its dorsal portion shows a
thickening of the epithelium which probably represents a transi-
tory thymus bud, disappearing with the closure of the pouch.
The ectodermal duct is a very much attenuated tube, but has
a longitudinal cellular ridge projecting into its lumen from its
medial wall (fig. 6).
306 CHARLES EUGENE JOHNSON
The third pouch also has severed its connection with the ecto-
derm and appears as an elongate, rather thick-walled longitudinal
vesicle, extending from the tip of the anterior horn of the hyoid
to a point opposite the middle of the posterior horn. The
cephalic end of the pouch lies medial to the anterior horn, while
the caudal end is lateral to the posterior horn. In length, the left
pouch extends through nineteen sections (285y), the right through
seventeen sections (255u). A very short pharyngeal stalk or
entodermal duct, now closed, extends through the sixth to the
eighth sections, inclusive, on the left and through the fifth to the
seventh on the right. On each side the pouch is crescentic in
cross-section (fig. 7), but anterior to the pharyngeal stalk the
convex side is ventral while posterior to the stalk it is dorsal.
The walls of the vesicle are generally of uniform thickness
anterior to the pharyngeal attachment, but here and there the
epithelium shows a tendency to fold, and at the anterior end
solid buds of cells have formed; likewise on the ventrolateral
surface of the vesicular wall there is a conspicuous ridge, formed
evidently by local proliferation, extending from the anterior end
of the pouch to its pharyngeal stalk. This ridge is symmetrical
on the two sides of the body and, together with the cell prolifera-
tion noted on the anterior wall of the pouch, is apparently the
beginning of thymus formation. Caudal to the pharyngeal
stalk the ventral wall of the pouch is decidedly thicker than the
dorsal, and from the dorsolateral wall there projects outwardly
a solid cellular peg which evidently represents the point of separa-
tion from the ectoderm.
The fourth visceral pouch is detached and far removed from
the surface epithelium. It is a small, more or less rounded
vesicle, with irregular surface contour and with slit-like cavity.
The ventrolateral wall is thickened, especially in its middle
portion. The entire vesicle extends through eight sections
(120u). It is attached to the ultimobranchial vesicle by a short,
narrow stalk which contains the last traces of a cavity. The
two sides of the body exhibit practically identical conditions.
A differentiation into thymus and parathyreoid portions is not
with certainty recognizable.
BRANCHIAL DERIVATIVES IN TURTLES 307
Regarding the fifth pouch, the gap in my series between the
present stage and the preceding is too great to indicate what has
taken place in the meantime. In the present specimen there is a
small mass of cells lying between the fourth pouch and the ulti-
mobranchial vesicle, just behind the point of connection between
these two; the mass has the appearance of undergoing degenera-
tion, and it is possible that it represents the remnants of the fifth
pouch.
The ultimobranchial body of the left side is typical for the stage
under consideration—an elongate tube lying lateral to and
parallel with the trachea. It is largest in its middle portion and
tapers more or less towards the ends. The walls are of uniform
thickness and the enclosed cavity is sharply defined. Proximally,
the vesicle narrows rapidly in approaching its connection with the
fourth pouch, and from this point on it becomes merely an at-
tenuated pedicle connecting the two vesicles as a unit with the
pharynx. Close to the entodermal wall this stalk is about to be
constricted off, but within it a pinhole cavity is visible.
The next step is based upon a 10.5-mm. Chelydra, a 6-mm.
Chrysemys, and a 9-mm. Trionyx. In Chelydra the third
visceral pouch has been transformed into an elongate, compact
mass. ‘The anterior two-thirds is considerably larger than the
caudal third and it contains a vestige of the original cavity,
around which the innermost cells retain in slight degree their
epithelial character. Anteriorly, and to a less extent in other
parts, the mass sends out a number of solid mounds of cells, which
give it a somewhat lobular appearance. The smaller caudal
mass is a continuation of the medial part only of the anterior mass.
It is cylindrical and, like the anterior part, contains a trace of
the earlier lumen. In brief, the conditions just described simply
mean that the third pouch at this stage shows definite differentia-
tion into an anterior thymus body and a posterior parathyreoid
body, representing, respectively, dorsal and ventral portions of
the original visceral pouch. In the specimens of Chrysemys
and of Trionyx the third pouch is developmentally slightly more
advanced, but otherwise it presents conditions similar to those
just described.
308 CHARLES EUGENE JOHNSON
The fourth pouch has by this time also developed into an
almost entirely solid body, club-shaped in form, the tapering end
directed forward and slightly marked off from the posterior part,
as if it represented a rudimentary thymus. The pouch extends
through eleven sections (175), the three middle sections alone
containing evidence of the former cavity. The caudal end is in
close proximity to the ultimobranchial body from which it ap-
parently has just become separated. The ultimobranchial vesicle
of the left side (fig. 9) shows a very considerable increase in size
and is expanded so as to be nearly circular in cross-section, but
it has the same smooth-walled appearance as in preceding stages.
In greater part the wall shows three or four tiers of nuclei, but
in some places there is only one. Its anterior extremity bears
a small cellular peg which evidently fixes the point of separation of
the fourth visceral pouch.
The conditions of the right side in this embryo deserve notice
in that there apparently is complete absence of the ultimobranchial
body; it is the only instance in my series where this occurs. A
slender cellular stalk, similar to that of the left side, extends from
the pharynx to the fourth pouch, to which it furnishes a short
pedicle, and then ends only three sections beyond this point,
without discernible evidence of an ultimobranchial vesicle.
However, the limits between what constitutes the ultimobranchial
vesicle proper and the part which represents more or less of the
drawnout portion of the pharyngeal wall cannot in any case be
exactly determined, and therefore, in view of the conditions found
in subsequent stages relative to the point of connection between
the fourth pouch and the ultimobranchial vesicle, it is still possible
that the latter is potentially present, though in a very rudimen-
tary form, in the distal portion of the entodermal stalk.
The embryo Chrysemys, in corresponding stage of develop-
ment, shows a condition of the branchial derivatives similar to
that of Chelydra, with minor variations. The second visceral
pouches have identical tube-like extensions (ectodermal ducts),
but these are without cellular buds or areas of proliferation. The
third pouch is somewhat more advanced. Its anterior portion is
a solid mass of more or less lobular appearance, the original
BRANCHIAL DERIVATIVES IN TURTLES 309
cavity having been obliterated as far back as the pharyngeal
stalk. Caudal to this point the pouch has still a conspicuous
lumen, but it becomes solid again in the posterior half. In its
entirety the third pouch does not exhibit such clear conditions as
in Chelydra, and it is uncertain from available material whether or
not any particular portion of its wall may be considered as
initiating the process of organ formation, such as appears to be
the case in Chelydra. In sections through the region of its
pharyngeal connection the vesicle has the same crescentic form
as in Chelydra and, on one side at least, the laterai wall is notice-
ably thicker, but, because of the solidification of the pouch ante-
riorly, the original relation or the significance of this thickening
cannot be determined. The fourth visceral pouch has a broader
connection with the ultimobranchial vesicle and the latter is
well developed on both sides of the body, although that of the left
is by far the larger.
In another 10.5-mm. Chelydra a variation in connection with
the fourth pouch and the ultimobranchial body should be noted.
On the right side a relatively large ultimobranchial vesicle
is present. It is spindle-shaped and extends through fifteen
sections, having a diameter in its widest part of approximately
one and a half times that of the trachea; its walls are thick
and the lumen clear-cut. Its anterior end lies just outside
the mesenchymal coat of the oesophagus and reaches the level
of the parathyreoid III. With this ultimobranchial body the
fourth pouch derivative as yet maintains a slender cellular
connection (fig. 11), but, instead of being situated at the anterior
end of the vesicle, where it is found in most cases, it here lies
at the posterior end. How this relation may have been brought
about is not evident, but it possibly may be accounted for by
assuming that, after the ultimobranchial vesicle had separated
from the pharynx, that portion of its neck proximal to the
junction of the fourth pouch, in which the limits of the ultimo-
branchial vesicle proper are indefinite, continued to develop,
while the part distal to the junction suffered regression or had,
perhaps, been rudimentary from the outset. On the left side of
_ the body the relations are of the usual kind. The ultimobranchial
310 CHARLES EUGENE JOHNSON
vesicle extends through twenty-five sections; while its walls
are thicker than in the preceding embryo—indicative, as a rule, of
an earlier stage— it shows a more advanced condition in that they
bear a number of secondary evaginations of various sizes as well
as numerous solid protrusions or sprouts. Both kinds are es-
pecially large and conspicuous about the anterior end of the
vesicle, while minor ones occur somewhat distal to its middle
section.
In a 6-mm. Chrysemys, representing approximately the same
developmental stage as the foregoing embryo, a further variation
with respect to the fourth visceral pouch and the ultimobranchial
vesicle occurs. The left fourth pouch has been converted into
a compact cellular mass with even surface contour and without
trace of lumen, and is attached in the usual manner by a solid
stalk near the anterior end of the ultimobranchial vesicle. The
last named, except for its smaller size, is similar to that of
the 10.5-mm. Chelydra. The right fourth pouch derivative is
much longer than the left (830 as against 240) and its middle
portion is expanded into a vesicle of nearly the same diameter as
the ultimobranchial vesicle itself (fig. 18), into which it opens by
a passage extending through five sections; and the ultimo-
branchial vesicle is unusually large for this side, beng somewhat
more than half the length and width of the left one.
A 9-mm. Trionyx is the youngest specimen of this genus n my
possession. In general development it agrees well with the
preceding specimen of Chrysemys. The derivatives of the third
visceral pouch reveal no noteworthy differences from those of
corresponding stages of Chrysemys or Chelydra; but the fourth
pouch derivative and the ultimobranchial vesicle show distinct
variations from the conditions in those genera. On the left side
the two bodies in question have the usual position relative to
each other and have a cellular connection, but in form they are
of somewhat different type. The ultimobranchial vesicle is
much more advanced in development than that of either Chry-
semys or Chelydra of corresponding age in that a large portion of
it has already been transformed into solid cord-like cell-clusters,
while elsewhere it bears spherical, hollow outgrowths from its
BRANCHIAL DERIVATIVES IN TURTLES Slt
walls. These growth processes have been most active in the
anterior portion of the vesicle, but are present in varying degree
throughout its length. In the midst of the proliferating mass,
however, the walls are sufficiently intact to show what had been
the general form and size of the vesicle at the height of its develop-
ment, and in these respects it bears closer resemblance to Chry-
semys than to Chelydra, as it evidently attains neither the large
size nor the thin-walled condition of the latter. The right
ultimobranchial vesicle is a thin-walled tubular structure whose
epithelium consists of one or two layers of flattened, loosely ar-
ranged cells, evidently in process of retrogression. The fourth
pouch derivatives are both characterized by a highly vesicular
condition, quite in contrast to the usual solid cellular mass in
corresponding stages of the other two genera, but a tendency
toward which was seen in the 6-mm. Chrysemys. The walls
_of these vesicles retain, in part, their early sharply defined epithe-
lial form, in part contain foldings and thickenings due to cell
proliferation. The tendency of the fourth pouch derivative in
Trionyx to assume a vesicular form occurs in later stages and
appears to be a distinctive feature of this genus.
Figure 14 represents a wax-plate reconstruction of the bran-
chial derivatives of the left side of an embryo Chelydra of 9.5-
mm. carapace length. The thymus and the parathyreoid III
maintain their earlier linear arrangement and partly encircle the
carotid artery. The fourth pouch derivative, still attached to
the ultimobranchial vesicle, lies medial to and occupies the inter-
val between the systemic and the pulmonary arch (the latter
omitted in the model). The ultimobranchial vesicle has attained
relatively enormous proportions, the maximal in my series,
having a diameter approximately one-half that of the oesophagus.
Only on its anterior and anterodorsal surfaces do the sections
reveal cellular outgrowths and extensions from the otherwise
smooth wall of the vesicle. Its fellow of the opposite side is
relatively insignificant and the fourth pouch of this side is also
much inferior in size and is furthermore completely detached from
the ultimobranchial body.
3i2 CHARLES EUGENE JOHNSON
An embryo Chrysemys and one of Trionyx of 8-mm. and
9-mm. carapace length, respectively, show a general develop-
mental stage corresponding to the preceding embryo Chelydra.
In Chrysemys the derivaties of the third visceral pouch together
form a more or less rounded three-lobed mass, partly encircling
the carotid artery from the dorsal side (on the left), or from the
medial side (on the right). Two larger anterior lobes constitute
the thymus, while the third lobe, smaller and situated posteriorly,
is the parathyreoid body, the two still having cellular continuity.
The parathyreoid here is lateral to the thymus instead of caudal,
as in Chelydra, possibly due to a growth or shifting caudad of the
thymus. The fourth pouch derivative and the ultimobranchial
body have the same relative positions asin Chelydra. The latter
body here likewise attains its maximal size as a vesicle, but is
relatively and absolutely much smaller and has the general form
of a cylindrical tube. The vesicle of the opposite side is
rudimentary.
In Trionyx the thymus and its associated parathyreoid III
have the same tandem arrangement as in Chelydra. The fourth
pouch derivative may lie against the medial side of the systemic
arch, or between this vessel and the pulmonary arch, opposite
the bifurcation of the trachea. On both sides of the body the
walls of this derivative are somewhat thickened, but maintain
an even epithelial arrangement about a relatively large central
cavity, as in the earlier 9-mm. stage. The right ultimobranchial
vesicle is very rudimentary; the left one is even smaller than that
of Chrysemys, and is profusely covered with cellular excrescences,
especially in its posterior portion.
LATER DIFFERENTIATION
In the well-advanced embryos just described the various
branchial derivatives have been identifiable, largely or entirely
by their respective histories and place relations. Actual struc-
tural differences in the thymus, the parathyreoids, and the
fourth pouch derivatives are, even in the oldest of these embryos,
wanting or at least uncertain in the sections. The form of the
dominant ultimobranchial vesicle renders this organ unmistak-
BRANCHIAL DERIVATIVES IN TURTLES ola
able, but, especially in Trionyx, on the side where the vesicle is
rudimentary, the fourth pouch derivative may at times assume
a very similar form, so that the two may be distinguished with
certainty chiefly by their relative position.
The following account is based upon an embryo Chrysemys of
11-mm. carapace length, one of 15-mm. carapace length, and one
at hatching; two embryos of Trionyx of carapace length of 9
mm. and 13 mm., respectively, and two of Chelydra of carapace
length of 15 mm. and 16 mm., respectively.
The thymus and parathyreoid bodies are now readily distin-
guishable from each other, both as to structure and staining
properties. The thymus has taken on the characteristic lym-
phoid appearance and stains deeply. The parathyreoid, on the
other hand, exhibits its usual cord-like, epithelial cell masses,
with invasions among them of mesenchymal tissue; these features
together with the relatively greater amount of cytoplasm in the
cells and their less deeply staining nuclei contrast this organ
sharply with the thymus. In regard to the relation of the thymus
to the carotid artery, Chrysemys and Chelydra are in accord and
differ from Trionyx. In the former two the artery is situated
laterally, having changed from an earlier, more ventral position.
In Trionyx the vessel courses along the medial surface of the
gland, but in an earlier stage it was near the ventral surface. In
both groups, if a large series were examined, a considerable
amount of variation would no doubt be found in the degree of
rotation of the thymus about the artery. The parathyreoids are
apparently also quite variable, within certain limits, as to their
position in the later stages. In Trionyx, where they are some-
what less advanced than in the other two forms, the organ of the
left side lies on the ventromedial, while that of the right lies on
the ventrolateral surface of the thymus, slightly anterior to its
caudal end. In Chrysemys the parathyreoid III is on the medial
side of the thymus, more or less deeply imbedded and separated
from the carotid by a considerable mass of thymous tissue;
in Chelydra its situation is lateral or dorsolateral upon the thymus
adjacent to the carotid in the younger specimen of this genus,
but in the older it is found to have been shifted somewhat and
314 CHARLES EUGENE JOHNSON
has become partly imbedded in the thymus (figs. 21, 22). Re-
garding the growth changes in the parathyreoid in these later
stages my series is too small to furnish definite answer, but from
measurements in Chrysemys it seems that, while the thymus
increases greatly, the parathyreoid III suffers a cessation or
retardation of growth in size between the stage of 15-mm. or
16-mm. carapace length and that of hatching.
In the embryo Chrysemys of 11-mm. carapace length the fourth
pouch derivative shows structural and staining characteristics
identical with those of the parathyreoid III. It lies somewhat
isolated from the derivatives of the third pouch and I find no
evidence of thymus tissue in connection with it on either side
of the body. The derivative of the fourth pouch, therefore,
at least from the evidence in this case, is a parathyreoid body
only, but it is quite possible that a thymus sometimes is developed
also. In the present specimen the parathyreoid IV has suffered
little if any change in position from that of this derivative of
earlier stages, being situated upon the dorsclateral surface and
slightly caudal to the anterior end of the ultimobranchial body;
lateral to it appears the posterior tip of the thymus III. The
parathyreoid IV of the right side, which is somewhat larger than
its fellow, still has the rudimentary ultimobranchial body at-
tached to its ventral surface. As stated in connection with the
9-mm. Trionyx, the fourth pouch derivative was inclined to be
more vesicular than in the other two genera during the early
stages, and the same tendency appears in the older embryos
now concerned. In the smaller of these (9-mm. ec. 1.) it has an
appearance not unlike that of the ultimobranchial vesicle, but is
smaller. The body of the right side especially is large and thin-
walled (fig. 17) and caudally has developed three secondary
out-pouchings from the main vesicle, giving to the whole still
more the character of an ultimobranchial body. In the older
embryo (13-mm. c. 1.) the bladder form is even more pronounced,
but here this feature may involve only a part of the entire organ.
Thus, on the left side, the fourth pouch derivative consists of a
ventromedial solid mass and a dorsolateral bladder-like portion
in which the wall is extremely thin and apparently in process of
BRANCHIAL DERIVATIVES IN TURTLES ary
disintegration, while on the right side there is a single much
enlarged cyst in which the dorsal and posterior walls alone bear
thickenings or proliferating cell masses (fig. 19). The walls of
-these bladder-like expansions of the fourth pouch derivative at
this stage do not, as a rule, possess the clear-cut epithelial ar-
rangement of their cells nor the smooth even contour of their
inner and outer surfaces which characterized the earlier stages.
The cells are notably crowded and jumbled, with here and there
dissociated cells intruding into the central cavity.
But while it appears that in Trionyx the fourth pouch deriva-
tive is characterized by the tendency to cyst formation from
its early stages and upward, a similar condition, and one which
was not foreshadowed in the last-described stage (9.5-mm.
ce. 1.) of this genus, occurs in the Chelydra embryos of 15-mm.
and 16-mm. carapace length (figs. 20, 23). The greatest develop-
ment of the vesicular portion is found in the smaller of the two
embryos, where it not only exceeds any of the corresponding
vesicles in Trionyx, but approaches closely the size of the larger
ultimobranchial vesicle in the same embryo. It will be observed
from the figures that only a part of the fourth pouch derivative is
involved in the cyst, the whole being, as in Trionyx, composed of
a glandular and a vesicular part. In the younger embryo the
glandular part lies upon the ventrolateral wall of the bladder
portion, while in the older specimen it lies upon the ventromedial
and the dorsolateral surface, of right and left sides, respectively.
At some points the cyst wall has reached a thinness bordering on
the breaking-point, where the cells form a single layer and as-
sume a mesothelial appearance. In all cases the cyst portion has
cellular continuity with the glandular body, although, as in
figure 20, the connection may at times be reduced to a very slender
stalk.
The significance of the vesicular portion of the parathyreoid
IV is not clear. As to its origin, however, it seems quite certain,
from the conditions observed in Trionyx, that it is a part of the
original cavity and wall of the fourth visceral pouch. The
question will suggest itself whether it may represent a portion of
the ultimobranchial vesicle which has separated, along with the
JOURNAL OF MORPHOLOGY, VOL. 36, NO. 2
316 CHARLES EUGENE JOHNSON
fourth pouch, and later manifests itself in the tendency to cyst
formation that is so characteristic of that body. Again, it
might conceivably be interpreted as a vestige of some other
derivative of the fourth pouch, such as a thymus. Opposed to
the first of these views, if not entirely to the second, is the fact
that in both embryos of Chelydra (15-mm. and 16-mm. ec. 1.),
although on the right side only, an exactly similar vesicle occurs
in connection with the parathyreoid III (fig. 21). In the younger
specimen the cyst is largely surrounded by thymous tissue. In
the later series of Chrysemys the parathyreoid IV gives no evi-
dence of cyst formation, but in an embryo of 15-mm. carapace
length parathyreoid III contains an excentric cavity of moderate
size whose wall is a single layer of cells, sharply differentiated
from the surrounding tissue. In an embryo at hatching there is
what appears to be a trace of such a cavity in the corresponding
gland; in the parathyreoid IV evidence of such condition is
doubtful.
The ultimobranchial body in all of the later stages mentioned,
except that of hatching, shows merely a continuation of the proc-
ess of reduction of the vesicle, begun in some of the younger
embryos. In the present older specimens the vesicle is either
completely broken down into a mass of diminutive vesicles and
solid cell masses more or less spherical or cord-like in form, as in
Chelydra of 16-mm. carapace length (fig. 23); or the main cyst
is studded with sprouts and is extensively broken up and reduced
in size, as in an embryo Chrysemys of 11-mm. carapace length.
The process of reduction and transformation of the original
vesicle apparently takes place, chiefly, by two methods: by the
formation through evagination and separation (and perhaps
also simply by constriction) from the main body, of smaller
cysts of varying sizes and forms, and by the outgrowth and de-
tachment from its wall of solid cellular sprouts. In the sprouts
the cells at first have a radial or epithelial arrangement in section,
and while in some of them an actual lumen may appear, in
others such is seemingly not the case. The secondary vesicles
undergo further reduction in the same manner as the parent
structure. In the older specimen of Chelydra (16-mm. ec. 1.)
BRANCHIAL DERIVATIVES IN TURTLES 317
and in Trionyx of 13-mm. carapace length the ultimobranchial
body assumes a structure resembling that of the thyreoid in the
same specimens, but, nevertheless, distinct and readily dis-
tinguishable from it by the complete absence of colloid within the
vesicles, by the comparatively small number of such vesicles or
tubules, as well as by their irregular form, thicker walls, and
often ill-defined lumina. A rudimentary right ultmobranchial
body is present in all of the later stages described and it under-
goes parallel differentiation with that of its much larger fellow.
In Chrysemys at the time of hatching the ultimobranchial
body has assumed an appearance very much like that of the
parathyreoid of the same embryo, namely, a rather lightly
staining lymphoid structure in which traces of the earlier ar-
rangement and grouping of the cells are clearly recognizable
only in a few places. The position of the body remains the same.
Deeply imbedded within the left ultimobranchial body lies the
parathyreoid IV, which, however, is surrounded by a thin con-
nective-tissue capsule of its own. The ultimobranchial body of
the right side consists of a small mass of tissue on the medial
side of and partly investing the parathyreoid IV; in structural
differentiation it is like its fellow of the opposite side.
SUMMARY
1. The development of the branchial derivatives was studied
in turtles of the genera Chelydra, Chrysemys, and Trionyx.
2. The persisting thymus arises from the dorsal portion of the
third visceral pouch. In the corresponding portion of the second
visceral pouch there is a cellular bud which is interpreted as a
rudimentary, transitory thymus. |
3. The ventral portion of the third visceral pouch gives origin
to a persisting parathyreoid.
4. The fourth visceral pouch gives rise to a persisting para-
thyreoid, but so far as available material indicates there is
no indisputable evidence that a persisting thymus arises from
this pouch. A rudimentary thymus which is transitory probably
occurs.
318 CHARLES EUGENE JOHNSON
5. The fifth visceral pouch seems to disappear soon after it
attains its greatest development, which in Chelydra was found to
be in embryos of 7.5 mm. to 9 mm. greatest length.
6. A conspicuous ultimobranchial vesicle is usually present
on each side in the early stages, but the one on the right, as a rule,
soon reaches limitations in growth and becomes greatly exceeded
in size by its fellow of the opposite side. The body on the
right may apparently at times be wholly lacking. Where both
are present, they appear to undergo parallel differentiation, at
least up to the time of hatching. The relatively huge dimen-
sions sometimes attained by the dominant ultimobranchial
vesicle is a striking feature.
7. In turtles the fourth and fifth visceral pouches and the
ultimobranchial diverticulum originate in a single conspicuous
evagination from the lateral pharyngeal wall. In this evagina-
tion the fourth pouch is the first to be differentiated; next ap-
pears the ultimobranchial diverticulum, and lastly the fifth pouch
may be distinguished, which is closely associated with the ultimo-
branchial diverticulum and is very small.
8. The fourth pouch and the ultimobranchial diverticulum
become separated as a unit from the pharynx, but remain con-
nected with each other until a comparatively late stage in their
development.
9. The fourth pouch in subsequent development exhibits
more or less of a tendency toward cyst formation. This seems
to be manifested earlier in Trionyx than in the other two genera
studied; but in later stages very large cysts, relatively speaking,
were found in connection with the fourth pouch in Chelydra.
In Chelydra such tendency was observed also in connection with
the parathyreoid III. The significance of the cysts is not
clear.
10. At the time of hatching the thymus is a rather voluminous
body of oblong shape, and parathyreoid III is a relatively small
rounded body which is more or less deeply imbedded in the
caudal portion of the former gland.
11. Parathyreoid IV, similar in size and shape to the para-
thyreoid III, is usually found in close association with or partly
BRANCHIAL DERIVATIVES IN TURTLES 319
or wholly imbedded in the ultimobranchial body (on the left side) ;
or (on the right side, where the ultimobranchial body is rudi-
mentary) it may be adjacent to parathyreoid III, and with it
becomes partly surrounded by thymous tissue; or it may lie
further caudad in association with the ultimobranchial body.
12. The ultimobranchial body, by the time of hatching, has
been transformed almost completely into a lymphoid organ,
resembling the parathyreoids at this stage. It is in no way
associated with the thyreoid.
BIBLIOGRAPHY
BEMMELEN, J. F. van 1893 Ueber die Entwicklung der Kiementaschen und
der Aortenbogen bei den Seeschildkréten, untersucht an Embryonen
von Chelonia viridis. Anat. Anz., Bd. 8.
1886 Die Visceraltaschen und Aortenbogen bei Reptilien und Végeln.
Zool. Anz., Bd. 9.
Jounson, C. E. 1918 The origin of the ultimobranchial body and its relation
to the fifth pouch in birds. Jour. Morph., v. 31.
LiessnerR, FE. 1888 Hin Beitrag zur Kenntnis der Kiemenspalten und ihrer
Anlagen bei amnioten Wirbelthieren. Morph. Jahrb., 13.
Maurer, F. 1899 Die Schildriise, Thymus und andere Schlundspaltenderivate
bei der Eidechse. Morph. Jahrb., 27.
Meuron, P. pe 1886 Recherches sur le développement du thymus et de la
glande thyreoide. Dessertation, Geneve.
Peter, K. 1900-01 Mittheilungen zur Entwicklungsgeschichte der Hidechse.
II. Die Schlundspalten und ihrer Anlage, Ausbildung und Bedeutung.
Arch. f. mikr: Anat., Bd. 57.
Saint-Remy ET PRENANT 1903-04 Recherches sur le développement de dérivés
branchiaux chez les Sauriens et les Ophidiens. Arch. de Biol., T. 20.
Verpun, P. 1898 Dérivés branchiaux chez les vertebrés supérieurs. Thése,
Toulouse.
ABBREVIATIONS
A.a., aortic arches S.a., systemic arch
Ar.car., carotid artery Thy., thymus
Ao.r., aortic root Thr., thyreoid
Br., bronchus Tr., trachea
D.ao., dorsal aorta U.b., ultimobranchial vesicle or body
Oes., oesophagus Vag., vagus nerve
Par. III, IV, parathyreoids, derived V.a.4, fifth visceral arch
from the third and fourth pouches, Ves., vesicular portion of parathy-
respectively reoids
Ph., pharynx V.p. 2, 3, 4, , visceral pouches, second
Ph.div., pharyngeal diverticulum to fifth
PLATE 1
EXPLANATION OF FIGURES
1 Frontal section through the posterior pharyngeal region of an embryo
Chelydra serpentina 5 mm. long. X 80.
2 Frontal section through the corresponding region of an embryo Chry-
semys marginata 6.5 mm. long, showing developing fifth visceral arch and the
differentiated fourth and fifth visceral pouches. X 80.
3 Same embryo as figure 2, section taken farther ventrally, showing relation
of fifth pouch to ultimobranchial vesicle. X 80.
4 Frontal section from an embryo C. serpentina 7.5 mm. long, passing
through the main body of the ultimobranchial vesicle and showing also progres-
sive development of the fifth visceral pouch. X 80.
5 Frontal section from an embryo C. serpentina 9 mm. long, showing
connection between fourth visceral pouch and ultimobranchial vesicle and also
a further step in development of the fifth visceral pouch and corresponding vis-
ceral arch. X 80.
320
BRANCHIAL DERIVATIVES IN TURTLES PLATE 1
CHARLES EUGENE JOHNSON
321
PLATE 2
EXPLANATION OF FIGURES
6 Transverse section through the ectodermal duct of the second visceral
pouch in an embryo Chelydra 9.5 mm. long. X 300.
7 Section through the third visceral pouch of the same embryo as figure 6,
showing early step in the development of the thymus. X 230.
8 Transverse section through the fourth pouch derivative of an embryo
Chelydra with carapace 9.5 mm. long. X 300.
9 Transverse section through the left ultimobranchial vesicle of an embryo
Chelydra 10.5 mm.long. X 230.
10 Transverse section through the developing thymus of the right side of an
embryo Chelydra 10.5 mm. long. X 230.
11 Transverse section through the fourth pouch derivative and the ultimo-
branchial body of the right side of an embryo Chelydra 10.5 mm. long. X 300.
12 Transverse section through a part of the left lateral wall of the left ultimo-
branchial vesicle and attached fourth pouch derivative, from an embryo Chelydra
with carapace 9.5 mm. long. Same embryo as figure 8. X 300.
322
BRANCHIAL DERIVATIVES IN TURTLES PLATE 2
CHARLES EUGENE JOHNSON
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PLATE 4
EXPLANATION OF FIGURES
17 Transverse section through the fourth pouch derivative and the ultimo-
branchial vesicle of the right side of an embryo Trionyx sp. with carapace 9 mm.
long. X 2380.
18 Transverse section through the same structures of the right side of an
embryo Chrysemys 6 mm. long. X 230.
19 Transverse section through the fourth pouch derivative of the right side
of an embryo Trionyx with carapace 13 mm. long. X 230.
BRANCHIAL DERIVATIVES IN TURTLES PLATE 4
CHARLES EUGENE JOHNSON
PLATE 5
EXPLANATION OF FIGURES
20 Transverse section through the parathyreoid IV and the caudal portion
of the thymus of the right side of an embryo Chelydra with carapace 16 mm. long.
x 50.
21 Same embryo. : Section through parathyreoid III and the thymus of the
right side. X 50.
22 Same embryo. Section taken farther anteriorly than that of figure 21,
showing thymus and parathyreoid III of left side. > 50.
23 Transverse section through the ultimobranchial body and the para-
thyreoid IV of the left side; from same embryo as figures 20 to 22. X 75.
24 Transverse section through the left ultimobranchial body and the para-
thyreoid IV of an embryo Chrysemys at hatching. X 50.
(Jy)
No
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BRANCHIAL DERIVATIVES IN TURTLES ; PLATE 5
CHARLES EUGENE JOHNSON
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Resumen por el autor, Horace W. Stunkard.
Los neurémeros primarios y la segmentacién de la cabeza.
La literatura sobre la segmentacion de la cabeza de los verte-
brados presenta grandes diferencias, tanto de observacién como
de interpretacién. Después de publicada una serie de trabajos
antiguos sobre los neurémeros, Locy (’95) y Hill (00) han de-
scrito la segmentacién primaria del sistema nervioso de Ambly-
stoma, Squalus, el pollo y otras formas. Los investigadores han
dudado a menudo de la exactitud de las observaciones de Locy
y Hill, y una repeticién de su trabajo, usando medios de eximen
tan semejantes a los suyos como es posible, demuestra que los
“neurOmeros primarios” no pueden considerarse como meta-
méricos. Las divisiones mediales observadas en la placa neural
de Amblystoma y consideradas por Griggs (10) como neuré-
meros verdaderos se deben en gran parte, si no totalmente, a la
segmentacién del mesodermo, y por consiguiente deben con-
siderarse tan solo como rasgos de importancia secundaria. Los
neurdmeros primarios de Locy y Hill, asi como los de Griggs y
otros autores que han estudiado el neuromerismo, son de tamano
irregular, en ntimero inconstante, de posicién asimétrica y no
pueden servir como criterio bien establecido de la metameria de
la cabeza de los vertebrados.
Translation by José I’. Nonidez
Cornell Medical College, New York
AUTHOR’S ABSTRACT OF THIS PAPER ISSUED
BY THE BIBLIOGRAPHIC SERVICE, JANUARY 16
PRIMARY NEUROMERES AND HEAD SEGMENTATION
HORACE W. STUNKARD
New York University
TWENTY FIGURES
The problem of the segmentation of the vertebrate head, old as
Oken and Goethe, has possibly attracted as much interest and
incited as much investigation as any other one question in
vertebrate morphology. The first investigators advanced a
theory based upon superficial external features the sutures of
the skull. Subsequent workers have investigated every struc-
ture enclosed within the skull. with the hope that light may be
thrown upon the obscurity and uncertainty enveloping the evolu-
tion of the head. This complex, intricate structure manifests
evidences of past ages; remnants and vestiges of the long period
of developmental history still persist, but the character of the
evidence, the complications, omissions, and reversals have
baffled all attempts at solution.
Huxley (58) overthrew the vertebral theory of the skull,
Balfour (’78) introduced mesodermal head cavities as criteria of
segmentation and clues to the number and relationship of the
cephalic somites, and Gegenbaur (’87) added cranial nerves and
visceral arches as segmental criteria. Van Wijhe (’86), (’89) con-
sidered the dorsal ganglia of importance and formulated criteria
to determine the true segmental nerves. He regarded the
olfactory and optic nerves as parts of the brain and not of seg-
mental value.
Von Baer (’28) noticed symmetrical folds in the hindbrain of
the chick; Dohrn (’75) related them to the mesodermal somites,
and Beraneck (’84) to the cranial nerves. Balfour reported that
the first gives rise to the cerebellum, and considered it doubt-
ful whether the other constrictions have any morphological
331
JOURNAL OF MORPHOLOGY, VOL. 36, NO. 2
BE HORACE W. STUNKARD
importance. Von Kupffer (’85) observed a ‘primary metamerism’
in the neural tube of Salamandra atra embryos which appeared
before the segmentation of the mesoderm, and Orr (’87), studying
the embryology of the lizard Anolis, noticed a number of symmet-
rical constrictions in the lateral walls of the hindbrain, “giving
the walls in horizontal section an undulated appearance.”
Kupffer called these ‘medularfalten’ and Orr adopted for them
the name neuromeres. This author formulated the first criteria
for determining the identity of the neuromeres. He described
two in the primitive forebrain, one in the midbrain, and six in
the hindbrain. McClure (’90), working on embryos of Amblys-
toma punctatum, Anolis sagroei, and the chick, found ‘‘a con-
tinuous and symmetrical series of neuromeres increasing in
size anteriorly, which extend from the lateral walls of the em-
bryonic brain, throughout the entire length of the neuron.” He
believed that the primary forebrain contained two neuromeres,
that the midbrain consisted of two neuromeres, and that the
third and fourth nerves were the nerves of these somites. Froriep
(91) found neuromeres prior to the segmentation of the meso-
derm, but did not attach any segmental importance to them, and
later (’92) decided they were the results of underlying meso-
blastic somites. He found the constrictions in the median
part of the cephalic plate, while the neural tube is still open,
four in Salamandra maculosa and five in Triton cristatus.
Waters (’92) confirmed the observations of McClure, and found
three segments in the forebrain. Eycleshymer (’95) observed
certain markings in the neural folds which might be interpreted
as neuromeres, yet he noted that their arrangement was decidedly
irregular and the structures were probably due to the action of
killing reagents. The transverse markings in the neural plate
he regarded as due to the formation of the myomeres.
The tendency to regard the neuromeres as segmental struc-
tures reached a definitive stage with the work of Locy (795).
This author reviewed the work on neuromeres exhaustively.
He made observations on Squalus acanthias, Amblystoma,
Diemyctylus, Rana palustris, Torpedo ocellata, and the chick.
In all these forms he described neuromeres in very early stages,
PRIMARY NEUROMERES AND HEAD SEGMENTATION 333
as soon as the neural folds are established, and before there is any
division of the mesoderm into protovertebrae. In the open
neural groove, the neuromeres of the hindbrain are, he stated,
merely the more apparent constrictions of a neuromerism that
involves the entire neural plate. He traced the neuromeres to
the anterior end of the medullary groove and those earliest
formed without a break into the later stages, identifying them
with the neuromeres of the closed neural tube. In the chick he
described neuromeres visible in the blastoderm of the twelfth
hour of incubation, and stated that this segmentation extends into
the primitive streak. In Amblystoma, at the stage with a
broadly expanded neural plate and widely open neural groove,
he found “the neural folds divided throughout their length into
a series of segments with no especial distinguishing features
between those of the head and those of the body region. The
median plate included between the neural ridges is smooth at
this stage; at a slightly later period, however, while the groove is
still widely open, the median plate exhibits very faint trans-
verse markings.” He pointed out that these median divisions
do not correspond with those in the neural ridges, and he attached
no morphological significance to them. He claimed that in all the
forms studied “The cells in the neural segments are character-
istically arranged, even in the earliest stages, and their arrange-
ment and structure would indicate that they are definite differen-
tiations of cell areas, not merely mechanical undulations.”
Locy summarized his work on neuromeres by stating that they
cannot be artifacts, that they arise before there is any segmental
division of the mesoderm, and so cannot be dependent upon the
latter. He concluded that neuromeric segmentation is more
primitive than mesodermic segmentation, and for this reason
may well serve as a basis for the study of the segmentation of the
head.
Neal (’98) was unable to verify Locy’s statements in Squalus.
He found the edges of the plate slightly and irregularly lobed,
but the lobes on the opposite margins of the plate did not corre-
spond either in number or position, nor did they show any definite
relation to the mesodermal somites. Regarding these ‘segments’
334 HORACE W. STUNKARD
as the results of unequal growth along the margin of the neural
plate, he contended that “‘it is obviously not necessary to regard
such irregularities of the edge of a rapidly expanding plate of
tissue as of morphological importance. <A disassociation of cells
or rapid proliferation of cells, which certainly does occur in this
region, would lead to such phenomena.”’ Neal found it im-
possible to trace definite segments into the later stages, for in
these stages, before the closure of the neural tube, in the majority
of specimens little or no evidence of segmentation along the
cephalic plate could be seen. In Squalus acanthias he found the
posterior boundary of the cephalic plate coincides with the pos-
terior boundary of encephalomere VI, opposite which the auditory
invagination takes place. Showing discrepancies in Locy’s
statements regarding the position of the auditory vesicle and the
posterior limit of the cephalic plate, Neal says, “‘I can see no
escape from the conclusion that he (Locy) has not traced neural
segments accurately up to the time they form neuromeres.”
Furthermore, Neal warned against formulating conclusions
from observation of a single organ system and applying them to
the phylogenesis of the vertebrate head. He contended that
primitively there existed a correspondence between neuromerism,
mesomerism and branchiomerism, and the problem of phyletic
cephalogenesis is to explain the present. lack of correspondence.
In Squalus he found five mesomeres alternating with six neuro-
meres in the otic and preotic region.
Hall (1900), working on Salmo and chick embryos, confirmed
the statements of Locy. He reported complete agreement as
regards the number and position of the neural segments in the
trout and chick embryos. The forebrain has three and the
midbrain two segments which, in the earliest stages, do not
differ in any essential features from those of the medulla. They
antedate the historic divisions, forebrain and midbrain, and
precede the optic evaginations. The primary neuromeres were
constantly and normally present in the early stages of all the.
embryos examined by him. Speaking of the external and corre-
sponding internal constrictions which separate the segments, he
says that in the early stages these grooves encircle the encephalon,
PRIMARY NEUROMERES AND HEAD SEGMENTATION 335
but in later stages the primary segmentation is confined to its
base and lateral walls, owing to the neural expansion and the
appearance in the dorsal region of a thin roof. He pointed out
that in the position occupied by the third, fifth, and sixth seg-
mental grooves, deep interna! constrictions appear that form the
posterior limits respectively of the forebrain, midbrain, and
cerebellum; that all the primitive grooves disappear during
embryonic growth, those of the forebrain first, those of the mid-
brain second, and lastly those of the hindbrain. In the chick,
when the neural folds close to form the neural tube, the walls of
the latter expand, not uniformly, but intrasegmentally, and the
position of the internal grooves is thus passively elevated upon
crests. Contrary to the statement of Locy, he found that in
younger embryonic stages of the chick and also of the trout, the
histology is very simple, the radial arrangement of cells is absent,
the nuclei do not recede intrasegmentally from the inner surface
of the brain but are uniformly distributed. In these stages he
reports that the only criteria by which he has counted neural
segments were external and corresponding internal grooves.
Concerning the value of various segmental criteria, Hill stated
that mesomeres are found only in elasmobranchs, amphibians,
and reptiles, and added that in elasmobranchs, the only group
in which their development has been traced, their study has
led to a greater divergence of opinion and more conflicting views
than is generally supposed. He dismissed branchiomeres with
a quotation from Minot that the gill clefts are not segmental
and concluded therefore that the branchial nerves are not in
segmental order. He argued that Neal’s (’98) conclusions were
based on negative evidence and that he had observed the seg-
ments where Neal failed to find them.
Johnston (’05) accepted the number of neuromeres described
by Locy as necessary to account for all the nerves and sense
organs connected with the brain, and stated that observations,
then incomplete, on embryos of Amblystoma punctatum seemed
to confirm Locy’s work. He contended that the nervous system,
acting in the réle of a connecting and coérdinating system, might
well act as a key for the interpretation of the facts secured by a
study of the other structures.
336 HORACE W. STUNKARD
Von Kupffer (’06) reviewed the work of Locy and Hill and
maintained that the question is still unanswered. Concerning
Hill’s work on the neuromeres of the chick, he says, “‘ Mit einiger
Ueberraschung werden wohl allgemein die Abbildungen auf-
genommen worden sein, mit denen Hill seine Beobachtungen
iiber die Primiiren Neuromeren bein Hiihnchen belegt. Es
macht den Eindruck, als wenn das subjective Moment die
Fiihrung des Zeichenstiftes doch wohl etwas zu stark beeinfiusst
hitte’’; and (p. 248) ‘Ich kann diese Angaben, mangels gleich
ausdehnter Beobachtungen, zwar nicht bestatigen aber ich will
sie nicht beanstanden.”’ Neal (714) translated ‘mangels’ in
this last sentence to mean ‘in spite of,’ which somewhat alters the
original meaning.
Filatoff (07) argued that neuromeres are mechanical results,
due to growth in a restricted space. He rejected Hill’s contention
that neuromeres are the chief and only certain criteria upon
which to build a judgment concerning the primitive metamerism
of the head, and agreed with Neal (98) and Koltzoff (’01) that
the proper method by which to attack the problem is to establish
an agreement between the neural segments and the somites,
nerves, and gill clefts.
Wilson and Hill (’07) could not accept the conclusions of Locy
and Hill, and maintained that Hill had not adequately met the
contention of Nea! (’98).
Belogolowy (10) maintained that the neuromeres are only
form changes of uncertain nature and irregular appearance,
possibly the results of mechanical factors, and that they are of
most uncertain value as criteria of the segmentation of the head.
Griggs (10) sought again to establish neuromerism as a basis
for determining the segmentation of the head. He described
four neuromeres in the procephalic part of the open neural plate
of Amblystoma embryos, and in a few specimens of later stages
noted neuromeres which appear posterior to the four procephalic
lobes, but the history of these posterior neuromeres could not be
traced nor their number or arrangement determined. He agreed
with Locy that in the early stages the plate and neural crests
are net segmented in the same way; he found occasional slight
PRIMARY NEUROMERES AND HEAD SEGMENTATION Bee
beadings or lobes on the neural crests, but did not regard them
as of morphological importance. These lateral lobulations he
found vary both in number and arrangement and as the neural
crests close over the plate all signs of segmentation behind the
procephalic lobes disappear. He described three distinct grooves,
the anterior and posterior germinal depressions and the ‘blasto-
groove’ which appear in the location later occupied by the
neural groove. Griggs stated that the primary neuromeres
described by Locy were not apparent in any of the embryos
which he examined. He concluded that the median transverse
grooves separate the true neuromeres, that the first contributes
to the formation of the forebrain, the second and third to the
formation of the midbrain, and the fourth to the formation of the
anterior part of the cerebellum.
Smith (12) described grooves which appeared very early in
the neural place of Cryptobranchus embryos, one regularly
antedated the others, and this I believe corresponds to the
transverse cephalic groove of Griggs. Anterior to this groove he
noted six transitory furrows and posterior to it an undetermined
number, but expressed the suspicion that these grooves are
connected with the formation of the mesodermal somites. In
early stages of the formation of the neural folds he observed
occasional transverse grooves, but stated that they are often
irregular and bear no definite relation to the segments of the neu-
ral plate. He argued that the true segments are to be found
between the transverse grooves of the neural plate, and pointed
out that in the region of the mesodermal somites the transverse
grooves of the plate are in line with the intersomitic grooves and
the neuromeres are in line with the somites. He was unable to
follow the various structures of the neural plate into the defini-
tive divisions of the embryonic and adult brain.
_ Graper (713) reviewed the literature on neuromeres exten-
sively; he criticised Hill’s ‘‘iiberraschenden und von niemand
bestitigten Zeichnungen”’ and was unable to confirm his obser-
vations in the chick.
Neal (14) argued that the hindbrain neuromeres manifest a
segmentation that cannot be ‘explained upon purely mechanical
338 HORACE W. STUNKARD
grounds, but contended that the differences in observation and
the divergent conclusions of investigators who have examined
neuromeres militate against the confidence of Loecy, Hill, John-
ston, Griggs, and others who hold that through the study of
neuromerism the primitive segmentation of the head will be
ascertained. Quoting Dr. Bashford Dean, he stated that in the
forebrain of Bdellostoma there appear two, three, or four neuro-
meres on one side or the other, never paired; in the midbrain
there is any number from one to eight; while in the hindbrain the
number varies from three to twenty-four, differmg in number on
different sides, a difference of ten having been noted in the right
and left sides of the same individual. He added that he had
examined hundreds of Squalus embryos in an attempt to con-
firm Locy’s results, and only two or three showed symmetry or
regularity in the segmentation of the edges of the neural plate,
while the beaded thickenings were not only asymmetrical, but
quite variable in different specimens. He maintained that the
primary brain vesicles and not their secondary subdivisions are
homologous with the hindbrain neuromeres and that the corre-
spondence in number of primary brain vesicles, myotomes, and
cranial nerves argues strongly for the metameric value of these
structures.
Smith (’14) observed transverse markings in the procephalic
plate of Desmognathus fusca embryos which were very transient,
varied in position in different individuals, and which he was
unable to trace through from one stage to another in living
specimens. In the posterior part of the plate the markings were
more uniform, persisted longer, and were subject to but slight
variation. In some specimens they corresponded closely to
the outpocketings in the medullary folds, but not in other
individuals. He. also described plications in the medullary
folds which appeared early and persisted until fused and ab-
sorbed in the expanding prosencephalon. These lateral irregu-.
larities did not correspond to the median grooves and he ascribed
their formation to mechanical factors. He pointed out that it
would be easily possible to select from the material a series which
would show a uniform development and fate of these foldings, but
PRIMARY NEUROMERES AND HEAD SEGMENTATION 339
after the examination of a large number of specimens decided
that they had no definite significance or fate. He regarded the
folds in the medullary plate as normal, but not constant, and no
evidence was found in the cephalic portion of the plate of divisions
to which a segmental value should be assigned.
Neal (18) has admirably summarized the evidence for and
against the metameric importance of neuromeres. Adducing
evidence from a thorough study of the problem of head develop-
ment, he contends that the neuromeres of the spinal cord are
passive results of the mechanical pressure of the adjacent meso-
dermic somites; that the rhombomeres have arisen in correlation
with the visceral arches with which they are functionally con-
nected; and that the only structures anterior to the medulla
which may be considered as segmental are the primary forebrain
and midbrain segments.
A number of other investigators have worked on different
phases of the head-segmentation problem and a more extended
review of the literature may be found in the bibliographies of the
papers cited here.
It is apparent from the disagreement in the results of former
investigators that the nature, number, and significance of the
neuromeres are far from determined. While neuromeres have
been frequently observed in many animals, and widely dis-
cussed, the conception of their value as segmental criteria has
been largely developed by Locy, Hill, Johnston, and Griggs.
It is extremely difficult, if not impossible, to correlate the obser-
vations and interpretations of the various authors, but the
repeated observation as to some kind of division in the neural
crests and open neural plate is sufficient to warrant further in-
vestigation. At the suggestion of Prof. J. 8. Kingsley, the writer
has studied the early stages in Amblystoma and the chick. The
work was begun in 1914 and earried on for two years in the
zoological laboratory of the University of Tllinois. It was
interrupted for two years because of military service, but was
continued and completed in the biological laboratory of New
York University. The writer wishes here to express to Professor
Kingsley his appreciation for the many helpful suggestions
340 HORACE W. STUNKARD
received in the course of the study. An attempt was made to
determine whether in Amblystoma a segmentation of the neural
crests is regularly and uniformly present, and to compare this
division with that of the neural plate. Further, to determine,
if possible, which, if either, is of metameric significance. The
persistent doubt regarding the accuracy of the observations of
Loecy and Hill on chick embryos makes a reinvestigation and
confirmation of their work very desirable.
The study of Amblystoma was made upon several hundred
embryos, collected near Champaign-Urbana, Illinois. The entire
series of changes involved in the formation and closure of the
neural tube was repeatedly observed under the binocular. To
make more careful observation, parts of the neural crests and
medullary plate were dissected and observed from all angles and
with various means of illumination. For material to supple-
ment the study of living specimens, embryos at all stages of
development from the wide-open to closed neural tube were
killed in various fluids and sections were cut in transverse,
frontal, and sagittal planes.
In Amblystoma, as the blastopore narrows to a small oval
structure, a distinct longitudinal groove forms anterior to it.
In a few specimens the groove appears to extend to the lip of the
blastopore, but in the large majority of embryos when the groove
is first formed a short distance separates it from the blastopore.
In some embryos the groove extends anteriorly and posteriorly
in a continuous manner, so that with the closing of the blastopore,
it forms the definitive neural groove. In other specimens,
however, another faint groove, usually shorter than the first,
may appear anterior to it. This observation agrees with that of
Griggs (10), although the appearance of the grooves does not
show the regularity or constancy reported by him. The first of
these grooves he termed the posterior germinal depression and
that anterior to it the anterior germinal depression. The groove
formed by the concresence of the lateral lips of the blastopore
he called the blastogroove. There is considerable variation in
the uniformity and regularity with which these grooves appear,
often separate germinal grooves are entirely absent and the
PRIMARY NEWROMERES AND HEAD SEGMENTATION 341
neural groove forms without the previous appearance of separate
depressions. Griggs stated that it is sometimes impossible to
distinguish between the anterior depression, posterior depression,
and neural groove. The depressions described by Griggs appear
in the same position as the neural groove, and I see no good
reason for considering them as anything other than stages in
the formation of the neural groove itself. The anterior end of the
neural groove is marked by a depression, the anterior pit of
Griggs, which becomes deeper, extends anteriorly, and, with only
slight variations in the process of development, becomes the
infundibulum. In these early stages it is sometimes possible to
distinguish in the neural groove faint alternating lighter and
darker areas, which in a few specimens suggest a segmental
condition, but observation of a large number of embryos shows
such Variation and irregularity as to preclude such an interpre-
tation.
Lateral longitudinal depressions at the sides of the neural
plate could not be distinguished before the appearance of the
neural crests. With the thickening of the ectoderm to form the
crests, these structures are slightly elevated and a lateral
linear depression is visible, not only on the median, but often
also on the lateral side of the neural crest. The neural ridges
increase in size and length, growing anteriorly, posteriorly, and
dorsally until they become continuous in front of and behind
the neural plate. As the neural crests grow dorsally, the anterior
part of the neural fold rises prominently, and the embryo has the
appearance shown in figure 1. At this stage the blastopore has
closed to a narrow slit and the neural groove extends from the
blastopore almost to the anterior part of the neural fold. On
either side, just caudal to and within this anterior part of the
fold, there is visible occasionally a small depressed circular or
oval deeply pigmented area. These depressions were described
by Eycleshymer, and, according to him, are the initial stages of
the paired eyes.
In many of the embryos a few (usually three to five) faint
transverse grooves appear in the anterior part of the medullary
plate, but they are not constant in number or regular in position.
342 HORACE W. STUNKARD
In some specimens other similar divisions appear posterior to
these, but they are less distinct and gradually fade out pos-
teriorly so that their number could not be determined. Nor-
mally, the transverse grooves first appear at the lateral edges of
the plate and extend toward the neural groove. Frequently one
appears on either side before the others and since these first
ones are at a corresponding level, their fusion forms a furrow
which I regard as the transverse cephalic groove of Griggs.
There is considerable irregularity in the formation of the grooves,
however; often those of the two sides are formed at different
levels and do not meet at the median line. The areas between
the grooves are then irregular in size and shape; frequently they
are almost triangular as the transverse grooves converge or
meet, either at the neural groove or at the lateral edge of the
plate.. The transverse grooves in the two sides of the neural
plate of the same embryo do not regularly correspond, and this
lack of correspondence is manifest in the figures of Griggs and
other authors. Only in the occasional and unusual specimen is
there present the regular arrangement described by Griggs. The
neural folds close rapidly and it is possible to observe the changes
that take place during the process. It is a significant fact
that the grooves do not always retain precisely their original
aspect during the closing process. Some of the grooves shift
slightly or fade out entirely and other grooves appear in different
positions.
Divisions of the neural plate caused by the transverse grooves
could not be clearly or satisfactorily demonstrated in sections,
but such study shows that, with the appearance of the transverse
grooves, the mesoderm has developed to a stage where it is
assuming a segmented condition, and I regard the formation
of the transverse grooves as due to the formation of the meso-
dermal somites. I am convinced that certain of the trans-
verse grooves coincide with the divisions between somites, and
I am inclined to believe that it is true of most if not all of
the transverse grooves. It is possible, however, that grooves
are also due to associated mechanical factors, pressure produced
by the multiplying cells and the infolding of the neural crests.
PRIMARY NEUROMERES AND HEAD SEGMENTATION 343
In the lateral ridges a beaded appearance is sometimes present,
but in no case did it show the regularity described by Locy. The
lobulations along the neural crests are often entirely absent, and,
when present they do not show definite regularity, either in
size or arrangement. The number of lobes varies from two or
three to as many as fifteen on one side, and little if any corres-
pondence could be detected between the lobulations of opposite
sides of the same embryo. Sections of the crests show the cells
to be distributed uniformly with occasional slight irregular
groupings, but there is no evidence of a segmental arrangement.
These aggregates or clusters of embryonic cells are not differen-
tiated into regular areas, but appear to be centers of rapid cell
proliferation. Before the neural folds close the forebrain and
midbrain are clearly outlined by thickened enlargements, and
by the time of complete fusion, the three primary brain vesicles
are distinctly defined. The crests close rapidly and in essential
_respects these observations confirm the description given by
Kycleshymer (95). The median divisions disappear with the
closure of the neural folds, and no definite relation between them
and the brain vesicles could be determined. Sections of many
embryos seem to indicate that their fate is not uniform, but
differs in different individuals.
For the study of chick embryos, several hundred eggs were
incubated, and over one hundred embryos were obtained for
study, giving a series of stages from the formation of the primi-
tive streak to the formation of the brain and spinal cord. Most
of the embryos were removed at the stage when the neural groove
is open, as this, according to Locy and Hill, is the most favorable
period at which to observe the primary neuromerism of the
nervous system. In the study of the living embryo most of the
work was done with a binocular although both dissecting and
compound microscopes were used. For illumination, trans-
mitted light, as well as reflected light from an electric are, a gas
light, and also direct sunlight were used. Following Locy’s
suggestion that ‘‘a dead black background is of course the best
surface for observing anything of this kind by reflected light,”
a circle of dead black paper was placed under the specimen in the
344 HORACE W. STUNKARD
aie
Figs. 1 to 7 and 12 to 14, camera-lucida drawings of Amblystoma embryos,
showing successive stages of development, the so-called primary neuromeres,
and the neuromeres of later stages.
Fig. 1 Early stage in the formation of the neural folds, showing the anterior
thickening.
Fig.2 Embryo showing neural groove, the lobulations along the neural folds,
and transverse grooves of the neural plate.
Fig. 3 Embryo with no evidence of segmentation in the neural folds and
regular transverse grooves of the neural plate.
Fig. 4 Embryo showing irregular character of the divisions of the neural
folds and neural plate.
Fig. 5 Embryo showing expanded anterior part of the neural plate, with
irregular divisions of the neural folds and neural plate.
Fig.6 Embryo elongated, with partial fusion of the neural crests.
Fig. 7 Complete fusion of neural crests and appearance of the brain vesicles.
PRIMARY NEUROMERES AND HEAD SEGMENTATION 345
Fig. 12 Frontal section through the open neural crests of an embryo, showing
irregular lobulations of crests and indefinite cell arrangement.
Fig. 13 Frontal section through the developing brain vesicles of embryo,
showing the later neuromeres.
Fig. 14 Cross-section of embryo at same state of development as figure 13,
showing mesodermal segmentation.
346 HORACE W. STUNKARD
bottom of the watch-glass. The neural tube was swept clean
of all surrounding tissues by the use of dissecting needles and
fine brushes, the sides of the neural tube were dissected and
also sections were cut of the roof and floor. In order to get
shadows, specimens were tilted and rotated to secure all angles
and degrees of illumination. Kleinenberg’s, Bouin’s, and Gil-
son’s killing fluids were used, and some of the embryos were
examined after faintly staining them with borax carmine and
Conklin’s picro-haematoxylin. To supplement the study of
whole and dissected specimens, sections were cut in transverse,
frontal, and sagittal planes and stained with Ehrlich’s acid
haematoxylin and Heidenhain’s iron haematoxylin. ~
No indication of anything that could be interpreted as seg-
mentation could be observed in the primitive streak, or before
the neural folds were clearly outlined as elevated ridges. At
this stage along the elevated margins of the medullary plate
~ certain lobulated irregularities are formed, giving a beaded
appearance to the crest, and these structures are present with
more or less uniformity in most embryos up to the closure of the
neural tube. They are, however, irregular in number in differ-
ent embryos and do not correspond in the two sides of the same
individual; their limits are often so obscure that they cannot be
determined with certainty, and the wide variation in their rela-
tive position makes it impossible to correlate them, either in the
two sides of the same embryo or in different embryos. They
differ greatly in size; often in the same embryo there are two or
three on one side while the corresponding region of the opposite
side will consist of a single lobe, or perhaps the edge will be
smooth, showing no lobulation. The variation in size, together
with the uncertain position and desultory arrangement suggest
strongly that this marginal lobulation is due entirely to differ-
ences in rate of cell proliferation at different points along the
rapidly expanding wall of tissue.
As the neural crests increase in size, they rise rapidly and
begin to fold over toward the median plane. At this stage
especial care was exercised to detect any indication of segmenta-
tion in the medullary folds or in the plate between the folds.
PRIMARY NEUROMERES AND HEAD SEGMENTATION 347
Occasional faint lines could be distinguished, but their position
and appearance were so irregular and variable that no seg-
mental importance could be attached to them.
Segmentation of the neural folds was reported by Hill (’00),
who described the marginal segments as separated by constric-
tions that ‘‘in early stages completely encircle the encephalon,
and in later stages are confined to its base and lateral walls.”
In the examination of large numbers of embryos, I have found on
the external surface of the neural folds faint constrictions that
appear as lines when the best shadow effects are obtained, but I
fail to find the regularity described and figured by -Hill. On
the contrary, the grooves are irregular in number and position,
and often a single groove will divide to form two. The grooves do
not regularly encircle the encephalon, but a groove will often
fade out at some point and slightly anterior or posterior to it
another groove will appear, so that the number of constrictions
is different for the two sides. These constrictions are so faint
that they can be traced only with difficulty, and in no specimen
approximate the condition shown in Hill’s figures. Further-
more, dissection shows that internal grooves do not regularly
correspond with external constrictions. I find external grooves
are present at these stages with no corresponding internal con-
strictions and internal grooves with no corresponding external
constrictions. Later in ontogeny Hill says the internal grooves
are elevated upon the apices of internal ridges, but the groove at
the apex of the internal ridge is not present with sufficient con-
stancy to be of value in determining the constrictions that are
of segmental importance. That there are grooves in addition
to those considered by Hill to be segmental, he admits when he
says, page 423, ‘‘secondary divisions that frequently are present
would eventually be confused with the primary ones.” But he
gives no criteria by which to distinguish between primary and
secondary constrictions, and in his figures certain ones are ex-
aggerated as ‘primary,’ while others are suppressed as ‘secondary.’
He states that in these early stages the only criteria by which he
determined segments are the external and corresponding internal
grooves, and that he considers the internal ridge as a secondary
Figs. 8 to 11 and 15 to 20, camera-lucida drawings of chick embryos, showing
successive stages of development, the so-called primary neuromeres and the
neuromeres of later stages.
Figs. 8 and 9 Embryos of three somites, dorsal view, drawn from living
specimens, showing the early beaded appearance of the neural crests.
Figs. 10 and 11 Embryos of eight and thirteen somites, respectively, dorsal
view, drawn from living specimens, showing the brain vesicles and later
neuromeres.
348
PRIMARY NEUROMERES AND HEAD SEGMENTATION 349
modification. Observation of a large number of embryos affords
no evidence to support the statement of Hill that ‘‘eleven con-
strictions are present on both inner and outer surfaces of the
open neural groove, that they are constant in number and
nearly equal in size and that they appear earlier in ontogeny
than the historic encephalic divisions, forebrain, midbrain, and
Hh Oy
Sh pel
oa‘.
HO,
be dance
Poe
Ona
ve,
oa
~e
on,
cent
Fig. 15 Frontal section through the neural crests of an embryo of three
somites, showing the irregular lobulations of the crests, absence of corresponding
external and internal grooves, and indefinite cell arrangement.
hindbrain.” Hill reported that the third and fifth grooves are
deeper than the others and mark the posterior limits of the fore-
brain and midbrain. While it is true that with the appearance
of the so-called secondary division of the neural tube into fore-
brain, midbrain, and hindbrain, the limits that separate them are
clearly marked, the present study fails to confirm his statement
350 HORACE W. STUNKARD
‘that an earlier segmentation is incorporated into this division
as follows: in the forebrain three primary somites; in the mid-
brain two; and in the hindbrain, six or six and one half if the
portion of segment twelve that lies in front of the first somite is
added to the latter.”’
19.
Fig. 16 Frontal section through the right neural crest of an embryo of five
somites, showing same features as figure 15.
Fig. 17 Sagittal section through the closing neural tube of an embryo of
ten somites, showing same features as figure 15.
Fig. 18 Sagittal section of the floor of medullary tube of an embryo of five
somites, showing same features as figure 15.
Fig. 19 Frontal section through the developing brain vesicles, showing the
later neuromeres.
Fig. 20 Same section as figure 19, showing cell arrangement in the right wall
of the hind brain just anterior to the otic invagination.
PRIMARY NEUROMERES AND HEAD SEGMENTATION 351
As the neural crests approach each other, the primary brain
vesicles and neuromeres of the hindbrain are well defined, and
fusion of the folds first occurs in the region of the midbrain
vesicle or slightly posterior to it. After the closure of the
neural tube there are clearly six segments anterior to the
auditory invagination. These brain vesicles and neuromeres of
the hindbrain are so well: known that further description is un-
necessary. In these divisions there is present the definite, char-
acteristic cell arrangement designated by Orr as distinguishing
true neuromeres. In the open neural groove there is no arrange-
ment of cells in the medullary ridges or floor that even suggests a
segmental condition. The cells are evenly distributed and do
not manifest any tendency toward groupings that would give
morphological significance to the grooves which serve as the
basis of the primary neuromerism of Locy and Hill.
DISCUSSION
A survey of the literature on the subject of head segmentation
shows most unusual differences, both in observation and inter-
pretation. These observations are based on the study of differ-
ent morphological features, and it has been impossible satis-
factorily to explain the discrepancies and differences reported.
In the study of neuromerism in urodeles, Kupffer, Froriep,
Eycleshymer, Neal, Locy, Griggs, Smith, and others have de-
scribed as many as eleven and as few as three segments in the
cephalic region. Locy considered the divisions of the neural
crests as segmental, and did not regard the divisions of the
cephalic plate as of metameric importance. Kupffer, Froriep,
Eycleshymer, Griggs, Smith, and others have agreed that in
any consideration of neuromeric segmentation, the divisions of
the medullary plate are of primary importance, but these authors
have not agreed as to their number or metameric significance,
and most regard them as due to the segmentation of the meso-
derm. Locy pointed out that the median divisions do not
correspond with those in the neural ridges; he reports four or
five divisions in the median plate and ten or eleven segments
in the neural ridges of the same region. He says, page 530,
a0e HORACE W. STUNKARD
‘Whether we find the median plate smooth in Amblystoma or
faintly segmented depends on the stage at which the examina-
tion is made, and we recognize that the appearances in any one
egg are not constant throughout the open groove stage; further,
that eggs of closely related animals are by no means necessarily
similar at corresponding stages.” According to Griggs, the
beaded appearance of the neural crests was not apparent in any
of the embryos of this stage examined by him, and he argues
for the metameric significance of the median divisions. Locy
and Griggs both attempt to establish neuromerism as a basis
for determining the segmentation of the head, but their results
are mutually exclusive and contradictory.
Locy’s statement that primary neuromeres are visible in the
blastoderm of the chick at the twelfth hour of incubation, just as
the head fold is first outlined, and that they extend into the
primitive streak, finds absolutely no support in any of the
material of the present investigation. His further statement
that ‘‘the cells in these segments are characteristically arranged,
even in the earliest stages, and their arrangement and struc-
ture would indicate that they are definite differentiations of cell
areas’? was denied by Hill, and in the present study evidence to
support this statement of Locy is also entirely wanting. Neal
(98) called attention to the fact that ‘none of the reproductions
of Locy’s photographs, with two possible exceptions, show a
segmentation of the neural folds in either the trunk or embryonic
rim.” He might well have added that none show neuromeres of
both sides and that the same embryo was not photographed
twice, in two different positions (which probably would be
necessary) that the neuromeres of the two sides might be com-
pared. In fact, Locy’s photographs, in my opinion, deny rather
than confirm his statement. He admits that his drawings ‘‘are
a little too distinct’’ and “the exactness has been exaggerated.”
Hill’s figures of the chick have called forth exclamations of
surprise and astonishment on all sides. He figures constrictions
of his primary neuromerism persisting in embryos with a closed
neural groove, but I have been unable to observe such a condi-
tion. It is a significant fact that the cell arrangement of the
PRIMARY NEUROMERES AND HEAD SEGMENTATION Boe
forebrain and midbrain at this stage does not show such segmen-
tation. In the primary neuromeres, he admitted that the seg-
mental arrangement of cells is absent, and argued that the radial
cytological condition which later appears is due to the intraseg-
mental expansion of the walls. Thus the definite structure of the
later neuromeres he attempted to explain on purely mechanical
grounds. If, however, these definite and constant structural
features be merely mechanical effects, one wonders how he can
hope to substantiate the transitory, indefinite, and irregular
beadings of the early stages as a primary neuromerism of phylo-
genetic importance.
Study of the neuromeres of the later stages has also led to
great diversity of opinion. The neuromeres of the medulla are
definite structures with characteristic morphological features.
It is an open. question whether or not they are homologous with
the divisions of the neural tube anterior and posterior to them.
The divisions of the spinal cord are undoubtedly formed by the
pressure of the adjacent mesodermic somites, and anterior to the
otic invagination the number and character of the somites are
far from established. It is in the anterior part of the neural
canal that the evidence is most scanty and indefinite. Neal
(18) argues that the primary brain vesicles are the true neuro-
meres of the region, and in the opinion of the writer his argument
is clear and comprehensive. If the central nervous system of the
primitive vertebrate were segmented, with the enlargement of
the brain there would be an enlargement of the segments. The
brain segments enlarge laterally and dorsoventrally, and it seems
only natural that they should enlarge anteroposteriorly. It
certainly is as reasonable to suppose that individual segments
would expand anteroposteriorly as to account for the elongation
of the brain by fusion of segments and the backward migration of
the cephalic region with the concomitant incorporation of addi-
tional segments in the brain. While gill clefts, visceral arches,
and epibranchial organs are cenogenetic, still they may be seg-
mental, being predetermined in position by nerves, blood vessels,
septa, and other segmental structures of the invertebrate. The
later brain is highly developed, with great specialization of parts,
354 HORACE W. STUNKARD
and ontogeny affords such fragmentary and inconclusive evi-
dence of phylogeny that neuromerism alone can hardly explain
the development of the head. The study of highly specialized
forms like the chick must appear of less importance than that of
more primitive forms, or at least of forms in which primitive
conditions persist. In this connection, Neal (718) has poimted
out that neuromeres are more conspicuous in the embryos of
higher, than they are in embryos of lower chordates, and this
would hardly be expected if they are vestiges of a primitive
neuromerism.
In ontogeny, segmentation regularly appears first im the
mesoderm and the segmentation of the mesoderm is more con-
stant and regular than segmentation in other tissue. Segmenta-
tion of other tissue normally results from and is in correspondence
with segmentation of the mesoderm. Mesomeres are uniformly
present in the lower chordates, and to disregard mesodermal seg-
mentation is therefore to overlook an item of paramount im-
portance in any study of head segmentation. In the ancestral
vertebrate there was undoubtedly a correspondence of meso-
meres, neuromeres, cranial nerves, and branchial organs, and
all of these structures must be considered in an explanation of the
present lack of correspondence.
The present study has shown that in Amblystoma and the chick
at least, the structures described by Locy and Hill as primary
segments cannot be regarded as metameric. Investigators have
repeatedly questioned the accuracy of the observations of Locy
and Hill, and a repetition of their work, using as far as possible
identical means of examination, has in the present case not only
failed to verify their observations, but disclosed a quite different
condition. The three morphological features upon which neuro-
merism can be based, marginal beadings, external and internal
grooves, and cell arrangement, all fail to give evidence to con-
firm the primary neuromerism of Locy and Hill. Neal could
not confirm Locy’s statements concerning Selachian embryos
and Ihave been unable to confirm Locy’s observations on Amblys-
toma or Hill’s on chick embryos. In my opinion, the so-called
‘primary metamerism’ is based upon incorrect observation and
PRIMARY NEUROMERES AND HEAD SEGMENTATION 355
cannot be accepted. The median divisions observed in the
neural plate of Amblystoma are largely if not entirely due to
segmentation of the mesoderm, and so ean be regarded only as
features of secondary importance. The primary neuromeres of
Locy and Hill, as well as those of Griggs and other students of
neuromerism, are irregular in size, inconstant in number, asym-
metrical in position, and cannot serve as trustworthy criteria
of the metamerism of the vertebrate head.
BIBLIOGRAPHY
Von Barr, K. E. 1828 Ueber Entwicklungsgeschichte der Tiere. Kénigsberg.
Batrour, F. M. 1878 A monograph on the development of elasmobranch
fishes. London.
BeLtogotowy, J. 1908 Zur Entw cae der Kopfnerven der Végel. Ein
Beitrag zur Morphologie des Nervensystems der Wirbeltiere. Bull.
Soc. Imp. Nat. Moscow, Hft. 3 und 4.
1910 German reprint, Moscow.
BERANECK, E. 1884 Recherches sur le développement des nerfs craniens chez
les lizards. Rec. Zool. Suisse, T. 1.
Dorn, A. 1875 Der Ursprung der Wirbelthiere und das Princip des Functions-
wechsels. Genealogische Skizzen. Leipzig.
EycLesHyMER, A.C. 1895 The early development of Amblystoma, with obser-
vations on some other vertebrates. Jour. Morph., vol. 10.
Finatorr, D. 1907 Die Metamerie des Kopfes von Emys lutaria. Zur Frage
iiber die korrelative Entwicklung. Morph. Jahrb., Bd. 38.
Froriep, A. 1891 Zur Entwickelungsgeschichte der Kopfnerven. Verh. Anat.
Gesell. Miinchen.
1892 Zur Frage der sogenannten Neuromerie. Verh. Anat. Gesell.
Wien. :
GEGENBAUR, C. 1887 Die Metamerie des Kopfes und die Ww irbeltheorie des
Kopfskelettes. Morph. Jahrb., Bd. 18.
GrRaPeR, L. 1913 Die Rhombomeren tae ihre Nervenberichtungen. Arch.
mikr. Anat., Bd. 83.
Grices, L. 1910 Early stages in the development of the central nervous system
of Amblystoma punctatum. Jour. Morph., vol. 21.
Hinz, C. 1900 Developmental history of the primary segments of the verte-
brate head. Zool. Jahrb., Abt. f. Anat. u. Ont., Bd. 13.
Huxtey, T. H. 1858 The Croonian lecture: On the theory of the vertebrate
skull. Proc. Roy. Soc. London., vol. 9.
JOHNSTON, J. B. 1905 The morphology of the vertebrate head from the view-
point of the functional divisions of the central nervous systen:. Jour.
Comp. Neur., vol. 15.
von Kuprrer, C. 1885 Primiire Metamerie des Neuralrohrs der Vertebraten.
Sitzungsber. math. physik. Kl. Miinchen.
1906 Die Morphogenie des Centralnervensystems. Handbuch vergl.
u. exp. Entwick. Wirbeltiere. Tena.
356 HORACE W. STUNKARD
Locy, W. A. 1895 Contribution to the structure and development of the verte-
brate head. Jour. Morph., vol. 11. ;
McCuvure, C.F. W. 1890 The segmentation of the primitive vertebrate brain.
Jour. Morph., vol. 4.
Neat, H. V. 1898 The segmentation of the nervous system in Squalus acan-
thias. A contribution to the morphology of the vertebrate head.
Bull. Mus. Comp. Zool. Harvard, vol. 31.
1914 The morphology of the eye muscle nerves. Jour. Morph.,
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1918 Neuromeres and metameres. Jour. Morph., vol. 31.
Orr, H.B. 1887 Contribution to the embryology of the lizard. Jour. Morph.,
voljd.
SmitH, B.G. 1912 The embryology of Cryptobranchus. Jour. Morph., vol. 23.
SmitH, P. E. 1914 Some features in the development of the central nervous
system of Desmognathus. Jour. Morph., vol. 25.
Van WisHE, J. W. 1882 Uber die Mesodermsegmente und die Entwickelung
der Nerven der Selachierkopfes. Naturk. Verh. K. Akad. Wiss.,
Amsterdam.
1886 Ueber Somiten und Nerven im Kopfe von Végel und Reptilien-
embryonen. Zool. Anz., Bd. 9.
1889 Die Kopfregion der Kranioten beim Amphioxus, nebst Bemer-
kungen iiber die Wirbeltheorie des Schidels. Anat. Anz., Bd. 4.
Waters, B. H. 1892 Primitive segmentation of the vertebrate brain. Quart.
Jour. Micr. Sci., vol. 33.
Witson, J. T., anD Hitt, J. P. 1907 On the development of Ornithorhynchus.
Phil. Trans. Roy. Soc., London, vol. 199.
AUTHOR'S ABSTRACT OF THIS PAPER ISSUED
BY THE BIBLIOGRAPHIC SERVICE, MARCH 27
THE ORIGIN OF BILATERAL SYMMETRY IN THE
EMBRYO OF CRYPTOBRANCHUS
ALLEGHENIENSIS
BERTRAM G. SMITH
Department of Anatomy, New York University and Bellevue Hospital Medical
College
THIRTY-THREE FIGURES
: CONTENTS
Anabysiaror the problemct sisic35. 6/6 c:2- selgs = eeas.2 92% St Othe Dae eae 358
Begpermients 4nd) ODSeGVALIONS.. th). 2. a jigsb ol doit oincdieweles ss ag adeae aol dante 360
A. The possible influence of gravity upon the direction of the median plane 361
Beebe entrance-path-of the spermatozoon........2)..0.....2...00sseesece 362
1. Relation toitbe plane of first cleavage.....22..0...1). sce. ccees- 365
2. Relation to the median plane of the gastrula.................... 365
C. The relation of the first cleavage furrow to the median plane of the
CENA FAUT Eno ae cy PSR ick wether REN RCI EERO CR ne ner tree ae eee a 367
(Pen ricnuatlOnyEXpehriMmentsysis.5.0 42 saan eis k lee eee ae ae SSE. 367
Dareons leretion, Witla Silk NECAG SJ). clus 6 & oese Seen Pais Paldue odie see 368
SERS UAL PROX) CEMMEM GSES pot War ee 5 ec Ph oer ASE ogcvaPs\cSpe iene (ora ove Seierarvie's a 370
Pen RE Ct ORMETA PEIRCE se nat 5 oni Meh) cio lopc, ofaeina cit thenancria; wate awaneys wr 373
BEERS AEE Y. GL) Gne AHICTORICTES |.35 0/25 Wtde id obit hast Va. Gy ack oe ts 374
D. The excentricity in the superficial cleavage pattern of the early blastula 374
1. Relation to the median plane of the gastrula............. ........ 376
2. elation to the first cleavage furrow...3.-.... sce. .cclecevcnndenses 377
3. Relation to the entrance-path of the spermatozoon............... 379
E. The excentricity manifested by the internal structure of the early
|G 2S, UE: ee ee Soe MO ees Ce Yee Ie Peed Are, Sot MES | Ne a ES aed 379
1. Relation to the excentricity in the superficial cleavage pattern.... 382
2. kelation to the plane’ of first'cleavage:. /..-.2.5..0i. sce cee e seen 383
F. The bilateral symmetry of the superficial cleavage pattern in the lower
lemisphererofsthe late blastular.= byes bis ae bat Gace oe aes teats 383
1. Relation to the median plane of the gastrula.................... 384
Z. Relation fo. the first cleavage furrow =........6< 6505.20 -0000006- 000% 385
G. The bilateral symmetry manifested by the internal structure of the
Meee MDA LAs irr ceee 21S: oye Sa eS Ta s Sehd a oy eieeas s Soe ab ines 386
1. Relation to the bilateral symmetry of the superficial cleavage
DN GUCLI cee ease oy Perey Here Nee CIEE Pato on are Giana ruatrays tol iere Bes Yea ao abo eros 388
2. Relation to the bilateral symmetry of the early gastrula............ 388
357
358 BERTRAM G. SMITH
DISCUSSION... Jhiaws thats Red ob ous Cee oer cee. cae Oe OR eee eee 390
A. Bilateral organization previous to fertilization....................... 390
B. Influence of the spermatozoon and of environmental factors,.......... 391
C. Relation of the first cleavage furrow to the median plane of the embryo. 392
D. The bilateral: symmetry ‘of the blastula..22...:.0 5: os. sen Soe 394
E. Embryonic axes in relation to bilateral symmetry.................... 396
DUMIMUATY..... 0. UAE EYES Eek Sa Cee tee eee Oba ot nt aE Oe eee eee 397
Bibhioprapliy? co. as sos Seo eciem ee oh deaeb mere yt eins ote 2a eee 398
ANALYSIS OF THE PROBLEM
In the higher animals, the most obvious features of organiza-
tion are those expressed by the terms bilateral symmetry, antero-
posterior differentiation, and dorsoventral differentiation. Since
bilateral symmetry is always accompanied by the other features
mentioned, the expression bilaterality is alone sufficient to desig-
nate the type of structure under consideration.
Bilaterality is a feature of such fundamental importance in the
organization of the embryo that we should expect it to appear
very early in the ontogeny, and the problem of tracing its origin
is one of considerable interest in relation to theories of heredity
and development. In particular one desires to know the earliest
manner of expression of the definitive bilateral symmetry of the
embryo, and whether the hereditary factors that are undoubtedly
at work can be modified by external influences.
In a previous communication (Smith, ’12, IT) it has been shown
that the polarity of the late ovarian egg of Cryptobranchus
allegheniensis, as expressed by its telolecithal character, es-
tablishes approximately the principal axis of the embryo: the
anterior end forms about 40° from the animal pole, the posterior
end quite accurately at the vegetal pole. But a careful study of
the ovogenesis recorded in another paper (Smith, 712, I) has not
revealed any feature of the ovarian egg that enables us to dis-
tinguish right and left, dorsal and ventral surfaces; in the mature
but unfertilized egg the condition is one of radial symmetry
and axial differentiation, with no trace of bilateral symmetry.
Subsequent observations, made by examining in toto preparations
of the ovarian eggs cleared in various oils, have not altered this
conclusion. Even the polarity of the egg is not visibly expressed
BILATERALITY IN CRYPTOBRANCHUS 359
in its organization from the beginning, but arises during ovo-
genesis.
In the gastrula stage, the bilateral symmetry of the embryo is
expressed in a perfectly obvious manner. Our problem then
requires us to look for the beginnings of bilateral symmetry in
the cleavage stages, or possibly in the fertilization stage, and leads
us to consider every deviation from strict radial symmetry that
suggests the beginning of definitive bilateral symmetry. The
following features must be considered in their relation to the posi-
tion of the dorsal lip of the future blastopore, and as a check on
the results it is desirable that these same features should be
considered in their relation to each other:
1. The direction of the entrance-path of the spermatozoon.
2. The direction of the first cleavage furrow, which defines an
axis of biradial symmetry in the cleavage pattern of the third and
later cleavage stages.
3. The excentric development of the micromeres shown by the
superficial cleavage pattern of the early blastula.
4, The excentric development manifested by the internal struc-
ture of the early blastula.
5. The bilateral symmetry of the superficial cleavage pattern
in the lower hemisphere of the late blastula.
6. The bilateral symmetry manifested by the internal structure
of the late blastula.
One desires to know the nature of the factors at work in pro-
ducing bilateral symmetry or determining the direction of the
median plane, when the egg is developing in its natural environ-
ment. The egg of Cryptobranchus is fertilized immediately
after spawning. Under the influences of light and gravity,
streaming movements may be induced in the cytoplasm of the
fertilized but unsegmented egg, which modify the results due
to the operation of internal factors; in the egg of the frog such
complications have been observed by various investigators.
Another circumstance that must be taken into account is
the well-known fact that in the amphibian egg the direction of
the early cleavage furrows may be changed by mechanical
pressure.
360 BERTRAM G. SMITH
In nature, the developing egg of Cryptobranchus is shielded
from the light; the factor of pressure also is negligible. In
experimental procedure these factors are best dealt with by elimi-
nating them so far as possible from the conditions of the experi-
ment. The only external influence that appears to be normally
related to the life of the egg, in such a way that it might affect
bilateral symmetry, is gravity. So long as the eggs are retained
in the body of the female their orientation is variable and incon-
stant, but since there is comparatively little axial differentiation
during this period, it is not likely that even under the most
favorable conditions of orientation could gravity exert any
appreciable influence on the organization of the egg. If the egg
is at all susceptible to the influence of gravity in determining the
direction of the median plane, the most favorable conditions are
presented immediately after spawning and fertilization. At this
time the cytoplasm accumulates rapidly about the animal pole.
The newly laid egg is closely invested by the gelatinous envelope,
and does not freely orient itself with the animal pole uppermost
until after sufficient water has been absorbed to cause the capsule
to become turgid and spring away from the egg—a process re-
quiring from one to two hours. During this period, in which
the polar axis may make any angle with the vertical, gravity
might possibly rearrange the contents of the egg in such a way
as to affect the direction of the median plane of the future embryo.
EXPERIMENTS AND OBSERVATIONS
All the experiments with living material were carried out in
a cellar, where the temperature was favorable for the develop-
ment of the eggs and the light could be controlled. So far as
possible the eggs were shielded from the light; they were handled
as gently as possible to avoid the disturbing effects of mechanical
manipulation.
Some of the experiments about to be described involve placing
the egg in a definite position and keeping it there for a considera-
ble period of time. Trials of various methods showed that it is
sufficient and most expedient to orient each egg in water in a
separate watch-glass and leave it in a situation where it will not
yy. wae. Ca * eae oe EO ——————
BILATERALITY IN CRYPTOBRANCHUS 361
be disturbed. In the stages under consideration the egg or
embryo is devoid of cilia; observation of landmarks furnished by
artificial markings show that during cleavage the eggs do not
undergo any perceptible rotation on a vertical axis. The inertia
of the heavy egg and contact of its lower surface with the sub-
stratum make it easy to guard against disturbances sufficient
to affect the position of the egg. In the first two or three sea-
sons’ work, the Syracuse watch-glasses used for these experi-
ments were placed on a massive walnut table resting on a gravel
foundation and against a stone wall; in later experiments, com-
prising the greater part of the work, they were placed on the level
top of a concrete wall. To make up for the loss by evaporation,
water was each day added gently by means of a pipette. The
probability of error increases, of course, with the length of time
involved in the experiments. Wherever possible, the results
were checked by other methods.
In certain experiments the eggs were removed from their gelat-
inous envelopes immediately after being taken from the uterus,
thereby allowing them to orient themselves, in water, at once
with the animal pole uppermost. This procedure eliminates,
during the fertilization period, the possible influence of gravity in
determining the direction of the median plane. The eggs used
in most of the experiments were taken from nests, where spawning
and fertilization took place in a natural manner; hence it was
necessary to test the possible influence of gravity under condi-
tions that sometimes occur in nature.
A. The possible influence of gravity upon the direction of the median
plane
For this experiment eggs taken from the uterus of a ripe female
were artificially fertilized without removal from their envelopes.
Twenty-one eggs were placed each in a separate watch-glass
without water and oriented with their polar axes in a horizontal
position and parallel to each other; the animal pole was directed
away from the observer. After allowing time for the capsule to
adhere to the glass, a little water was added gently by means of a
pipette. In the course of one or two hours the capsules absorbed
362 BERTRAM G. SMITH
sufficient water to allow the eggs to rotate slowly until the animal
pole was uppermost. Eighteen eggs survived to the gastrula
stage. Figure 1 shows the direction of the principal axis of
each embryo in the advanced gastrula stage; it is evident that
there is no preponderance of any particular direction. So far as
it goes, this experiment indicates that gravity acting on the egg
DVEOOO
VOODOO F
OOOOUS
Fig. 1 Digrams showing the results of an experiment to test the possible
influence of gravity in determining the direction of the median plane of the
embryo of Cryptobranchus allegheniensis. The newly fertilized eggs were placed
with their polar axes in a horizontal position and parallel to each other; the
animal pole was directed away from the observer (i.e., toward the top of the page
in the figure). After the absorption of water by the envelopes, each egg rotated
slowly, in response to gravity, through 90° in such a manner as to bring the
animal pole uppermost. The diagrams show the eggs in polar view at the time of
gastrulation; the arrow, pointing anteriorly, indicates the direction of the median
plane of the gastrula.
immediately after fertilization is not a factor of any importance
in determining the direction of the median plane of the embryo.
B. The entrance-path of the spermatozoon
Various observations indicate that penetration by the sperma-
tozoon induces profound cytological changes in the egg. In a
previous paper (Smith, *12, I, figs. 7 to 12) I have described
certain external phenomena consequent upon the entrance of
BILATERALITY IN CRYPTOBRANCHUS 363
the spermatozoon; these changes were very clear and striking
in one spawning of eggs, though less obvious in others. The
internal phenomena alone are sufficient to show that the sperma-
tozoon immediately exerts a very decided influence: a wave of
condensation of the cytoplasm and finer yolk globules always
marks the progress of the spermatozoon (Smith, 12, I, figs. 41 to
46). These features suggest that forces are at work comparable
to those that produce the gray crescent of the frog’s egg.
In order to investigate the relation of the entrance-path of
the spermatozoon to other features of the developing egg, an
attempt was made to control the direction of entrance of the
spermatozoon, as follows: Unfertilized eggs were taken from
the uterus of a ripe female, the accessory envelopes were
removed and each egg immersed in water in a separate Syracuse
watch-glass. Under these conditions the egg at once orients
itself with the animal pole uppermost. Each egg was then
fertilized with milt applied by means of a fine pipette to the right
hand edge of the germinal dise or blastodisc. Great care was
exercised to guard against any subsequent change in the position
of the egg. The seminal fluid of Cryptobranchus is decidedly
viscous and difficult to handle in small quantities, consequently
the control of the direction of entrance of the spermatozoon
cannot be assumed to be very accurate.
A number of circumstances combined to make the task of
carrying out of this experiment decidedly troublesome and labori-
ous. It was necessary to capture a large number of adult speci-
mens in order to get a few females whose eggs were in precisely
the right condition for fertilization. Less difficulty was experi-
enced in obtaining males in condition for breeding, nevertheless
some failures were due to the unripe or spent condition of the
males. The operation of removing the egg envelopes and apply-
ing the seminal fluid requires some time, and unless the eggs are
fertilized within a few minutes after their removal from the
uterus, they fail to develop. Of the eggs experimented upon
about 84 per cent either failed to develop or segmented in an
irregular and probably abnormal manner; very few reached the
gastrula stage. It is probable that this heavy loss was due in
BERTRAM G. SMITH
OOODODO-
DOSOOO-
ODODOOO-
DOODDOO-
OODORGE-
QBOOOQOO-
DOOODOO-
DODDDO-
Fig. 2 Diagrams showing the direction of first cleavage in forty-eight eggs
of Cryptobranchus allegheniensis in which an attempt was made to control the
direction of entrance of the spermatozoon. The eggs are shown in polar view; the
seminal fluid was applied to the edge of the germinal disc on the right-hand side
of the egg.
BILATERALITY IN CRYPTOBRANCHUS 365
large part to the removal of the envelopes, for it is comparatively
easy to secure artificial fertilization and normal development
when the envelopes are not removed. Excessive polyspermy, to
which the eggs are peculiarly exposed after the removal of their
envelopes, may account for many cases of abnormal development
or failure to develop. In order to control the direction of fertili-
zation it is necessary to apply the seminal fluid at some distance
from the center of the blastodisc; no doubt it often happened that
the spermatozoon entered the egg too far from the animal pole,
and was unable to penetrate the yolk in order to reach the egg-
nucleus. In order to obtain results from a sufficient number of
eggs, the work was carried on each breeding season for five years.
Over three hundred eggs were subjected to this experiment; the
number does not include eggs that were rejected at once because
of obvious inaccuracy in the control of the direction of applica-
tion of the spermatozoa.
1. Relation to the plane of first cleavage. Forty-eight eggs
segmented in a normal manner; in these eggs the direction of
first cleavage with reference to the probable direction of entrance
of the effective spermatozoon is shown in figure 2. It is evident
that there is a decided tendency for the first cleavage furrow to
come in approximately at right angles to the entrance-path of the
spermatozoon.
2. Relation to the median plane of the gastrula. Sixteen eggs
survived to form normal gastrulae. In these eggs the relation of
the median plane of the gastrula to the probable direction of
the entrance-path of the spermatozoon is shown in figure 3.
The results indicate that there is no uniformity in the relation
between the entrance-point of the spermatozoon and the plane of
bilateral symmetry of the gastrula.
In interpreting the data, a slight complication arises from the
fact that these eggs which survived to the gastrula stage included
nearly all those exceptional cases in which the first cleavage
furrow departed from the general rule of forming approximately
at right angles to the direction of application of the seminal
fluid. As an aid in studying this aspect of the situation, the direc-
tion of first cleavage in each egg is indicated in the diagram. If
366 t BERTRAM G. SMITH
we assume that the control of the direction of entrance of the
spermatozoon was faulty and that the direction of first cleavage
affords an index to the real direction of fertilization, we still find
WDOS
D Cb 29 C9
DESI ODS
¥Fig.3 Diagrams showing the direction of the median plane of the gastrula in
sixteen eggs of Cryptobranchus allegheniensis in which an attempt was made to
control the direction of entrance of the spermatozoon; the direction of first
cleavage, also, is indicated. The eggs are shown in polar view; the seminal fluid
was applied to the edge of the germinal disc on the right-hand side of the egg.
In each egg an arrow, pointing anteriorly, shows the direction of the median
plane of the gastrula; the position of the blastopore is indicated by a broken
curved line.
that there is no uniform relation between the entrance-path of
the spermatozoon and the median plane of the gastrula.
Indirect evidence in support of this conclusion is furnished by
the results set forth in the next section. For it will be shown that
BILATERALITY IN CRYPTOBRANCHUS 367
there is no fixed relation between the direction of the first cleavage
furrow and the median plane of the gastrula. Since a fairly
definite relation has been established between the direction of first
cleavage and the direction in which the spermatozoon enters the
egg, it follows that there is no fixed relation between the entrance-
path of the spermatozoon and the median plane of the gastrula.
C. The relation of the first cleavage furrow to the median plane of the
gastrula
Does the first cleavage furrow separate the material for right
and left halves, or for anterior and posterior ends of the embryo?
Beginning with the third cleavage stage, biradial symmetry is
a conspicuous feature of the superficial cleavage pattern, and in
the lower hemisphere serves as a ready means for identifying
the early cleavage furrows up to the gastrula stage (figs. 4 to
13, 26 and 27). Is the cleavage determinate, and is this biradial
symmetry a mode of expression of the definitive bilateral sym-
metry of theembryo? Since the axis of biradiality is marked by
the first cleavage furrow, in answering this question it is necessary
to consider only the relation of this furrow to the median plane of
the gastrula.
1. Orientation experiments. The question was first investigated
by the simple device of orienting, without removing the envelopes,
a large number of eggs in the first cleavage stage in such a manner
that the planes of first cleavage were parallel, and leaving them
in this position to develop to the gastrula stage. . At the time of
first cleavage the egg naturally and freely orients itself within
the envelope in such fashion that the animal pole is kept upper-
most. This experiment was performed on several different
occasions, using in all more than a hundred eggs; nearly all sur-
vived to the gastrula stage. The results, which were recorded
by means of diagrams, fail to show any tendency for either the
first or the second cleavage furrow to coincide with the median
plane of the gastrula. It seems unnecessary to publish the data,
since the point is conclusively established by the more exact
experiments that follow.
368 BERTRAM G. SMITH
2. Constriction with a silk thread. Shortly after the appearance
of the first cleavage furrow, the accessory egg envelope was
removed and a silk cord tied around the egg in such a manner as
Figs. 4 to 7 Surface views of the upper hemispheres of four eggs of Crypto-
branchus allegheniensis in early cleavage stages, showing the biradial character
of the cleavage pattern. Figs. 4 and 5, stage 4; fig. 6, stage 5; fig. 7, stage 6.
The figures were drawn with the aid of acamera lucida. X7.
to constrict it slightly in the direction of the first cleavage furrow.
The blastomeres were by no means entirely separated, and the
egg developed as a whole; the device served merely to mark the
direction of the first cleavage furrow. The operation is a delicate
BILATERALITY IN CRYPTOBRANCHUS 369
one, and even after it had been accomplished with every appear-
ance of success, the egg usually collapsed in a later cleavage
stage. Out of a considerable number of eggs treated in this way,
Figs 8 to 11 Surface views of the lower hemispheres of four eggs of Crypto-
branchus allegheniensis, showing the biradial character of the cleavage pattern
Fig. 8, stage 5; fig. 9, stage 6; fig. 10, stage 7; fig. 11, stage 9. The figures were
drawn with the aid of acamera lucida. X 7.
only nine lived to form gastrulae; the appearance of the lower
hemispheres of these eggs in the gastrula stage is shown in figure
14, It will be observed that in two of these eggs the median
plane of the gastrula coincides approximately with the plane of
370 BERTRAM G. SMITH
first cleavage, in two others the median plane is at right angles to
the plane of first cleavage, while in the five remaining eggs the
median plane is oblique to the plane of first cleavage: a result
that is about what we might expect if the relation between these
two planes is held to be purely a matter of chance.
3. Staining experiments. A method of making permanent
marks with Nile-blue sulphate on the living egg of Crypto-
branchus has been described in a previous paper (Smith, 714).
Since the stain is slightly toxic, certain precautions must be
es =
asceeenes
=
me,
os:
SEES Sine
Se ENS NEES
Figs. 12 and 13 Equatorial views of two eggs of Cryptobranchus alleghenien-
sis in advanced segmentation stages, showing the biradial character of the cleav-
age pattern in the lower hemispheres. Fig. 12, stage 8; fig 18, stage 9. The
figures were drawn with the aid of acamera lucida. X 7.
observed. The egg is removed from its accessory egg envelope,
but retains the chorion (the structural equivalent of the vitelline
membrane of the frog’s egg). The egg is then immersed in
water in a Syracuse watch-glass, and a rather strong aqueous
solution of the stain applied with a capillary pipette in such a
manner as to make the smallest possible distinct spot. After
about thirty seconds the excess of stain is removed with a pipette
of larger caliber and the dish flooded with fresh water. Not more
than one egg is placed in each watch-glass, and the water is
changed several times during the first day, and once a day there-
after. With few exceptions, eggs so treated develop normally.
BILATERALITY IN CRYPTOBRANCHUS 371
A microscopical examination of the stained substance of the egg,
twenty-four hours after the application of the stain, showed that
the cytoplasm, and not the yolk granules, takes the stain.
PDE
OPC
DDD
Fig. 14 Nine eggs of Cryptobranchus allegheniensis in the early gastrula
stage, inverted to show the vegetal hemisphere; the plane of first cleavage has
been marked by tying a silk thread around the egg. The figures were drawn
with the aid of a camera lucida.
In order to mark the direction of first cleavage, eggs were taken
shortly before the appearance of the second cleavage furrow;
at this time the first cleavage furrow had not quite reached the
equator. Two small spots were made on opposite sides of the
oe BERTRAM G. SMITH
GVO OGODN
DDOBBR
DESBED
DDDD OD
ODO DDDY
OOODDD
BOBO OOD
Fig. 15 Diagrams showing the relation between the direction of the first
cleavage furrow and the median plane of the gastrula in forty-two eggs of Crypto-
branchus allegheniensis. The vertical line indicates the direction of the first
cleavage furrow, which was marked with Nile-blue sulphate; the arrow, pointing
anteriorly, indicates the direction of the median plane of the gastrula.
BILATERALITY IN CRYPTOBRANCHUS 3/3
egg where the plane of first cleavage intersects the equator;
these spots remained perfectly distinct in the early gastrula
stage. To determine accurately the relation between the first
cleavage furrow and the median plane of the gastrula, it is
necessary that the egg be examined immediately after the
beginning of gastrulation; for during the progress of gastrulation
one of the spots may, in certain cases, become involved more
than the other in the shifting of material toward the median
line, which has been described as a phenomenon of concrescence
(Smith, 714). By taking observations promptly after the first
appearance of the dorsal lip of the blastopore, this source of
error may be avoided entirely.
Fifty-five eggs were thus treated; forty-two survived to the
gastrula stage. In these forty-two eggs (fig. 15) there was no
uniformity in the direction of first cleavage with respect to the
median plane of the gastrula. Experience with the method here
employed inspires one with so much confidence in the accuracy
of the data obtained that the results of this experiment alone
might be taken as a conclusive answer to the question under
consideration.
4. Direct comparison. As already stated, the biradial symme-
try of the cleavage pattern in the lower hemisphere enables one
to identify first and second cleavage furrows in the late cleavage
stages (figs. 26 and 27). It is sometimes possible, with the aid
of a good dissecting microscope, to identify the first and second
cleavage furrows in the region of the vegetal pole even after the
appearance of the blastopore (figs. 28 and 29); this enables one
to make a direct comparison between the direction of the first
cleavage furrow and the median plane of the gastrula.
The first cleavage furrow was identified in the early gastrula
stage in twenty-seven eggs. In six eggs the first cleavage furrow
extended approximately in the median plane of the embryo, in
eleven eggs it was oblique to the median plane, and in ten eggs it
crossed the median plane approximately at right angles. The
validity of these results depends of course on a correct identifica-
tion of the first cleavage furrow. All cases that seemed doubtful
were discarded, but there remains the possibility that one might
374 BERTRAM G. SMITH
occasionally be deceived in distinguishing the first from the second
cleavage furrow. One can hardly attribute to this method the
degree of accuracy inherent in the two preceding methods.
However, the results tend to confirm the conclusion that there is
no constant relation between the direction of first cleavage and the
median plane of the embryo.
5. Instability of the micromeres. In studying the problem of
orientation of the early cleavage furrows it is necessary to bear
in mind the extensive shifting of the micromeres and consequent
torsion of cleavage furrows that takes place from the beginning
of second cleavage throughout the remaining early cleavage
stages (Smith, ’12, II). The portions of the early cleavage
furrows that traverse the region of macromeres are relatively
stable, but, on account of the instability of the micromeres dur-
ing the early segmentation period, upper and lower portions
of a meridional cleavage furrow may come to lie in different
directions.
After keeping a living egg under constant observation during
the early cleavage stages and sketching the cleavage pattern at
frequent intervals, it is possible upon comparing these sketches
to trace the first two cleavage furrows through the region of
micromeres up to the sixth and sometimes the seventh generation
of blastomeres. This has been done in a number of instances,
and it has been found in every case that the path of a given cleav-
age furrow becomes very irregular. The most extensive shifting
occurs during the early stages, beginning with second cleavage;
later, as the micromeres become smaller, the distances involved in
these movements are not so great.
D. The excentricity in the superficial cleavage pattern of the early
blastula
Since the study of conditions arising during cleavage necessi-
tates a rather precise designation of stages, we shall have occasion
to refer to these stages by the serial numbers adopted in an earlier
paper (Smith, ’12, II) devoted to the external development; the
entire segmentation period is divided into ten stages.
BILATERALITY IN CRYPTOBRANCHUS ao
At first, cell division proceeds most actively at the animal
pole; consequently we may regard this pole as the center of a
primary area of cellular activity. In stages 5 and 6 (illustrated
by figs. 16 and 17 of the present paper), also in the stages that
immediately follow, the frequent occurrence of a secondary area
of accelerated cell division has been noted (Smith, 712, II). On
opposite sides of the circular area occupied by the micromeres,
these cells are unequal in size and number; the smaller micro-
meres give evidence of more recent division, since the cleavage
3
3/
\/ 16
Figs.16and17 Surface views of the upper hemispheres of two eggs of Crypto-
branchus allegheniensis in early blastula stages (stages 5 and 6, respectively),
showing excentric development of the micromeres. The figures were drawn with
the aid of a camera lucida. xX 7.
furrows that bound them are superficially deeper and more open,
as is always the case with newly formed furrows. In other words,
the region of most active cell division is no longer confined
to the vicinity of the animal pole, but extends from that pole a
short distance along a meridian. A line drawn from the center
of the secondary area of accelerated cell division through the
animal pole defines an axis of excentricity in the superficial
cleavage pattern of the micromeres.
The condition is really one of bilateral symmetry, but to avoid
unwarranted implications I have tried to describe it without using
this term. The question naturally arises whether this excen-
376 BERTRAM G. SMITH
tricity in the superficial cleavage pattern of the early blastula is
an expression of the definitive bilateral symmetry of the embryo.
As we shall see, a somewhat similar condition exists in the lower
hemisphere of the late blastula (figs. 26 and 27), and this is un-
doubtedly an expression of the definitive bilateral symmetry;
but between these two stages, early and late, respectively, there
intervenes a period in which it is more often impossible to detect
any deviation from strict radial symmetry in the superficial
cleavage pattern. Consequently, we should not assume that
there is genetic continuity between these two similar phases that
occur at widely separated stages; they require separate investiga-
tion. In this section we shall consider only the problematical
bilaterality of the early blastula.
1. Relation to the median plane of the gastrula. a. Orientation
experiments. Eighty-three eggs. showing excentricity in the
cleavage pattern of the early blastula (stages 5 to 7, inclusive)
were oriented, each in a separate watch-glass, without removal
from their envelopes. Seventy-five eggs lived to the gastrula
stage. The results, which were recorded by means of diagrams,
indicate that there is no constant relation between the axis of
excentricity in the superficial cleavage pattern of the early
blastula and the median plane of the gastrula. This result was
wholly unexpected and difficult to reconcile with the impres-
sions gained through the study of the internal development; con-
sequently, the subject was investigated again by another method.
b. Staining with Nile-blue sulphate. A method more accurate
than the preceding is to remove the envelopes and mark the axis
of excentricity by means of a vital stain. The marking was
readily accomplished by applying a small drop of Nile-blue sul-
phate to the side of the egg on which the larger micromeres oc-
curred. The axis of excentricity is thus defined by an imaginary
line drawn from the point marked, through the animal pole. To
insure accuracy in the identification of this axis, each egg was
examined with a dissecting lens, and those failing to show marked
excentricity were rejected. The results were recorded promptly
at the very beginning of gastrulation, to avoid possible errors due
to concrescence.
BILATERALITY IN CRYPTOBRANCHUS one
Seventy-seven eggs in stages 5 and 6 were marked as above
described; only twenty-three survived to the gastrula stage.
This heavy loss was due to the fact that the staining fluid was
inadvertently made too strong. The results for the twenty-three
eggs that survived are shown by the first twenty-three diagrams,
occupying the upper part of the page, in figure 18.
Forty eggs in stage 7 were marked in the same manner. In
this stage the roof of the blastocoele is thin and translucent in a
region which, as a rule, is slightly excentric with respect to the
polar axis of the egg, and lies toward the side possessing the larger
micromeres. The smaller micromeres now extend further from
the animal pole than do the larger micromeres. Of the eggs
marked, twenty-five survived to the gastrula stage. The results
are shown by the last twenty-five diagrams, occupying the lower
part of the page, in figure 18. .
These experiments confirm the conclusion reached by the
method of orientation. Whatever the origin and significance
of the excentricity in the cleavage pattern of the early blastula,
it is clearly of no value in foreshadowing the direction of the future
median plane of the embryo. If it is indeed causally related to
the development of the definitive bilateral symmetry of the
embryo, then in its incipient condition this bilaterality must be
an unstable thing, subject to a shifting of one of its axes of differ-
ential cellular activity.
It would be interesting to apply the same tests to stages 8 and
9, but in these stages the excentricity in the superficial cleavage
pattern is not so easily recognizable, particularly in living
material.
2. Relation to the first cleavage furrow. This comparison was
made by identifying the first cleavage furrow in eggs that showed
marked excentricity in the superficial cleavage pattern of stages
5 to 7, inclusive, using preserved material. In these stages the
first cleavage furrow in the region of macromeres can be iden-
tified with certainty. The results for twenty-three eggs may be
ummarized as follows: in nine eggs the axis of excentricity
coincides approximately with the plane of first cleavage; in seven
eggs the axis of excentricity is oblique to the plane of first cleavage
Aa,
SEXED
Fig. 18 Diagrams showing the relation between, a) the axis of excentricity in
the superficial cleavage pattern of the micromeres of the early blastula and,
b) the median plane of the gastrula in forty-eight eggs of Cryptobranchus alle-
gheniensis. Each diagram represents the upper hemisphere of an egg; the small
round spot on the side toward the top of the page indicates a mark made with
Nile-blue sulphate on the side of the egg possessing the larger micromeres; the
arrow, pointing anteriorly, indicates the direction of the median plane of the
gastrula.
QPBGS6SGEeQ
SC6QA8CEGE8
378
BILATERALITY IN CRYPTOBRANCHUS 379
and in the seven remaining eggs the axis of excentricity is approxi-
mately at right angles to the first cleavage furrow. Evidently
there is no constant relation between the two features con-
sidered.
3. Relation to the entrance-path of the spermatozoon. In the
experiments already described in which an attempt was made
to control the direction of entrance of the spermatozoon, excen-
tricity in the cleavage pattern of the early blastula was observed
in fifteen eggs. No doubt a much larger number of cases of
excentricity would have been found had all the eggs been kept
under continuous observation. In these fifteen eggs (fig. 19)
there was an evident lack of uniformity in the direction of the
axis of excentricity with respect to the probable direction of the
entrance-path of the spermatozoon.
E. The excentricity manifested by the internal structure of the early
blastula
In order to study the internal development of Cryptobranchus
during the cleavage stages, entire eggs were embedded in paraffin
and cut into vertical serial sections (i.e., sections taken in planes
parallel to the polar axis of the egg). Of these sections the ones
passing approximately through the center of the egg are desig-
nated as meridional. In a large number of cases the eggs were
not oriented with reference to any structural features other
than polarity. In examining sections of such eggs in stages 5 to
9, inclusive (early blastula to very late blastula), the writer was
quickly impressed with the fact that the internal development of
the micromeres is excentric with respect to the animal pole. On
one side of the axis of polarity the micromeres composing the roof,
and especially the lateral wall, of the blastocoele aré smaller and
more numerous, richer in cytoplasm, poorer in yolk, and in the
later stages usually extend a little farther from the animal pole
than on the opposite side. In favorable cases this inequality is
revealed by a single meridional section (figs. 20 to 25); in other
cases it is less readily recognizable through a mental reconstruc-
tion of the entire series.
380 BERTRAM G. SMITH
The impression gained through the study of sections is that
this differentiation is of the same general character throughout
the entire blastula stage, having its beginning in stages 5 and 6,
progressing rapidly in stages 7, 8, and 9, and continuing in some-
what different form through stage 10 (very late blastula). The
condition is one of bilateral symmetry, and its development ap-
OONOE
VAIDOS-
VOTO:
Fig. 19 Diagrams showing the direction of the axis of excentricity in the
superficial cleavage pattern of the upper hemisphere of the early blastula in
fifteen eggs of Cryptobranchus allegheniensis fertilized by seminal fluid applied
to the edge of the germinal disc on the side indicated by the arrows on the right-
hand margin of the figure. The arrow drawn through each circle indicates the
axis of excentricity; the head cf the arrow is placed on the side occupied by the
larger and less numerous micromeres.
pears to be fundamentally a single continuous process. That
the direction of differentiation of the micromeres, with reference
to the positions of the more stable macromeres, is unchanged
during this long period of development is of course not to be
assumed without proof. In the early stages, before the excentric
differentiation is well established, it seems likely to be subject to
changes in direction; in the later stages it assumes an aspect of
BILATERALITY IN CRYPTOBRANCHUS 381
greater stability. We shall here consider the relation of the
excentric internal structure of the early blastula to some other
features of organization.
A aus e =
FO NereN
SIR
Siva
20 21
Figs. 20 to 25 Meridional sections taken in the median plane of bilateral
symmetry of eggs of Cryptobranchus allegheniensis in early to late blastula
stages. S, segmentation cavity or blastocoele. Fig. 20, stage6; fig. 21, stage 7;
figs. 22 and 23, stage 8; figs. 24 and 25, stage 9. The figures were drawn with the
aid of acamera lucida. X 8.
382 BERTRAM G. SMITH
1. Relation to the excentricity in the superficial cleavage pattern.
It is a very natural supposition that the excentricity in the super-
ficial cleavage pattern is merely the outward expression of the
excentric development of the micromeres revealed by the study of
the internal structure. To test this hypothesis, sixteen preserved
eggs in stages 5 to 8, inclusive, were split (with a razor blade)
along the axis of excentricity in the superficial cleavage pattern
and the internal structure examined with a lens. In every egg
but one the roof and lateral walls of the blastocoele showed ex-
centric development of the micromeres after the fashion pre-
viously observed in serial sections, and in twelve eggs the median
plane of excentricity in the internal structure coincided with the
plane of splitting.
The point was further investigated by means of specially
prepared serial sections. Nineteen eggs in stages 6 to 8, in-
clusive, showing marked excentricity in the superficial cleavage
pattern, were oriented in paraffin, and sectioned in planes parallel
to the axis of excentricity. In seventeen eggs the meridional
sections showed approximate coincidence in the direction of
excentric development of the micromeres as manifested by exter-
nal and internal features, respectively, while in the two re-
maining eggs the meridional sections gave no evidence of excentric
development.
Therefore we conclude that the excentricity manifested by the
internal structure of the early blastula is definitely correlated with
the excentricity in the superficial cleavage pattern; these two
features are different aspects of the same thing. The experi- -
mental evidence has shown that there is no constant relation be-
tween the axis of excentricity in the superficial cleavage pattern
of the early blastula (stages 5 to 7, inclusive) and the median
plane of the gastrula; we must now accept the same conclusion
for the excentricity in the internal structure.
Incidently, the study of sections shows why, in stages 8 and
9, the excentric development of the micromeres is not so clearly
expressed by the superficial cleavage pattern as it is by the deeper
structure. In these stages there occurs a rather uniform flatten-
ing of the superficial layer of micromeres, which masks the
BILATERALITY IN CRYPTOBRANCHUS 383
changes within; the outer layer of cells is becoming epithelial in
character.
2. Relation to the plane of first cleavage. In the following
experiments the first cleavage furrow was identified in the vegetal
hemisphere only.
Thirteen preserved eggs in stages 5 to 8, inclusive, were split
with a razor along the plane of first cleavage. In three eggs the
first cleavage furrow coincided with the axis of excentricity as
revealed by the internal structure, in seven eggs the first cleavage
furrow was oblique to the axis of excentricity, and in the three
remaining eggs the first cleavage furrow extended at right angles
to it. So far as they go, these results indicate that there is no
fixed relation between the plane of first cleavage and the axis
of excentricity in the internal structure.
The matter was further investigated by means of serial sections.
Eleven eggs in stages 5 to 8, inclusive, were cut into sections par-
allel to the first cleavage furrow; in nine of these eggs the merid-
ional sections showed unequal development of the micromeres
on opposite sides of the polar axis, while in the two remaining
eggs the meridional sections gave no evidence of such differentia-
tion. ‘This result considered alone might be taken to indicate a
tendency for the axis of excentricity to coincide with the plane
of first cleavage; but this conclusion is nullified by the results of
the preceding test, also by the one which follows: of eleven eggs
in stages 5 to 8, inclusive, sectioned at right angles to the first
cleavage furrow, six showed excentricity in the meridional sec-
tions, while in five the meridional sections were lacking in this
feature.
F. The bilateral symmetry of the superficial cleavage pattern in the
lower hemisphere of the late blastula
In the late blastula (stages 9 and 10) bilateral symmetry is
usually recognizable in the cleavage pattern of the lower hemi-
sphere. The vegetal pole, marked by the point of intersection of
the first and second cleavage furrows, is excentrically situated
within the area occupied by the macromeres: a more rapid
multiplication of cells has occurred on one side of the egg, where
384 BERTRAM G. SMITH
micromeres and transitional cells approach nearer the vegetal
pole (figs. 26 and 27). In most eggs the transition from small to
large cells is more gradual on the side where the more rapid
multiplication of cells occurs; on the opposite side it is character-
ized by a rather abrupt line of demarcation. These features im-
pose a phase of excentricity and bilateral symmetry upon the
previously existing biradial symmetry of the cleavage pattern
of the lower hemisphere. A meridian drawn through the vegetal
eae e tens
Se
aSem
Figs. 26 and 27 Surface views of the lower hemispheres of two eggs of Crypto-
branchus allegheniensis in late blastula stages (stage 10, early and late phases,
respectively). In each egg the lower pole as determined by gravity lies at the
center of the figure; the vegetal pole, at the intersection of the first two cleavage
furrows, is slightly above this point and excentrically situated within the macro-
meres. The upper part of each figure represents the side on which the blasto-
poreistoappear. The figures were drawn with the aid of acamera lucida. X 7.
pole and the center of the area occupied by the macromeres
defines the axis of excentricity and bilateral symmetry in the
cleavage pattern.
1. Relation to the median plane of the gastrula. In many eggs
preserved in the early gastrula stage the cleavage pattern of the
lower hemisphere is well-defined; it is sometimes possible to
identify first and second cleavage furrows and thus to locate
the vegetal pole at their intersection (figs. 28 and 29). The ex-
centric position of the vegetal pole within the region of macro-
meres and the bilateral phase of the cleavage persist into the
BILATERALITY IN CRYPTOBRANCHUS 385
gastrula stage. These features are usually better expressed than
in the eggs shown in the figures, which were chosen because the
distinctness of their early cleavage furrows enabled them to be
drawn with a camera lucida. The dorsal lip of the blastopore
serves as a landmark for determining the true median plane of
bilateral symmetry of the gastrula, and in every case studied
the axis of excentricity and bilaterality of the superficial cleavage
pattern lies in this median plane; this was clearly made out in
Rta Rey
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Figs. 28 and 29 Surface views of the lower hemispheres of two eggs of Crypto-
branchus allegheniensis in the early gastrula stage, showing the biradial charac-
ter of the cleavage pattern which enables one to distinguish first and second
cleavage furrows. In figure 28 the first cleavage furrow lies approximately in
the median plane of the gastrula; in figure 29 it lies nearly at right angles to this
plane. The drawings were made witb the aid of acamera lucida. X 7.
about a dozen eggs. We conclude, therefore, that the definitive
bilateral symmetry of the embryo is indicated by the cleavage
pattern of the late blastula; the earliest stage in which bilateral
symmetry thus becomes unequivocally expressed is the one
designated stage 9.
2. Relation to the first cleavage furrow. On account of the
biradial character of the cleavage pattern of the lower hemi-
sphere, it is often possible to distinguish the first from the second
cleavage furrow in the vicinity of the vegetal pole of the late blas-
tula. Observation of a considerable number of eggs makes it
certain that the direction of the first cleavage furrow bears no
constant relation to the axis of bilaterality in the cleavage pattern.
386 BERTRAM G. SMITH
G. The bilateral symmetry manifested by the internal structure of the
late blastula
When a living egg is examined in a very late blastula stage, the
roof and lateral walls of the blastocoele are found to be slightly
translucent. When such an egg is immersed in water and held
between the observer and the source of light, it can usually be
seen to possess a very definite bilateralsymmetry. On one side
Fig.30 Meridional section taken in the median plane of bilateral symmetry of
an egg of Cryptobranchus allegheniensis in a very late blastula stage (late phase
of stage 10). S, segmentation cavity or blastocoele; Y, yolk. The drawing was
made with the aid of acamera lucida. X 15.
of the large blastocoele the roof is more translucent than on the
opposite side and contrasts more abruptly with the opaaue
yolk. The condition is more clearly shown in meridional sec-
tions, but in order to observe the exact condition described, one
must be careful to obtain an egg killed when it is just ready to
begin gastrulation. When the plane of the section is sagittal,
the contrast between the two sides is usually obvious enough
(figs. 30 and 31). Not only has the roof of the blastocoele
BILATERALITY IN CRYPTOBRANCHUS 387
become slightly thinner on one side, which we shall see becomes
the dorsal side of the embryo, but on this side the floor of the
blastocoele dips abruptly downward to form a small crevice,
while on the opposite side it curves gradually upward. On the
thinner side of the roof of the blastocoele, the cells are more
columnar in form; on this side of the egg, below the level of the
blastocoele, the micromeres usually, but not always, extend a
EBT
c\
Fig. 31 Meridional section taken in the median plane of bilateral symmetry of
an egg of Cryptobranchus allegheniensis in a very late blastula stage (late phase
of stage 10). S, segmentation cavity or blastocoele; Y, yolk. The drawing was
made with the aid of acamera lucida. X 15.
little further toward the vegetal pole. When the plane of the
section is transverse, the structure revealed is symmetrical.
So far as one can judge from the study of sections, this differ-
entiation of the very late blastula is genetically continuous with
the excentric development of the micromeres in earlier stages,
certainly as early as stages 8 and 9 (figs. 22 to 25). In these
earlier stages the roof or lateral wall of the blastocoele is decidedly
thicker on the side where the more rapid multiplication of cells
JOURNAL OF MORPHOLOGY, VOL. 36, NO. 3
388 BERTRAM G. SMITH
occurs; nevertheless, there are reasons for believing that this is
the side that eventually becomes the thinner (dorsal) side.
For in the very late blastula stage (figs. 830 and 31), the side with
the thinner wall is in the more advanced stage of differentiation,
as evidenced by the columnar character of its cells and the greater
progress made in the extension of the cap of micromeres over the
yolk cells. In the earlier stages mentioned, the depression or
crevice in the floor of the blastocoele, if present at all, is located
on the side of greater thickness and more advanced differentia-
tion. Another evidence of a reversal in the relative thickness
of opposite sides of the roof of the blastocoele is that in an in-
termediate stage (early phase of stage 10) they are equal in thick-
ness, and can then be distinguished only by a careful study of
the character of the cells and by the position of the crevice, which
in this stage is a fairly pronounced and constant feature. The
thinning-out of the originally thicker portion of the roof of the
blastocoele is apparently accomplished in part by a migration
of cells from its inner surface, away from the median plane, in
part by a process of circumcrescence. Since the plates were
prepared, I have sectioned additional material in which the gap
between figure 25 and figure 30 is more satisfactorily bridged
and the above conclusions confirmed.
1. Relation to the bilateral symmetry of the superficial cleavage
pattern. When an egg in a late blastula stage is sectioned along
the axis of bilaterality indicated by the superficial cleavage
pattern of the lower hemisphere, this axis is found to coincide in
direction with the median plane of symmetry of the internal
structure. Thus the bilateral symmetry of the cleavage pattern
is but the external expression of the more fundamental symmetry
of the internal organization of the egg.
2. Relation to the bilateral symmetry of the early gastrula. In
the living egg, it may be observed that the blastopore begins to
form just below the equator, on the side where the lateral wall of
the blastocoele is more translucent, but at a lower level than
the floor of the blastocoele. The study of sections of the be-
ginning gastrula shows that the bilaterality of the late blastula
is carried over into the gastrula stage (figs.32and33). Internally,
BILATERALITY IN CRYPTOBRANCHUS 389
the first evidence that gastrulation is about to begin is the fact
that the cells lying just below the crevice on the dorsal side of the
egg, where the segmentation cavity dips downward, become
rounded or oval in outline, preparatory to immigration or in-
vagination.
The morphological evidence seems sufficient to connect the
bilateral symmetry of the late blastula (stages 8 to 10) with the
definitive bilateral symmetry of the gastrula, since it is very
improbable that sudden changes in the direction of the bilateral
organization of the blastula should occur after this condition is
well established.
The meridian that bisects the beginning blastopore defines
the median plane of bilateral symmetry of the gastrula, which
ultimately becomes the sagittal plane of the embryo. As more
fully described in previous contributions (Smith, 712, II; and
14), the anterior end of the embryo forms in this meridian about
40° from the animal pole, while the posterior end forms in the
region where the blastopore closes, at the vegetal pole. Conse-
quently, the dorsal side of the embryo forms mainly from mate-
rials which in the early gastrula stage lie between the beginning
blastopore and a point situated some distance above it, in the
thinner portion of the roof of the blastocoele. In thelateblastula
and early gastrula, this dorsal region coincides with the region
of greatest cell activity, which is not confined to the thin portion
of the roof of the blastocoele, but extends below the level of the
blastocoele into the equatorial region on the side of the egg
where the blastopore begins to form. This extension or shifting
of the original area of excentric cellular activity is an expression
of the circumcrescent or epibolic phase of gastrulation. The
opposite and less active side of the egg, where the now thickest
portion of the roof of the blastocoele joins the yolk cells, is
ventral. Hence the differentiation which first establishes the
definitive bilateral symmetry of the blastula is dorsoventral in
direction, and constitutes a new embryonic axis secondary to the
principal or polar axis of the egg. In other words, radial symme-
try with axial differentiation gives place to bilateral symmetry
as soon as a new axis of differentiation is established approxi-
mately at right angles to the first.
390 BERTRAM G. SMITH
DISCUSSION
A. Bilateral organization previous to fertilization. In the egg
of Cryptobranchus allegheniensis we find polarity arising dur-
ing ovogenesis and bilaterality established only after cleavage
has reached an advanced stage. The writer’s observations
give no support to the idea sometimes advanced that ‘“‘bi-
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aia.
CKD
LDS
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CY
ay
Fig. 32 Sagittal section of a beginning gastrula of Cryptobranchus allegheni-
ensis. B, blastopore; S, segmentation cavity or blastocoele; Y, yolk. The
drawing was made with the aid of a camera lucida. X 15.
laterality as well as polarity are inherent characters of the
protoplasm and persist from generation to generation” (Bartel-
mez, 712). In many species of animals the egg is bilaterally
organized before fertilization, but it has never been shown that
this bilateral organization is present as such from the beginning
of ovogenesis. The available evidence favors the theory of nu-
clear determination, as opposed to the hypothesis of cytoplasmic
inheritance.
BILATERALITY IN CRYPTOBRANCHUS 391
B. Influence of the spermatozoon and of environmental factors.
Various observers (Roux, ’83, ’85; Schultze, 00; Morgan and
Boring, 03) have shown that following fertilization and previous
to cleavage the egg of the frog is bilaterally organized, and
that this bilateral symmetry foreshadows the definitive bilateral
symmetry of the embryo. In the fertilized egg the bilateral
eorels
CRIS
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Fig. 33 Sagittal section of a beginning gastrula of Cryptobranchus allegheni-
ensis. B, blastopore; S, segmentation cavity or blastocoele; Y, yolk. The
drawing was made with the aid of acamera lucida. 15.
organization expresses itself superficially in the formation of a
gray crescent on the site of the future blastopore. Since the
crescent is formed on the side opposite the point of entrance of
the spermatozoon (Roux, ’83, ’85, ’87, ’03; Schultze, ’00), Roux
maintained that the point of entrance of the spermatozoon
determines the direction of the median plane of the embryo.
That the egg is not dependent upon the spermatozoon for
the establishment of its bilateral symmetry is shown by the
392 BERTRAM G. SMITH
fact that in many species of animals the egg develops bilaterality
in advance of fertilization, and many eggs are capable of develop-
ing parthenogenetically into organisms possessing the bilateral
symmetry characteristic of the species. Consequently, there is
no necessary relation between the fertilization meridian and the
plane of symmetry.
The relationship which has been shown to exist in certain cases must
therefore depend upon a certain time relationship in the course of the
two processes. The influences radiating from the spermatozoon estab-
lish a gradient from its original excentric position, which may influence
the direction of the plane of symmetry in which there is also a gradient,
if its determination is synchronous, as in the frog (Lillie, 719).
In the egg of the frog, external influences such as gravity
and light acting at the time of fertilization may exert an addi-
tional modifying influence in determining the direction of the
median plane (Jenkinson, ’09, Appendix A). In the egg of
Cryptobranchus, neither the direction of sperm entrance nor the
influence of gravity are factors of any appreciable importance in
the determination of bilaterality.
In the frog’s egg, the first cleavage furrow usually passes
through the entrance-point of the spermatozoon (Newport, 754;
Roux, 785, ’87; Schultze, 700). According to Roux (’87), it is
the entrance-path of the spermatozoon that determines the posi-
tion of the gray crescent, while it is the latter part of the sperm-
path, the ‘copulation-path,’ that determines the direction of
first cleavage. Since the entrance-path and the copulation-path
do not always lie in the same direction, we have here an explana-
tion of the fact that, in the egg of the frog, the first cleavage
furrow sometimes fails to coincide with the plane of symmetry
(Jenkinson, ’09, pp. 248, 307 and 308). In the egg of Crypto-
branchus the first cleavage furrow tends to form at right angles to
the fertilization meridian. J have not been able to follow satis-
factorily the latter part of the sperm-path in Cryptobranchus,
but it seems likely that the condition is the same as in the egg of
the axolotl (Fick, ’93), where the copulation-path forms at right
angles to the entrance-path.
C. Relation of the first cleavage furrow to the median plane of the
embryo. Recent observers agree that the cleavage of the am-
BILATERALITY IN CRYPTOBRANCHUS 393
phibian egg is indeterminate in the sense that there is no causal
connection between the direction of early cleavage furrows and
the median plane of the embryo. This view has gained ground
in spite of the fact that in particular species there is an ap-
proximate coincidence between either the first or the second
cleavage furrow and the median plane.
In the frog’s egg the plane of first cleavage tends to coincide
with the median plane of the future animal, though the relation
is far from exact (Newport, ’54; Roux, ’85, ’87; Morgan and
Boring, 703; Jenkinson, ’09, pp. 165-168 and Appendix A).
Brachet (’03, 05) demonstrated experimentally that each of the
first two blastomeres of the segmenting egg of the frog is capable
of producing an entire embryo only when the plane of first cleav-
age coincides with the previously determined plane of bilateral
symmetry; right and left halves, dorsal and ventral sides, anterior
and posterior ends, are predetermined in the undivided egg, and
in normal development it makes no difference how the egg is
cut up by the early cleavage furrows. McClendon (’09, ’10)
was able to remove completely one of the first two blastomeres
of the egg of the tree-frog Chorophilus triseriatus. A large
number of eggs were thus operated upon; in a considerable
number of cases the remaining isolated blastomere gave rise to a
complete normal embryo, and some of these lived to the larval
stage. These results, considered in connection with the findings
of other investigators, led this author to conclude that each of the
first two blastomeres is totipotent only when the first cleavage
furrow bisects the gray crescent.
In the newt Diemyctylus viridescens, Jordan (’93) found that
in the majority of cases the first cleavage furrow forms at right
angles to the direction of the future median plane. Jordan
does not interpret this relation to mean that there is any causal
nexus between the two. Spemann (’01-’03) found that in the
newt Triton cristatus, the first furrow is usually (two-thirds to
three-fourths of all cases) at right angles to the sagittal plane,
and separates the material for the dorsal and ventral halves of
the embryo; only occasionally (one-fourth to one-third of all
cases) do sagittal plane and first furrow coincide. Herlitzka
394 BERTRAM G. SMITH
(96, ’97) had previously shown that by constricting these eggs
in the two-cell stage by means of a noose of fine hair tied around
the egg in the plane of the first furrow, it was sometimes possible
to obtain two complete embryos of rather more than half size.
_Spemann showed that this result could be obtained only in those
occasional cases where the first cleavage furrow coincides with the
median plane of the embryo.
In the living egg of Necturus, Eycleshymer (04) was able to
keep the first cleavage furrow in the lower hemisphere under
observation until the blastopore appeared. In the twenty-two
eggs studied there was no fixed relation between the median plane
of the embryo and the early cleavage furrows. The absence of
a constant relation between the first cleavage furrow and the
median plane of the gastrula has been demonstrated for the egg
of Cryptobranchus by a variety of methods as recorded in the
present paper.
In an extensive series of observations on the living, segment-
ing eggs of Ambylstoma, Diemyctylus, Rana, and Bufo, Jordan
and Eycleshymer (’94) found that the first and second cleavage
furrows undergo extensive torsion. This phenomenon seemed
to the authors suff.cient basis for the conclusion that the early
cleavage planes and the embryonic axes have no vital connec-
tion, and that the coincidence, where it exists, is of no funda-
mental significance. Jf we assume that material for right and
left halves of the body is segregated on opposite sides-of either
the first or the second cleavage furrow, then as a consequence of
the shifting of micromeres we shall later find some of this material
crossing the median line. Eycleshymer’s (’04) later study of
cleavage in the living egg of Necturus revealed a similar irregu-
larity. Extensive shifting of micromeres and torsion of cleavage
furrows occurs in the early stages of segmentation of the egg of
Cryptobranchus.
D. The bilateral symmetry of the blastula. While the direction
of the early cleavage furrows in the amphibian egg is thus shown
to be without causal relation to the median plane of the embryo,
bilaterality is indeed sooner or later made manifest in the cleavage
pattern as a consequence of more rapid cell division on one side
BILATERALITY IN CRYPTOBRANCHUS 395
of the region of micromeres. In the frog’s egg, Morgan and
Boring (’03) have noted that the pigmented cells on the gray
crescent side of the egg are slightly smaller from the beginning
than the other pigmented cells. Eycleshymer (’98, ’02, 04,
715) has shown that in the blastula stages of Amblystoma,
Necturus, Rana, Acris, and Bufo there is a secondary or excentric
area of smaller cells, which, roughly speaking, lies within a sector
of the circular area occupied by the micromeres, and that this area
of accelerated cell division always lies on the side on which the
dorsal lip of the blastopore is to appear.
The primary area of cellular activity, at the upper pole of the amphib-
ian egg, forms the basis of the cephalic end of the embryo. The
secondary area of cell activity, on the blastoporic side of the egg, forms
the basis of the greater portion of the posterior half of the embryo.
These two areas constitute an embryonic tract, from which arise at
least two-thirds of the embryo. The posterior end of the embryo is
formed by a coalescence of the lateral portions of the blastoporic mar-
gins (Hycleshymer, ’98).
In Eycleshymer’s earlier writings emphasis is placed on the
occurrence of this area of accelerated cell division in the late
blastula stage, as illustrated by his figure of Amblystoma (Ey-
cleshymer, ’98, fig. 100); but in his later investigations he found
that in several species excentric development is present in the
early blastula, sometimes as early as the fourth or fifth cleavage
stage. These two similar conditions, appearing respectively
early and late in the amphibian blastula, he regarded as geneti-
cally continuous. I have been able to confirm Eycleshymer’s
observation concerning the early appearance of excentric develop-
ment in the micromeres of Necturus; but in Cryptobranchus,
experimental results make it necessary to distinguish between
the problematical significance of the excentricity of the early
blastula and the undoubted significance of the bilateral symmetry
of the late blastula. According to Lillie (’08, pp. 42 and 47),
the axis of excentricity in the early cleavage pattern of the
pigeon’s egg bears no constant relation to the median plane of
the embryo. It is undoubtedly true that the excentric develop-
ment of the blastoderm which truly marks the beginning of
396 BERTRAM G. SMITH
dorsoventral differentiation appears at different stages in different
species of animals.
Schultze (00) was probably the first to describe the bilateral
organization of the late blastula of the frog, and Ishikawa (’08,
709) has described a similar condition in the developing egg of
the giant salamander of Japan.
In the segmenting egg of the frog, Bellamy (’19) has demon-
strated a primary area of high susceptibility to the action of
reagents, in a meridian that bisects the gray crescent and near
the center of the pigmented hemisphere; also a secondary area of
high suséeptibility in the equatorial region immediately above the
gray crescent, hence just above the site of the dorsal lip of. the
future blastopore.
E. Embryonic axes in relation to bilateral symmetry. In the
ovarian egg of Cryptobranchus, the first visible differentiation
having reference to the form of the adult is manifested in what we
eall polarity: an active pole, rich in cytoplasm, is differentiated
from an opposite and relatively inactive pole concerned mainly
with the storage of food materials. At this stage of develop-
ment the structure in any plane taken at right angles to the polar
axis is radially symmetrical. Eventually, this polar axis deter-
mines approximately the principal axis of the embryo, or the
axis of anteroposterior differentiation.
The further step necessary for the establishment of bilateral
symmetry is the appearance of a secondary axis, the dorsoventral
axis, extending at right angles to the first. In the egg of Crypto-
branchus this secondary axis becomes apparent through the
unequal development of opposite sides of the roof of the blasto-
coele; the region of more active cell division becomes the dorsal
side of the embryo.
It is perfectly obvious that these two axes supply all the differ-
entiations necessary to establish a condition of bilateral symme-
try, for the plane determined by the intersection of these two axes
divides the egg into halves which were originally alike, and which
are modified in a corresponding manner by the differentiation
that proceeds along the secondary axis.
BILATERALITY IN CRYPTOBRANCHUS 397
To explain the maintenance of bilateral symmetry throughout
the subsequent development of the embryo, it is necessary to
suppose that later differentiations are conditioned and controlled
by the differentiations along the two axes already established;
in particular, differentiation along the mediolateral axes of the
body does not proceed independently, but only in subordination
to the more potent and regulatory anteroposterior and dorso-
ventral differentiation.
It would seem simplest to assume that right and left halves of the
body are not at all self-differentiated, but that they are conditioned by
the other body axes. In this way the establishment of two axes each
with two distinct poles (anterior and posterior; dorsal and ventral)
would fully suffice to determine the bilaterality of the organism, be-
cause, if we presuppose a similar interaction of analogous anlagen, those
lying in the third axis and giving rise to the right and left halves of the
body should naturally arrange themselves so that they would represent
mirror images of each other, when they occupy the same position in
relation to the two differentiated axes (Przibram, ’11).
SUMMARY
1. The polarity ‘of the egg of Cryptobranchus allegheniensis
arises during ovogenesis and establishes approximately the direc-
tion of the anteroposterior axis of the embryo.
2. The organization of the mature but unfertilized egg is
characterized by radial symmetry with differentiation along the
polar axis, but with no evidence of bilaterality.
3. Gravity acting at right angles to the polar axis of the egg
during the fertilization period is without perceptible effect in
determining the direction of the median plane of the embryo.
4. The direction of entrance of the spermatozoon is not a
controlling factor in determining the direction of the median
plane of the embryo.
5. The first cleavage furrow forms approximately at right
angles to the direction of the entrance-path of the spermatozoon.
6. The direction of first cleavage bears no fixed relation to the
direction of the median plane of the embryo.
7. In the early blastula stages, the direction of excentric de-
velopment of the micromeres bears no constant relation to the
direction of the median plane of the embryo.
398 BERTRAM G. SMITH
8, In the late blastula, bilateral symmetry is manifested by both
the superficial cleavage pattern and the internal structure, and
this condition is undoubtedly an expression of the definitive
bilateral symmetry of the embryo.
9. The bilateral symmetry of the late blastula is the conse-
quence of dorsoventral differentiation imposed upon the pre-
existing radial symmetry and anteroposterior differentiation of
the egg.
10. The subsequent development of the two lateral halves of
the egg is conditioned and controlled by the differentiation
which proceeds along the two axes (anteroposterior and dorso-
ventral) already established; these two axes suff.ce to determine
and maintain the bilateral symmetry of the embryo.
BIBLIOGRAPHY
BarTeLMEZ, Geo. W. 1912 The bilaterality of the pigeon’s egg. Jour. Morph.,
vol. 23, no. 2.
1918 The relation of tre embryo to the principal axis of symmetry in
the bird’segg. Biol. Bull., vol. 35, no.6, Dec.
Bettamy, ALBERT WitL1am 1919 Differential susceptibility as a basis for
modification and control of early development in the frog. Biol.
Bull., vol. 37, no. 5, Nov.
Bracuet, A. 1903 Sur les relations qui existent chez le Grenouille entre le
plan de penetration du spermatozoide dans l’oeuf, le premier plan de
division et le plan de symetrie de la gastrula. Comptes Rendus de
l’Assoc. des Anat., Sess. 5.
1905 Recherches experimentales sur l’oeuf de Rana fusca. Archives
de Biologie, T. 21.
1906 Recherches experimentales sur l’oeuf non segmenté de Rana
fusca. Arch. fiir Entwickelungsmech., B. 22.
Conxuin, E. G. 1917 The share of egg and sperm in heredity. Proc. Nat.
Acad. Sci., vol. 3.
EyctesHymMEr, A.C. 1898 The location of the basis of the amphibian embryo.
Jour. Morph., vol. 14.
1902 The formation of the embryo of Necturus, with remarks on the
theory of concrescence. Anat. Anz., B.21.
1904 Bilateral symmetry in the egg of Necturus. Anat. Anz., B. 25.
1915 The origin of bilaterality in vertebrates. American Naturalist,
vol. 49.
Fick, R. 1893 Ueber die Reifung und Befruchtung des Axolotleies. Zeitschr.
fiir wiss. Zool., B. 56.
HeruitzKa, A. 1896 Contributo allo studio della capacita evolutiva dei due
primi blastomeri nell’ uovo di tritone (Triton cristatus). Arch. fir
Entwickelungsmech., Bd. 2.
BILATERALITY IN CRYPTOBRANCHUS 399
HeruitzKa, A. 1897 Sullo sviluppo di embrioni completi da blastomeri isolati
di uova di tritone (Molge cristata). Arch. fiir Entwickelungsmech.,
Bd. 4.
IsHrkawa, C. 1908 Ueber den Riesen-salamander Japans. Mitteilungen der
Deutschen Gesellschaft fiir Natur- und Vélkerkerkunde Ostasiens,
Bd. 11, Teil 2.
1909 Note on the gastrulation of the giant salamander, Megaloba
trachus sieboldii. Proc. Seventh Internat. Zool. Congress, Boston,
Aug. 19-24, 1917.
JENKINSON, J.W. 1909 Experimental embryology. Clarendon Press, Oxford.
JorDAN, EpwinO. 1893 The habits and development of the newt, Diemyctylus
viridescens. Jour. Morph., vol.8.
JoRDAN, E.O., AnD EycLesuyMeER, A.C. 1894 On the cleavage of the amphibian
ovum. Jour. Morph., vol. 9.
Linutz, FRANK R. 1906 The development of the chick. Henry Holt & Co.
1919 Problems of fertilization. The University of Chicago Press.
McCuenpon, J. F. 1909 On the totipotence of the first two blastomeres of the
frog’s egg. American Naturalist, June.
1910 The development of isolated blastomeres of the frog’s egg.
Am. Jour. Anat., vol. 10, no. 3, July.
Morean, T. H., snp Bortne, Atice M. 1903 The relation of the first plane of
cleavage and the gray crescent to the median plane of the embryo of the
frog. Arch. fiir Entwickelungsmech., Bd. 16.
Newport, G. 1854 On the impregnation of the ovum in the amphibia; and on
the early stages of the development of the embryo. (Third series.)
Philos. Trans. Royal Soc. London, vol. 144.
PRzIBRAM, Hans 1911 Experiments on asymmetrical forms as affording a clue
to the problem of bilaterality. Jour. Exp. Zo6l., vol. 10.
Roux, W. 1883 Ueber die Zeit der Bestimmung der Hauptrichtungen des
Froschembryo. Leipzig.
1885 Ueber die Bestimmung der Hauptrichtungen des Froschembryo
im Ei und iiber die erste Theilung des Froscheies. Breslauer Artzl.
Zeitschr.
1887 Die Bestimmung der Medianebene des Froschembryos durch
die Kopulationsrichtung des Eikernes und des Spermakernes. Arch.
fiir mikr. Anat., Bd. 29.
1893 Ueber die ersten Teilungen des Froschei und ihre Beziehungen
zu der Organbildung des Embryos. Anat. Anz., Bd.8.
1903 Ueber die Ursachen der Bestimmung der Hauptrichtungen des
Embryos im Froschei. Anat. Anz., Bd. 23.
ScuvuttzE, O. 1900 Ueber das erste Auftreten der Bilateralen Symmetrie im
Verlauf der Entwicklung. Arch. fiir mikr. Anat., Bd. 15.
Smita, Bertram G. 1912 The embryology of Cryptobranchus allegheniensis,
including comparisons with some other vertebrates. Part I: Intro-
duction; the history of the egg before cleavage. Jour. Morph., vol. 23,
no. 1. Part II: General embryonic and larval development, with
special reference to external features. Jour. Morph., vol. 23, no. 3.
1914 An experimental study of concrescence in the embryo of Crypto-
branchus allegheniensis. Biol. Bull. vol. 26.
Spemann, H. 1901-1903 Entwickelungsphysiologische Studien am Triton-Ei.
I, 11, II. Arch. fiir Entwickelungsmech., Bde. 12, 15, 16.
Resumen por la autora, Edith Pinney.
La supresiOn inicial del desarrollo en los 6évulos de fecundaci6én
cruzada.
I. Cruzamientos con el édvulo de Fundulus.
II. Cruzamientos reciprocos entre Ctenolabrus y Prionotus.
Las observaciones sobre las mitosis de la segmentacién tem-
prana en 6vulos de hibridos peces indican la existencia de factores
especificos que operan durante la anafase de segmentacién. La
division normal de los cromosomas no se lleva a cabo en muchos
6vulos hibridos. Mientras que el trastorno posee caracteristicas
generales, tales como la aglutinacién, retraso en el movimiento
de las mitades de los cromosomas y su falta de separacién, las
cuales resultan en la distribucién desigual de la cromatina en los
blastémeros hijos, la distribucién actual de los elementos cro-
maticos varia considerablemente. La autora interpreta dicho
trastorno, por consiguiente, como la expresién de la falta de
coordinacion entre factores del desarrollo, tal vez cambios espe-
cificos de viscosidad, del citoplasma del 6vulo y de la cromatina
del espermatozoide. Las pruebas acumuladas no prestan apoyo
a la hipétesis que supone que las cualidades hereditarias espe-
cificas de los cromosomas individuales juegan papel importante
en la reaccién. Este concepto de la naturaleza de los factores
que producen comportamiento anormal de la cromatina en los
6vulos hibridos de los peces esté en armonia con los resultados
del desarrollo obtenidos en varios cruzamientos que hasta el
presente no han podido explicarse basdndose en las relaciones
taxondmicas. La comparacién de los resultados de la hibri-
dacién en los peces demuestran que si dos especies se cruzan
reciprocamente sin trastorno en la mitosis de segmentacidn, los
évulos de estas dos especies reaccionardin de un modo semejante
cuando se fecunden con el mismo esperma extrafio. Este re-
sultado se aplica a todos los cruzamientos llevados a cabo con
peces hasta el presente, asf como los efectuados en los equinoder-
mos. Ademas, verifica las expectaciones de nuestra hipétesis de
que el comportamiento anormal de los cromosomas de los évulos
hibridos depende de la coordinacién entre los sistemas que
cambian fisicamente en el citoplasma del é6vulo y la cromatina
del espermatozoide.
Translation by José F. Nonidez
Cornell Medical College, New York
AUTHOR’S ABSTRACT OF THIS PAPER ISSUED
BY THE BIBLIOGRAPHIC SERVICE, APRIL 17
THE INITIAL BLOCK TO NORMAL DEVELOPMENT
IN CROSS-FERTILIZED EGGS
I. CROSSES WITH THE EGG OF FUNDULUS
II. RECIPROCAL CROSSES BETWEEN CTENOLABRUS AND PRIONOTUS
EDITH PINNEY
Lake Erie College
SEVENTEEN FIGURES (TWO PLATES)
There is a marked absence of specific fertilization qualities
among the germ cells of teleosts, such as has been demonstrated
in the eggs of certain invertebrates. It is true that the per cent
of fertilized eggs in fish hybridization varies widely, both in dif-
ferent crosses and in the same crosses at different times, but no
quantitative study of this variation has been made. There is,
however, a specificity factor present in fish eggs which has been
met with in the eggs of echinoderms (9). It expresses itself in a
disturbance of the mitotic process during the first cleavage
anaphase and forms the first critical block to development.
All of the nuclear phenomena following fertilization up to the
time that the equatorial plate is formed in preparation for the
first cleavage are normal in all of their visible morphological
aspects. During the anaphase, however, abnormalities arise.
In an earlier paper (15) it was shown that the abnormalities
in development in certain crosses could be traced to these abnor-
malities in the mitosis of early cleavage. Two general types of
mitotic behavior during the first cleavage division were observed
and described; one type being normal, division of the chromatin
occurring with undisturbed mitotic precision; the other type
being abnormal, showing a number of irregularities in the accurate
division of the chromosomes, such as unequal distribution,
fragmentation, and, possibly, elimination. That this early
401
402 EDITH PINNEY
mitotic disturbance was not the cause of all of the abnormalities
appearing during the course of development in fish hybrids is
quite certain, since hybrid eggs which undergo normal mitosis
in early cleavage often fail to develop (12). The cause of the
later-appearing derangements still remains to be determined. The
two problems are not necessarily identical and the present paper
will be limited to a consideration of the abnormal mitoses imme-
diately following fertilization and their significance.
In the paper referred to above (15) I suggested that the suc- —
cess of the first cleavage mitosis depends upon certain specific
physical conditions of the substratum, namely, the egg proto-
plasm. The new crosses described in this paper strengthen
that interpretation and, by extending the field for comparison,
throw light also, I believe, upon some hitherto rather ob-
scure and puzzling results of fish hybridization. I refer to the
absence of any correlation between developmental results and
taxonomic relationships.
For a detailed account of the behavior of the chromatin in
the Ctenolabrus crosses with Fundulus heteroclitus, Menidia
menidia notata and Stenotomus chrysops, the reader is referred
to my earlier paper (15). The methods used in the present
investigation are also fully discussed there. I will confine the
descriptive part of this paper to new crosses or new observations
on previously described crosses.
I. DESCRIPTION OF CROSSES WITH FUNDULUS
1. Fundulus heteroclitus 9 X Ctenolabrus adspersus &
This cross was discussed in my earlier paper. At that time
I had only a few preparations of my own. These taken in con-
junction with the figures published .by Morris (11) and compared
with my own preparations of normal Ctenolabrus eggs, formed
the material basis of my conclusions in regard to this cross.
In order to clear up any doubt to which the former limitations
in material might give rise, I wish to report here upon a new
lot of preparations which furnish abundant evidence that the
Ctenolabrus chromatin in the Fundulus eggs is very unequally
DEVELOPMENT IN CROSS-FERTILIZED EGGS 403
distributed in the first anaphase, as well as in subsequent divi-
sions. Figures la and 1b are from an egg in the first cleavage,
as are also figures 2a and 2b. Figure 3, a and b, and figure 4
show the anaphase of the second cleavage. The conditions
displayed here are so typical for all of the sections studied that
it seemed superfluous to multiply the evidence by many draw-
ings of practically the same thing. The only variation between
first anaphase spindles in these eggs is in the amount of undivided
chromatin which lags at or near the equator. This variation
indicates that, whether chromatin is actually eliminated or not,
and there is much reason to think that it is, the foreign chroma-
tin which does remain in the egg is distributed in a variety of
ways. Undivided chromosomes can pass to one pole only and,
therefore, one of the first two blastomeres lacks some chromo-
somes which the other contains. Thus extreme variation between
blastomeres arises and is increased during early cleavage. It
is a significant fact that the behavior of the chromatin during
the first anaphase is duplicated during the second cleavage.
Perhaps a word might be added here in description of the
lagging masses of chromatin. Those which are nearer the daugh-
ter groups of chromosomes resemble in contour and size the
smaller chromosomes of the foreign species (15). Other masses,
usually lying on or near the equator of the spindle, cannot be
reconciled with single chromosomes of either species. They
resemble more nearly the undivided chromosomes of the early
metaphase stages. Some of the masses show rod-like projec-
tions which suggest that perhaps two or even more chromosomes
have adhered to each other during their partial journey to the
pole. The appearance may be due, however, to nothing more
than a collision such as frequently occurs in normal anaphases.
Figure 3a shows what is plainly a split chromosome, the halves
of which are still adherent, passing to one pole. I have yet to
observe an anaphase figure in this cross at these early stages in
which there is no lagging chromatin.
JOURNAL OF MORPHOLOGY, VOL. 36, NO. 3
404 EDITH PINNEY
2, Fundulus heteroclitus 2 x Prionotus carolinus 3
In this combination there is the same abnormal behavior
on the part of the sperm chromatin in the early mitoses that is
seen in the cross just described. Figures 5 to 8, inclusive, show
first and second cleavage anaphases. All of the chromosomes
crowded at the ends of the spindle could not be included in
the drawing without altering their spatial relations. The lag-
ging chromatin, however, has been depicted as accurately as is
possible with such minute objects. There can be no doubt that
here again we would obtain a great range in variation in the
chromatin content of early blastomeres. As before, the second
cleavage anaphase repeats the abnormal behavior of the first.
Observations upon the prophase stages of the first cleavage
were not made, but it seems reasonable to assume that they
resemble the prophase stages of the second cleavage. After
the first cleavage two normal-appearing nuclei are reformed, the
centrosome divides, the asters which are to function in the second
cleavage appear, and their growth proceeds normally. This
process is beautifully clear in the Fundulus egg. That the rays
of the aster are formed by the rearrangement of the cytoplasmic
reticulum, as described by Wilson (17) for the sea-urchin egg,
is quite obvious, and one feels convinced that, however much
the structural appearances in fixed material differ from the actual
state of the living egg, the relation between astral system and
cytoplasm is the same in both. When the astral rays have ex-
tended well out into the cytoplasm, approximately half-way to
the cell wall, division of the chromatin, which meanwhile has
formed the equatorial plate, begins. It is during this ensuing ana-
phase stage that abnormalities arise. The point I wish to make
is that if the second anaphase which is abnormal is preceded by
perfectly normal processes as far back as the first anaphase
which was likewise abnormal, then we may assume that it in
turn is preceded by a normal prophase. Morris gives figures
which show the early stages preceding the first cleavage ana-
phase in the cross Fundulus 9 xX Ctenolabrus @ to be normal
(11). The same sort of observations were made by Godlewski
DEVELOPMENT IN CROSS-FERTILIZED EGGS 405
on a cross between members of two different classes of Echino-
derms (5).
It must be remembered that in speaking of normal processes
I refer only to the visible, morphological changes which follow
each other in orderly succession in the course of cleavage. This
cycle is uninterrupted and the changes themselves appear nor-
mal. If any deviations from the normal course of affairs are
present, they are far too slight to be recognized even in a very
careful study.
In order to meet the possible objection that this characteris-
tic lagging and clumping is not typical of this and the foregoing
cross, I should perhaps emphasize the fact that the peculiarities
described appear in every egg observed at this stage. That
it is not an artifact, due to poor fixation, is proved by the
fact that preparations of other crosses with the same egg
in which the same technique was used show normal anaphases
as consistently as these exhibit abnormal anaphases. If these
appearances were artifacts, we should not expect to find this
regularity in their occurrence. It should also be mentioned in
this connection that in all of the crosses with Fundulus eggs, the
eggs are taken from several females and placed in the same dish.
When the sperm is added the eggs are stirred about so that in
taking out a pipette full of eggs to fix it is certain that the eggs
are well mixed and that those fixed in any stage originate from
several females. The character, then, which determines the
behavior of the chromatin during the anaphase is not an indi-
vidual character, but is common to the species.
3. Fundulus heteroclitus 2° & Menidia menidia notata ov
The behavior of the chromatin in this cross has been described
by Moenkhaus (10), and the facts have become such a familiar
part of our cytological knowledge that any further description
of the details of cleavage are unnecessary. My purpose in re-
peating his observations was to determine whether the condi-
tions he describes are common to all hybrid eggs of this cross.
My preparations resemble his descriptions and figures so closely
and so consistently that there is left no room for doubt that -here
406 EDITH PINNEY
we have a cross in which this block to normal mitosis of the ana-
phase period is absent. Figures 9 and 10 are from my material
and present first and third cleavage conditions during the criti-
eal period. Division is normal.
4. Fundulus heteroclitus 9 X Stenotomus chrysops ¥
Of this cross I am able to give only second-cleavage figures
(11, a and b, and 12, a and b). These show the usual condi-
tions in straight-fertilized eggs. There is no lagging or clumping,
and I feel that one may safely conclude that the same conditions
prevail during the first cleavage. I base this inference upon
observations made on other crosses reported here and else-
where (15).
Figure 12 shows the two sections of one spindle. The chromo-
somes were so well separated that it was possible to count them.
There are forty-six at either pole. In figure lla the individual
chromosomes have been drawn spread out laterally, so that all
of the rods of each group are not reproduced in their actual posi-
tions. Such a drawing illustrates how much more can be gained
from a study of the preparations than is indicated by the drawings
themselves. In this figure the longer Fundulus chromosomes
are easily identified, as are the smaller Stenotomus elements.
The rods of medium length cannot with certainty be ascribed
to either species. There is a definite grouping on this second-
cleavage spindle. Evidently, the plane of the section has passed
to one side of the Fundulus group, as all of the long rods are in
one section. The smaller chromosomes in figure 12 b belong to
the Stenotomus group.
This cross, therefore, shows the type of behavior that is char-
acteristic of self-fertilized eggs. It falls in the same group as
the cross with Menidia.
II. RECIPROCAL CROSSES BETWEEN CTENOLABRUS ADSPERSUS
AND PRIONOTUS CAROLINUS
In all of the reciprocal crosses with the cunner which I had
studied up to this time there was a marked difference in the
chromatin behavior during the early anaphases. In the crosses
DEVELOPMENT IN CROSS-FERTILIZED EGGS A407
in which the egg of Ctenolabrus was used normal mitotic divi-
sion was the rule. The eggs of Stenotomus, Fundulus, and
Menidia, however, when fertilized with the sperm of Ctenolabrus
showed the typical abnormality during the anaphase cleavage
which is described above. It was, therefore, a matter of interest
to find a cross with this species in which the reciprocals were
alike in their early mitotic behavior, as is the case in the cross
between Ctenolabrus and Prionotus. The behavior is normal
in both eggs. Nothing earlier than second-anaphase figures of
both of these crosses were observed. From much observation
of other crosses I feel convinced that the second cleavage mitosis
resembles the first very closely, and that therefore in these recip-
rocal crosses no elimination of chromatin or abnormalities in
mitosis occur during the first cell division.
I thought that perhaps some evidence on this point might
be gained from a study of polar groups of chromosomes after
actual division had occurred. With this in mind, I attempted
to estimate the chromosomes to be expected in polar groups and
then determine whether second-anaphase groups fulfilled this
expectation. The sources of error in such a study are numerous,
and the estimates that I have made can only claim to be approxi-
mate. Counts of both polar and lateral views of anaphase
groups place the number of chromosomes in the normal Priono-
tus egg near fifty. My earlier counts of the species Ctenolabrus
give indications that the number there is about forty-four. We
should then expect between forty-five and fifty in the hybrid
eggs. Actual counts are as follows:
52 53 56 49
’ 51’ 56’ 59’ not counted
55 58 55
58’ 50’ 50°
The unexpectedly large number here is probably due to the
sectioning of single chromosomes. No entire spindles or even
single polar groups were found. Obviously, such material is
not adapted to accurate counting. As evidence, while it may
show that no great elimination of chromosomes has occurred,
as regards irregular distribution of chromosomes it has no value.
Ctenolabrus 2 xX Prionotus &
Prionotus @ X Ctenolabrus 0,
408 EDITH PINNEY
The best evidence of regular division is obtained from early
anaphases. Such a stage is drawn in figure 13, a and b. There
is no abnormality. Mitosis ,here is wholly orthodox. Corre-
sponding daughter chromosomes on their way to opposite poles
can be identified.
As seen from this figure and figures 14 and 15, Prionotus
chromosomes are indistinguishable in a Ctenolabrus egg. The
only difference between the two species of elements lies in the
probable presence of more hooked-shaped chromosomes in
Ctenolabrus, but it has never been determined that shape is
a constant feature in fish chromosomes. Figures 16 and 17 are
from the cross Prionotus @ xX Ctenolabrus ¢.
SUMMARY OF DATA
The following table presents, in a summarized form, the data
for all of the crosses thus far studied. In case of the cross,
Menidia @ xX Fundulus <, the results stated here are those
CTENOLABRUS FUNDULUS STENOTOMUS MENIDIA PRIONOTUS
°) g L°) 2 .°)
Ctenolabrus ~ x Early mi- | Early mi- | Early mi- | Early mi-
tosis ab- tosis ab- tosis ab- tosis
normal normal normal normal
Fundulus Early mi- x Early mi-
tosis tosis
prevail- norma!
ingly
normal
Stenotomus o& | Early mi- | Early mi- x
tosis tosis
normal normal
Menidia Early mi- | Early mi- x
tosis tosis
normal normal
Prionotus @ Early mi- | Early mi- x
tosis tosis ab-
normal normal
DEVELOPMENT IN CROSS-FERTILIZED EGGS 409
reported by Moenkhaus (10). I have not verified his results.
Figure 23 of his paper was earlier interpreted by me as indicat-
ing lagging in this hybrid. The figure, however, shows only
one pole of the spindle and there is nothing to indicate the posi-
tion of the equatorial plane, which fact I overlooked in my diag-
nosis. His other figures are free from the undivided masses of
chromatin which are characteristic of the irregular mitosis found
in some crosses. This behavior justifies its inclusion in the group
of normally reacting crosses.
DISCUSSION
1. The significance of the early mitotic disturbance in hybrid eggs
Teleost eggs clearly exhibit a factor which regulates the
activity of the sperm chromatin in cleavage. The nature of the
mitotic disturbances which have been described above would
indicate that the immediate factor was a physical condition of
the egg cytoplasm, probably the normal state of fluidity or vis-
cosity characteristic of the division phase. The possibility that
physical factors of the sperm take part in this reaction should
not be excluded, but in the progress of cleavage the egg cyto-
plasm plays a more active réle, while the elements contributed
by the sperm are relatively passive (15). In this sense the egg
determines the cleavage phenomena, the process of chromosome
separation as well as the rate of the antecedent processes.
The results of heterogeneric hybridization in echinoderms are
analogous to those in fishes. We have several accounts of the
cytological events following cross-fertilization in these forms,
and in all of them, with one or two exceptions, the description
of the behavior of the chromatin resembles very closely that
given for fishes (1, 4, 5, 7, 8, 16).
In order to be convinced that the phenomena are similar in
both forms, the reader has only to compare the figures given
here for the two Fundulus crosses in which the sperm of Cteno-
labrus and Prionotus was used and the figures already published
by the writer of the mitotic process in eggs of Stenotomus and
410. EDITH PINNEY
Menidia fertilized by the sperm of Ctenolabrus (15) with the
figures given by Herbst (7) for Sphaerechinus ° xX Strongylo-
centrotus &, by Baltzer (1) for the eggs of Strongylocentrotus,
Echinus, and Arbacia fertilized by Sphaerechinus sperm, and
by Tennent (16) for the reciprocal crosses between Arbacia and
Toxopneustes.
The behavior of the chromatin in the cross between Echinus
acutus @ xX Echinus esculentus <, reported by Doncaster and
Gray (4), shows slight differences from those described by the
foregoing investigators. In the latter cross the chromosomes
form vesicles and are eliminated during the anaphase. The
vesicles make their appearance in the prophase, which may in-
dicate that the origin is different from that causing the lagging
occurring in other forms. It appears that lagging was also
observed by these authors in connection with the cross Echi-
nus acutus @ xX Echinus milearis 3, for of this cross they say,
“In the hybrid eggs we found that some of the chromosomes
developed vesicles, but no other elimination occurred except
the possible non-division of certain chromosomes, about which
we are uncertain.”
Godlewski (5) reports chromatin elimination in his interclass
cross between Echinus @ and Antedon o&. The Antedon
chromosomes take part in the first mitosis, but are eliminated
later. Counts of polar views of anaphase groups are given as
evidence. The figures accompanying this paper are unsatis-
factory in that they give no evidence as to the manner in which
elimination is accomplished.
In the cross studied by Kupelwieser (8) we have an extreme
ease of elimination, due probably to factors operating earlier
in development.
The actual elimination of chromosomes from the mitotic
mechanism rests on more conclusive evidence in the case of
echinoderms than it does in that of fishes.
Unequal distribution of paternal chromatin unquestionably
occurs in both, although aside from its casual mention by Don-
caster and Gray (4), Herbst (7) is the only investigator to report
this for echinoderms. Baltzer’s figures, however, show unmis-
DEVELOPMENT IN CROSS-FERTILIZED EGGS 411
takable cases of undivided chromosomes passing to one pole
(1). It is more difficult to decide in the case of the figures given
by Tennent (16), since the matter is complicated by the presence
of the large V-shaped chromosomes of Toxopneustes.
The evidence from fish crosses does not support the idea that
the chromosomal behavior is highly specific. If only certain
paternal chromosomes were affected, as Baltzer claims does
occur in certain of his crosses (1), then we would expect to find
in every anaphase of the first cleavage the same picture. This
is not the case. The amount of lagging varies. It also occurs
in the cleavage following the first and always at the anaphase.
In Baltzer’s crosses, as his figures show, mitotic disturbances
appear in all of the early cleavages including the fourth. I
have elsewhere (14) emphasized the difficulties in the way of
identifying many of the extruded chromosomes in echinoderm
crosses. If elimination or unequal distribution depends upon
the specific nature of individual chromosomes, we would expect
more variability in the character of the disturbance, more regu-
larity in the extent of its occurrence during any one mitotic
phase, as well as an expression of its specificity during other
periods of the mitotic cycle. The fact that it appears only
during the anaphase, that it is variable in extent, and that it
occurs in the second, third, and fourth cleavages as well as in
the first is in harmony with the suggestion that the behavior is
the result of a general physical reaction between the egg cyto-
plasm and the sperm chromatin. |
All of the figures referred to strengthen the impression that
the disturbance is due to a general physical reaction involving
the entire sperm chromatin and the egg cytoplasm and is not
the result of a differential action of the egg components toward
individual paternal chromosomes due to the specific differences
existing in the hereditary quality of the latter. According to
modern ideas of heredity, the chromosomes determine taxo-
nomic characters. In so far they are specific. Their relation in
cell division is the same for all. If some paternal chromosomes
undergo abnormal distribution or elimination and others escape,
it is a result of chance rather than of specific hereditary qualities.
412 EDITH PINNEY
The whole phenomenon is one that is concerned with develop-
mental rather than with hereditary factors.
If, as was originally suggested by Biitschli (18, 2), protoplas-
mic currents are concerned in the movements of chromosomes,
we might have in the specific character of such currents the
physical factor necessary to regulate chromosome division in these
cases. The results of Chambers (3) and Heilbrunn (6) are highly
suggestive in this connection. Both of these workers have
demonstrated changes in the viscosity of the cell protoplasm
during mitosis. Specific viscosity differences in the eggs at the
anaphase period may cause the abnormal division of chromo-
somes occurring in some heterogeneric hybrids. The conditions
of viscosity that prevail during the cleavage of these hybrids are,
I believe, the normal conditions always present in the egg and
are not deviations from the normal caused by the foreign sperm.
I infer this from the fact that the egg cytoplasm exerts a differ-
ential effect toward the two sorts of chromosomes which it con-
tains. The egg chromosomes divide normally. The sperm
elements show abnormal behavior.
There are two exceptions to this. Doncaster and Gray (4)
consider that the abnormally behaving chromatin in the cross
Echinus acutus @ x Echinus esculentus & is of maternal ori-
gin. The phenomenon described by them for that cross, how-
ever, is of an apparently different nature and need not be con-
sidered in this category. The other exception occurred in the
cross, Arbacia 9 X Toxopneustes <, in which Tennent (16)
observed the elimination of chromosomes of both species from
the nucleus. These eggs were, however, given rather ‘drastic
treatment to cause penetration of the foreign sperm. The eggs
stood in sea-water for four hours and were then treated with
alkaline sea-water. If this treatment in any way changed the
egg cytoplasm, the results are no longer inconsistent, but follow
the expectations of the hypothesis expressed here. The point
could be tested perhaps by self-fertilizing Arbacia eggs treated
in the same manner.
DEVELOPMENT IN CROSS-FERTILIZED EGGS 413
2. Chromosome behavior and taxonomic relationships
Whatever the nature of the physical condition governing the
mitotic processes at the critical anaphase stage, the condition
itself is no doubt an expression of the specific chemical composi-
tion of the egg cytoplasm. This chemical composition may
be more highly specific than the physical state which it condi-
tions, that is, it is conceivable that the egg protoplasm of two
species of fish may differ chemically and yet resemble each other
so closely in their physical characters that they may react alike
in crossing. In other words, protoplasmic relationships between
species are not necessarily correlated with the physical condi-
tions present in their germ cells at corresponding morphologi-
cal stages. This, I believe, explains the fact that the results
of heterogeneric hybridization show no correlation with taxo-
nomic relationships.
While the presence or absence of abnormal mitosis in early
cleavage is not correlated with taxonomic relationships, a com-
parison of the crosses made shows some indication of an under-
lying relationship based on the egg’s behavior in this respect
which is independent of species affinities. For instance, refer-
ence to the tabular summary above shows that when the germ
cells of Menidia and Fundulus combine reciprocally, develop-
ment is not hindered by this block to normal mitosis, although
it may, and usually does, meet with some disturbing factor later
on. Both of these eggs exhibit such a block to the sperm of
Ctenolabrus; that is, both show the same behavior to the same
foreign sperm. Further, the reciprocal crosses of Ctenolabrus
and Prionotus show similar behavior in that both proceed nor-
mally. The egg of Fundulus produces the same reaction in the
sperm of both of these species. I should like to have made fur-
ther tests by crossing Menidia @ with both Prionotus @ and
Stenotomus <, but unfortunately Menidia eggs were not ob-
tained in 1921. One of course hesitates to draw conclusions
from so little evidence, but the facts are certainly significant.
A review of the reactions of echinoderm eggs in hybridiza-
tion reveals certain similarities of behavior which favors the
414 EDITH PINNEY
same interpretation. Lillie (9, p. 191) has tabulated the results
of Baltzer’s crosses. From his table it is seen that Echinus and
Strongylocentrotus cross reciprocally with no elimination. The
eggs of both species eliminate Sphaerechinus chromosomes at
the first cleavage. The egg of Sphaerechinus, on the other hand,
tolerates the male chromatin of either species. In addition
the two sorts of eggs eliminate Arbacia chromosomes, but not
until the blastula stage is reached.
In connection with these echinoderm crosses one should re-
member that cross-fertilization was only possible after treating
the eggs with alkaline solutions. The primary effect of this
treatment is to alter the normal cortical reaction of the egg to
the foreign sperm. If it affects the physical character of the
cytoplasm, it is more than probable that it does this in a uni-
form manner so that the same relative conditions obtain in the
treated eggs as would exist in eggs that were not treated.
Norman (13) showed a difference between the eggs of Cteno-
labrus and Fundulus, forms which behave differently in crossing,
by subjecting them to the action of heat: 30°C. was suff.cient
to stop segmentation in Ctenolabrus, while it required a tempera-
ture of 38°C. to produce the same effect in Fundulus. This
indicates some specific difference in the cytoplasm of these two
eggs. It is not unreasonable to suppose that other fish eggs
would show different points of susceptibility to heat in this
regard. Whether such susceptibility points would follow the
taxonomic affinities of species or show independent variation
would be an interesting point to determine in this connection.
The evidence so far accumulated seems to me to point to the
variation in the physical factors controlling mitosis as one basis
upon which the lack of correlation between developmental suc-
cess in fish hybrids and taxonomic relationships can be explained.
If this view is correct, it should be possible to reproduce these
phenomena experimentally in both straight-fertilized eggs and
in crosses. In that direction lies the hope of further analysis.
-DEVELOPMENT IN CROSS-FERTILIZED EGGS 415
LITERATURE CITED
Bauzer, F. 1910 Ueber die Beziehung zwischen dem Chromatin und der
Entwicklung und der Vererbungsrichtung bei Echinodermenbastarden.
Archiv. f. Zellforschung, Bd. 5.
Birscuur, O. 1900 Bemerkungen iiber Plasmastrémungen bei der Zell-
theilung. Archiv. f. Entwickelungsmechan., Bd. 10.
CHAMBERS, R. 1917 Microdissection studies. II. The cell aster; a rever-
sible gelation phenomenon. Jour. Exp. Zod6l., vol. 23.
Doncaster, L., anp Gray, J. 1913 Cytological observations on the early
stages of segmentation of Echinus hybrids. Quart. Jour. Mic. Sei.,
vol. 58.
GoptrewskI, E. 1906 Untersuchungen iiber die Bastardierung der Echini-
den und Crinoidenfamilien. Archiv. f. Entw. Mech., Bd. 20.
HeI~prunn, L. V. 1920 An experimental study of cell-division. I. The
physical conditions which determine the appearance of the spindle
sea-urchin eggs. Jour. Exp. Zo6l., vol. 30.
Herest, Curt 1909 Vererbungstudien VI. Archiv. f. Entw. Mech.,
Bd. 27.
Kupetwiseser, Hans 1909 Entwicklungserregung bei Seeigeleiern durch
Molluskensperma. Archiv. f. Entw. Mechan., Bd. 27.
Linuiz, F.R. 1919 Problems of fertilization. Univ. of Chicago Press.
Mornxuavs, Wo. J. 1904 The development of the hybrids between Fun-
dulus heteroclitus and Menidia notata, with especial reference to the
behavior of the maternal and paternal chromatin. Am. Jour. Anat.,
vol. 3.
Morris, Maraaret 1914 The behavior of the chromatin in hybrids be-
tween Fundulus and Ctenolabrus. Jour. Exp. Zodl., vol. 16.
Newman, H. H. 1915 Development and heredity in teleost hybrids.
Jour. Exp. Zodl., vol. 18.
Norman, W. W. 1896 Segmentation of the nucleus without segmentation
of the protoplasm. Archiv. f. Entw. Mechan., Bd. 3.
Pinney, Evira 1911 A study of the chromosomes of Hipponoe esculenta
and Moira atropos. Biol. Bull., vol. 21.
1918 A study of the relation of the behavior of the chromatin to
development and heredity in Teleost hybrids. Jour. Morph., vol. 31.
TENNENT, D. H. 1912 Studies in cytology. II. The behavior of the chro-
mosomes in Arbacia-Toxopneustes crosses. Jour. Exp. Zodl., vol. 12.
Witson, E. B. 1895 Archoplasm, centrosome and chromatin in the sea-
urchin’s egg. Jour. Morph., vol. 11.
1901 Experimental studies in cytology. II. Some phenomena of fer-
tilization and cell-division in etherized eggs. Archiv. f. Entw.
Mechan., Bd. 18.
EXPLANATION OF PLATES
All figures were drawn with the aid of a camera lucida. The optical equip-
ment consisted of a 1.8 oil-immersion objective with a no. 8 ocular. Thedraw-
ings, made at table level, at a magnification of 1600 X, are reproduced as drawn.
It is not possible to produce a drawing in two planes which will show the objects
as adequately and convincingly as they appear when studied in three planes.
Added to this is the difficulty of drawing such minute objects as fish chromo-
somes accurately. The drawings are as faithful a representation of the facts
as can perhaps be boped for under these conditions.
PLATE 1
EXPLANATION OF FIGURES
1to4 Fundulus heteroclitus 9 X Ctenolabrus adspersus o’.
1,aandb An anaphase of first cleavage in two sections. Some chromosomes
omitted for clearness. All of the lagging chromatin is included in the drawings.
Chromosomes of Fundulus are more numerous in a. Ctenolabrus chromo-
somes appear in b.
2,aandb A late anaphase in two sections. Two types of chromosomes can
be recognized. There is lagging, but the amount of chromatin involved is less
than that in figure1. Not all of the chromosomes were drawn.
3, aandb Two sections of one spindle. One split chromosome is seen pass-
ing to the lower pole of a. The hooked chromosomes in a are characteristic of
Ctenolabrus.
4 One section of a fairly late anaphase of second cleavage, showing an acute
case of irregular distribution of chromatin. This is typical for the rest of the
two spindles present in this egg.
5to8 Fundulus heteroclitus 9 X Prionotus carolinus ~. The chromosomes
of both species can be identified in these drawings. The Prionotus type are
short rods. There are fewer hooks than were found in Ctenolabrus.
5 An early anaphase of second cleavage. Only one of the two sections of the
spindle is given. Marked lagging.
6 The middle section of a first-cleavage anaphase that appeared in the prepa-
rations in three sections. Some chromosomes omitted.
7 Anearly anaphase of the second cleavage. Marked lagging.
8 An older spindle of the same stage. Only one section drawn. Both types
of chromosomes are seen at the poles.
416
LATE 1
DEVELOPMENT IN CROSS-FERTILIZED EGGS
EDITH PINNEY
Se SSS
= SE
= ene RE —
emt oe ee a eres == es ad
=
= SaaS ae
SS Sars. ee
amen ee eS
al o en ieee
= a 5) a Ses
I =m a = ——
—~aa) eS So.
ae =
Se ee —— ee pony ee
——__—_————eer—™ —
SSS SE — = Pee ‘=
= ar ee Oe ——
— Se Ge eat eee —_- eee e
<-4¢= Se ee — Sa
oa Yipee ee — eS
—— = —_none—- S
[2) SSS SS ae
=
ee
SS a
—— ee —————
= grt — == eee
— = = = es
—— —S == —
—— — oe eee Se eee — a a
— —_ = —_— @: = ——_ —— ——, —
ee ee ee eee (ees
Sw = ee ———
iS
417
3a
PLATE 2
EXPLANATION OF FIGURES
9 Fundulus heteroclitus 2? X Menidia menidia notata ™. First-cleavage
anaphase. A few of the Fundulus rods have been displaced by the knife. Only
one section drawn.
10 Third-cleavage anaphase of the same cross. Many chromosomes omitted.
No lagging.
11, a and b Fundulus heteroclitus @ xX Stenotomus chrysops &*. Two sec-
tions of an anaphase of second cleavage. All of the chromosomes are drawn.
There are forty-six at either pole. Those in a have been spread laterally in
drawing.
12, a and b Second-cleavage spindle of the same cross. No lagging. Some
chromosomes omitted in drawing.
13, a and b Ctenolabrus adspersus 9 X Prionotus carolinus o&. Two sec-
tions of an early anaphase of the third cleavage. The daughter halves of divid-
ing chromosomes could be easily identified not only for those drawn, but in the
case of those omitted from the drawing.
14 A later anaphase of the same cross. Third cleavage. No lagging.
15 Same as 14.
16 Prionotus carolinus @ X Ctenolabrus adspersus o&. Second cleavage.
No lagging.
17 Samecross. Later anaphase of second cleavage. Normal mitosis.
418
- _ = =
—_—_Yy -=_— =~ tae ———
ee ee 6 Sg
4 Sg ae
= ee = —
———a Ce eee oe
= — ~— - —
—s —>—$—. nee Se =
a a Scone ——
a nw —_—_——_ = ——
a — —
—— ge
<= — Se =
== oe = BS —
So Ss ES i
— So a a= =
—— =—-— _ _.
—_—=— SS ——___—_ So SS
SS ee, ee ae 7
See a — eee
(Se aay a eee Se.
SS —— ————— —— oso
a Le — ee yee Se
SS Se, Se
2 eS eee
—- — # Se ———_
et _Y a SE
SSC SS a ——
=o f. ee
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—————— EF eo _ SS ea—
——— — oe ee o—— =
———— ES —=—F cs ———
a SS <= [os]
male
ack
ein
Resumen por el autor, Oliver P. Hay.
Sobre la filogenia del caparazén de los Testudinata y las relaciones
de Dermochelys.
El presente trabajo renueva una discusién comenzada en 1899
sobre las relaciones de la tortuga con caparazon coridceo, Dermo-
chelys, con las otras tortugas. La primera posee en la parte
dorsal del caparazon siete filas de grandes placas 6seas; en la
parte ventral cinco filas solamente. El estudio de otros miembros
del mismo 6rden demuestra que estas filas estan representadas
por el mismo ntimero de escudos cérneos; algunas de las filas
han sido halladas solamente en unas pocas especies. En casos
raros existen elementos 6seos debajo de estos escudos.
Las tortugas mds antiguas posefan un caparaz6n externo (el
de Dermochelys) y uno interno (el de las tortugas ordinarias).
Dermochelys hered6 el caparazén externo perdiendo la mayor
parte del interno; las otras tortugas perdieron el externo quedando
solamente vestigios. Varios autores se han opuesto a esta teoria,
especialmente Verluys y sus discipulos. En el presente trabajo
el autor intenta responder a sus criticas, llamando la atencién
acerca del caparazon del género Chelys, en el cual ha encontrado
huesos distintos debajo de los escudos cérneos de las cinco filas
superiores (media, primera lateral y periférica) y debajo de dos
de las filas del caparazén ventral (segunda fila a partir de la
linea media). A consecuencia de esto el otro érden de los
Testudinata consta de dos sub-6rdenes, Athecae y Thecophora.
La presencia de otros huesos dérmicos es de dificil explicacién.
Pueden ser equivalentes a los huesos encontrados en Dermoche-
lys entre las filas de los huesos mds grandes.
Translation by José F, Nonidez
Cornell Medical College, New York
AUTHOR’S ABSTRACT OF THIS PAPER ISSUED
BY THE BIBLIOGRAPHIC SERVICE, MAY 22
ON THE PHYLOGENY OF THE SHELL OF THE
TESTUDINATA AND THE RELATIONSHIPS
OF DERMOCHELYS
OLIVER P. HAY
Associate of the Carnegie Institution of Washington
ONE TEXT FIGURE AND TWO PLATES
Some years ago the testudinate genus Dermochelys was an
object of interest to the writer, and he discussed the structure
and origin of its peculiar shell and the systematic position of the
animal (Amer. Naturalist, vol. 32, 1898, pp. 929-948. The
Fossil Turtles of North America, 1908, p. 23). Since that time
several important papers on the subject have been published,
especially by Dr. J. Versluys and his students. The writer
wishes to take up again briefly the subject. Inasmuch as Doc-
tor Versluys’ paper, ‘‘Uber die Phylogenie des Panzers der
Schildkréten und wber die Verwandtschaft der Lederschild-
kréte Dermochelys coriacea’”’ (Palaeont. Zeitschr, Bd. 1, 1914,
S. 321-347), furnishes a résumé of the results obtained by him-
self and his coworkers, this paper only will be directly considered.
Doctor Versluys rightly emphasizes the importance of Dermo-
chelys, recognizing that either it represents a very old lateral
branch of the testudinate stem or that in its shell it presents a
remarkable example of a rapidly divergent development. He
concludes that the view has been confirmed which makes of
Dermochelys a not very distant relative of the Cheloniidae.
Dermochelys is regarded by Doctor Versluys as belonging to
the Cryptodira for two principal reasons. The first is that the
neck is bent in a vertical plane, as in the Cryptodira, instead of
a horizontal one, as in the Pleurodira; the second, that the indi-
vidual vertebrae conform in the shapes of their articular ends to
the arrangement in the Cryptodira. As to the first proposition
421
422 OLIVER P. HAY
it may be said that the primitive testudinates had relatively
undifferentiated cervicals and short necks which could be bent
equally well in all directions. A retraction of the head for de-
fense, first between the fore legs and later into the shell by bend-
ing the neck in a vertical plane, is the action that has been adopted
by the great majority of turtles, not only the Cryptodira, but
also the Trionychoidea. The method of protecting the head
resorted to by the Pleurodira is a special one, and must have
been the result of special conditions. Of living species of turtles
about four-fifths bend the neck in a vertical plane, only one-fifth
in a horizontal. Of known extinct species apparently many
more than four-fifths belong to Cryptodira and Trionychoidea.
Hence if Dermochelys was derived from an independent branch
of the protestudinates, there are certainly more than four chances
out of five that the species would have adopted the habit of
bending the neck in a vertical plane.
To Versluys’ second proposition one may reply that it is not
true that the forms and the order of succession of the cervicals
are as fixed in the Cryptodira, as might be supposed from his
statement. The reader may consult Vaillant’s paper on this
subject (Ann. Sci. Nat., ser. 6, vol. 10, art. 7, pp. 1 to 106, pls.
25 to 31).
Variations in the form of the articular surfaces are found in
the cervicals of other groups of turtles. In the pleurodires
they are constructed so as to permit easy movement in a hori-
zontal plane; but there exist deviations from the general plan.
In the Trionychoidea (essentially Cryptodira) the typical ar-
rangement is for all except the first and last to be convexoconcave.
Probably no one is able to say what advantages result to the
cryptodires in having the fourth so generally biconvex, with
those in front of it convexoconcave and those behind it concavo-
convex. Versluys (p. 325) has suggested that it is in adapta-
tion to the strong curvature of the neck during retractions of
it; but in the trionychids the curvature is excessive, and here
all the vertebra, except the first and the last, are convexoconcave.
That it is a matter of indifference one can hardly believe. We
seem to be justified in concluding that the forms of these cervi-
PHYLOGENY OF SHELL OF TESTUDINATA 423
cals may be modified to suit the requirements of the creatures,
and we need not suppose that these modifications required great
periods of time. If indeed Pyxis has all of its cervicals procoe-
lous, we can hardly conclude that its ancestors back to the primi-
tive turtles had such cervicals.
However the ancestors of Dermochelys took their origin, the
neck was, and has probably always been, short. If they formed
one of the two divisions resulting from the first cleavage of the
order they may have very early taken to a habitual aquatic
existence. Leading such a life and possessing short necks, it is
improbable that they would have developed side-bending necks.
Having the same number of cervicals, each composed of the same
primary elements, and experiencing the same needs in sustaining
the head in swimming and in protecting it as did the Cheloniidae,
there seems to be no reason why exactly the same kind of cervi-
cals should not have been produced. If slight differences at
first existed, we must suppose that these would have been elimi-
nated in time, unless we believe that heredity prevailed over
adaptability.
It will be impracticable to consider all of the seven structures
which Doctor Versluys discusses as showing a probable close
relationship between Dermochelys and the Cheloniidae. One
may grant that his arguments possess force, without admitting
that they subvert other considerations. Some of the structures,
as the intertrabecula and the pouches in the nasal passages, are
of obscure origin and purpose and in need of further investiga-
tion. As regards the intertrabecula, may it not have been
possessed by the protestudinates and transmitted by them to
the Thecophora and the Athecae alike? It may later have been
lost by most members of the former group. Relatively few
testudinates have been examined for this structure, and the
discovery of it in any one species of Cryptodira outside the
Cheloniidae, in any of the Pleurodira, or of the Trionychoidea
would be fatal to the conclusion that has been drawn from its
presence in the Cheloniidae and Dermochelyidae.
As to the structure of the roof of the mouth, the palatine bone,
and the position of the choanae, one might easily admit all that
424 OLIVER P. HAY
Doctor Versluys affirms, without admitting that his conclusion
follows. A secondary palate is a possession of some turtles of
all the higher divisions of the order, and there is hardly a possi-
bility that these secondary structures have been derived in all
cases from a common source. The early representatives of the
Athecae were probably swamp- or coast-frequenting species
and they may have subsisted on hard food; the mastication of
this may well have developed a secondary palate. Having later
taken more and more to life in the sea and to soft food, the palate
may have gradually degenerated to its present state.
We are indebted to Doctor Versluys for the finding of a large
parasphenoid bone in Dermochelys (Zool. Jahrb. Anat., Bd.
28, S. 283-294) and his discovery appears to be confirmed by two
disarticulated skulls in the U. 8S. National Museum. Inasmuch
as this bone has not been recognized in any of the other sea tur-
tles, Versluys concluded that there was no close relationship
between the Cheloniidae and Dermochelys. Certainly, if the
latter genus had been derived from any of the Cheloniidae, we
might expect that some of the Cretaceous members would possess
a parasphenoid.
On the part of those who believe that Dermochelys and its
allies have been derived from the chelonioid Cryptodira, much
importance has been given to the fact that the eighth cervical
in both the Cheloniidae and Dermochelys forms an articulation
with the nuchal, and Doctor Versluys makes allusion to it.
To the writer it appears that this articulation has lost its impor-
tance as a mark of kinship. From Versluys (p. 322, footnote)
we learn that Menger has discovered that the nuchal is a com-
posite bone, one layer of which may have been derived from
the ribs of the hindermost cervical. This could hardly have
come to pass without a close connection of the neural arch of
that vertebra with the nuchal. Jaekel (Palaeont. Zeitschr.,
Bd. 2, S. 102) has found that in his Stegochelys (Triassochelys)
the spinous process of the eighth cervical (Jaekel’s first dorsal),
as well as that of the succeeding vertebra, is attached without
suture to the nuchal. In the great majority of these reptiles
the connection has been dissolved; in the sea-inhabiting mem-
bers of the group it has, for special reasons, been retained.
PHYLOGENY OF SHELL OF TESTUDINATA 425
Versluys (p. 326) holds the view that, since the Cryptodira
possess the thecophore shell inherited from the Amphichelydia,
the primitive ancestor of Dermochelys must also have possessed
such a shell, and by this there appears to be meant a practically
complete shell such as that of the Cheloniidae. The present
writer holds, however, that Dermochelys was not derived from
the Amphichelydia and has therefore nothing to do with the
eryptodires. The common progenitor of the Athecae and the
Thecophora possessed the elements of the armor found now in
Dermochelys; likewise, perhaps in a rudimentary form, the ele-
ments which constitute the carapace and the plastron of the
other existing turtles. Proceeding from this common condition,
the Thecophora lost the superficial skeleton, but developed the
deeper-seated one, while in the Athecae the inner one became
more and more reduced.
Versluys appears to be in doubt whether or not the epithecal
armor of Dermochelys was secondarily developed. He is in-
clined to regard it as composed partly of new elements, partly of
old. The median and costal rows of enlarged scutes of the leather-
back may, he thinks, be new structures, and he refers to those
epithecal bones found alternating with the neurals in Toxochelys
and the more numerous ones of Archelon. He thinks it possible
that new epithecal bones might arise under the horny scutes
at their center of growth. This appears to be a reasonable
proposition. It would provide for rows of four or five bones;
but how would Doctor Versluys account for the approximately
fifty bones in each of the seven rows of the carapace of Dermo-
chelys? Where did all the little plates of bone originate that fill
the spaces between the rows? If it be assumed that the species
of Toxochelys were developing a new epithecal shell, two ques-
tions may be asked: 1) Why should they have been providing
for themselves a new armor whilst the old one was yet in good
order? 2) Those epithecal! neural bones had a tendency to
coossify with the underlying neurals. How could a new shell
be produced under such circumstances? As old useless elements
1 These have been called by Wieland epineurals, but the term had long before
been applied to very different bones in the fishes.
426 OLIVER P. HAY
one can see why they might coossify with the neurals; other-
wise, not.
The marginal rows of osseous elements in the armor of the
leatherback are regarded by Versluys as being equivalent to the
peripheral bones of the thecophores and both as belonging to
the epithecal skeleton. In Archelon a supramarginal bone has
been found to articulate with two of the peripherals. The supra-
marginal is an epithecal bone; therefore, argues Versluys, the
peripherals are likewise epithecal bones. However, one might
insist with equal right that these peripherals are thecal elements
because in the great majority of turtles they articulate with the
costal plates and with the nuchal. Horny scutes alternate in
the same way with both the costal plates and the peripherals.
Versluys recognizes that in the case of the other scutes they cor-
respond with epithecal bones that have disappeared; but he
appears to believe that the scutes overlying the peripherals
form an exception. It would be very remarkable if the scutes
once coincided with the epithecal elements and later came to
alternate with them as they do with the thecal bones. We
ought at least to have satisfactory evidence that such a change
has been effected.
Inasmuch as the plastral bones, omitting the epiplastrals
and the entoplastron, are derived from gastralia, the peripherals
of each side may possibly have originated from an outer longi-
tudinal row of gastralia.
Those investigators who have access to skeletons of the South
American pleurodire Chelys are invited to make a study of its
shell. In the United States National Museum there is a mounted
skeleton which presents some features which appear to have a
bearing on the relationships of the various groups of the Testudi-
nata. This skeleton has the catalogue number 29545 and the
record shows that the animal came from Caicara, Venezuela.
As is well known, there is, on the lateral keels of the species of
this genus, near the hinder border of each costal scute, an ele-
vation, or boss. In the skeleton mentioned there is found on
each of the bosses of the second scute areas, right and left, a cap
of thin bone which is joined suturally to the underlying costal
PHYLOGENY OF SHELL OF TESTUDINATA 427
bone. These plates of bone are thin, about 25 mm. long, and
about half as broad. On the bosses of the other scute areas no
such bones are found, but the summits of these bosses present
adequate evidence that they were once capped by similar thin
plates. It appears probable that some of these plates were lost
in the preparation of the skeleton; others may have been ab-
sorbed during the life of the animal.
On the bosses situated on the neural bones and near the hinder
end of the vertebral scutes no thin bones distinct from the neu-
rals are found, but on each boss there is a rough and pitted sur-
face which suggests that such a bone was once there. Coming
now to the borders of the shell, we may examine the projecting
points of the peripheral bones, those points which are situated
at the rear of the various marginal scutes. No bones distinct
from the peripherals are there found, but there are indications
that such bones may have been present. On several of these
points, or bosses, are found pitted surfaces, to each of which
appears to have been joined by suture a bone of considerable
size. On the plastron of the skeleton referred to are surfaces
which suggest the former presence of thin superficial bones, and
these are situated at the center of growth of each plastral scute.
The one on each pectoral scute is very large and rough. If the
bone was once there it may have been lost during the maceration
of the shell.
From the American Museum of Natural History, New York,
through the courtesy of its Department of Herpetology, the
writer has received three shells of the genus Chelys. One of
these, having the number 7167, is disarticulated. On this last-
mentioned shell the following observations have been made.
On the fifth neural (fig. 2) there is a triangular patch of thin
bone which is joined to the underlying neural by suture, but
which in places around the edge appears to be coossified with
the neural. The area occupied by it is about 16 mm. long and
at the rear 15 mm. wide. The upper surface of this bone is
rough and pitted. The thin plate has the appearance of being
partially absorbed. The sulcus bounding the third vertebral
scute lies behind the area described and on the sixth neural. ‘The
428 OLIVER P. HAY
presence of this bone confirms the conclusion that was reached
regarding these bones on the neurals of the specimen in the
United States National Museum.
On the third neural of this specimen, at the rear of the second
vertebral scute, there is an area which is rough and pitted, but
no overlying plate of bone is found. This has probably been
completely absorbed. A smaller similar area is seen at the rear
of the first vertebral scute, on the first neural. Near the rear of
the fourth vertebral scute, on the peak of the high ridge there
is found, lying also partly on the seventh neural and partly on the
eighth, a patch which is very uneven and deeply pitted; but
if there was ever an overlying plate of bone there it is now gone.
On the hinder part of the narrow ridge of the surface occupied
by the vertebral scute is a long rough tract, but no overlying
bone is found.
Coming now to the costal bones, attention will be given first
to the fourth of the right side, that costal into which is inserted
the buttress of the right hypoplastron. Capping the summit of
the boss forming a part of the lateral keel and near the rear of the
second costal scute area is a plate of bone (fig. 3) distinctly sutured
to the underlying costal. It is about 15 mm. long and nearly
as wide. Where it comes to the suture between the third and
fourth costal bones, it is nearly 4 mm. thick. On the corre-
sponding elevation of the left fourth costal there is a pitted area
similar in size and shape to that on the right side, but the cap of
bone has either been absorbed or has fallen off during macera-
tion. One cannot doubt that it was at some time present. Com-
ing forward to the boss at the rear of the first costal scute area,
on the second costal bone, we find a rough and deeply pitted
area much like that found on the fourth costal, but no plate of
bone caps it. The impression is again given that this plate has
been lost in maceration. It appears to have extended forward
on the first costal bone. On the corresponding boss on the right
side is a surface in size and shape like that of the left side, but it
is smoother. The bosses near the rear of the third and fourth
costal scute areas indicate that they may once have been fur-
nished with thin plates of bone, but of these there are now no
traces.
PHYLOGENY OF SHELL OF TESTUDINATA 429
Turning, now, our attention to the peripheral bones, we find,
at the peaks of the tooth-like processes along the border, areas
so similar to those found on the neurals and costals that we can
hardly doubt that they were once covered each by a thin bone.
These may have been lost during preparation of the skeleton. On
the left fourth peripheral (fig. 4) there is a fragment of one of
these bones sutured to the peripheral. It is only 10 mm. long
and 4 mm. wide, but evidently it was once about 15 mm. long
and 5 mm. wide. A part of it appears to have been absorbed.
On the upper surface of the peripheral an impressed area ex-
tends 8 mm. from the edge, and the bone mentioned appears to
have once covered this area. The latter does not show well in
the figure; but on the lower face of the peripheral the impressed
surface is larger and deeper. On no other peripheral is there
found a separate bone, but the surfaces for receiving them are
usually distinct, sometimes conspicuously so. Figure 5 of the
plate presents a view of the border of the first and of a part of
the second right peripherals of carapace 7167. The view is
partly from below. The lines radiating from the letters, a, a,
call attention to the rough surfaces which appear to have sup-
ported bony plates. Similar surfaces are present even on the
projecting points of the pygal bone. These appear to have been
spread out as thin laminae over the upper surface as far forward
as the sulcus in front of the marginal scutes.
Another carapace (no. 6596) appears to have belonged to an
old captive individual, and the borders of the shell are consid-
erably worn, especially over the hind legs. No bones corre-
sponding to the superficial ones above described are observable,
but their former presence is in some places distinctly indicated.
On the front of the nuchal scute area (fig. 6, a) there is, however,
a bone 16 mm. long from side to side and 3.5 mm. wide. This
is placed at the center of growth of the nuchal scute. The
third carapace (no. 5911), apparently belonging to a species
different from the others, appears to present no features that
add to or subtract from what has been observed in the others.
Interesting results are secured in a study of the plastra. That
of the specimen no. 7167 must first receive attention, and a
430 OLIVER P. HAY
figure of it is presented (fig. 9). Beginning at the rear, there is
found on the right xiphiplastral (e) a thin plate of bone now 30
mm. long and 10 mm. wide, but it was evidently once 6 mm.
longer. The greatest thickness is 3 mm. On the left side this
bone is missing, but the surface to which it was articulated is
distinct. These bones, as in other cases, are situated at the
center of growth of the corresponding scutes. Coming forward
to the femoral scutes, it is found that nearly the whole of the
outer border of each is occupied by two epithecal bones (d, d).
One of these lies on the xiphiplastral, the other on the hypoplas-
tral; but on the left side the hinder of the two bones has scaled
off. The length of the two bones is 62 mm.; the breadth 11 mm.
Along the hinder border of the right abdominal scute, at the
lower end of the bridge (c), there is a thin bone 22 mm. long, 10
mm. wide, and 4 mm. thick at the hinder end. On the left side
there is no corresponding bone, but a small scar marks its posi-
tion. On the hinder border of each pectoral scute at the upper
end of the bridge (6, 6) is a large plate of bone, the length being 45
mm., the width 20 mm., the greatest thickness 5mm. At the
outer hinder corner of the humeral scutes there is hardly any
indication of the epithecal bones that might be looked for there.
On the right side is a rough surface where the little plate was
probably once seated. On the gular of each side is a rough sur-
face where evidently a plate of bone was once attached. The
scar on the right side is 20 mm. long and 11 mm. wide; the one
on the left side is narrower (a, a).
One might expect to find some evidences of the presence of
an epithecal bone within the area of the intergular scute, but
none is certainly found. From the plastron of no. 6596 most of
the epithecal bones have been lost. Those on the femoral scutes
were not so large as in no. 1167. On the abdominal scute areas
traces of them are mostly gone. On the pectorals the epithecal
bones are large. On the right side the bone is missing, but there
is a deeply pitted surface where it was lodged. On the left side
the bone consists of two pieces, the intermediate part having
probably been absorbed. The two pieces taken together measure
38 mm. in length; the rear piece is 23 mm. wide. The borders
PHYLOGENY OF SHELL OF TESTUDINATA 431
of those scute areas and a part of that of the humerals appear
to have been covered by epithecal bones; if so, the latter have
disappeared. No bones or surfaces worthy of note appear at
the centers of growth of the gulars and the intergular. On the
plastron of no. 5911 no epithecal bones corresponding to those
mentioned are found, but plain traces of most of them are pres-
ent. They appear to have been thinner and usually to have
been absorbed. Nearly the whole free edge of the epiplastra
within the intergular scute area of the specimen in the National
Museum is occupied by two or three rough surfaces to which
were probably attached epithecal plates.
Some months after the preceding paragraph had been written,
Dr. L. Stejneger found: in his collection nearly all of the horny
scutes which had been removed from the shell of the mounted
specimen, no. 29545, above mentioned. These confirm the
writer’s conjecture that the bones interesting us had been lost
from the skeleton in the course of preparation. Three verte-
bral scutes are preserved. On the inner surface of the first one,
at the point where the bone is to be looked for, there is a patch
of tissue 10 mm. long and 3 mm. wide; but, when it is thoroughly
moistened and then treated with hydrochloric acid, no reaction is
seen. The bone salts had probably been absorbed. The second
and third vertebral scutes are not preserved. On the fourth
there is a very distinct bone 14 mm. long and about 10 mm. wide.
Above, it is partly exposed by abrasion of the horny scute. On
the fifth scute there is distinct bone forming a patch 27 mm.
long and 8 mm. wide. It is partly exposed on the upper surface.
All of the costal scutes are preserved except the left second. Each
of the first costal scutes bears on the under surface a large and
thick patch of bone. That on the left side is 21 mm. long and
13 mm. wide. The bone of the right side is partially exposed
above; that of the left side is not. As stated above, the plates
of bone belonging under the second costal scutes remain on the
mounted:skeleton. The left third costal scute retains its plate
of bone, 21 mm. long and 7 mm. wide. When a piece of it was
removed and put in acid abundant gas was liberated. The scute
of the right side also has its bone. Neither this nor that of the
432 OLIVER P. HAY
left side has the horny scute eroded from the surface. The bone
beneath each of the fifth costal scutes is small. When a frag-
ment was dug out and treated with acid gas was liberated.
About fifteen of the marginal scutes are present. Of these
nearly all retain patches of bone which correspond to the pro-
jections along the border of the carapace. These bones are
partially exposed outwardly by the wearing away of the pro-
jections against objects during the movements of the animal.
It has not been convenient to determine the position of all these
scutes on the margin. One however, is the left eleventh; another
apparently the right twelfth. One, probably the ninth left,
seems to have a strip of bone 25 mm. long, which formed the
edge of the carapace under that scute. At this point may be
mentioned the nuchal scute. At the middle of its front border
there is a fragment of bone which responds readily on the appli-
cation of acid.
The scutes of the plastron are present and they bear on their
inner surfaces those patches of bone which the writer judged
from the marks on the mounted skeleton must have been pres-
ent. As these are better displayed on specimens described below,
nothing more will be said about them.
Now must be described another set of bones, the meaning of
which is yet to be determined. ‘These are small, thin, flat plates
which are likely to be indicated anywhere on the surface that
was covered by the horny scutes. Often the plates themselves
are present and, after the bone is moistened, may be picked out
of their resting places. In other cases they appear to have fallen
out during maceration. Sometimes they have evidently become
coossified with the surrounding bone; sometimes there is present
only ascar which seems to show that long before the death of the
animal the plate had been absorbed. Occasionally it is difficult
to determine whether or not a depression in the bone represents
one of these plates. The latter are usually more or less nearly
circular or polygonal, but are sometimes irregular in form. A
full-sized illustration of the lower face of the right fifth and sixth
peripheral bones of no. 6596 of the American Museum of Natural
History is here presented (fig. 7). A little above and to the
PHYLOGENY OF SHELL OF TESTUDINATA 433
left of the center of the figure is a little bony plate marked by a
conspicuous border. This was taken from its resting place and
returned. Near it, on the right hand, is a larger patch, slightly
lower than the general surface and in which there was once a
little five- or six-sided plate. Near the upper left-hand corner is
a pretty large irregular.and rather indistinct surface which rises
onto the scute area in front of it. The appearance indicates
that the plate of bone which occupied it had long been absorbed.
On its right again there is a little plate which has become pretty
thoroughly coossified with the bone around it. At the lower
end of the figure are two plates whose outlines are rather indistinct.
A good many similar areas are found scattered here and there
over the surface of the carapace of no. 6596. Also on the cara-
pace 5911 a few such areas are found. On the disarticulated
carapace 7167 many shallow pits are found which appear to have
been filled by little plates of bone; but these may have come
away with the horny scutes at the time of maceration. On this
shell they appear to be clustered especially around the bosses
of bone belonging to the various scute areas, but they are found
also elsewhere. They do not appear to be due to any abnormal
condition of the bone, and they were certainly buried under the
horny scutes.
Many of these small plates which are distributed without
order are found on the flat part of all of the three plastra from
the American Museum. On no. 7167 (fig. 9) a number of these
are seen fixed in their pits. In other cases they are gone, ab-
sorbed or lost in maceration. On the plastron of no. 6596 have
been many such plates. A few remain, but of others only their
impressions are left. An oval one is 10 mm. long; another appar-
ently occupied by a single plate is still larger. On the plastron
of no. 5911 are seen shallow depressions in which had rested bony
plates, some of them of considerable size.
After the greater part of this paper had been written, still
another specimen of Chelys was put into the writer’s hands for
examination. This had been in the Zoological Park for some
months. It had never been known to take any food, and it
probably died of starvation. Since a hole is found bored through
434 OLIVER P. HAY
the hinder edge of the shell, it is judged that the animal had
been kept in captivity before it was brought to this country.
The length of the carapace is 400 mm. After maceration and
cleaning, an examination has been made of the shell. On the
carapace not as many of the scute areas have furnished epithe-
cal bones at the centers of growth of the scutes as was hoped.
Nevertheless, a thin cap of bone was found on the rear of the
third vertebral scute and a small bone at the rear of the second
right marginal scute and another on the left. Distinct evidence
of similar bones occurs at other points where they might be ex-
pected to occur. On the plastron there is a scar on the right
side of the front edge of the intergular where there may have
been a plate of bone. On nearly the whole of the front of the
right gular there is a surface (a) from which a bone was cer-
tainly lost during maceration. No plates of bones are found on
the outer hinder angles of the humeral scute areas. On» the
outer hinder angle of the plastral portion of each of the pectoral
(b) and the abdominal (c) scutes of both sides is found a large
patch of thin bone. All of these bones give evidence of more or
less absorption and removal. On the outer border of each fem-
oral scute area, at about its middle, is a thin bone (d) 30 or
more mm. long. This appears to correspond to the anterior of
the two bones found on the femoral areas of the specimen shown
on plate 1. The hinder one had probably long before been
absorbed. On the anal scute areas no similar bones are present,
but a scar (c) on the one of the left side may indicate the former
existence of a plate.
The most conspicuous feature of this shell is the numerous
smaller plates scattered irregularly all over the surface of both
the upper and the lower sides. Figure 8 shows some of these
of nearly the natural size on the left side of the first vertebral
scute area and on parts of the adjoining scutes. Here the little
bones are yet present, each in a depression in the costal bone.
Nearly all of these bones are polygonal. All over the shell are
presented areas where there were evidently once little flakes of
bone, but these are now gone, only little pock-like scars remain-
ing. The figure of the plastron shows the number and size of
PHYLOGENY OF SHELL OF TESTUDINATA 435
the bones (fig. 1). In two cases the depression holding the
plate makes a hole through the shell, but this is only where they
lie in the course of a sulcus where the bone is thin. These little
bones have a yellowish appearance, being thus somewhat dif-
ferent from those of the other specimens. Nevertheless, they
give the usual reaction with acid, and under the microscope they
show the haversian canals and the lacunae.
What interpretation is to be put on these flakes of bone it is
difficult to say. It has appeared possible that they are repre-
sentatives of the mosaic of bony plates which are found between
the keels in Dermochelys. So far as the writer now sees, the
principal argument against this explanation is the irregularity
of distribution. It has been suggested by some scientific friends
that they are produced by parasites, but of this the writer has
seen no evidence.
Still another shell of Chelys has been found in the collection
of the U. 8. National Museum. This has the catalogue num-
ber 8602 and is recorded only as having come from Amazon
River. On this specimen there are no traces of either the plates
of bone which underlie the center of growth of the various horny
scutes, nor of those smaller plates which are scattered irregu-
larly over the shell. How to account for the condition the writer
does not know. Unless there is great variation in Chelys fim-
briata, this specimen must belong to another species than that
of the mounted one. It is possible that now and then an individ-
ual fails to reproduce such useless vestigial structures. At
least the writer believes that this case does not invalidate his
explanation of the presence of the bones found at the centers of
growth of the scutes. If now and then a cat should fail to have
the vestigial first upper molar, this would not prove that in
other cases this molar had not been inherited from the original
felids.
Our study of the shells of Chelys has therefore resulted in
demonstrating the presence of epithecal bones which in the
writer’s opinion, correspond to those of the median, first lateral,
and the marginal keels of the carapace and of the outer lateral
keels of the plastron of Dermochelys; besides numerous smaller
JOURNAL OF MORPHOLOGY, VOL. 36, No. 3
436 OLIVER P. HAY
flakes of bone which possibly correspond to the plates which
form the mosaic between the keels of Dermochelys. No traces
of the supramarginal and inframarginal keels are found. The
presence of the bones of the marginal keels, as shown by dis-
tinct sutural surfaces and by the actual bones, suffices to prove
that the peripheral bones of Cryptodira and Pleurodira are not
epithecals, but belong to the same category as the costal plates,
the neurals and the nuchal.
Dr. Otto Jaekel described in 1915 (Palaeont. Zeitschr., Bd.
2, S. 88-112) a remarkable and finely preserved turtle from the
Trias of Germany. He is to be congratulated on having the
opportunity to study such an important specimen and on his
results. Unfortunately, the part of the Zeitschrift which con-
tains the conclusion of his paper has not been received at Wash-
ington. Some remarks will be made here on that part at hand.
Doctor Jaekel named this animal Stegochelys dux; but, inas-
much as this generic name was preoccupied, he later proposed
instead the name Triassochelys (Abel, Die Stamme der Wir-
beltiere, 1919, pp. 386-392, figs.)
In case Doctor Jaekel means, as he doubtless does, that he
has been able to furnish corroborative evidence that the plastron
of the Testudinata is composed of the clavicles and the inter-
clavicle and of abdominal ribs (gastralia), his statement is read-
ily accepted; but certainly there was previously little doubt
about its composition. The present writer in 1898 (Amer.
Naturalist, vol. 32, p. 934) assumed this view and made no claims
of originality therefor. In the writer’s paper referred to, he
attempted (p. 946) to determine the number of gastralia that had
entered into the formation of the plastron. This number, three
or four pairs, is indeed small; and naturally, in case the num-
ber recorded by Jaekel, about twenty-five in each of the
anteroposterior rows, is confirmed, the writer’s calculations will
be discredited.
The type of Triassochelys was evidently a fully mature, prob-
ably an old animal; and, like many of the ancient testudinates,
it appears to have had most of the various bones of the shell
thoroughly coossified. With the exception of the sutures between
PHYLOGENY OF SHELL OF TESTUDINATA 437
the gastralia, none appears to be with certainty described. The
plastron appears to have been solidly united with the carapace
and no suture appears to separate the gastralia along the mid-
line. Under such conditions, how can it be assumed that there
were no hyoplastra, no mesoplastra, no hypoplastra, and no
xiphiplastra? Is it probable that this turtle, which in most
features resembles so closely other well-known forms, differed
from them all in having none of the ordinary plastral bones,
except the front ones, but instead of these a plastron composed
of distinct and little modified gastralia?
Jaekel finds that the gastralia of Triassochelys diverged as
they passed from the bridges toward the midline, and he gives
an explanation of the divergence. If, now, this plastron repre-
sents a primitive condition from which, through segregation
and consolidation of the gastralia, were produced definitive
plastrals, how are we to explain the fact that in those turtles
which possess mesoplastrals the sutures between the plastral
bones converge as they are followed toward the midline? They
appear, therefore, not to have followed the sutures between the
gastralia, but to have struck across them at varying angles.
There can be no doubt that Triassochelys is closely related
to Proganochelys. In this Triassic turtle Fraas (Jahresh. Ver.
vaterl. Naturk., vol. 55, 1899, p. 416, pls. VII and VIII) con-
vinced himself that there was present a pair of mesoplastrals,
greatly expanded at the outer ends. It seems that later Doc-
tor Jaekel (Placochelys placodonta, 1907, p. 59) succeeded in
shaking Fraas’s confidence in his determinations; but it appears
to the present writer that the probabilities are in favor of their
approximate correctness. How Jaekel’s observations are to
be harmonized with the views here expressed the writer does
not at present comprehend. It may be noted in passing that
Doctor Jaekel was in error when he stated that Fraas believed
that there were in Proganochelys two pairs of mesoplastrals.
Doctor Jaekel concluded that in Triassochelys the pectoral
scutes were missing. ‘There appear to be no sufficient reasons
for this conclusion. The great scutes which bound the notches
for the fore legs are surely pectorals. In front of these scutes
438 OLIVER P. HAY
there is abundant room for humerals, gulars, and even intergu-
lars. The last-mentioned two pairs of scutes are applied to the
epiplastra and the front of the entoplastron, as may be seen in
figures of the Pleurosternidae and Baénidae (Hay, Fossil Tur-
tles of N. A., 1908). These bones in Triassochelys were evidently
small, and the gulars and intergulars were correspondingly small.
To the writer it seems quite probable that the front of the plas-
tron of Jaekel’s specimen broke off along the humeropectoral
sulcus.
Doctor Jaekel tells us (p. 106, fig. 9) that in his Triassochelys
there are on each side of the carapace seventeen peripheral
bones and that the marginal scutes correspond to these in num-
ber and in their boundaries. These are statements of such
importance scientifically that they ought to be supported by un-
questionable evidence. Although Doctor Jackel states that these
peripherals are very distinctly set off from each other and from
the costals, he does not say that the bone sutures are present.
Unless the sutures are to be seen, the limits of the bones are
indeterminable. The condition of the shell in general indicates
that the sutures are closed. What sets the areas off from one
another is probably only the sulci between the marginal scutes.
Indeed, Jaekel (p. 199, fig. 23) informs us that such is the case.
If the reader will examine the figures in the writer’s work of
1908, referred to above, which illustrate the structure of the
Baénidae (apparently not distant relatives of Triassochelys),
or will take a look at a shell of one of the Chelydridae or a shell
of Chelys, he will find that the sulci between the marginal scutes
cross the borders of the carapace at the notches, while the
bone sutures cross between the notches. In the Baénidae there
are often some small apparently supernumerary scutes at the
front of the carapace. These appear to correspond to the little
scutes which Doctor Jaekel has counted as the first and second
in his series. At the rear of the carapace of Baéna the supracau-
dal scutes have been suppressed, along with the pygal bone.
In Triassochelys these supracaudal scutes are present, but much
reduced in size. In this way we may account for the unusual
number of marginal scutes in Triassochelys. In that animal
PHYLOGENY OF SHELL OF TESTUDINATA 439
there were, however, in all probability not more than eleven
peripheral bones on each side.
Fraas (op. cit., p. 409, fig. 1; reproduced by Jaekel) has indi-
cated the presence of twenty or more marginal scutes in Pro-
ganochelys; but if there were really present lines which marked
out the boundaries between these areas, some of them were
probably bone sutures; others sulci between the marginal scutes.
The results sought after in this paper may be summed up as
follows:
1. The neck of the leatherback has not been inherited from
the eryptodires, but has been independently developed.
2. The evidences relied on to connect the leatherback with
the chelonioid sea-turtles, living or extinct, are by no means
compelling.
3. Vestigial bones have been discovered in the Thecophora
which correspond to those of the following keels in Dermochelys:
the upper median (Toxochelys, Archelon, Chelys); the costal
(Chelys); the supramarginal (Archelon), the marginal (Chelys),
and the first lateral of the plastron (Chelys). The supramarginal
keels are represented in many species by scute areas also. The
inframarginal keels are known to us only from scute areas on
the bridges. The lower median keel may be retained in the
unpaired intergular of the Pleurodira, the intercaudal (Abel
op. cit. p. 410, fig. 319) and occasional unpaired scutes in other
turtles.
4. By the presence of vestigial bones on the peripherals at
the points whence the marginal scutes expand it is shown that
these peripherals are not to be homologized with the marginal
bones of Dermochelys, but that they belong to the thecal armor.
5. The occurrence of the various elements representing the
epithecal armor in species scattered about in nearly all the large
groups of turtles, and most of them provided with good solid
shells, appears to show that these elements are vestiges of an
armor of a common ancestor and not the beginnings of a new
epithecal one.
6. The retention of the epithecal covering by Dermochelys,
the loss of most of the thecal shell, and the possession of many
440 OLIVER P. HAY
other structural peculiarities indicate that the ancestors of this
turtle early parted company with the rest of the order.
7. The order of Testudinata is composed of two suborders,
Athecae and Thecophora.
Doctor Versluys has presented a figure which was designed to
show his conception of the composition of the carapace of the
Text Figure
primitive testudinate. The present writer has taken the liberty
to modify the figure so that it shall present in a way his own
views regarding the structure of the carapace of this interesting
and theoretical animal. In addition to the epithecal bones shown
on the carapace, the tail, and the neck, the writer has indicated
a number on the head which underlay its horny plates. It
appears evident that Jaekel’s Triassochelys possessed a num-
PHYLOGENY OF SHELL OF TESTUDINATA 44]
ber of such bones scattered over its skull, but at its stage of life
these had doubtless become consolidated with the underlying
bones.
It may be that the costal plates ought to be represented as
coming down to the peripherals. It appears to be assumed
that fontanelles in the carapace are the result of reduction of the
costal plates and peripherals and that this reduction, as well
as a flattening of the whole body, is due to an aquatic existence;
but we have lately learned that an African species of Testudo
has suffered a nearly complete loss of its shell and has at the
same time become excessively flattened (C. R. Acad. Paris,
vol. 170, 1920, p. 263). It appears not unreasonable to suppose
that in the most primitive turtles the costal plates had not yet
joined the peripherals; perhaps not yet the neurals.
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443
PLATE 2
EXPLANATION Of FIGURE
9 Lower surface of plastron of Chelys. No. 7167, Amer. Mus. Nat. Hist.
Showing epithecal bones on the scute areas. a, a, on areas of gular scutes; b, b, on
areas of pectoral scutes; c,c, on areas of abdominal scutes; d, d, on areas of
femoral scutes; e, e, on area of anal scutes.
444
PHYLOGENY OF SHELL OF TESTUDINATA
PLATE 2
= OLIVER P. HAY
445
Resumen por el autor, Alden B. Dawson.
La topografia de la cloaca del macho de Necturus en relacién
con las glindulas cloacales.
El orificio externo de la cloaca del macho de Necturus es una
hendidura longitudinal franjeada por dos labios poco desarr-
ollados los cuales en su extremo caudal llevan un par de papilas
blandas. Los labios estén mas modificados a consecuencia de
la presencia de numerosas fisuras transversas. En _ posicién
inmediatamente dorsal al orificio cloacal esta la cimara cloacal
o vestibulo que se continua cranialmente en el tubo cloacal. El
piso de este ultimo tiene forma de artesa honda, con la mucosa
sureada por crestas delgadas y paralelas las cuales se interrumpen
caudalmente convergiendo en las papilas altas y delgadas pre-
sentes a los lados de la cAmara cloacal. El techo esta modificado
también por la presencia de un sureco medio profundo y a cada
lado del tubo cloacal, entre esta depresién dorsal y la ventral,
existen dos surcos longitudinales.
La eavidad cloacal esta’ por completo rodeada por masas de
elindulas tubulares largas y tortuosas. La gran masa media
ventral se conoce con el nombre de gldandula cloacal. Sus
tubulos se abren en las cimas de las crestas paralelas y en los
Apices de las papilas delgadas internas. Dos masas de tubulos,
las glindulas abdominales pares, estan situadas ventrolateral-
mente a la cimara cloacal y sus tibulos desembcean en la super-
ficie media de las papilas externas pares. Dorsalmente existe
una masa glandular media, la glindula media. Esta glandula
presenta por lo menos cuatro diferenciaciones, que se distinguen
histol6gicamente por el cardcter del epitelio que tapiza sus
tubulos. Existe una pequefia masa media cranial, una masa
media caudal muy grande y dos masas laterales. Todos los
tubulos de la masa de la glandula pélvica se abren en el techo
del tubo cloacal. El autor considera un método posible de
formaciOn de un espermatdéforo.
Translation by José F. Nonidez
Cornell Medical College, New York
AUTHOR’S ABSTRACTS OF THIS PAPER ISSUED
BY THE BIBLIOGRAPHIC SERVICE, MAY 1
THE CLOACA AND CLOACAL GLANDS OF THE MALE
NECTURUS
ALDEN B. DAWSON
Department of Anatomy, Loyola University School of Medicine
THREE PLATES (SIXTEEN FIGURES)
INTRODUCTION
At present the mating habits of Necturus are not definitely
known. Strong circumstantial evidence indicates (Kingsbury,
’95) that fertilization is accomplished by the deposition of sperma-
tophores and the reception of the spermatozoa which are borne
upon the summits of the deposited spermatophores into the
cloaca of the female. The time and the exact manner of in-
semination are not known. An abundance of spermatozoa was
found by Kingsbury (’95) in the spermathecae of six females
_ which he examined during the late fall and winter. Females
examined by the writer in October and March were found also
to have large numbers of sperms in their spermathecae. Ac-
cording to Smith (11), fertilized eggs are deposited chiefly dur-
ing May and June. .
Although our information on the time and manner of fertili-
zation is still incomplete, it seems highly probable that sperma-
tophores are produced by the male Necturus. The matrix of
the spermatophores is probably a product of the cloacal wall
acting in conjunction with the surrounding masses of tubular
glands. The degree of glandular activity in this region should
furnish therefore some clue as to the probable time of spermato-
phore deposition. With this in mind, a study of the cloaca was
undertaken. Owing, however, to the complexity of the internal
configuration of the cloaca, the complicated relations of the clo-
acal wall to the tubules of the surrounding gland masses, and the
many varying types of tubules encountered, the comparative
study of the glandular activity at different times of the year had
447
448 ALDEN B. DAWSON
to be postponed until the limits of the different masses of glands
had been definitely determined. Accordingly, the present report
deals primarily with the various masses of tubular glands in
their relation to one another and to the topography of the cloaca.
In a later communication it is planned to describe the variations
which occur in the glands during the different seasons of the year
and to follow the changes undergone by the several types of
‘cells during the production of secretion.
Only adult males were used in this study. The material was
dissected out and fixed in either formalin, Zenker’s fluid, or
Bouin’s fluid. Serial sections, transverse and longitudinal,
were made of the entire cloacal mass, including the cloaca proper
and the surrounding glands. The tissue was stained with haema-
toxylin and eosin, Van Gieson’s picro-acid fuchsin and Mallory’s
stain for connective tissue.
LITERATURE
We are indebted to Heidenhain (’90) for the first detailed and
accurate description of the cloaca of a male urodele. He de-
scribed three kinds of cloacal glands in the male Triton, the so-
called cloacal gland, the pelvic gland, and the abdominal gland.
Before this but two types of glands were recognized. Zur Mih-
len (93), who worked on Triton, Salamandra, and Siredon, con-
firmed in the main the findings of Heidenhain. Kingsbury
(95), in the course of an extended study of the cloacas of female
Diemyctylus, Plethodon, Desmognathus, Amblystoma, and
Necturus, discussed, incidentally for purposes of comparison,
the structure of the cloacas and the adjacent glands of the males
of these different genera. In Necturus, Kingsbury did not make
a sufficiently careful study of the glands to enable him to deter-
mine whether the abdominal gland is present.
EXTERNAL APPEARANCE OF THE CLOACA
The external opening of the cloaca of the male Necturus is
simply a longitudinal slit bordered by two inconspicuous lips
which, at their caudal ends, give rise to a pair of low rounded
papillae (fig. 1, ext.p.). The lips are modified further by
CLOACAL GLANDS OF MALE NECTURUS 449
numerous transverse fissures and, immediately caudad to the
paired external papillae, there is a distinct transverse crescentic
groove. A ventral enlargement extending laterally along the
cloacal slit and cranially toward the region of the pelvic girdle
marks the extent of the large cloacal gland.
INTERNAL TOPOGRAPHY OF THE CLOACAL CAVITY
For purposes of description, the cavity of the cloaca may be
considered as consisting of two portions, an enlarged caudal
chamber or vestibule opening ventrally to the exterior by way
of the cloacal slit and a narrower cephalic, tubular portion
connecting the cloacal chamber with the rectum (figs. 4, 5,
Chohes clits):
The internal configuration of the cloaca is decidedly complex,
but in an undistended condition the cavity exhibits a very defi-
nite and constant form. The various depressions, folds, papil-
lae, etc., which go to produce the complicated pattern of the
cavity serve therefore as landmarks of the different regions into
which the tubular glands discharge their secretion.
Before entering upon the more detailed description of the
several regions of the cloaca, brief mention will be made of the
most conspicuous modifications of the cloacal wall. The ventral
side of the cloacal tube has the form of a deep, narrow trough,
the mucosa of which is thrown into high, thin ridges (figs. 4, 5,
12, 13, 14, v.tr., v.r.). Caudally, in the region of the cloacal
chamber, the ventral ridges are interrupted and merge into tall,
slender papillae (figs. 4, 5, 15, int.pp.). Dorsally the cloacal
tube contains a deep median groove (figs. 4, 5, 18, 14, md.gr.)
and on its sides between the dorsal groove and the ventral
trough are two well-defined longitudinal furrows (fig. 13, lt.fur.).
The cephalic end of the cloacal tube presents the simplest
condition, and the transition from rectum to cloaca occurs with-
out any very evident change in structure. The urogenital
ducts open dorsolaterally into the extreme cephalic end of the
tube. They terminate separately in a pair of prominent papil-
lae which project ventrally from the bottoms of two pit-like
depressions (figs. 4, 5, 7, ug.p.). The urinary bladder opens
450 ALDEN B. DAWSON
medially into the ventral side of the cloaca, almost opposite the
more dorsal urogenital papillae (figs. 4, 5, 8, wr.bl.o.).
Caudad to the orifice of the urinary bladder a prominent longi-
tudinal fold projects from the midventral wall, and on either side
of it other smaller irregular folds can be distinguished (fig. 5,
mo.f., v.f.). The main fold continues caudally for a short dis-
tance as a single fold, but soon becomes doubled and is eventually
broken up into the thin ridges which cover the walls of the ven-
tral trough (figs. 5, 9, 10, v.tr., v.r.). Laterally the wall of the
cloacal tube is also modified by two low folds which extend, on
either side, from the regions of the urogenital papillae caudally
to the cephalic ends of the longitudinal lateral furrows, with
whose dorsal walls they merge (figs. 4, 9, li.f.). Furthermore,
when the cavity of the cloaca is laid open by a longitudinal ven-
tral incision so that the dorsal portion of the cloacal tube is ex-
posed, the lateral folds, with the aid of the median dorsal groove
and lateral furrows, are seen to mark off a Y-shaped area, the
stem of which extends cephalad toward the region of the urogeni-
tal papillae (fig. 4, Y).
Another striking feature of the cephalic portion of the cloacal
tube is the presence of large numbers of melanophores in the
underlying connective tissue. No other portion of the cloacal
cavity exhibits a like pigmentation, although a few scattered
melanophores can occasionally be seen in other regions.
The ventral trough, longitudinal lateral folds, and median
dorsal groove already referred to, are found in the more caudal
portion of the cloacal tube. The ridges of the ventral trough
are relatively high and thin. They run almost parallel, but
diverge slightly as they approach the cloacal chamber. The
number of ridges present is quite constant, the average being
thirty-two, although thirty-four ridges can occasionally be
counted (figs. 13, 14, v.r.). The longitudinal lateral furrows
and median dorsal groove do not exhibit any conspicuous modi-
fications and, gradually growing shallower as they pass cau-
dally, are eventually obliterated in the region of the cloacal
chamber.
CLOACAL GLANDS OF MALE NECTURUS 451
The cloacal chamber itself is relatively simple in form, two
rather deep ventrolateral recesses being the only modifications
of interest in this study (figs. 4, 5, 15, vl.rec.). The long, slender
papillae, found on the floor and ventral portions of the walls of
the chamber, are also present in the ventrolateral recesses. The
papillae in the recesses, however, are usually short. Both the
internal papillae and the ventral ridges are highly vascular,
being permeated by blood channels of considerable size.
THE WALL OF THE CLOACA
The wall of the cloaca, especially in its cephalic portion, closely
resembles that of the rectum. Mucous and muscular layers are
readily recognized (figs. 7, 8). No serous coat, however, is
present, but the outermost layer consists of areolar tissue which
blends with the connective tissue of the adjacent structures.
The presence of large numbers of long tubular glands, which
surround and open into the cloacal cavity, has resulted in a
great thickening and extensive modification of practically the
entire cloacal wal] and of the three coats comprising it, but
the tunica muscularis has suffered the greatest displacement.
a. The tubular glands
The grouping of great numbers of tubular glands in the cloacal
wall has resulted in the production of a large glandular mass
about the cloaca, which, for the lack of a better term, will be
designated as the cloacal gland mass.° This mass lies caudad
to the pelvic girdle and occupies a large median ventral area.
It is enclosed in a connective-tissue sheath which apparently is
a modified portion of the median ventral septum which more
caudally separates the hypaxial muscles of the tail (fig. 3, ™m.v.s.).
The dorsal portion of the mass extends close to the trunk-tail
vertebrae and laterally is bounded in part by the unmodified
trunk-tail myotomes and in part by three pairs of slender caudal
muscles (mm. ischiocaudalis, caudalifemoralis, and caudalipu-
boischiotibialis, Wilder, ’12) which are attached to the posterior
appendicular skeleton (fig. 2). Cranially, the dorsal gland mass
extends to the posterior ends of the mesonephroi and to the caudal
JOURNAL OF MORPHOLGGY, VOL. 36, NO. 3
452 ALDEN B. DAWSON
margin of the pelvic girdle. Dorsocaudally, the common sheaths
of the three pairs of caudal muscles and the unmodified median
ventral septum limit the mass.
The ventral portion of the gland mass is continued into the
loose subcutaneous connective tissue, extending laterally beyond
the median area bounded by the hypaxial muscles and, cephali-
cally, to cover the surface of the caudal portion of the pelvic
girdle.
In the cloacal mass of the urodeles studied (Heidenhain,
°90; Zur Miihlen, 793; Kingsbury, 795) at least three distinet
types of tubule have been recognized. They are arranged in
definite groups and are known as the cloacal, pelvic, and abdomi-
nal glands, respectively. In Necturus both the cloacal and
pelvic glands are greatly developed. The homolog of the ab-
dominal gland can also be recognized, but it is relatively small
and separated into two compact lateral masses (figs. 2, 3, 16,
abd.gl.).
The cloacal-gland tubules form the large median ventral por-
tion of the cloacal mass (figs. 11 to 15, cl.gl.). The tubules are
long and straight. They extend in a cranial direction and end
blindly. The mouths of the tubules open both on the summits
of the thin ridges covering the ventral trough of the cloacal tube,
and on the tips of the slender internal papillae which fringe the
cloacal chamber. On the ridges the tubules terminate in low
conical elevations which are arranged longitudinally to form two
parallel rows. The terminal elevations which compose these
double rows on each ridge are not placed opposite to each other,
but have a regular alternating arrangement. The tubules which
are connected with the internal papillae also exhibit a regular
arrangement, usually two and occasionally three opening to-
gether at the tip of each papilla.
The large group of tubules comprising the dorsal portion of
the cloacal mass has been designated as the pelvic gland. They
are sharply separated from the ventral cloacal tubules by two
lateral connective-tissue septa (fig. 2, c.t.s.). The pelvic-
gland tubules, in contrast with the relatively straight cloacal
tubules, are distinctly convoluted. They extend dorsocranially,
CLOACAL GLANDS OF MALE NECTURUS 453
do not branch, and end blindly. In fresh material they appear
opaque, while tubules of the cloacal gland usually appear clear.
The difference in appearance is due to the different character
of the secretion in their lumina.
All of the tubules of the pelvic gland open into the dorsal
portion of the cloacal tube. They are arranged in four groups:
a small cephalic medial group of short tubules (fig. 10, plv.gl.’’) ;
further caudad, two symmetrical, lateral groups of somewhat
longer tubules (figs. 11, 12, plv.gl.’’’), and a very large median
caudal group of long, greatly convoluted tubules (figs. 11, 12,
13, 14, plv.gl.’). The caudal portion constitutes the greater
part of the pelvic gland. The tubules which form the lateral
differentiations of the pelvic gland are comparatively few in
number. They are distributed cephalocaudally on either side
of the cloacal tube and lie close to the lateral septa (c.t.s.) which
separate the main mass of the pelvic gland from the more ven-
tral cloacal gland.
The different groups of pelvic tubules are not distinctly sep-
arated from one another in any portion of the gland, but are
distinguished by the character of their glandular epithelium.
Owing to the great variety of secretory phases exhibited by the
different tubules, it is not always easy to determine with certainty
whether the tubules under consideration are of an entirely dif-
ferent character or are merely different phases of activity of
the same kind of tubule. It is with some hesitation, therefore,
that I have distinguished a median cephalic group, since cephalad
to the main mass of the caudal division of the pelvic gland the
tubules of its lateral differentiations approach the middorsal
line, and in serial sections are seen to be intermingled with the
more caudal tubules of the cephalic group. However, so far
as my histological study has progressed at this time, there ap-
pears to be good evidence that the tubules of the groups under
discussion, although intermingled where they come in contact,
possess secreting cells of two distinct types. The tubules of
the median caudal division, on the other hand, can be readily
recognized at all times.
454 ALDEN B. DAWSON
All pelvic tubules terminate in low papillae. At the bases of
these papillae shallow circular depressions are usually observed
recalling the structure of the circumvallate papillae of the tongue.
In some regions papillae are indistinct and only barely recogniz-
able. The tubules of the large median caudal division, for the
most part, open upon the walls of the median dorsal groove
(figs. 13, 14, 15). The more cranial tubules of the lateral pelvic
differentiations open on the dorsal walls of the so-called longi-
tudinal furrows (figs. 12, 13), while the most caudal ones are
found to open upon a middorsal region, the caudal end of the
stem of the Y-shaped area previously described (figs. 11, 12).
The short tubules of the median cephalic group terminate on
the middorsal region which forms the cranial portion of the stem
of the Y-shaped area (fig. 10).
In comparison with the cloacal and pelvic glands, the abdomi-
nal gland in Necturus appears almost vestigial. It is divided
into two masses which lie near the caudal end of the cloacal ori-
fice and dorsolaterally to the paired external papillae (figs. 2, 3,
abd.gl.). The tubules which compose this gland are short and
greatly convoluted and possess a characteristic epithelium which
distinguishes them definitely from the other tubules of the cloa-
cal mass (fig. 16). They open mainly on the medial surfaces of
the external papillae, but a few are also found to open along the
inner margins of the cloacal lips.
b. Muscular layers and dorsal ganglion
The muscular coat consists of two layers of smooth muscle
which, in the extreme cephalic portion of the cloacal tube, are
sharply differentiated into an inner circular and an outer longi-
tudinal layer (figs. 7,8). Further caudad, however, this definite
arrangement is more or less disturbed by the presence of a large,
dorsal, ganglionated plexus and the numerous tubular glands.
The dorsal ganglion represents a local enlargement of a por-
tion of the sympathetic nervous system, being apparently a
caudal continuation of the myenteric plexus of the intestinal
tube (figs. 9, 10). From the ganglion small bundles of nerve
fibers pass caudally to the various cloacal glands.
CLOACAL GLANDS OF MALE NECTURUS 455
Heidenhain (’90) observed a like mass of nerve tissue occupy-
ing a somewhat similar position in the male Triton and, accord-
ing to him, it is found only in the males. He was in doubt as to
the function of the ganglion, but interpreted it as being a ter-
minal enlargement of the ganglionated plexus associated with
the kidneys, and suggested that it might be a portion of the
adrenal system which is more or less diffuse in urodeles. ‘‘ Wo-
hin diese Ganglienmassen zu rechnen sind (Nebenniere?), ist
mir unbekannt” (p. 190). In some specimens of Necturus I
have found scattered cells which exhibit a specific affinity for
chromium.
The tubular glands extend deep into the cloacal wall, of which,
as has been already stated, they form the most conspicuous part.
In the dorsal portion of the wall the tubules of the pelvic gland
obliterate the sharp differentiation of the muscularis into two
layers, and the muscle fibers are irregularly arranged and inter-
woven, forming with the intermingled connective tissue a dense
fibromuscular stroma in which the secreting tubules are imbedded.
Some of the muscle cells of the stroma, however, are arranged
circularly about the numerous tubules to form delicate muscu-
lar tunics.
The tubules of the cloacal gland, on the other hand, while as
large and closely packed as those of the pelvic gland, do not pro-
duce such a decided rearrangement of muscle fibers, so that, in
the ventral portion of the cloacal wall, a circular as well as a
longitudinal layer can usually be distinguished. The tubules
pierce only the circular muscle layer and are imbedded in a fibro-
muscular stroma similar to that described for the pelvic gland.
Each tubule is also surrounded by a delicate layer of circularly
arranged muscle cells. The ventral portion of the longitudinal
coat of muscle fibers is not invaded by the cloacal tubules, but
persists as a compact layer, arranged as a flat sheet to cover the
ventral or external surface of the cloacal gland (figs. 11, 12, 13,
14). In the region of the cloacal chamber the definite arrange-
ment of the smooth muscle into layers is gradually lost and the
fibers are mingled with the connective tissue of the cloacal wall.
456 ALDEN B. DAWSON
c. Epithelium of cloaca
The epithelium lining the cloaca of the male Necturus is not
simple in any region. In the cephalic portion it is two-layered,
consisting of a superficial columnar or cuboidal layer and a deep
somewhat flattened replacing layer. In certain areas the outer
layer of cells is ciliated; in others, the outer cells are of the tall
mucous type, and in still others, they are unmodified. More
caudad the number of cell layers is gradually increased until at
the margins of the cloacal aperture a stratified epithelium simi-
lar to that of the external surface of the body is found. No
Leydig cells, however, are present within the cloaca, although
they occur in considerable numbers in the epidermis a short
distance from the cloacal orifice.
Dorsally, in the region of the paired urogenital papillae, a
small ciliated area is found. More cephalad, toward the rec-
tum, the epithelium is non-ciliated and of the mucous type.
Caudad, on the portions of the dorsal wall through which the
pelvic tubules open, i.e., on the Y-shaped area, the median dor-
sal groove and the dorsal walls of the lateral furrows, the super-
ficial cells, for the most part, are unmodified, resembling in their
staining reactions the cells which in other areas possess cilia.
Locally, however, groups of tall clear cells, typically mucous
in appearance, are found. It seems possible, therefore, that the
unmodified cells may be young or rejuvenating mucous cells.
Ventrally, in the region of the orifice of the urinary bladder,
the cloacal wall is covered by a mucous epithelium, but more
caudad ciliated cells are found, chiefly along the summits of the
longitudinal folds. Also scattered patches of ciliated epithelium
link up the ventral ciliated portions with the dorsal ciliated area.
The high, thin ridges of the ventral trough are for the most part
covered with a two-layered ciliated epithelium, but the conical
elevations, on which the cloacal tubules terminate, have a mu-
cous epithelium. The transition from one type of epithelium to
the other is abrupt.
The slender internal papillae, through which the cloacal tu-
bules open, with the exceptions of small areas at their bases, do
CLOACAL GLANDS OF MALE NECTURUS 457
not have a ciliated epithelium. The more cephalic papillae
are covered by a two-layered mucous epithelium, while those
nearer the external aperture have a stratified epithelium of
three to four layers similar to that covering the unmodified
wall of the cloacal chamber and the cloacal lips.
DISCUSSION
In Necturus, spermatozoa are regularly found within the
cloaca of the female. The transfer of spermatozoa from the
body of the male to that of the female is supposedly accomplished
by means of spermatophores. In Cryptobranchus, however,
fertilization is external and the sperms are expelled into the water
without the formation of spermatophores (Smith, ’07). In
Diemyctylus (Jordan, 701) and Amblystoma (Wright and Allen,
709) spermatophores are deposited and the female by her own
activity must ensure the entrance of the spermatozoa into her
cloaca. In the Tritons and in Desmognathus (Wilder, 713)
the transfer of the spermatophore is accomplished by a venter
to venter copulation.
Just what role the greatly developed cloacal glands of the
male Necturus play in the mingling of the sexual products it is
difficult to say. The success of the spermatophore method de-
pends largely on the proximity of the female. In some urodeles
specialized integumental glands are believed to attract the oppo-
site sex. No such glands have been found in the integument of
Necturus (Dawson, ’20). Some of the cloacal glands, accord-
ingly, may perform this function. The abdominal glands, on
account of their superficial position, would not apparently enter
into the spermatophore formation. They may, therefore, liber-
ate chemicals which diffuse through the water and attract the
female or, if the spermatophores are transferred directly to the
female by a venter to venter copulation, these glands, together
with the external papillae on whose median surfaces they open,
may assist in overcoming the diff.culties involved in sperm trans-
fer in the water, the external papillae forming a kind of intromit-
tent organ.
458 ALDEN B. DAWSON
The configuration of the cavity of the cloaca, the arrangement
of the glands and the positions of the ciliated areas make the
theory of spermatophore formation in Necturus seem plausible
and tend to stimulate speculation. The mucous secretion of
the cloacal gland when liberated into the ventral trough would
be gradually moved caudally by the cilia on the ridges and would
eventually collect in the cloacal chamber and the ventrolateral
recesses projecting from it. In this position the mass of mucous
material would probably be increased by additional secretion
from the cloacal tubules which terminate on the internal papillae.
Dorsolaterally in the cloacal tube, the ripe sperm would be ex-
pelled from the urogenital ducts and the median dorsal ciliated
area would carry them back until they became mingled, first,
with the secretion of the median cephalic portion of the pelvic
gland and later with the secretions of the lateral and caudal
portions of this same gland. By this time the sperms would be
in the dorsal groove and far enough caudad to be caught up by
the moving mass of mucous secretion which is propelled caudad
by the cilia of the ventral ridges. In this manner a spermato-
phore, having as a base a mass of mucous secretion and bearing
on its dorsal surface spermatozoa mingled with secretion from
the pelvic gland tubules, might be formed. The final solution
of this problem must await direct observation in the field.
Early writers attempted to homologize the cloacal glands of
urodeles with the prostate and bulbo-urethral glands of the
higher mammals. Any such homology has been denied by Hei-
denhain (’90) and Kingsbury (’95). In attempting to discover
homologies it seems unwise to begin with what are obviously
specializations of some more simple arrangement, and the pros-
tate and bulbo-urethral glands doubtless represent such special-
ization. A more primitive condition is seen in both monotremes
and marsupials, in which urethral glands, tubular glands occur-
ring in the wall of the urogenital canal, are abundant. In mono-
tremes there is a common cloaca with a primitive penis project-
ing slightly from its ventral wall. From this simple organ it is
believed the typical penis of mammals has been derived and it is
also regarded as homologous with the intromittent organ of
CLOACAL GLANDS OF MALE NECTURUS 459
turtles and crocodiles which develops from the ventral wall of
the cloaca. The cloaca of urodeles is doubtless homologous with
the cloaca of both reptiles and mammals, and from the ventral
portion of this the special organ of copulation has been evolved.
The cloacal glands of urodeles and the urethral glands of mono-
tremes and marsupials perform the same function, i.e., furnish
a fluid or semifluid vehicle for the spermatozoa, but this simi-
larity of function is not sufficient to establish the homology, since
we have many instances of similar structures performing the
same function in different groups of vertebrates, but they are
not homologous. However, even if the homology between the
cloacal glands of urodeles and the urethral glands of lower mam-
mals cannot be established, it is at least interesting to note that
in such widely separated groups of vertebrates the same type of
gland has been evolved in a similar position to serve apparently
similar needs.
BIBLIOGRAPHY
Dawson, A. B. 1920 The integument of Necturus maculosus. Jour. Morph.,
vol. 34, pp. 487-589, 6 pls.
Hemennalin, M. 1890 Beitrige zur Kenntnis der Topographie und Histologie
der Kloake und ihrer driisigen Adnexa bei den einheimischen Tritonen.
Arch. f. mikr. Anat., Bd. 35, S. 173-266, Taf. 10-13.
JorpaNn, E. O. 1891 The spermatophores of Diemyctylus. Jour. Morph..,
vol. 5, pp. 263-270.
Kinessury, B. F. 1895 The spermathecae and methods of fertilization in some
American newts and salamanders. Trans. Am. Micr. Soc., vol. 17,
pp. 261-304.
Smitu, B.G. 1907 The life history and habits of Cryptobranchus alleghenien-
sis. Biol. Bull., vol. 13, pp. 5-39. .
1911 Nestsand larvae of Necturus. Biol. Bull., vol. 20, pp. 191-200.
Wiuper, H. H. 1912 The appendicular muscles of Necturus maculosus. Zool
Jahrb., Suppl. 15 (Festschrift fir J. W. Spengel, Bd. 2), S. 383-424,
Taf. 23-27.
Wiper, I. W. 1913 The life history of Desmognasthus fusca. Biol. Bull.,
vol. 24., pp. 251-341.
Wricut, A. H., anp Auten, A. A. 1909 Early breeding habits of Amblystoma
punctatum. Am. Nat., vol. 43.
Zur Mtuuen, ALEX, v. 1893 Untersuchungen iiber den Urogenitalapparat der
Urodelen. Dissert., Dorpat, 62 pp.
DESCRIPTION OF PLATES
ABBREVIATIONS
abd.gl., abdominal gland
c. mu.. circular muscle layer
cl. ap., cloacal aperture
cl. ch., cloacal chamber
cl. gl., cloaca] gland
cl.gl.a., cloacal gland. area of
cl.lp., cloacal lip
cl.lp.f., cloacal lip, fissures of
cl. t., cloacal tube
c.t.s., connective-tissue septum
d.g., dorsal ganglion
ext.p.. external papilla
int.pp., internal papillae
lt.f., lateral fold ;
lt.fur., lateral furrow
l.mu., longitudinal muscle layer
md.gr., median dorsal groove
md.g.w., median dorsal groove, wall of
mv.f.. median ventral fold
mv.s., median ventral septum
mes., mesonephros
m.cf., muscle caudalifemoralis, im-
pression of
m. epit., muscle caudalipuboischio-
tibialis, impression of
m.isc., muscle ischiocaudalis,
pression of
pl.gl.’ pelvie gland,
division
pl.gl.’, pelvic gland, median cranial
division
pl.gl.’’’, pelvic gland, lateral division
p., peritoneum
rectum, rectum
l.c.gr., transverse crescentic groove
ug.d., urogenital duct
ug.p., urogenital papilla
ur .bl., urinary bladder
ur.bl.cav., urinary bladder, cavity of
ur.bl.o., urinary bladder, orifice of
ur.t., urinary tubules
vl.rec., ventrolateral recess
v.f., secondary ventral folds
v.r., ventral ridges
v.tr., ventral trough
Y. Y-shaped area
im-
median caudal
PLATE 1
EXPLANATION OF FIGURES
1 Ventral view of the pelvic region of a male Necturus, showing the super-
ficial topography of the cloaca.
2 Lateral view of the entire gland mass.
had been hardened in aleohol.
3 Dorsal view of the entire gland mass.
had been hardened in alcohol.
4 Dorsal view of the cloacal cavity.
slightly to one side of the midventral line, and laid open.
Dissected from a specimen which had been hardened in
completely divided.
alcohol.
Drawn from a live animal in May.
Dissected from a specimen which
Dissected from a specimen which
The cloaca was slit longitudinally,
The cloacal gland is
5 Ventral view of the cloacal cavity, laid open by a longitudinal incision
along the middorsal line.
Pelvic gland is completely divided.
Dissected from a
specimen which had been hardened in aleohol.
460
CLOACAL GLANDS OF MALE NECTURUS
PLATE 1
ALDEN B. DAWSON
{61
PLATE 2
EXPLANATION OF FIGURES
6 An outline sketch of figure 5, showing the position and plane of section of
the sections represented in figures 7 to 17. The number-at the end of each line
corresponds with the number of the plate figure representing that level.
7 Transverse section cutting the cloaca at the level of the urogenital papillae.
8 Transverse section cutting the cloaca at the level of the orifice of the
urinary bladder.
9 Transverse section of cloaca cutting the caudal end of the mesonephros
and passing through the dorsal ganglion in the region of its greatest extent.
10 Transverse section of the cloaca, showing the tubules of the cranial differ-
entiation of a pelvic gland.
11 Transverse section of cloaca through the cranial ends of the lateral furrows
and ventral trough, showing the tubules of the lateral differentiations of the
pelvic gland.
CLOACAL GLANDS OF MALE NECTURUS
ALDEN B. DAWSON
7
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463
PLATE 2
PLATE 3
EXPLANATION OF FIGURES
12 Transverse section of the cloaca immediately anterior to the cranial end
of the median dorsal groove.
13. Transverse section of the cloaca showing the median dorsal groove,
lateral furrows and ventral trough.
14. Transverse section through the cloaca caudal to the extent of the lateral
furrows and immediately cranial to the chamber of the cloaca.
15 Transverse section of the cloacal chamber, showing the ventrolateral
recesses.
16 Transverse section through the caudal end of the cloacal slit showing the
paired masses cf abdominal gland tubules.
464
PLATE 3
CLOACAL GLANDS OF MALE NECTURUS
ALDEN B. DAWSON
465
Resumen por el autor, H. Hibbard.
Inclusiones citoplasmicas en el 6vulo de Echinarachnius parma.
Una comparacion entre el citoplasma de los huevos de Echi-
narachnius fecundados por sus mismos espermatozoides y los
fecundados con espermatozoides de Arbacia no ha demostrado
la existencia de diferencias visibles. El autor ha llevado a cabo
un estudio del citoplasma del 6vulo antes de la fecundacién y
en diferentes intervalos después de esta. Ha podido comprobar
la existencia de tres tipos de inclusiones: 1) Deutoplasma en
forma de gotitas de grasa situadas cerca del nticleo y también
en forma de esferas vitelinas muy numerosas esparcidas por el
citoplasma; 2) Mitocondrias, y 3) Grandes precipitados de
material coloide coloreable con la hematoxilina ferruginosa
después de la fijacién en licor picroacético o en sublimado acético.
Estos ultimos corptisculos pueden encontrarse en el édvulo no
fecundado cuando su citoplasma esta en estado soluble (sol) pero
cesan de formarse cuando el citoplasma se transforma en una
gelatina (gel) durante su preparacién para la primera divisi6n.
No se conoce nada mas acerca de la naturaleza de estos precipi-
tados. Existen pruebas que indican que las gotas de grasa
situadas cerca del nucleo se fragmentan en pequenisimas gotitas
que se esparcen por la célula y producen las mitocondrias, y que
estas a su vez son instrumentales en la formaci6én de los cor-
pusculos vitelinos. Estos ultimos desaparecen gradualmente
cuando son absorbidos por el 6vulo durante los procesos de la
segmentacion.
Translation by José F. Nonidez
Cornell Medical College, New York
AUTHORS ABSTRACT OF THIS PAPER ISSUED
BY THE BIELIOGRAPHIC SERVICE, MAY 22
CYTOPLASMIC INCLUSIONS IN THE EGG OF
ECHINARACHNIUS PARMA
HOPE HIBBARD
Bryn Mawr College
ONE TEXT FIGURE AND FOUR PLATES (TWENTY-FOUR FIGURES)
CONTENTS
Lou. HED OIE DED Bete ie ert tae acide MSR aan ari ee ition a peta ger dea 467
Preparation of material............ aE, TASS 5 RSE SS SEIRD TEE) PEN 469
BOTS MNT OLD ae Or POR eRe Ra a a ood hy ad res} Lacy e aysiers a1 Aote vices Whoa eens @ seeds 472
AMD CULO ASIN CHITCIUSTONS eine: cles nahin 6 Aahares 6 pisqhe n opchertiale aitencs oilaeh 473
We IDEM Oats attic Senge urna eR er Acai aia ig ade cei Bae Pick Mente Nah 473
eC OPM OMNES Pre ROT e eA ALa a RLS nhs SATIRE OR Beas DHE, 475
SON EMee Wit Less (Yiolke ite. se Ales «ek amnesia ee o 2a cba eae tak 475
1B DTTC OCTET 7S epee ee Pe Ne Ore ee 477
[ty Ths SIs OY! aN aNaG 1 yt i ee ea Ra apa ey ic tip BD 477
vege id BROMO ETS ACV hae 1 8 ac RM, a Ga Lee EP Ee CTL OD 479
LIVEGUSE Tides Gil Wee to le Mas Bt eed ts UR REALE S CESS ORE A Oe MRED oe 4B eee 481
SEES! pO SS A Fo Se i ieee cone | ae ne 483
inom Maree sek aeweryedc + cobibiete syste Pe eee RRC AROS tele creer Oe ene 484
INTRODUCTION
The cytoplasm of the egg has attracted a great deal of atten-
tion among cytologists during the last few years, in contrast to
the almost universal attention paid to the nucleus before that
time. A great many observations on cytoplasmic inclusions
have been made, but there is a distinct lack of codrdination of
the results of such work. Cowdry, in his valuable contribution
to the literature on mitochondria, has summed up and correlated
the observations and conclusions of various authors regarding
these structures. Numerous other bodies occurring in the cyto-
) plasm have been reported, but usually investigators have given
merely a description of the morphology and staining reactions
of these bodies. There have been, however, attempts to con-
sider cytoplasmic inclusions in the light of the physiology of the
467
JOURNAL OF MORPHOLOGY, VOL. 36, NO. 3
468 HOPE HIBBARD
cell and to trace the interrelations of distinct bodies through
different stages. The possible transformation of mitochondria
into secretion granules, pigment, yolk, etc. (Cowdry gives a
list of eighty such things into which mitochondria have been
reported to change), and the cycle described by Schreiner (’15),
in which fragments of the nucleolus wander into the cytoplasm,
‘unite into vegetative threads, and break up into secondary
granules which are ultimately transformed into fat drops, are
instances of these attempts to consider visible structures as
steps in the physiological processes of the cell.
The processes of metabolism in the cell necessarily include ac-
tivities of both nucleus and cytoplasm. The part played by the
nucleus is not fully understood. Some investigators have de-
scribed particles of material passing out of the nucleus into the
cytoplasm. These particles have been regarded as chromatin
(Schaxel, 711; Danchakoff, ’16), or as fragments of the nucleolus
(Schreiner, 715; Nakahara, 717; Walker and Tozer, ’09; Har-
gitt, 719, ete.). According to other investigators, the nucleus acts
on the cytoplasmic substrate by liberating enzymes, which
diffuse through the nuclear membrane and permeate the cyto-
plasm. Tennent (’20) has found in Arbacia eggs fertilized by
Moira sperm, precipitates in the cytoplasm which are interpreted
as the result of enzymes from the nucleus brought in by the
foreign sperm.
The present work was undertaken in the hope of demonstrat-
ing more exactly the relation between the nucleus and the cyto-
plasm by comparing the cytoplasmic contents of an egg fertilized
by sperm of its own species with that of an egg fertilized by
sperm of another species. It was thought possible that the cyto-
plasm when acted on by two different types of nuclear enzymes
might show visible differences. In the study of the particular
cross made, Echinarachnius < Arbacia, no such visible differences
between the self-fertilized and the cross-fertilized eggs have been
found. This does not invalidate the conclusion that the nucleus
gives out enzymes into the cytoplasm. It probably indicates
that in the particular cross used here the enzymes of the foreign
sperm were so much like those of the species sperm that no-
INCLUSIONS IN EGG OF ECHINARACHNIUS 469
visible differences in effect occurred. It should be remembered,
however, that minor chemical variations may easily occur with-
out giving rise to visible differences.
Attention was then directed to the cytoplasmic contents of
the egg before fertilizaton and the changes which occur after
fertilization and during the early stages of development. The
study of the cytoplasm of the egg has certain peculiar advantages
over the study of the cytoplasm of tissue cells. To be sure,
one does not find secretion granules or other structures associated
with specialized function, but there are instead those substances
necessary for processes of development and differentiation. By
the use of various methods, several types of inclusions have been
demonstrated and certain conclusions regarding their part in the
general metabolism of the cell have been reached.
The work was undertaken at the suggestion of Dr. David
Hilt Tennent and pursued under his direction. It is a great
pleasure to express my appreciation of his constant and stimulat-
ing supervision throughout the course of the investigation.
PREPARATION OF MATERIAL
The material for this work was collected, fixed, and imbedded
at Woods Hole during July and the early part of August, 1920.
The particular eggs used were those of the sand-dollar, Echina-
rachnius parma. They were fertilized by Echinarachnius sperm or
Arbacia sperm, and in all cases parallel series were kept of the
self-fertilized and cross-fertilized eggs. Just (’19) has shown that
when the eggs are normally shed into sea-water they may be
cross-fertilized without special treatment. However, it is very
rarely that Echinarachnius females can be obtained at Woods
Hole which will shed their eggs. To stimulate them to shed,
the test is clipped around the circumference with scissors and the
animal placed aboral face down on a watch-glass. Although
this was done with individuals from practically every lot of sand-
dollars brought in from June 28th to August 4th, only one animal
was obtained during that time which shed eggs. Therefore, it
was necessary to open the test and shake out the ovaries in sea-
470 - HOPE HIBBARD
water. The eggs of this form are mature when shed or when they
easily shake out of the ovary, and may be fertilized by species
sperm as soon as they are clean, but eggs need further treatment
before cross-fertilization. To clean from bits of ovarian tissue
and coelomic fluid, the water in the finger-bowl in which the
ovaries have been shaken is stirred, then allowed to settle for a
brief period and the supernatant water poured off. The finger-
bowl is then refilled with fresh sea-water and the process re-
peated several times. In this way the heavier eggs which settle
are kept, while the lighter debris is poured off. Since these
eggs were not normally fertilizable by Arbacia sperm, it was
necessary to resort to some artificial means of breaking down the
cortical resistance. As in other eggs, there are three methods
of breaking down this resistance, namely, 1) by staling the eggs,
2) by over-insemination, and, 3) by the use of alkali. For.
the purpose of this work the third method was employed. To
determine the optimum concentration of alkali, two experiments
were performed. Varying amounts of n/10 NaOH in one case
were added to sea-water, and n/10 KOH in the other. After
fertilization by Arbacia sperm, the percentage of development
was recorded. Very little difference was found between the
NaOH and the KOH. The standard strength of twenty drops
of n/10 NaOH per 150 ce. of sea-water was adopted as the pro-
portion of alkali yielding the best results. The alkaline sea-
water was added to the cleaned eggs from which most of the water
had been poured, and as soon as possible the sperm suspension
was added and the contents of the dish well mixed. As soon as
the eggs had settled, the supernatant liquid containing excess
sperm and alkali was drawn off by means of a suction flask con-
nected as figured.
By this method all the liquid save about 8 cc. could be quickly
withdrawn, leaving the eggs undisturbed on the bottom of the
finger-bowl. Fresh sea-water was then added. After self-fer-
tilization the supernatant water was similarly withdrawn and
replaced by fresh sea-water.
In any one series of eggs preserved the eggs from only one
female were used and they were fertilized by sperm from one
INCLUSIONS IN EGG OF ECHINARACHNIUS A471
male of the same species or from one male Arbacia. In every
case three finger-bowls were kept. In one, kept as a control, were
placed eggs, 150 cc. of sea-water, and no sperm; in the second,
eggs fertilized by species sperm, and in the third, eggs fertilized
by Arbacia sperm by the aid of alkali. A sample of unfertilized
eggs was preserved, and from the second and third finger-bowls
samples were preserved at varying intervals after insemination.
For example, series 4 was fixed every fifteen minutes, series 9
every twenty minutes, series 17 every ten minutes, etc. The
original bowls were kept until the following day and examined to
make sure that no contamination had occurred before actual
insemination, as proved by the failure in every case of the eggs in
the control to form fertilization membranes or to cleave. In
order to prevent chance fertilization, hands, instruments, and
dishes were washed in fresh water before opening each animal.
In addition, the animal itself was rinsed in fresh water and then
in sterilized sea-water before being opened. Frequently all
dishes, pipettes, and instruments were put into a large’ kettle
and boiled.
The fixing fluids used were picro-acetic (saturated aqueous
picrie acid 95 parts, glacial acetic acid 5 parts), sublimate-acetic
(saturated aqueous corrosive sublimate 100 parts, glacial acetic
acid 5 parts), Bouin’s fluid, Allen’s warm modified Bouin (Bouin
50 ee. urea 1 gram, chromic acid 0.75 gram) made up immediately
before using, Perenyi’s fluid, Meves’ fluid (Lee, Vade-Mecum,
7th ed., p. 328), Champy’s fluid (3 per cent potassium bichromate
7 parts, 1 per cent chromic acid 7 parts, 2 per cent osmic acid
4 parts), Cajal’s fluids, Helly’s Zenker-formol, strong Flemming,
472 HOPE HIBBARD
and Flemming without acetic acid. In the last-named fluid
one series was fixed for one day and another for seven days.
The fixation in Perenyi and Cajal was very poor, and therefore
the material was discarded. All the material was imbedded at
Woods Hole in soft paraffn, then taken to Bryn Mawr, reim-
bedded, sectioned, and stained. The sections were for the most
part 4u in thickness.
The stains employed were Heidenhain’s iron hematoxylin,
Auerbach’s acid fuchsin-methy] green, lithium carmine and Lyons
blue, basic fuchsin and methylene blue, Benda’s alizarin and
erystal violet, safranin, safranin and gentian violet and orange
G, and for special tests, sudan JII and Ziehl’s carbol-fuchsin.
Samples of material fixed in solutions containing osmic acid were
also mounted unstained. Of these stains, the iron hematoxylin
and the Benda stain proved the most satisfactory and were the
most widely employed. In making up the alizarin the direc-
tions given in Guyer’s Animal Micrology were followed rather
than those given by Benda himself or by Cowdry in describing
Benda’s method. Guyer gives the following formula for Benda’s
solution of sulphalizarinate of soda: 1 part of saturated aqueous
solution of stain to 80-100 parts of water. Benda’s own direc-
tions are to add 1 part of a saturated alcoholic solution of the
stain to 80-100 parts of water. Both methods were tried but
Griibler’s sulphalizarinate of soda was found to be practically
insoluble in aleohol. The stain made from the saturated aqueous
solution of the dye gave excellent results.
OBSERVATIONS
As has been mentioned above, there were no visible differences
between the self-fertilized and the cross-fertilized eggs. Any
given method of fixation followed by the same stains gave identi-
cal results in the two cases. In order to compare them the better,
sections of self-fertilized eggs and sections of cross-fertilized eggs
were mounted side by side on the same slide. This insured ex-
actly the same degree of staining.
A number of structures were found in the cytoplasm following
different methods of fixation and staining. All the bodies
INCLUSIONS IN EGG OF ECHINARACHNIUS 473
found could be demonstrated in the unfertilized egg, but some of
them changed or disappeared during subsequent stages of de-
velopment. Where there were progressive changes in the cleay-
age stages, a whole series was mounted on one slide. Thus any
variation due to differences in. technique of staining was elimi-
nated, since all stages of the same series received the same
treatment.
Gatenby (19 b) and others have classified cell inclusions in two
main groups: first, inert inclusions like deutoplasm and, second,
active or living inclusions like mitochondria. As deutoplasm are
classed fat, glycogen, yolk, etc. The egg of Echinarachnius
parma contains a considerable amount of fat. The glycogen, if
there had been any, would have been dissolved out by the tech-
nique employed in preparing this material. The cytoplasm is
packed with spherical or plate-like bodies of nutritive material
which is identified as yolk. Active inclusions in the form of
mitochondria have been demonstrated. There is still another
type of structure found in these eggs. It is an inert inclusion and
yet is not deutoplasm. It will be considered under the heading
‘Precipitations.’ The occurrence of these substances will be
considered more in detail.
A. Deutoplasmic inclusions
1. Fat. It is known (Partington and Huntingford, ’21) that
fat droplets reduce osmic acid to osmium dioxide and assume,
therefore, a dense black appearance after the use of a fixing
fluid containing osmic acid. Accordingly, eggs which had been
fixed in Fleming, Flemming without acetic, or Meves’ fluid were
mounted unstained and examined for fat. No fat was found in
the material which had been fixed for seven days, but in the
Flemming and in the Flemming without acetic material which
had been fixed for eighteen to twenty-four hours there were
numerous black bodies. Figure 1 shows an unfertilized egg
fixed in Flemming without acetic and mounted unstained. There
are in the cytoplasm large blackened masses surrounded by fine
droplets of blackened material of uniform size, and in some cases
there are clumps of fine droplets without any central larger drop.
474 HOPE HIBBARD
This emulsified condition is a characteristic of fats. It seems
probable that the original large drop of fat is being split up
into smaller parts and that the scattered fine particles of
blackened material throughout the cell have been formed by such
emulsification of larger masses.
Sudan ITI, a specific stain for fat, was used on this material,
but gave no decisive results because the drops had been previously
blackened and naturally could not be stained red. A further
proof of their fatty character was obtained by soaking the sec-
tions for twenty-four hours in oil of turpentine. After this treat-
ment the black droplets were completely dissolved out. Since
turpentine is a fat solvent, the material which was removed was
probably fat.
The large groups of fat droplets are slightly more numerous in
the region of the nucleus than they are in the more distant parts
of the cytoplasm. ‘This is of interest in the light of the views of
Schreiner, Popoff, and others, that granules of nuclear origin
pass through the membrane and give rise to fat droplets. The
granules are believed to come from the nucleolus. ‘The evidence
given here of the accumulation of fat near the nucleus shows noth-
ing more than the fact that they are associated with some kind
of nuclear activity. It is also true that an occasional oocyte
has been found on the slides in which there is always a large nu-
cleolus present which is entirely absent in the ripe egg.
The continued splitting of the large drops into minute droplets
and the dispersal of the latter through the cytoplasm is illustrated
in figures 2 and 3. These show eggs from the same series from
which figure 1 was drawn, in stages twenty-five minutes and one
hour and forty minutes, respectively, after insemination. As
development proceeds, there is a gradual decrease in the amount
of blackened material present. As the cell prepares for the first
division the fat droplets are much fewer, and in the two-celled
stage none are visible. It is highly probable that these cells do
not show as much fat as is present in the living egg, because some
of it must have been dissolved out in the processes of preparation,
but the fact that a definite series of changes can be demonstrated
is a true indication of what actually occurs.
INCLUSIONS IN EGG OF ECHINARACHNIUS 475
2. Glycogen. As was mentioned formerly, no glycogen was
demonstrated in these eggs. No material was fixed by any of
the methods for the preservation of glycogen.
3. Nutritive plates (yolk). By far the most conspicuous and
unusual inclusions are those which are best demonstrated after
fixation in Flemming without acetic, Meves’ or Champy’s
fluids, and staining in iron hematoxylin or Benda’s alizarin and
crystal violet. These bodies are shown in figures 1 to 6, 12, or
19 to 24. In figure 12, drawn from an egg fixed in Champy’s
fluid, they are distinctly plate-like and much larger than in any
other lot of material. The more usual appearance is shown in
figures 19 to 24, where they are smaller and less distinctly plates.
Their fate indicates that they are nutritive in function. They
do not, however, respond to all the usual tests for yolk. At
first their staining reaction seems to mark them as mitochondria,
for in the series illustrated in figures 19 to 24 the fixation is
Flemming without acetic and they are stained a deep violet with
the Benda stain. They do not always give this reaction, how-
ever. The series described above was fixed for seven days. If it
be compared with the series shown in figures 4 to 6, which is also
Flemming without acetic, but fixed for eighteen to twenty-four
hours only, it will be seen that the large bodies are there in both
cases, but they differ in staining capacity. They take the violet
stain strongly after seven days’ fixation, but are pink after one
day’s fixation. The behavior of these two series when stained
with iron hematoxylin shows great dissimilarity also. In the
first case the plates are black and in the second they do not stain.
Since they are so striking in appearance in some series of eggs,
their apparent absence after other fixatives was unaccountable
until it was found that in practically every case the material of
which these plates are formed could be shown in the cytoplasm
even though not in the form of such distinct separate bodies.
For instance, when stained in iron hematoxylin after fixation in
modified Bouin, the cytoplasm had a decidedly reticular ap-
pearance with minute black granules throughout (fig. 18); in the
same material stained in Benda’s stain (fig. 15), or in iron hema-
476 HOPE HIBBARD
toxylin and basic fuchsin, the cytoplasm had a mottled appear-
ance. <A distinct difference between the cytoplasm of the center
of the egg in which the division figure lies and the peripheral
cytoplasm may be seen. ‘This regional differentiation corre-
sponds to the distribution of the nutritive bodies fixed so distinctly
in Flemming without acetic. This shows that the nutritive
material is present after modified Bouin fixation. Similar
results were found after sublimate-acetic fixation. After strong
Flemming followed by safranin, gentian violet, and orange G,
this nutritive material takes up the orange stain more strongly
than any other cell constituent. Therefore, while certain fixing
fluids are decidedly more favorable for the demonstration of
these plates, the material is not dissolved completely by the other
reagents used.
Yolk is the only substance ordinarily found in the cytoplasm
of the egg in great quantities comparable to this material. But
these plates do not respond to all the tests for yolk given by Miss
Beckwith (’14). For instance, yolk turns black after Flemming
fixation and iron-hematoxylin stain. These bodies are not black.
Yolk is definitely fixed by picro-acetic and sublimate-acetic.
Gatenby (’19 b) states that in some animals yolk discs take a
deep violet after Benda’s stain. He points out that this staining
reaction may be due to protein in the yolk in addition to lecithin.
The yolk plates in the egg of Echinarachnius, however, do not
invariably stain a deep violet, but do so only after prolonged
fixation. Some proof of the nature and function of these bodies
may be obtained by tracing them through the early cleavage
stages to the blastula. They become gradually fewer in number
and in the blastula they are almost gone. Figures 19 to 24 show
progressive stages in which they have become fewer. Figure 24
illustrates a stage four hours after insemination in which there are
spaces left which the large plates formerly occupied and in which
many of the minute granules are stained violet. This may
be due to the breaking up of the larger masses by a process of
digestion. The disappearance of this material is not confined
to those series which have been fixed in Flemming without acetic
Meves, and Champy. Figure 11 (picro-acetic) shows a much
INCLUSIONS IN EGG OF ECHINARACHNIUS 477
more spongy and vacuolated cytoplasm than the earlier stages
of the series, which indicates a loss of some substance. Figures
16 and 17 show a similar phenomenon in modified Bouin material.
Therefore, these plates are of a nutritive character and are used
up by the cell during processes of development.
They contain a certain amount of fat, as is shown by staining
with sudan III. Fifteen minutes’ staining with alcoholic sudan
III gives them a very decided salmon-pink color. After soaking
for twenty-four hours in oil of turpentine, the sections stained in
sudan III gave no color whatever, even when the stain was
allowed to act for thirty minutes. There was evidently some fat
there which was removed by the turpentine. After treatment
with turpentine the plates retained their characteristic form and
appearance except that they lacked the capacity to take up sudan
III. The nutritive plates are probably of complex chemical
structure.
While the nutritive plates do not in all cases behave like yolk
with regard to staining reactions, yet their obvious function and
their shape and distribution point to the conclusion that they
must be yolk.
From their method of origin these yolk plates may be linked up
with the other cell inclusions. This point will be considered
after the discussion of mitochondria.
B. Inving inclusions
Mitochondria. In addition to the above deutoplasmic in-
clusions, there are also active, living constituents which are dis-
tinct from the ground cytoplasm. These are the mitochondria.
The stain which differentiates them most successfully from other
cell inclusions is Benda’s alizarin and crystal violet. They show
up a deep violet against a background of neutral pink. Iron
hematoxylin blackens them, but it also blackens other cell
granules. With basic fuchsin and methylene blue they are red,
with safranin they are red. It is possible to demonstrate mito-
chondria after fixation in Flemming, Flemming without acetic,
Meves, Champy, and modified Bouin. They are not found
478 HOPE HIBBARD
after ordinary Bouin fixation. ‘The shape and distribution of
mitochondria in the eggs of Echinarachnius are shown in figures
13 to 17 which were drawn from a series of eggs fixed in modified
Bouin and stained in Benda’s alizarin and crystal violet, and in
figures 4, 5, and 6 which were drawn from a series fixed in Flem-
ming without acetic followed by the Benda stain.
The study of this material indicates that the mitochondria
bear a definite relation to the fine uniform fat droplets described
above. In figure 4 there is shown an unfertilized egg fixed in
Flemming without acetic and stained according to Benda’s
method in which the mitochondria were deep violet. They are
shown as black granules in the figure. The egg also contains
granules which appear the same in size, shape, and distribution
but which take the violet stain with varying degrees of intensity
or which may be quite pink like the ground cytoplasm. There
is also a continuous variation in color from the small fat droplets
which are brown from osmic-acid impregnation, through similar
granules which are less and less brown, to pink granules. Itseems
probable, therefore, that the small fat droplets which are formed
by emulsification of larger fat drops change gradually, as indicated
by differential staining, into the bodies which take a deep violet
stain after Benda’s method and are identified as mitochondria.
In the modified Bouin material, the mitochondria are stained
a deep violet with the Benda stain. They are the granules illus-
trated in figures 13 to 17. These same eggs when stained with
iron hematoxylin show many more black granules than can be
identified as mitochondria. Figure 18 shows such an egg thirty
minutes after insemination which contains a larger number of
black granules than the number of mitochondria shown in figures
14and15. No granules stain with sudan III after this fixation.
The relation between the mitochondria and the nutritive plates
may be considered here. The great mass of plates is already
formed by the time the egg is ripe, but there is some indication
of how they may be formed. In the eggs from which figures 4,
5, and 6 were drawn the mitochondria were stained violet and the
nutritive plates pink. In figure 4 and more especially in figures
5 and 6 some of the mitochondria show pale centers. In fact,
INCLUSIONS IN EGG OF ECHINARACHNIUS 479
sometimes the inside distinctly took the pink alizarin stain.
This is interpreted as showing the formation of the nutritive
plates, or yolk, from mitochondria. The nutritive material
accumulates at the center and increases until it is nearly as large
as one of the numerous yolk plates. When this occurs the mito-
chondrial remnant.is found as a delicate violet rim around the
surface of the plate, or there may be in addition a larger bit of
the violet-stained material clinging to one side.
There have thus been followed the fat drops which are emulsi-
fied into minute fat droplets and are distributed through the cyto-
plasm where they are probably transformed by some kind of
synthesis into mitochondria. These mitochondria in turn build
up within themselves the large nutritive plates which furnish
energy for the cleavage processes. ‘The line which is drawn be-
tween active and inactive inclusions is, in the case of the egg of
Kchinarachnius parma, purely arbitrary. All transitional stages
between deutoplasmic granules like fat, and mitochondria have
been demonstrated.
C. Precipitations
After the use of picro-acetic and sublimate-acetic fixatives,
. striking bodies which stain strongly in iron hematoxylin are found
in the cytoplasm. These are the large black masses surrounded
by clear areas shown in figure 7. They are invariably at the
centers of open spaces, which leads to the conclusion that they
are condensed or precipitated from material once occupying the
entire space. After fixation in sublimate-acetic they tend to
assume a slightly more elongated form than after picro-acetic as
figured. Such irregular precipitations are found in all unfertilized
eggs fixed either in picro-acetic or sublimate-acetic. Proof that
eggs containing them are normal is found in the subsequent
history of some series where practically 100 per cent development
followed fertilization. It may be argued that these precipitations
are the result of the action of the fixing fluids, since, as has been
pointed out by Mathews (Physiological Chemistry, p. 120 and
p. 1086), the salts of metals.and picric acid have the power to
precipitate proteins. The following facts prove that this cannot
480 HOPE HIBBARD
be the entire explanation of the matter. These masses are actual
precipitations of material in the normal cell, for in later stages
these fixing fluids do not form such precipitations. Figures
7 to 11 show that the bodies have become fewer as development
has proceeded. By the time the first division is well under way
all the large masses have disappeared and there is no indication
of the larger spaces in which they lay. They do not again reap-
pear after the first division.
Similar precipitated masses may be found following other
methods of fixation, but the precipitations do not actively take up
any of the stains employed; they are always like the diffusely
stained ground cytoplasm. Figures 13, 14, and 15 illustrate
their appearance following modified Bouin fixation. In these
eggs, stained in alizarin and crystal violet, they were of the pale
pink color of the great mass of cytoplasm, and therefore not so
conspicuous as when stained in iron hematoxylin after picro-
acetic or sublimate-acetic fixation. In some eggs they looked
almost like bacteria, but they failed to stain with Ziehl’s
carbol-fuchsin. Also their behavior in later stages could not
be accounted for if they were bacteria.
As to the nature of these bodies, nothing is known beyond
the fact that they are precipitations of colloidal material in the
cytoplasm. It is probable that these bodies are of the same
character as the rods observed by Tennent (’20) in the egg of
Arbacia fertilized by Moira sperm, after fixation in sublimate-
acetic.
In the unfertilized egg there is no uniformity in the orientation
of the precipitated masses, but as soon as the spindle for the
first division begins to form, they begin to be oriented parallel
to the astral radiations extending through the cytoplasm. ‘They
appear as if swept about by the currents of more fluid protoplasm
flowing in toward the focus of the aster, until they present the
least surface in opposition to the direction of flow. Such a
flowing in of more fluid protoplasm to the centers of the hyaline
areas (such as are shown in figure 10) as the protoplasm goes into
a state of gelation at division, was demonstrated by Chambers
(19). In this connection, Bowen’s observations on the division
INCLUSIONS IN EGG OF ECHINARACHNIUS 481
of mitochondria in Hemiptera (’20) may be mentioned. He
states that the fact that the mitochondria are oriented with
definite relation to the centrosome proves that they are under its
directive influence, and therefore there is a mechanism for exact
division of mitochondria in mitosis as well as for chromosomes.
Since the flowing of material described by Chambers occurs at
this time, it is inevitable that any inert masses lying in the cyto-
plasm should be swept into line, and their orientation by this
means need have no connection with any attractive force exerted
on them by the centrosome.
DISCUSSION
All of the inclusions found in the egg of Echinarachnius parma
have been found in the unfertilized egg and they have decreased
during cleavage. ‘This is wholly in accord with the nature of the
processes going on in the egg at this time. Its growth period is
past and it is about to start on a series of changes involving
great energy expenditure. While these changes are occurring
there is no opportunity for the aquisition of food material either
from maternal tissue or from the medium in which the cells are
living. All energy then must come from materials stored up
within the egg at the time it is set free from maternal tissues.
This does not in any way preclude transformations of some mate-
rials in the cytoplasm into others, and it is believed that such trans-
formations have been demonstrated. Here there are reduced to
their lowest terms the problems of the biochemist concerning
the synthesis of proteins, carbohydrates, and fats, for the large
amounts of the substances present in an animal body are merely
the sum total of all the minute particles of proteins, carbo-
hydrates, and fats synthesized and transformed in the single
cells. The exact steps in the synthesis of the complex chemical
compounds which serve as sources of energy in the physiology of
the cell are wholly undetermined. There are no reliable micro-
chemical tests for many of these substances and our interpreta-
tion of them according to their staining reactions may be quite
erroneous.
482 HOPE HIBBARD
Regaud has demonstrated that mitochondria are made up of
phospho-lipin and albumen. The transformation of mitochon-
dria into fat or of fat into mitochondria has been discussed by a
number of investigators, but the intermediate steps are unknown.
Dakin states that fatty acids are not directly transformed into
amino acids, but the evidence leaves open the possibility of such
a transformation through carbohydrates. In that case, is there
a carbohydrate stage in the formation of mitochondria or of yolk?
Evidence of such a stage would be removed by all ordinary
methods of technique in preparing sections. Perez (’03) found
in the adipose cells of Formica rufa a transformation of fat
globules into albuminous bodies by the digestion of the former.
A number of other authors have touched on this problem of
synthesis in the cytoplasm, but their conclusions are not in agree-
ment. According to Popoff (10), chromidia from the nucleus
pass out and change to fat. Saguchi (’20) working on the islet
cells in the pancreas, describes lipoid granules being formed
from mitochondria. Hollande (’14) observes fat formed from
granules near the nucleus and then a transformation of part of
the fat into albuminoid bodies. Beckwith (’14) found pseudo-
chromatin granules which developed directly into yolk spheres;
Schaxel (711) found chromatin emitted from the nucleus which
formed yolk spheres; Danchakoff (’16) found chromatin emitted
from the nucleus which synthesizes more chromatin in the cyto-
plasm. Ludford (’21) in Patella oogenesis found yolk to be
formed by Golgi bodies which were entirely distinct from the
mitochondria and the fragments of the nucleolus which he de-
scribed as being emitted from the nucleus. Nakahara’s results
(17) on Pieris, and Perez’ work on Formica rufa both point to a
transformation of fatty bodies into albuminous bodies. From
these many observations it is evident that there are transforma-
tions of visibly distinct bodies into one another in the living cell.
The great need at present is for some technique which will demon-
strate accurately the intermediate steps.
In this paper an attempt has been made to interpret those
chemical compounds in the cytoplasm of the egg of Echinarach-
nius parma which form distinct visible bodies and which can be
INCLUSIONS IN EGG OF ECHINARACHNIUS 483
stained with ordinary reagents, and to show the part played by
them in the processes of development initiated by fertilization.
SUMMARY
1. The cytoplasm of eggs of Echinarachnius parma when
fertilized by sperm of the same species shows no visible differ-
ences from that of eggs fertilized by Arbacia sperm.
2. Fat drops occur in the unfertilized egg. ‘These drops are
emulsified and the fine droplets of fat thus formed gradually
become used up or transformed during the early cleavage stages.
3. Small spherical mitochondria are found scattered through-
out the cytoplasm and there is evidence to show that they are
the direct products of the fine fat droplets.
4. The cytoplasm is packed with plates of nutritive material
which have some fatty component in their makeup. ‘They are
probably yolk. They are in some cases closely associated with
mitochondria, and it is probable that the mitochondria are
instrumental in their synthesis. These plates gradually become
fewer in number and in the blastula the cytoplasm is quite
spongy and full of vacuoles once occupied by the plates.
5. After certain fixatives, picro-acetic and sublimate-acetic,
large precipitations of colloidal material in the cytoplasm are
stainable with iron hematoxylin. Other fixing fluids preserve
them, but do not mordant them so that they actively take up
the stain. These precipitations are found in the unfertilized
egg when its cytoplasm is in a sol state. They cease being
formed as the cytoplasm becomes a gel during its preparation
for the first division.
JOURNAL OF MORPHOLOGY, VOL. 36, NO. 3
484 HOPE HIBBARD
BIBLIOGRAPHY
ALLEN, E. 1919 A technique which preserves the normal cytological conditions
in both germinal and interstitial tissue in the testis of the albino rat.
Anat. Rec., vol. 16.
Arnotp, J. 1914 Uber Plasmastrukturen und ihre funktionelle Bedeutung.
Jena.
BrecxwitH, C. J. 1914 Genesis of plasma structures in Hydractinia. Jour.
Morph., vol. 25.
Brenna, C. 1910 Farbung der Mitochondria. Enzyklopidie Mik. Technik.
Urban und Schwarzenberg.
BERLESE, A. 1901 Vorgiinge welche wihrend der Nymphosis der metabolischen
Insecten vorkommen. Zool. Anz., Bd. 24.
Bowen, R. H. 1920 Studies on insect spermatogenesis. I. The history of the
cytoplasmic components in the sperm in Hemiptera. Biol. Bull.,
vol. 38.
Cowpry, E. V. 1918 The mitochondrial constituents of protoplasm. Pub.
Carnegie Inst. Wash., No. 271.
CHAMBERS, R. 1919 Changes in the protoplasmic consistency and their rela-
tion to cell division. Jour. Gen. Physiol., vol. 2.
Dakin, H.D. 1912 Oxidations and reductions in the animal body. Longmans,
Green & Co.
DancHAKOFF, V. 1916 Studies in cell division and cell differentiation. I. The
development of the cell organs during the first cleavage of the sea-
urchin egg. Jour. Morph., vol. 27.
GatENBy, J. B. 1917 Cytoplasmic inclusions of germ cells. Parts I and IL.
Quart. Jour. Micr. Sci., vol. 62.
1918 Cytoplasmic inclusions of germ cells. Part II1l. Quart Jour.
Mier. Sci., vol. 63.
1919 a Cytoplasmic inclusions of germ cells. Parts]Vand V. Quart.
Jour. Micr. Sci., vol. 63.
1919 b Identification of intracellular structures. Jour. Roy. Mier.
Soc.
1920 On the relationship between the formation of yolk and the mito-
chondria and Golgi apparatus during oogenesis. Jour. Roy. Micr. Soe.
GuyErR, M.F. 1917 Animal micrology. Univ. of Chi. Press
Haraitt,G.T. 1919 Germ cells of coelenterates. VJ. Jour. Morph., vol. 33.
HouuanpeE, A. C. 1914 Formation endogénes des crystalloids. Arch. Zool.
Exp. Gen., T’. 53.
Just, E.E. 1919 The fertilization reaction in Echinarachnius parma. II. Biol.
Bull., vol. 36.
Luprorp, R. J. 1921 Contributions to the study of the oogenesis of Patella.
Jour. Roy. Mier. Soe.
Maturews, A.P. 1920 Physiological chemistry. Wm. Wood & Co.
NakaHArRA, W. 1917 Physiology of nucleoli in silk gland cells in insects, Jour.
Morph., vol. 29.
PARTINGTON AND HuNnTINGFORD 1921 The reduction of osmic acid by lipoids.
Jour. Roy. Mier. Soe.
INCLUSIONS IN EGG OF ECHINARACHNIUS 485
Perez 1903 Contribution 4 l’étude des métamorphoses. Bull. sci. France et
Belgique, vol. 37.
Poporr, M. 1910 Ein Beitrag zur Chromidialfrage. Festschrift zum sechzigs-
ten Geburtstag, R. Hertwig. Bd. I. Arbeiten aus dem Gebiet der
Zellenlehre und Protozoenkunde. Jena.
Sacucui, W. 1920 Cytological studies of Langerhans’ islets. Am. Jour. Anat.,
vol. 26.
Scureiner 1915 Uber Kern und Plasma Veriinderung in Fettzellen. Anat.
Anz., Bd. 48.
ScuaxeL, J. 1911 Das Zusammenwirken der Zellbestandteile bei der Eireifung,
Furchung und ersten Organbildung der Echinodermen. Arch. fiir
mik. Anat., Bd. 76.
1910 Die Eibildung der Meduse Pelagia noctiluca. Festschrift zum
sechzigsten Geburtstag. R. Hertwig. Bd. I. Arbeiten aus dem
Gebiet der Zellenlehre und Protozoenkunde. Jena.
Tennent, D.H. 1920 Evidence on the nature of nuclear activity. Proc. Nat.
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WaLKER AND TozER 1909 Observations on the history and possible function
of the nucleoli in the vegetative cells of various animals and plants.
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DESCRIPTION OF PLATES
All drawings, except figure 12, were made at table level by the aid of a camera
lucida, using a Zeiss 1.5-mm. oil-immersion objective, and a compensating ocular
no.4. This gave a magnification of 1250 diameters. Figure 12 was made slightly
above table level and its magnification was 1170 diameters.
Plates 1 and 3 were then reduced to 2? their size, giving a final magnification
of 500 diameters.
Plates 2 and 4 were reduced 3, making a final magnification of 625 diameters,
except for figure 12, which is 585 diameters.
PLATE 1
EXPLANATION OF FIGURES
(All eggs illustrated on this plate are from the same series.)
1 Unfertilized egg. Flemming without acetic, unstained.
2 Twenty-five minutes after insemination. Flemming without acetie,
unstained.
3 One hour and forty minutes after insemination. Flemming without
acetic, unstained.
4 Unfertilized egg. Flemming without acetic, alizarin and crystal violet.
5 Twenty-five minutes after insemination. Flemming without acetic,
alizarin and crystal violet.
6 One hour after insemination. Flemming without acetic, alizarin and
crystal violet.
486
PLATE 1
INCLUSIONS IN EGG OF ECHINARACHNIUS
HOPE HIBBARD
487
PLATE 2
EXPLANATION OF FIGURES
(Figures 7 to 11 are from the same series.)
7 Unfertilized egg. Picro-acetic, iron hematoxylin.
8 Twenty minutes after insemination. Picro-acetic, iron hematoxylin.
9 Fifty minutes after insemination. Picro-acetic, iron hematoxylin.
10 One hour after insemination. Picro-acetic, iron hematoxylin.
11 Three hours and one-half after insemination. Picro-acetic, iron hema-
toxylin.
12 One hour and ten minutes after insemination. Champy, iron hematoxylin
488
INCLUSIONS IN EGG OF ECHINARACHNIUS PLATE 2
HOPE HIBBARD
489
PLATE 3
EXPLANATION OF FIGURES
(All eggs illustrated on this plate are from the same series.)
13 Unfertilized egg. Modified Bouin, alizarin and crystal violet.
14 Fifteen minutes after insemination. Modified Bouin, alizarin and crystal
violet.
15 Forty-five minutes after insemination. Modified Bouin, alizarin and
crystal violet.
16 Two hours and forty-five minutes after insemination. Modified Bouin,
alizarin and crystal violet.
17 Eight hours and fifteen minutes after insemination. Modified Bouin,
alizarin and crystal violet.
18 Thirty minutes after insemination. Modified Bouin, iron hematoxylin.
490
PLATE 3
INCLUSIONS IN EGG OF ECHINARACHNIUS
HOPE HIBBARD
491
PLATE 4
EXPLANATION OF FIGURES
(All eggs illustrated on this plate are from the same series.)
19 Unfertilized egg. Flemming without acetic, alizarin and crystal violet.
20 One-half hour after insemination. Flemming without acetic, alizarin and
crystal violet.
21 One hour after insemination. Flemming without acetic, alizarin and
crystal violet.
22 One hour and one-half after insemination. Flemming without acetic,
alizarin and crystal violet. ¥
23 Twoand one-quarter hours after insemination. Flemming without acetic,
alizarin and crystal violet.
24 Four hours after insemination. Flemming without acetic, alizarin and
crystal violet.
INCLUSIONS IN EGG OF ECHINARACHNIU
HOPE HIBBARD
PLATE 4
er}
eo? .
Seertee &
493
AUTHOR’S ABSTRACT OF THIS PAPER ISSUED
BY THE BIBLIOGRAPHIC SERVICE, SEPTEMBER 11
THE CASTES OF TERMOPSIS!
CAROLINE BURLING THOMPSON
Department of Zoology, Wellesley College
NINE TEXT FIGURES AND TWO PLATES
CONTENTS
iM FROM TKOHIOTR Eo 5 Soke s MAME Uke COURS OO aa eNR Ie Tenet Pa crcl MTR ee ae ee ete 495
Miatentaleancurne TN OUSHeM ea oes os aa «ee rascal cesta events Sec ad ght 496
PE PCAGUES MAURIE Rt tie ene sates ce eee ee Rs te ene eet, She Me! 497
ieacmmopss angustieollis agen: (220 1355 Lees Ae. 497
[Dreswe lalorenesnilis o doaat ESO Sb So Ho ES ODM SORES ORES Olas SIR See ee oh aot aE: Ge eo 499
CRIS TORMy Ola ley An CTS hI COUNIS ewe Sati: cas pera tou nelle ae aise eis. sspe eee 500
Litas: SANG 7657 01 igCGy Hae pe soe 101 1 ie ep pees a eld EE 500
icmincedtadnlnot the mirstrorms.): UN LR e EeROe. OS ) BEAK. 509
ein enlarecd.adulissot, tberirst, LOM. ...c0. 3 den <heacciecth by ey ete e ad 510
Mieseconu Worn, ar Ean rusticOlliss. sr... 6s. < «2. yes oe as ees cn ohne OM a a oe 512
Sinema Ot hHe seeOmG LOM os... , sos om so es nee Abies s cadet ca OLS
The youns adult-of the second form.’).. 002.5. .0) 2 Ae at. 514
Lhejenlarcedvadult) of the second, form) .....: 6.0.0. 2 Vi eee ima ok 515
Benes oimago rm hve oust Olliss ss 6 sepa tes ie sas cas susvaagh ex. ao oteameeeede, occ ates 516
SUBEKSOLerLOlMUer AN SUSU COMMSE Mem. eabia, hofece atusepgiakiis = Ricncitukst Whol fopehes ores aus 519
eRe NIER SE, BC ee ET, SRO N Gee Bhpeembans © foe SEE E- 525
Caubnea Ria Sete eee YS Se ee eet ee ee cadet ONT Le SE I, SEAL. 529
eg MS eB See he ee TES Ee erro ead) a RRO ee RSs Le 3 530
INTRODUCTION
Termopsis angusticollis and T. nevadensis are found along the
Pacific slope of the United States and in British Columbia, and
the latter species extends into Montana (Banks and Snyder, ’20).
The two species frequently occur in the same locality and in
close proximity; at Pacific Grove, California, the writer has found
several colonies of the two species, less than a foot apart, in one
log. ‘Termopsis is the largest North American termite and also
the least injurious. The nests or galleries are found in partly
decayed wood in forests, usually just beneath the bark, and not in
1 Owing to the death of the author, proof of this article has been read by the
editor and Dr. T. E. Snyder.
495
496 CAROLINE BURLING THOMPSON
the deeper heart wood; very rarely in the woodwork of buildings,
and never in the earth.
In normal colonies of T. angusticollis and T. nevadensis four
stable types or castes may be found: 1) the first-form adults, with
long wings at the time of swarming, and later with the scales or
bases of the broken-off wings; 2) the second-form adults, with
very short wing vestiges which are much shorter than in second-
form individuals of other genera; 3) the wingless third-form adults;
4) the soldiers. The first and third forms and the soldiers are of
common occurrence, the second forms are comparatively rare.
In addition to these four stable castes, three additional types or
variations are occasionally found. These are: soldiers with wing
pads (fig. 9), second-form individuals with very minute wing
vestiges (fig. 7), and individuals closely resembling the third
form, but with wing vestiges that are merely narrow lateral
borders of the thoracic segments (fig. 8). The nymphs of all
castes occur in different stages of development according to the
season of the year. No true sterile worker caste is known to
occur in Termopsis, although the wingless third-form individuals
have been frequently described by different writers as ‘worker-like
forms’ and even as‘workers.’ The worker functions are performed
by the developing nymphs, especially those of the third forms,
and it is well known that the older reproductive forms do not
survive long in captivity unless in the company of nymphs. The
same is true of soldiers.
MATERIAL AND METHODS
Most of the material for this paper was collected by the writer
in the pine forests of Pacific Grove, California, in April and May,
1919, while a guest of the Hopkins Marine Station of Leland
Stanford University. I wish here to express my thanks for the
hospitality of the station during this time. For the identification
of my collections and for additional material the writer is indebted
to Dr. T. E. Snyder, of the Bureau of Entomology, U. 8. Depart-
ment of Agriculture. a
This study of Termopsis angusticollis has been made chiefly
by means of serial sections and dissections of organs after stain-
CASTES OF TERMOPSIS 497
ing the body in bulk and partly decolorizing. Stained whole
mounts of the heads, showing the brain and eyes, are fairly
successful, and some whole mounts of nymphs have been made,
but the stained whole mounts of the abdomen or entire insect that
have proved so useful in studying other termite genera have been
rarely successful with this genus. This may be due to the large
size, to the thicker skin, and the more abundant fat-body, but I
am inclined to attribute it in large part to the different chemical
composition of the Termopsis tissues, with consequent different
staining reaction from other termites.
In the following pages the material described is Termopsis
angusticollis, but, although T. nevadensis has been less thoroughly
studied, enough has been done to state that the morphology of the
two species is very similar.
THE CASTES
Termopsis angusticollis Hagen
T. angusticollis is the largest North American termite, the
body length of young first-form individuals, from the tips of the
mandibles to the end of the tenth abdominal segment, ranging
from 10 to 14 mm., and the head width from 2.1 to 2.8 mm.
The old enlarged first-form females attain a length of 15to17mm.,
and the males 14 to 15 mm. These figures show that the post
adult growth which takes place after mating is very slight in this
genus. The first-form males and females are active and of nearly
similar size throughout life, for the Termopsis queens never
acquire the relatively huge bulk attained by the queens of the
smaller genera of the higher termites, such as Reticulitermes,
Nasutitermes, etc. It seems surprising at first that the egg-laying
queens of such a large termite should be of relatively smaller size
and greater activity; this, however, is merely an evidence of
the primitive character of the genus. The small bulk and the
activity of Termopsis queens may be due partly to the more or
less exposed habitat in wood above ground, partly to the lack
of the true work caste, but chiefly to the fact that Termopsis, in
common with other lower termites, still retains some of the
ancestral independence of the non-social insects.
498 CAROLINE BURLING THOMPSON
The second form of T. angusticollis (fig. 6) with very short wing
vestiges extending only to the first? abdominal segment, and some
specimens with even shorter wing vestiges, is of infrequent occur-
rence. It is possible that this form has been overlooked by col-
lectors on account of its inconspicuousness and that itmay bemore
common than is now supposed. Among the many specimens of
reproductive adults collected from about fifty colonies in Pacific
Grove, Califorinia, I have only six individuals of the caste, two of
which were taken for third forms until my attention was called to
their very short wing vestiges by Dr. T. E. Snyder, and four
which were at first considered young first-form nymphs.
The wingless third-form individuals have a wide range in size,
seen not only in the abdomen, which increases but slightly with
age, but also in the head and thorax. The body length of young
egg-laying females of my collection varies from 11 to 13 mm., and
the width of the head of both sexes ranges from 2.5 to 3.7 mm.
Older third-form females with slightly enlarged abdomens measure
from 15 to 17 mm. long. A number of individuals closely resem-
bling the third form in structure, but with very minute wing
vestiges (fig. 8), have been found and will be referred to again.
The soldiers have a still greater size range, the body length,
from tip of mandibles to the end of the last abdominal segment,
varying from 15 to 25 mm., and all sizes may be found in a single
colony. Ina colony taken on May 12, 1919, a number of young
white soldiers, evidently just molted, were present. Examining
the material later, three of these young soldiers, one female and
two males, were found to have wing pads. The other young
soldiers of the same lot were normal. The histology of these
soldiers with wing vestiges will be described below. It will be
recalled that in the related genus Kalotermes soldiers with wing
vestiges are not uncommon, and occur in many species.
The soldiers with wing vestiges and the second- and _ third-
form individuals with minute wing vestiges are to be regarded as
evidences of the high degree of variability of this genus, especially
in the direction of the retention of primitive characters.
2 The sternite, or the ventral part, of the first abdominal segment of termites
is not developed, so that the true second segment appears to be the first, when
viewed from the ventral surface, and for convenience will be so termed in this
paper.
CASTES OF TERMOPSIS 499
DEVELOPMENT
The eggs of T. angusticollis are long, slender, and reniform.
In living eggs, and after fixation, two sizes may be noted: the small-
est eggs are about 1.3 mm. long; the larger ones range from 1.5 to
1.7 mm. Iam unable to state what these size differences may
imply.
The youngest nymphs of T. angusticollis that I have examined—2.2
to 2.6 mm. long, with eleven and twelve antennal segments—are all
alike in external appearance, but with a lens the two types of fertile or
reproductive nymphs and sterile or soldier nymphs may be seen. The
heads are of similar size, but the large brain, almost filling the head
cavity of the reproductive nymph, is clearly distinguished from the
smaller brain of the soldier nymphs, and correlated with the brain
structure is the whiter denser abdomen of the reproductive nymphs
and the more transparent abdomen of the soldier nymphs. In stained
and mounted specimens the larger sex organs of the reproductive
nymphs are in marked contrast to the smaller ones of the soldier nymphs
(Thompson, 719, p. 385).
The origin of the soldier, in late nymphal life, from a wingless,
rounded, worker-like nymph, has been described by several
writers. Lespés (55) described this origin of the soldier of L.
lucifugus, and Snyder (’15) saw it in L. flavipes, L. virginicus,
and T. angusticollis. Knower (’94) saw the nasutus of Eutermes
rippertii (?) (Nasutitermes pilifrons) molt from a worker-like
skin. The writer can add one more example of the late origin of
the soldier from a previous work-like phase in the species ‘Termop-
sis nevadensis. In May, 1919, a dedlated first-form male and
female of T. nevadensis, together with about fifteen wingless
nymphs, which were thought to be young thirdforms, were collected
at Pacific Grove, Califorinia, and placed in a small glass vial with
fragments of pine wood. The vial was brought to Wellesley in
July of the same year, and when the contents were examined a
white newly molted soldier was found in place of one of the wing-
less nymphs.
Fuller (’20), p. 248, writing of the origin of the soldier, states:
It is even very difficult to state exactly when the distinguishing
characters of soldiers and workers first become discernible. In certain
species examined it would appear that slight differences can be detected
500 CAROLINE BURLING THOMPSON
in the third instar. The transformation of a soldier takes place during
the third period of quiescency, so that after ecdysis the differentiation
is most decided. A difference between majors and minors of the same
caste is observable at the beginning of the fourth instar. The smaller
grow but little and become adult minors. The larger increase to twice
their length and several times their bulk and become adult majors.
THE FIRST FORM OF T. ANGUSTICOLLIS
The first form has three well-defined phases of development:
a) nymphs of the first form, with long wing pads and creamy
white bodies; 6) winged adults of the first form, with long wings
and dark brown bodies, length variable, 11 to 13 mm; c) the
older males and females the post adults of the first form, with
enlarged abdomen and the ‘scales’ or bases of the shed wings,
average length 15 mm., greatest length observed 17 mm.
The nymph of the first form
According to their age, these nymphs present two different
appearances, which are so diverse that they at first seem to belong
to two different castes. The younger, immature, nymphs of the
first form—(fig. 2) 10 to 12 mm. and under, with probably two
molts to undergo—may be distinguished from the mature nymphs
—(fig. 1) 11 to 138 mm., and with only one more molt—by the
appearance of the wing pads and the size and color of the com-
pound eyes. The immature nymphs have small colorless or pale
brown eyes and thin transparent wing pads, extending back to
the third abdominal segment; the mature nymphs have larger
dark brown eyes and thick wing pads with greatly convoluted
tissue and indistinct venation, extending back likewise to the third
abdominal segment. The mesonotum and metanotum are narrow
from side to side in the mature nymphs, a character found in the
first form adult, but are broad in the younger immature nymphs.
Both immature and mature nymphs of the first form may be
distinguished from mature second-form nymphs by the slightly
smaller compound eyes of the latter and its very short wing pads,
which extend only to the first abdominal segment.
The mature nymphs of the first form are found shortly before
the period of swarming, which occurs in California from May to
CASTES OF TERMOPSIS 501
October. The body color of the mature nymphs (fig. 1) is creamy
white, except for two dark areas at the base of the clypeus and at
the tips of the mandibles. The head has almost the typical adult
form, broadest behind the eyes, and tapering forward to the
a 6
Fig. 1 Termopsis angusticollis. a, mature nymphs of the first form, with
thick wing pads, wp., surface view; 0, immature nymph of the first form, with
thin wing pads, wp., Spencer oc. 6, obj. 32 mm., stage level, reduced one-half.
clypeus. A depressed area on the frontal surface marks the
position of the ‘frontal gland,’ but no external opening, or fontanel,
is visible. ‘The large compound eyes are dark brown and slightly
reniform (fig. 2, a). Twenty-five and twenty-six antennal
segments have been observed. The wing pads are thick and
502 CAROLINE BURLING THOMPSON
opaque, slender, though rather short in comparison with the
first-form nymphs of other genera—a characteristic of this genus.
In stained whole mounts (fig. 1, a) the thickened and greatly
convoluted embryonic tissue of the future wings, w.p., is clearly
seen, the veins and tracheae in process of formation. The meso-
and metathoracic tergites are narrow from side to side as in the
adult. ‘The notable features of the legs are the five tarsal seg-
ments, a primitive character, and the pulvillus or onychium
Fig. 2 Termopsis angusticollis. a, mature nymph of the first form, lateral
view of head; b, immature nymph of the first form, lateral view of head. Spencer
oc. 6, obj. 32 mm., stage level, reduced one-third.
between the claws. The lateral tibial spines (fig. 3, b) are almost
as large as in the adult, and are variable in number, from five to
one. Female nymphs are clearly recognized from males by the
larger seventh abdominal segment which, on the ventral surface,
practically covers the eighth. Styles or genital appendices are
present on the ninth segment in both sexes. The anal cerci
consist of five segments.
Other external points of difference between the mature and
immature nymphs, besides the differences in eyes, wing pads, and
CASTES OF TERMOPSIS 503
shape of thorax already mentioned, are the relatively broader
head and the smaller lateral tibial spines of the younger nymphs.
The most interesting features of the internal anatomy are in the
nervous system—brain, eyes, and frontal gland—and in the
digestive and reproductive systems.
The brain of Termopsis has the form characteristic of the lower
termites, namely, a great extension from side to side, caused by
the size and lateral position of the optic lobes, and the slighter
q
\ ‘
)
k
A g
Ay
Fig. 3 Termopsis angusticollis, a comparison of the metathoracic legs of the
four castes. a, first-form adult; 6, mature frst-form nymph; c, immature first-
form nymph; d, second-form adult; e, f, third-form adults; g, h, adult soldiers.
Spencer oc.6, obj. 32 mm., stage level, reduced three-fifths.
development of the mushroom bodies. The smaller mushroom
bodies of lower termites with their smoother rounded surfaces
may be recognized at a glance from the relatively larger and con-
voluted bodies of a higher termite, e.g., Reticulitermes.
As in other termites, the brain of Termopsis attains its greatest
size and complexity in the mature nymph and young adult of the
first form, these two phases being practically alike in brain
structure. Figure 4, a, represents an optical section of the brain
of a first-form adult; the huge optic lobes are correlated with the
504 CAROLINE BURLING THOMPSON
large compound eyes, and in sections the cell layers and the three
fiber masses are plainly seen. The mushroom bodies, though
relatively somewhat smaller than in the higher termites, attain
their greatest size in the individuals of this caste. The smooth
A MO
—-
Fig. 4 Termopsis angusticollis. A comparison of the brains and compound
eyes of the four castes. a, first-form adult; b, second-form adult; c, third-form
adult; d, adult soldier. mb, mushroom body; ol, optic lobe; ce, compound eye.
Spencer oc. 6, obj. 32 mm., stage level, reduced two-fifths.
CASTES OF TERMOPSIS 505
rounded surfaces seen in the mushroom bodies of all the other castes
(fig. 4, b,c,d) are slightly convoluted in the first form, the outer
lobe together with part of the inner, curving upward to a level
above the inner lobe. Sections show that the three cell groups of
the mushroom bodies described in the brain of Reticulitermes,
_ Thompson (’16), are also present in Termopsis, and that similarly
all the cells are small and of equal size. The relations of the
anterior and posterior roots of the mushroom bodies are also the
same as in Reticulitermes and the other termites studied by the
writer. The protocerebral lobes are connected by a broad ven-
tral, and two slender dorsal commissures. The central body is
largest in individuals of the first form. A frontanel nerve is
present in Termopsis, homologous with the frontanel nerve de-
scribed in Reticulitermes, Thompson (’16), running from the
basement membrane of the frontal gland down to the upper sur-
face of the ventral protocerebral commissure, in the same sec-
tions with the posterior roots of the mushroom bodies. There is
nothing essentially peculiar in the other parts of the brain.
The brain in the younger first-form nymphs (fig. 1, 6) differs
from that of the mature nymphs (fig. 1, a) in the smaller optic
lobes and the smooth rounded contour of the mushroom bodies.
In this caste these characters are due to the age of the individuals,
but the same characters occur as castal differences, as will be seen
in the description of the brains of the other castes and in figure 4.
The frontal gland. The ‘frontal gland’ of Termopsis is of the
type termed by Holmgren a non-glandular ‘Fontanel-platte.’
It consists, in the first form nymph (fig. 5, fg) of a broad but shal-
low depression of modified hypodermal cells covered above by
their own cuticle and resting upon a base of connective tissue
which tapers to a point, and from which the frontanel nerve
(Thompson, ’16) takes its way downward and inward between
the mushroom bodies to the protocerebral tissue. The hypoder-
mal cells (fig. 5, hy) of the head of Termopsis are tall and slender
epithelial cells, the supporting cells distinctly columnar in form
and interspersed with occasional gland cells, but on the frontal
surface within the area of the frontal gland the height of the
supporting epithelial cells is very greatly increased, and similarly
506 CAROLINE BURLING THOMPSON
the occasional gland cells are tall and slender, but the majority
of the cells are non-glandular. The same non-glandular structure
of most of the frontal gland cells may be noted also in the adults
of Termopsis, so that, together with the absence of an external
opening or fontanel, the frontal gland of Termopsis may be said
to be non-functional and merely a primitive form of the elaborate .
sac-like frontal glands of the higher termites. To draw an
embryological comparison, the frontal glands as seen in the first
forms of Termopsis and Reticulitermes, respectively, may be
compared in form to the neural-plate and neural-tube stages of
an embryo chick.
Fig. 5 Termopsis angusticollis. First-form adult, frontal section of the
head through the frontal gland and anterior part of the brain. fg, frontal gland;
hy, hypodermal cells. Spencer oc.6, obj. 16 mm., stage level, reduced one-third.
The noteworthy features of the abdomen are in the digestive
and reproductive systems and in the fat body.
In the digestive system, four enteric caeca, or outpocketings
between the gizzard and the mid-intestine, may be noted. They
are short slender tubes, two dorsal and two ventral, and all of
equal size in the reproductive castes, but unequal, two large and
two small, in the soldier. Imms (’19) has described five such en-
teric caeca in Archotermopsis, and states, page 165, that ‘‘these
structures have not so far been detected in any other Termite.”
The number of malpighian tubules in Termopsis is eight—the
same number as in Archotermopsis.
—
CASTES OF TERMOPSIS 507
In a young immature female nymph of the first form with thin
wing pads, about 10 to 12 mm. long, the reproductive organs
present the following appearance, as seen in figure 11. The ova-
ries contain many egg tubes, consisting of numerous eggs in linear
rows, the two ovaries being connected with each other by threads
of connective tissue, the terminal filament, not shown in figure
11, as it is easily broken in dissection. A few of the proximal
ova are enlarged, showing that the growth period has begun.
The oviducts are narrow as yet, but bear at their junction a small
trilobed seminal receptacle, or spermatheca. The colleterial
gland is much convoluted, but its tubules are of narrow diameter.
The reproductive organs of a mature female nymph of the first
form with thickened wing pads, 12 to 13 mm. long, are shown in
figure 12. The largest ova are nearly three times the size of those
of the immature nymph just described; many more enlarged eggs
are present, and the oldest, or proximal ones, are surrounded by
a pellicle of small cells. The oviducts are broader, the seminal
receptacle, sr, has grown, and the colleterial gland, cl, is larger as
a whole and has tubules of greater diameter.
In male nymphs of the first form a similar correspondence be-
tween age and size of the reproductive organs may be observed in
the two phases of young immature nymphs with thin wing pads
and mature nymphs with thick wing pads. In figure 16 the
reproductive organs of a mature male nymph of the first form are
shown. ‘The testes, ¢, consist of many short rounded lobes, the
vasa deferentia are slender and open into the basal part of the
paired seminal vesicles, sv, which are greatly branched and con-
voluted, the convolution increasing with age. The seminal
vesicles after their junction with the vasa deferentia open into
the ejaculatory duct which ends in a short muscular penis. In
stained whole mounts of the male reproductive organs, which
have been dissected out from the surrounding fat-body, zones of
developing sex cells in the testes are clearly seen even with a low-
power lens.
Sections of the male reproductive organs of a mature nymph of
the first form, made by sectioning the posterior end of the abdo-
508 CAROLINE BURLING THOMPSON
men, show that these organs are closely invested by the masses of
the fat body. The testis is divided into many lobes opening at
their lower or proximal ends into the central space which leads
into the upper enlarged end of the vas deferens. The testis lobes
are enveloped by a delicate outer layer of connective tissue which
also extends as septa into the interior, dividing each lobe into
separate portions, in which lie the groups or cysts of the male sex
cells. The youngest sex cells, the spermatogonia, form a terminal
zone at the upper, distal, ends of the lobes; proximal to these may
be seen zones of cells that are evidently in the first maturation
division, but with the chromosomes so massed together that no
exact determination of their number could be made; a still more
proximal zone of smaller cells in mitosis probably represents the
second maturation division, and toward the center enlarging sper-
matids in groups of four are recognizable. In the central space
many spermatids and a few evidently nearly mature spermatozoa
are present. The vas deferens is lined by slender columnar
cells, surrounded by a thin layer of connective tissue and muscle
fibers. The tubes of the seminal vesicles are lined with an epi-
thelium of tall slender cells with clear basal nuclei and prominent
nucleoli. These cells are evidently glandular, for dark secretion
granules occur in the cytoplasm and a fluid secretion, staining
yellow with iron haematoxylin and orange G, is foundinthelumen.
Muscle fibers and connective tissue form the outer part of the
walls of the seminal vesicles, and no spermatozoa have been
observed within the vesicles.
The fat-body in the mature first-form nymph forms a nearly
solid mass between the viscera and the body wall, completely
enveloping the sex organs.
3 Stevens (’05) has made a brief study of the spermatogenesis of Termopsis
angusticollis, presumably of the first form. She states that the spermatogonial
number of chromosomes is twenty-six, and that there is one unique feature in the
development, namely, that there is only a nuclear division and no division of
the cell body of the first and second spermatocytes, so that the four spermatids
resulting from a primary spermatocyte actually develop as four nuclei within a
single cell body, and appear throughout development in groups of fours.
CASTES OF TERMOPSIS 509
The winged adult of the first form
After the last nymphal molt the individuals of this caste have a
colorless body, but rapidly assume the dark brown adult pigmen-
tation. The large black compound eyes, the dark body pigment,
and the long filmy wings are the distinguishing characters of this
phase.
The wings of T. angusticollis are dissimilar in size, the fore
wing being slightly larger; there are also slight differences in
venation and in the humeral suture in the two wings.
Comstock (718, p. 1438) states: ‘‘In Termopsis there is a com-
plete humeral suture in the fore wing, as in Mastotermes, and in
the hind wing the anal area is crossed by a suture that appears to
be the beginning of a humeral suture.’”’ The writer finds that in
some specimens of T. angusticollis the humeral suture is incom-
plete in the fore wing, extending toward the costal margin only as
far as vein R 3 of Comstock, and not reaching the anal margin.
In this respect Termopsis resembles Archotermopsis, as, according
to Imms (719, p. 99), in Archotermopsis the ‘‘basal suture” ‘‘is
frequently incomplete in that it does not always extend to the
costal margin.” The completeness or incompleteness of the
humeral suture in different individuals is evidently one more
example of the great variability already noted in the genus
Termopsis.
As in other termites, the veins of the anterior or costal part of
the wing are fully chitinized, those of the poe part are but
faintly chitinized.
After the swarming or ‘colonizing flight’ (Snyder, Banks and
Snyder, ’20) is over and mates are chosen, the wings are shed,
breaking usually behind the humeral suture in the fore wings,
and leaving the bases or ‘scales’ attached to the thorax. The
short aerial life is then ended forever and the life within the
galleries of wood is begun.
Except for the increase in the size and maturity of the sex
organs, the internal anatomy of the winged adult is very similar
to that of the mature nymph.
510 CAROLINE BURLING THOMPSON
The enlarged adults of the first form
The young deiilated first-form individuals have a dark chest-
nut-brown head and thorax, and an abdomen with bands of light
brown alternating with white, which bands become more marked
as the abdomen enlarges with age. The head is broadest between
the eyes, tapering forward to the clypeus; the frontal surface be-
tween the eyes and caudal to the brain bears a wide and shallow
depression, plainly seen in profile view of the head, made by the
sinking in of the cuticle above the large but non-functional fron-
tal gland, which, in this species, has no external opening or fonta-
nel. The compound eyes are black and large, 0.48 by 0.16 mm.,
and slightly reniform. Just behind the suture of the clypeus and
nearly in line with the bases of the antennae are two light cres-
centic areas which, in the opinion of the writer, represent vestiges
of the two lateral ocelli, present in most other lower termites,
but not hitherto described in Termopsis. The view that these
are vestigial ocelli and not merely spots or flecks in the chitin is
based upon the study of dissections and sections of the head,
and upon the similar position of the ocelli in the related genera
Kalotermes and Hodotermes. In the latter genus, Jorschke (’14,
p. 219) describes, in a similar position, in the worker of Hodo-
termes vagans, two white spots or areas which he concludes are
rudimentary ocelli.
The antennal segments are frequently broken at the tip, but
in some apparently uninjured specimens twenty-six segments
have been noted.
The meso- and metathoracic segments are narrow from side to
side with a heavy median line and bear throughout life the stubs,
‘scales,’ of the broken-off wings.
The tibiae of all three pairs of legs bear lateral as well as ter-
minal spines, which are large and serrate. The lateral spines are
arranged on different tibial surfaces in the three pairs of legs,
always being found on the outer or anterior surface of the pro-
thoracic legs, on both outer and inner surfaces of the mesothoracic
legs, and on the inner surface of the metathoracic legs. The
number of spines is very variable, from five to one, and usually
CASTES OF TERMOPSIS aa
different in the legs of a pair. Imms (’20, p. 97) notes a similar
arrangement and variability of the lateral tibial spines of Archo-
termopsis. The size of the lateral tibial spines of Termopsis
varies with age, and to some extent in the different castes, as may
be seen in figure 3. The first-form adult (fig. 3, a) has the largest
lateral spines with one margin serrate; in the mature first-form
nymph (fig. 3, b) they are slightly smaller and not serrate, and
very small in the immature first-form nymph (fig. 3, c). In the
second-form adult (fig. 3, d) the lateral spines are very small, and
and still smaller in some third-form individuals (fig. 3, e) and in
some soldiers (fig. 3, h). In other third-form individuals and in
other soldiers (fig. 3, f, g) the spines are larger, though always
smaller than those of the first-form adult, and not serrate. Five
tarsal segments are distinctly seen on the inner surface of the legs,
but the second segment is usually not seen on the outer surface.
A large pulvillus or onychium is seen between the claws. In
these two points also Termopsis agrees with Archotermopsis.
The sexes of the adults are seen with even greater clearness than
in the nymphs, as the seventh sternite of the adult female com-
pletely covers the eighth, and the styles, genital appendices,
of the ninth segment are absent in the female, but present in the
male. The anal cerci of the tenth abdominal segment are dark
brown and consist of five segments. The abdomen is more en-
larged in older females than in males, but the activity and the
relatively slight postadult enlargement of this, the largest Ameri-
can termite, cause surprise and disappointment to those first
acquainted with the greatly distended abdomens of the queens
in the smaller genera, Reticulitermes and Nasutitermes.
The internal anatomy of the sex organs and fat-body only will
be described.
In enlarged deilated females, 13 to 15 mm. long (fig. 10), the
largest eggs observed in the egg tubes were only slightly smaller
than eggs that have been laid and had a similar yolk content.
The oviducts and especially the vaginal duct are very broad and
thick walled; the seminal receptacle, sv, has attained its greatest
size; the tubules of the colleterial gland, cl, are thick and dilated
and contain a fluid secretion. In the very oldest females fewer
and shorter egg tubes are noted.
Ly CAROLINE BURLING THOMPSON
In the enlarged deilated males, 12 to 14 mm. long (fig. 15), the
testes, t, are slightly smaller, especially in the very oldest in-
dividuals, than in the mature nymphs of the first form (fig. 16).
This is due to the shrinkage of the organ as most of its component
sex cells become transformed into spermatozoa and pass out into
the vas deferens, leaving behind eventually only connective-
tissue cells. The vasa deferentia are firm though slender, with
a definite lumen and are connected with the base of the seminal
vesicles on their dorsal surface. The seminal vesicles, sv, are .
greatly branched and convoluted, each tubule being broader and
more expanded than in the mature nymph. No spermatozoa
were found within the seminal vesicles of males of any age or
caste, and the lumen is filled by a fluid secretion, staining yellow
with iron haematoxylin and orange G. This shows that the semi-
nal vesicles of Termopsis are glandular in function, and doubtless
homologous with the colleterial glands of the female.
Sections of the testes of the older males of the first form show
the central space and the proximal portions of most lobes filled
with masses of metamorphosing spermatids and mature sperma-
tozoa in tangled clumps, not in packets. In the oldest individuals
very few spermatogonia remain in the distal ends of the lobes, and
there are many empty spaces crossed only by strands of connec-
tive tissue. The tubes of the seminal vesicles have a larger lumen
and a more copious secretion than in the nymphs.
The fat-body in very old individuals is still large, but is no
longer a solid mass, and contains more empty spaces than in the
nymphs and young adults.
THE SECOND FORM OF T. ANGUSTICOLLIS
Second-form individuals of T. angusticollis, with very minute
wing vestiges (fig. 6), are at present considered rare, and speci-
mens have been recognized with certainty only in the young adult
and enlarged adult phases. The writer is inclined to believe,
however, that this caste is less rare than has been supposed, and
that it has been mistaken on the one hand for a young nymphal
phase of the first form and on the other for an adult of the third
form. Specimens frequently have one or more of the tiny wing
CASTES OF TERMOPSIS alles
vestiges broken off, the scars remaining, and such scars or mutila-
tions are very commonly found on individuals which have been
thought to belong to the third form.
The nymphs of the second form
The nymphs of the second form are not known to me with ab-
solute certainty, although one specimen in my material might
Fig. 6 Termopsis angusticollis. Second-form adult, head and thorax, sur-
face view. wv, wing vestige; m, scar or mutilation. Spencer oe. 6, obj.32 mm.,
stage level, reduced one-half.
be either a nearly mature nymph or a young adult of this caste.
This specimen is about 10.5 to 11 mm. long. The body is
creamy white, the eyes pale pink with white rims, oval and
small. The meso- and metathoracic segments are broad from side
to side and bear very small wing vestiges, the second pair extend-
ing only over part of the second abdominal segment. The in-
ternal anatomy of the second-form nymph has not been studied,
514 CAROLINE BURLING THOMPSON
but, as in the case of the first form, is probably similar to that of
the young adult described below.
The: young adult of the second form
The body length of the young adult of the second form is about
11 to 12mm. The color of the body is pale yellow with darker
chitinized areas on the clypeus and mandibles. The head is
slightly broader than that of the first-form adult and tapers less
in front of the eyes. The eyes are pinkish with a white rim,
oval and not reniform, 0.2 by 0.1 mm., slightly smaller than the
eyes of young first-form nymphs of similar length with which
Fig. 7 Termopsis angusticollis. Second-form adult with unusually short
wing vestiges, thorax, m, scar or mutilation. Spencer oc. 6, obj. 32 mm., stage
level, reduced one-third.
these young adults are sometimes confused. No fontanel is
present. Two light crescentic areas, vestiges of the lateral ocelli,
are seen on the frontal surface of the head, in a position similar to
that in first-form individuals. The meso- and metathoracic
segments are very broad from side to side, evidently a primitive
character, as it will be recalled that this condition was noted in
the younger first-form nymphs. The wing vestiges are very
short and are variable in length, in some specimens (fig. 7) ap-
pearing merely as heavy lateral borders to the thoracic segments,
in others (fig. 6) as small scale-like vestiges that extend over part
or all of the second abdominal segment. The venation of these
tiny wing vestiges is similar to that of the adult in that the main
trunks of the homologous veins are present. Very often some
CASTES OF TERMOPSIS we
wing vestiges are broken off, leaving jagged scars on the edge of
the thoracic segments.
The legs of the second-form adult (fig. 3, d) are not larger and
heavier than those of the first form, as in the genus Reticulitermes,
the tibiae indeed are shorter, and the lateral tibial spines are mark-
edly smaller, varying in number from four to one. Five tarsal
segments are present, the second segment reduced in size, and a
pulvillus, onychium, is found between the claws.
The enlarged adult of the second form
The body length of the largest second-form queen of my col-
lection is 14 mm.; the males are smaller, 10 to 11mm. The color
of the body is pale yellow and the enlarged abdomen has the
characteristic bands of yellow and white. The compound
eyes are dull pinkish with white rims, and the vestiges of the
lateral ocelli may be noted. No fontanel is present. Some of
the wing vestiges are frequently broken off, leaving Jagged scars
(fig. 6, m). The enlargement of the abdomen is as much as, or
more than, in first-form queens, and the caudal end is blunt, not
pointed. The styles, genital appendices, of the ninth abdominal
segment are present in both sexes. The anal cerci consist usually
of four segments, and rarely of five.
Internal anatomy. The brain (fig. 4, 6) is smaller than in the
first form, differing chiefly in the reduced optic lobes and in the
smaller mushroom bodies with smooth contours, lacking the
convolutions of the first form. In the few heads sectioned no
frontal gland could be detected.
The female reproductive system is evidently well developed,
but owing to scarcity of material no preparations have been made.
The male reproductive organs are intermediate in size between
those of the first and third forms. Sections of the testes show
the male sex cells in the different phases of development, but except
for size the testes of the three reproductive castes appear much
alike.
The fat-body is large and fills most of the abdominal space
but appears loose and vacuolated with age as in the other castes.
516 CAROLINE BURLING THOMPSON
THE THIRD FORM OF T. ANGUSTICOLLIS
Like the other reproductive castes, the third form of T. angusti-
collis has the three developmental phases of nymph, young adult,
and older adult, but these phases are less marked, owing to the
negative structural characters of the caste, such as the lack of
wings, the paler body pigment, the less distended abdomen of
older individuals, etc.
Like the other castes again, the third form is very variable,
the body length of mature third forms ranges from 9 to 17 mm. the
width of the head from 2.5 to 3.6 mm., the form of the abdomen
from flat to distended. The true color of the head and body is
light straw colored, or almost colorless, but the woody contents of
the intestine give the abdomen of most specimens a dark muddy
appearance. The compound eyes are small, 0.2 by 0.1 mm.,
either colorless or pale pinkish with white rims. The number of
antennal segments is twenty-six or twenty-seven. The head
is much broader than in either first or second forms. The tho-
racic segments are either wholly wingless or, in many specimens
(fig. 8, wv), with narrow marginal thickenings which may be in-
terpreted as very minute vestiges of the ancestral wing pads
common to the species—another instance of variability.
The legs are not relatively larger than in the first form, as might
be expected with the broader head and body. In some in-
dividuals (fig. 3, e) the legs are actually smaller, with slender
tibiae and tiny lateral tibial spines; in other larger individuals
(fig. 3, f) the legs slightly surpass the size of the larger first forms,
but the spines are smaller. Five tarsal segments and an ony-
chium are present as in the other castes.
The abdomen is sometimes distended, though less so, relatively,
than in the first- and second-form adults; sometimes flat, as in
the soldier. It was at first thought that the third-form individual
with flattened abdomen might be sterile, or true workers, but a
careful dissection of many flattened specimens has proved that
the collapse of the abdomen frequently comes with age, after
many eggs have been laid, and after the fat-body deteriorates,
and that the rounded abdomen is usually, although not always,
characteristic of young individuals with large fat-masses and
CASTES OF TERMOPSIS on We
2 9
Fig.8 Termopsis angusticollis. Third-form adult with minute wing vestiges,
wv, head and thorax, surface view. Spencer oc. 6, obj.32 mm., stage level, reduced
one-half.
Fig. 9 Termopsis angusticollis. Young white soldier with wing pads, wp.,
head and thorax, surface view. Spencer oc. 6, obj. 32 mm., stage level, reduced
one-half.
518 CAROLINE BURLING THOMPSON
many unlaid eggs. ‘The wingless condition and active habits of
the third form of Termopsis have led many writers to refer to it
as a ‘worker,’ or at best as a ‘worker-like’ form, but because true
workers are sterile the term should not be applied to this invari-
ably fertile caste. Females are recognizable from males by the
larger seventh abdominal sternite; the styles, genital appendices,
are present in both sexes. The pale yellow anal cerci of the tenth
abdominal segment have frequently four segments, but sometimes
five, as in the first form.
Internal anatomy. The brain (fig. 4, c) varies with the individ-
ual in size, but even in the largest specimens is smaller then that
of the first form, on account of the reduction of the optic lobes;
the mushroom bodies are large, but lack the curved surfaces of
the first form. ‘The hypodermis of the frontal surface is not
differentiated to form a frontal gland in the specimens sectioned.
The four intestinal caeca are of equal size as in the other repro-
ductive castes.
The female reproductive system of a young third-form queen is
shown in figure 13. There are practically no differences in any
of the organs from those of a young first-form adult. A nearly
mature egg from an older third-form queen is shown in figure 14.
The male reproductive system (fig. 17) shows a great increase
in size in this caste. The huge testes, ¢, consist of a multitude of
short rounded lobes; the vasa deferentia, as seen in whole mounts,
are filled with spermatozoa, sp., the tubules of the seminal vesicles,
sv, are very long, stout, and convoluted. The testes sectioned
were very young, though of large size, and most of the sex cells
were in the condition of spermatogonia and spermatocytes,
with no spermatozoa as yet developed. All of the whole mounts
of older individuals showed spermatozoa in the testes and vasa
deferentia.
The fat-body is smaller in third-form individuals than in the
other reproductive castes, not always filling the abdominal spaces.
de)
CASTES OF TERMOPSIS 51
THE SOLDIER OF T. ANGUSTICOLLIS
The adult soldier of T. angusticollis is very variable in size
and color. Banks and Snyder (’20) state that the body length of
this caste ranges from 15 to 19 mm. and the writer has collected
some specimens 25 mm. long. In the darkest individuals most of
the head is black, the remainder of the body shading backward to
a light brown abdomen. In lighter individuals the color ranges
from dark brown on the head to the whitish abdomen. The head
is greatly elongated; the compound eyes are small, 0.08 by 0.04
mm., but are larger in young soldier nymphs; crescentic areas
are present on the frontal surface of the head similar to those
found on the second and third form, but even smaller, and like
them probably representing vestigial ocelli; the fontanel is lacking
in the soldier, as in all other castes of Termopsis angusticollis.
Twenty-five antennal segments have been counted; this number,
however, is probably variable.
Although the thorax is normally wingless, in one colony, taken
at Pacific Grove, California, in May, 1919, the writer found three
young white soldiers with short wing pads (fig. 9), together with
normal wingless young soldiers. It will be recalled that Heath
(03) also found a few soldiers with wing pads in this genus.
The abdomen is elongated and round in cross-section in some
individuals, short and much flattened in others. The legs are
large and stout; the lateral tibial spines are variable in number,
five to one, and in size, some individuals (fig. 3, g) having quite
large lateral spines, those always smaller than those of the first
form, other specimens (fig. 3, h) have very minute spines; five
tarsal segments are visible, and a small onychium. The anal
cercl have either four segments, like the second form, or five, as
in the first form. Styles, genital appendices, occur in both sexes,
and the sexes are readily distinguished by the larger seventh
abdominal sternite of the female.
Internal anatomy. The brain of the soldier is smaller than
that of the first and third reproductive forms, but is as large if
not larger than that of the second form. By comparing figure
4, a, b, c, d, it will be seen that the brains of the four castes of
Termopsis differ chiefly in the size of the optic lobes; a correlation
520 CAROLINE BURLING THOMPSON .
with the different-sized compound eyes, and that the most signifi-
cant parts, the mushroom bodies, are of nearly similar bulk in
the second and third forms and in the soldier, and are only slightly
larger in the first form. The brain of the soldier of Termopsis,
slightly smaller than two reproductive castes and about equal to
another, is, therefore, when compared with the soldier brains of
higher termites, a relatively large brain. In short, this soldier
brain has not varied far from the primitive type of the genus—
the first form—and lacks the degenerate or specialized features
characteristic of the soldiers of higher termites. The interesting
correlation between the size of this soldier brain and that of the
almost sterile reproductive organs will be pointed out below.
The hypodermis of the frontal surface of the head of the soldier
of Termopsis is not differentiated into a frontal gland as in the
nymphs and adults of the first form. The specimens sectioned
were young white soldiers, but with well-developed heads covered
by a fairly thick cuticle. No sections were made of adult soldier
heads on account of the very thick cuticle.
In the abdomen, the four enteric caeca of the soldier are not
of similar size, as in the reproductive forms, but the two dorsal
‘caeca are slightly larger than the ventral pair.
The female reproductive system. The reproductive system of
the female soldier of Termopsis angusticollis presents some of the
most interesting features of this study. In long-headed but
colorless soldier nymphs, with probably only one more molt to
undergo, the ovaries are smaller than in the nymphs of any of
the reproductive castes of about similar age. The egg tubes are,
however, numerous, but the eggs are small, only three or four eggs
at the lower or distal ends of the tubes having begun to enlarge
(fig. 20). These slightly enlarged eggs are surrounded by distinct
egg follicles.
The egg tubes of each ovary are continued forward in threads
of connective tissue which unite and form a delicate strand, the
terminal filament, which in turn runs to the anterior end of the
abdomen, spreading out into a brush of fibers at its point of
attachment. The terminal filament occurs in the ovaries of all
the castes of this species, but is seen to best advantage in the
CASTES OF TERMOPSIS WAN
soldier, and is shown in figure 19, tf. The oviducts are broad
and, in some individuals, are completely fused with a small but
well-formed seminal receptacle; in other individuals the oviducts
have not grown together with the seminal receptacle. The
colleterial gland is greatly convoluted, but the individual tubules
are slender. It will be seen from the foregoing description that
many of these young soldiers seem to give promise of fertility in
the adult phase. The adult female soldier is, on the contrary,
undoubtedly infertile. The reproductive organs of a large num-
ber of adult females have been dissected out after staining and
other individuals have been sectioned. In every case there is
evidence of the arrested development of the female reproductive
organs, either of the ova alone or of the ova and the ducts. In-
stead of the well-rounded ova, regularly spaced and with large
central nuclei, that were present in the soldier nymphs (fig. 20),
we note in the adult soldiers, shrunken eggs, irregularly spaced
with respect to one another and with smaller nuclei at one end
of the cell body (fig. 21). Furthermore, many adult individuals
were found in which the three embryonic fundaments of the
oviducts, the seminal receptacle, and the colleterial gland, had
failed to unite during development (fig. 18), so that the three
parts were entirely separate, which is always the case in the sterile
soldiers and workers of Reticulitermes and Prorhinotermes where
reproduction is impossible (Knower, ’01; Thompson ’20). Two
degrees of infertility, therefore, exist among the adult female
soldiers of Termopsis angusticollis: 1) individuals in which the
oviducts, the seminal receptacle, and the colleterial gland are
fully fused, so that sexual intercourse might be possible, but whose
ova have undergone an arrest in development (fig. 19); 2) individ-
uals without fusion of the above-mentioned ducts, so that sexual
intercourse would be impossible, and in addition an arrested de-
velopment of the ova (fig. 18).
In the young female soldier with wing pads found at Pacific
Grove, the ova were as yet normal, but the three fundaments of
the reproductive system were not united, so that this specimen,
if it had lived, would have fallen into the second category of
infertile female soldiers.
522 CAROLINE BURLING THOMPSON
These observations are not in agreement with the work of
Heath (03), who has recorded the case of a Termopsis soldier.
I quote below Heath’s account, (’03, p. 58):
In colonies where either the king or queen persists the substitute
royal individual is usually, so far as I know, an immature perfect insect,
but, where both have perished the substitute royalty may sometimes
contain a worker or a nymph or even a soldier capable of laying eggs.
Such monstrous forms are not infrequent in large orphaned nests, but
never apparently in colonies headed by the true royal pair. We may
also find winged soldiers, soldiers with mandibles of varying size, and,
as just mentioned, soldiers with wing pads, the straw-color characteristic
of substitute forms and with functional reproductive organs. These
last named insects are comparatively rare. I have had but three in my
possession. All of them laid eggs in captivity and in one case I followed
the development for a long period of time, but the young and the
nymphs and workers into which they became transferred, appeared in
all respects perfectly normal.
The male reproductive system. The testes of young white soldier
nymphs are smaller than those of the adult soldiers and the lobes
are shorter and stouter. Prominent zones of dividing cells may
be noted in stained whole mounts. In sections, groups of cells
which are evidently spermatogonia are found at the tips of the
lobes, proximal to these are masses of cells in division with the
chromosomes in tetrad form. Other groups of much smaller
cells in mitosis are doubtless in the second maturation division,
and in the more central: part of some lobes spermatids develop-
ing in groups of four may be recognized. In the central space of
the testes a few apparently mature spermatozoa are present.
There are no visible signs of degeneration or arrested development
in the testes of these young soldier nymphs. The seminal vesicles
however, are not normally developed, consisting of a few very
short and slender tubules whose epithelial cells lack the height of
the homologous cells of the three reproductive castes.
In the adult soldier the testes are larger than in the first-form
adults and nymphs, although much smaller than those of the
second and third forms, but the vasa deferentia are slender and
contracted, almost without lumen, and the seminal vesicles are
vestigial in structure. Few of the testes lobes are broad and
rounded, as in the reproductive castes, but are long and usually
CASTES OF TERMOPSIS 52a
slender (fig. 22), several small lobes merging into one larger lobe,
and finally all uniting at the base of the testis into one common
space, with which the vas deferens is connected. ‘The vasa de-
ferentia are attenuated lobes with thin walls, the same is true of
the small and poorly developed tubules of the vestigial sem-
inal vesicles. The paired character of the seminal vesicles is
especially well seen in the soldier.
Sections of the testes of the adult soldier show that there is a
complete breaking down of the inner ends of the lobes into a
central chamber or space, so that the base of the testis makes one
big sac with slender shrunken lobes leading to it, the remaining
spermatogonia and spermatocytes occupying a few cysts at the
tips of the lobes. In some adults the central space is filled by
masses of developing or possibly degenerating spermatids inter-
spersed with a few spermatozoa. In other, older, individuals the
spermatozoa predominate over the spermatids. A very few swol-
len spermatids and some spermatozoa were noted in the vas
deferens of one adult soldier. No spermatozoa were found in
the seminal vesicles. The slender tubules of the seminal vesicles
of the adult soldier, as seen in sections, consist of an outer layer
of slender epithelial cells less than one-half as tall as the similar
cells of the reproductive castes, and lacking the dark staining
secretion granules that are so abundant in the latter. Only a
very small amount of yellow staining secretion (iron haematoxy-
lin and orange G) is found in the lumen of the seminal vesicle
tubules of the soldier, in contrast to the copious secretion of the
reproductive forms.
From the facts just stated, it seems evident that, although
the testes of the adult soldier produce spermatozoa that are ap-
parently normal, the lack of the secretion of the seminal vesicles
may render these spermatozoa non-functional and thus cause the
sterility of the male soldier. A detailed cytological study of the
soldier spermatozoa may prove that they lack some morphological
feature or that they are entirely normal, but the attenuated vasa
deferentia and the degenerate seminal vesicles indicate that these
parts at least of the male reproductive system are vestigial.
An examination of the gonads of the young male soldier with
524 CAROLINE BURLING THOMPSON
wing pads shows the same conditions as in the young wingless
soldier nymphs. My conclusion is, therefore, that the male
soldier of T. angusticollis, as well as the female soldier, is actually
sterile, although near the ancestral state of fertility.
Current literature affords other instances of sterility, although
spermatozoa are formed by the testis.
Boring and Pearl (718), in a study of hermaphrodite birds,
have noted a case of sperm in the testis without correlated sex
behavior. In reference to the bird known as 1426, they say:
‘This is the most interesting of the Holland birds, absolutely
indifferent as to its sex behavior and yet with sperm in the testis,
and at least one corpus luteum remnant in the ovary, and the
ovary of a laying hen.”
Safir (’20), referring to the cause of sterility in the XO males of
Drosophila, states that the XO males do not inject sperm into
the female during copulation, but that when the XO males were
dissected the testes appeared perfectly normal in shape and color,
though of smaller size. When the testes were teased open the
bundles of sperm remained compact, and when separated artifi-
cially it was found that the sperm was non-motile. After many
dissections ‘“‘it became apparent that the immediate cause of the
sterility was the non-motility of the relatively scanty sperm.”
Safir also notes that the cell bodies of the primary and secondary
spermatocytes of Drosophila often fail to divide after the nuclear
divisions, forming giant multinuclear cells, the spermatids, which,
he believes, often die and disintegrate without the formation of
spermatozoa, but, in other cases, develop into the bundles of non-
motile spermatozoa. The resemblance of this spermatogenesis
to that of Termopsis, as described by Stevens (’05), gives rise
to interesting speculations.
The fat-body of the abdomen of soldier nymphs, though copi-
ous, is far less developed than in the nymphs of the first and
second reproductive forms. In the adult soldiers the fat-body is
reduced to a thin layer beneath the skin.
CASTES OF TERMOPSIS 525
DISCUSSION
To review briefly the more significant facts stated in the pre-
ceding sections we find that the colonies of T. angusticollis
have commonly four stable types or castes of individuals, the first,
second, and third forms, fertile; and the soldier, wholly or almost
sterile, with occasional and rare deviations or variations from one
or all of the castes. We find, further, that each caste is a complex
of evidently correlated characters which, on the whole, are well
defined, although the range of variability in Termopsis is unusu-
ally great.
The first form, with long wings or stubs, has, as a rule, the
highest type of structure, so that in general there is a gradation
of structure from the first form down through the other castes.
Examples of this are: the brain, the compound eyes, the frontal
gland (present only in the first form), the wings, the size of the
lateral tibial spines, the body pigment, the anal cerci. In fewer
cases there is gradation up from the first form, e.g., the size of the
head, of the testes, and of the soldier mandibles and legs.
The genus Termopsis is remarkable for the retention of many
primitive characters, both in habits and in structure. Among
habits may be mentioned the activity of the old egg-laying queens;
the relatively strong powers of flight of the winged first forms in
comparison with other termites genera; the lack of a true nest
except for the galleries in wood; the frequent presence of several
parent first forms in a colony.
The list of primitive structures is a long one. The great size;
the large number of antennal segments, twenty-six to twenty-
seven; the hypodermal plate that forms the non-glandular
frontal gland of the first form; the vestiges of the lateral ocelli;
the slightly reniform condition of the compound eyes of the first
form, especially in the nymphal phase; the incomplete humeral
suture of the wings; the primitive type of venation; the wing
vestiges of third form and soldier; the arrangement of the lateral
tibial spines on the three pairs of legs, similar to that of Archo-
termopsis; the five tarsal segments; the very slight post-adult
growth of the abdomen of the egg-laying queens; the four enteric
caeca of all castes; the eight malpighian tubules of the digestive
JOURNAL OF MORPHOLOGY, VOL. 36, NO. 4
526 CAROLINE BURLING THOMPSON
tract; the lack of the sterile worker caste found in the higher
termites the large brain and the nearness to fertility noted in
both male and female soldiers.
Two views are held today as to the origin of these complexes
of characters that we call the castes. In Italy, the classic theory
of Grassi and Sandias (’93—’94)—that the castes are the product
of their environment, the result of special feeding and the action
of protozoa—finds an ardent advocate in Dr. Carlo Jucci. Jucci
(20-21) has made certain interesting experiments in cutting off
the wings of developing nymphs, and has analyzed the excretory
products of the different castes, and, in a preliminary note before
the Academy dei Lincei, claims to have demonstrated the exist-
ence of the particular diet by which caste production was brought
about by Professor Grassi in his experiments.
The view that termite castes are hereditary, the product of
the germ plasm, found its first support in France from Prof. E.
Bugnion (71213), who advanced strong evidence drawn from
observations on the mushroom-feeding and other termites of Cey-
lon. In England, the morphological work of Dr. A. D. Imms
(19) on the primitive genus Archotermopsis gives valuable evi-
dence which will be quoted more fully below. InAmerica,support
for this view has come from the field observations and the breed-
ing experiments of Dr. T. E. Snyder (’15, 716), from Dr. Alfred
Emerson, whose work on South American termites is still unpub-
lished, and from the writer, whose opinions are based upon the
differentiations found in newly hatched termites, and morphologi-
cal data from the adult castes. Valuable indirect evidence is
derived from the studies of Dr. C. A. Kofoid and Miss Olive
Swezy on the protozoa of the intestinal tract of termites. Kofoid
and Swezy (719) state that all the castes of T. angusticollis are
infested by protozoa, and in this connection it should be remem-
bered that Grassi’s hypothesis postulates the absence of protozoa
in the reproductive forms, as the cause of their fertility, and,
conversely, the presence of protozoa in the sterile workers and
soldiers only.
The biologist who believes that termite castes are of heredi-
tary origin will next ask the mode of origin.
CASTES OF TERMOPSIS 527
Imms (19, p. 144) writes in regard to this question as follows:
“‘T maintain that there is no satisfactory evidence conclusively
proving that any particular type of nutrition, or the absence
thereof, is capable of producing such fundamental changes in
the external and internal morphology which characterize the
soldier caste. It has also been shown that the reduction in the
gonads is not an invariable attribute of the soldier, and that caste
production is not in any way related to the presence of intestinal
protozoa.”
On pages 147 to 149 Imms (’19) discusses his view of the origin
of termite castes by mendelian inheritance from the winged sexual
forms:
I propose to consider first a typical species of Termite comprising
monomorphic soldier and monomorphic worker castes and the ordinary
winged sexual forms. Let the symbols ASF represent the various alle-
lomorphs which express themselves collectively in the winged sexual
forms; F standing for the fertility determinant, and f for the absence of
that factor. The worker mutation I would explain as having arisen
by the loss of certain correlated allelomorphs, which we represent by S
and their absence by s. Similarly, by means of a second mutation,
involving the loss of another group of characters A, the soldier caste is
accounted for; the absence of A we will represent by a. The parental
forms will have the constitution AaSsFf, but the formula may be simpli-
fied by omitting the fertility determinant, which will be considered at a
later stage, since it bears no relation to the origin of caste. Now the
cross AaSs x AaSs affords an ordinary casein which two kinds of dif-
ferentiating characters are united, and the series contains nine different
forms among sixteen individuals (Bateson, 713, pp. 355 and 345).
These may be classified as follows: )
Soldiers Winged sexual forms Workers
2 AaSS 2 AASS 2 AASs
1 aaSS 4 AaSs 1 AAss
2 aaSs 2 Aass
Sterile gametic union
1 aass
‘The above hypothesis, involving the Mendelian inheritance of two
analogous mutations, appears to offer a simple explanation of the origin
of polymorphism among Termites. It, furthermore, accounts for the
persistence of castes, which are in themselves mostly sterile, securing
their representation in the germ-plasm of the species in each succeeding
generation.
528 CAROLINE BURLING THOMPSON
Thompson and Snyder (’19), attempting to answer the question
of the mode of origin of the termite castes, suggested that the
castes might be interpreted either as a series of fluctuating varia-
tions or as mutations ‘“‘comparable to the series of mutations
found in Drosophila.’’ To-day, the writer, influenced by the
recent work of Morgan and his school, especially by their interpre-
tation of the genetic behavior of Oenothera lamarckiana, be-
lieves that termite castes should be interpreted as comparable to
the offspring of Oenothera, as arising by segregation from a
heterozygous parent form. In modern terminology, therefore,
the termite castes are not mutants, in the sense of the progeny of
Drosophila, arising once for all from a mutating parent, and then
breeding true, but are rather segregants, in the sense of the off-
spring of Oenothera lamarckiana, arising generation after genera-
tion by the splitting and recombination of the genes of a hetero-
zygous parent form. My views on this point therefore, are in
general agreement with those of Imms, except in the use of the
term mutant, which cannot to-day be applied with exactness to
the recurrent termite castes.
With another theoretical point advanced by Imms I am unable
to agree. Imms (719, p. 146) says of the wingless third form of
Archotermopsis, which he terms the ‘worker-like’ form: ‘I con-
sider that they exhibit the first step in the evolution of the worker
caste.’ . . . . ‘‘At the same time they afford a clue to the
possible origin of the worker, which appears to have arisen as a
mutation of the nymphal stage and not of the winged adult.”
(Italics mine.) The view that the wingless sterile worker is
merely a physiological phase of the wingless fertile third form,
and one a step to the other, has also tempted the writer (Thomp-
son and Snyder, ’20), but a careful study of any termite genus
with both castes gives strong evidence that the two castes are
morphologically distinct. The fertile third form of higher ter-
mites, like the first form, is probably heterozygous, and produces
among its offspring sterile workers, but we lack as yet actual
proof of this. We do know, however, that the first form gives
rise to both third forms and workers. Imms’ statement, that
workers may have arisen as mutations of the nymphal stage, and
CASTES OF TERMOPSIS 529
not of the winged adult, has a flavor of the neoteinic or ‘substitu-
tion’ idea, which seems in disharmony with his other views and
which I am unable to support.
We know also to-day from the work of Morgan and his school
that many characters are controlled by a single factor and, con-
versely, that a character may be affected by several factors. The
many characters of a termite caste are undoubtedly correlated,
and probably linked in heredity; indeed a caste, like sex, may de-
pend upon one factor. We may make hypothetical mendelian
formulae, but as yet there are no exact data from breeding to test
by the formulae. We may talk about the expected results, but
we need the actual ones. We do not know the ratio between
the number of fertile and sterile forms. We are not absolutely
sure whether the second and third forms arise from parent first
forms or from parents like themselves, nor whether each repro-
ductive type is capable of producing the sterile workers and sol-
diers. A few breeding and field observations exist; for example,
Feytand (’12) states that the first brood in a new colony consists
only of sterile forms, workers; Snyder (’15) observed in several
genera that the first brood is composed of both workers and sol-
diers, and in a definite ratio, according to the species or genus;
but more data are needed, and must be obtained to complete the
evidence for the hereditary origin of the termite castes.
SUMMARY
1. The two species angusticollis and nevadensis of the genus
Termopsis are found on the Pacific slope and in the northwestern
United States.
2. The habitat is the decaying wood of forests, very rarely in
buildings, and never in the earth.
3. Four stable castes are of common occurrence; the first form,
the second form, and the third form are the fertile reproductive
castes; among the soldiers the females are sterile, the males are
probably also sterile, though near fertility. There is no true
sterile worker caste.
4. The sexes are differentiated externally in all castes.
530 CAROLINE BURLING THOMPSON
5. Three additional variations or types are of occasional oc-
currence. These are: second and third forms with very small
wing vestiges and soldiers with wing vestiges. There is no cor-
relation between the presence of wing vestiges and fertility,
but brain size and fertility are invariably correlated.
6. A plate-like non-functional frontal gland, without fontanel,
is present in first-form nymphs and adults.
7. Vestiges of the lateral ocelli are found in all the castes,
except, possibly the third form.
8. There is great variability in all organs, and even in the de-
gree of infertility of some female soldiers.
9. Termopsis is considered a very primitive genus, on account
of its many ancestral characters and its close resemblance to the
even more primitive genus Archotermopsis.
10. The castes of termites are regarded as segregants, arising by
mendelian inheritance from a heterozygous parent form.
Wellesley, Massachusetts
July, 1921
BIBLIOGRAPHY
Banks, N., AND SnypER, T. E. 1919 Revision of Nearctic termites with notes
on biology and geographic distribution. U.S. National Museum, Bull.
108.
Borine, AticE M., anp Prarnt, RayMonpd 1918 Sex studies. XI. Hermaphro-
dite birds. Jour. Exp. Zodl., vol. 25.
Buenion, E. 1912 Observations sur les termites. Differentiation des Castes.
Comp. Rend. Soe. Biol. Paris, I, T. 72.
Comstock, J.H. 1918 The wings of insects. Ithaca, New York.
Frytaup, J. 1912 Contribution 4 l’étude du termite lucifuge. Arch. d’anat.
micros., T. 13.
FULLER, CLAUDE 1920 Studies on the post-embryonic development of the
antennae of termites. Annals Natal Museum, vol. 4.
Grassi, B., anp Sanpias, A. 1893-94 Costituzione e sviluppo ‘della societa
dei Termitidae. Atti Acad. Gioenia disci. nat., Catania.
Heatu, Harotp 1903 The habits of California termites. Biol. Bull., vol. 4.
HoitmeGreN, N. 1909 Termitenstudien 1. Anat. K. Svenska Vetensk. Akad.
Handl., Bd. 44.
1911 Termitenstudien 2. System. K. Svenska Vetensk. Akad.
Handl., Bd. 46.
Iuus, A. D. 1919 On the structure and biology of Archotermopsis, together
with descriptions of new species of intestinal Protozoa, and general
observations on the Isoptera. Phil. Trans. Royal Soc. London,
Series B. vol. 209.
CASTES OF TERMOPSIS 531
JORSCHKE, HERMANN 1914 Die Facettenaugen der Orthopteren and Termiten.
Inaug. Dissert. Leipzig.
Jucci, C. 1920-21 Sulla differenziazione delle caste nella Societa dei Termitidi.
Rendicont. R. Accad. Naz. Lincei, vols. 29, 30.
Knower, H.McE. 1894 Origin of the ‘Nasutus’ (soldier) of Eutermes. Johns
Hopkins Univ. Bull., vol. 13.
1901 A comparative study of the development of the generative tract
in termites. Johns Hopkins Hospital Bull., vol. 12, nos. 121-123.
Kororp, C. A., AnD Swezy, O. 1919 Studies on the parasites of the termites.
ItolIV. Univ. of Cal. Publ., vol. 20, nos. 1 to 4.
Lespks, C. 1856 Recherches sur l’organisation et les moeurs du Termite
lucifuge. Ann. Sci. Nat. Zool., 4e ser., T. 5.
Morean, T.H. 1919 The physical basis of heredity. Philadelphia and London.
Moututer, H. J. 1918 Genetic variability, twin hybrids and hybrids, in a case
of balanced lethal factors. Genetics, vol. 111.
Sarin, 8. R. 1920 Genetic and cytological examination of the phenomena of
primary non-disjunction in Drosophila melanogaster.
Snyper, T.E. 1915 Biology of the termites of the eastern United States, with
preventive and remedial measures. U. 8. Dept. Agric., Bur. Ent.,
Bull. no. 94, pt. II.
Stevens, N. M. 1905 Studies in spermatogenesis with especial reference to
the ‘accessory chromosome.’ Carnegie Institution of Washington,
Publ. 36.
Tompson, C. B. 1916 The brain and the frontal gland of the castes of the
‘white ant,’ Leucotermes flavipes Kollar. Jour. Comp. Neur., vol. 26.
1917 Origin of the castes of the common termite, Leucotermes flavipes
Kol. Jour. Morph., vol. 30.
1919 The development of the castes of nine genera and thirteen species
of termites. Biol. Bull., vol. 36.
Tuompson, C. B., anp SnypER, T. E. 1919 The question of the phylogenetic
origin of termite castes. Biol. Bull., vol. 36.
1920 The ‘third form,’ the wingless reproductive type of termites:
Reticulitermes and Prorhinotermes. Jour. Morph., vol. 34.
DESCRIPTION OF PLATES
ABBREVIATIONS
cel, colleterial gland sv, seminal vesicle
ov, oviduct t, testis
sp, spermatozoa if, terminal filament
sr, seminal receptacle
PLATE 1
EXPLANATION OF FIGURES
All figures are drawn from semitransparent whole mounts of dissections of
Termopsis angusticollis. Spencer oc. 6, obj. 16 mm., stage level.
10 Female reproductive organs, first-form adult.
11 Female reproductive organs, immature first-form nymph.
12 Female reproductive organs, mature first-form nymph.
13 Female reproductive organs, third-form adult.
14 Nearly mature egg, third form.
532
CASTES OF TERMOPSIS PLATE 1
CAROLINE BURLING THOMPSON
533
PLATE 2
EXPLANATION OF FIGURES
All figures are drawn from semitransparent whole mounts of dissections of
Termopsis angusticollis. Figures 15 to 19 and 22, Spencer oc. 6, obj. 32 mm., stage
level.
Figures 20 and 21, Spencer oc. 6, obj. 16 mm., stage level.
Male reproductive organs, first-form adult.
Male reproductive organs, mature first-form nymph.
Male reproductive organs, third-form adult.
Female reproductive organs, adult soldier.
Male reproductive organs, adult soldier.
Ova from the proximal end of an egg tube, soldier nymph.
Ova from the proximal end of an egg tube, adult soldier.
Female reproductive organs, adult soldier.
Or
w
eg
PLATE 2
CASTES OF TERMOPSIS
CAROLINE BURLING THOMPSON
Ou
535
Resumen por el autor, Dean L. Gamble.
La morfologia de las costillas y procesos transversos de Nec-
turus maculatus.
En la mayor parte de los urodelos la cabeza ventral o capitu-
lar de la costilla se inserta sobre un proceso del arco neural,
dorsalmente a la arteria vertebral. En Necturus, lo mismo que
en los elasmobranquios, la cabeza ventral de la costilla se in-
serta, en todas las vértebras, excepto en las dos o tres primeras,
en el nédulo basal, ventralmente a la arteria vertebral. En la
poreién extrema anterior de la columna vertebral, el tabique
horizontal y las costillas estan opuestas a la base del arco neural.
En esta regién cada costilla se inserta en un proceso del arco
neural (el ‘‘portador de la costilla’’? de Goeppert) y no presenta
connexién alguna con el nédulo basal. Esta prueba presta apovo
a, la explicacién de Rabl sobre la insercién de las costillas de los
urodelos, la cual es opuesta a la explicacién mas generalmente
aceptada de Goeppert. Las costillas de la segunda y tercera
vértebras de Necturus se insertan sobre la columna vertebral
de un modo exactamente semejante al que se observa en los
otros urodelos. Ademas, los bastones dorsales y ventrales de
los procesos transversos en las primeras vértebras de Necturus
son homdlogos de semejantes estructuras en otros urodelos.
El] nédulo primitivo basal de Necturus, (el elemento basi-ventral
de Gadow) desarrolla un proceso lateral, la parapOfisis, y una
hemapOfisis ventral. En la regién del tronco, en un estado mds
joven, ambas existen. En estados ulteriores, en la regién del
tronco, la parapdfisis persiste y se une con la cabeza ventral
de la costilla, mientras que la hemap6fisis desaparece. En
la regién de la cola la hemapOfisis se alarga para formar el arco
hemal y la parap6fisis desaparece. En este respecto Necturus
corresponde casi exactamente a Polypterus.
Translation by José F. Nonidez
Cornell Medical College, New York
AUTHOR'S ABSTRACT OF THIS PAPER ISSUED
BY THE BIBLIOGRAPHIC SERVICE, JULY 17
THE MORPHOLOGY OF THE RIBS AND TRANSVERSE
PROCESSES IN NECTURUS MACULATUS!
D. L. GAMBLE
THIRTY-ONE FIGURES
A summary of the literature upon the transverse processes
and their relations to the ribs and haemal arches in the urodeles
results in contradictions with regard to the morphology of these
parts. An attempt to verify statements by a study of the
development of the parts concerned has resulted in the present
communication. Necturus was chosen as the type, partly for
convenience and partly because it was believed that it might
exhibit conditions primitive enough to serve as a basis for
interpreting the morphology of these structures in urodeles
generally.
For a clear conception of the problem, the following observa-
tions are pertinent: the ribs of urodeles exhibit several differences
when compared with the dorsal fish ribs. In elasmobranchs the
ribs are connected with the centrum by means of the basal
stump, which is located ventrad of the vertebral artery. In
most urodeles, however, the ventral head of the rib is attached
to a process of the neural arch above the vertebral artery. If
this latter attachment were brought about by the simple dorsal
shifting of the rib and basal stump, the relations of the vertebral
artery in regard to these structures would be unchanged.
Two interpretations of this condition obtain at present. One
is that, due tothe dorsal shifting of the horizontal septum and with
it the rib, the latter structure has simply lost its connection with
the basal stump and has become attached to the neural arch
(Rabl, ’92).
1 This problem was suggested by, and carried on under the direction of Dr. H.
D. Reed. The writer is not only indebted to him for his help and advice through-
out the work, but also for the use of his splendid series of Necturus larvae.
537
538 D. L. GAMBLE
The interpretation most generally accepted is that of Goeppert
(96), who attempted to show that the attachment of the rib to
the neural arch has come about in a complicated fashion. Goep-
pert was an advocate of the view that the rib is an outgrowth of
the basal stump, while Rabl thought of the same structure as an
independent element, having nothing to do genetically with the
vertebra. Goeppert therefore offered another explanation to
account for the new rib attachment, which was not incompatible
with his belief in the morphological relations of the two structures.
The following is a brief summary of Goeppert’s work on this
problem. In Necturus, the basal stump, at about the middle
of its length, sends off a dorsal process which he called the ‘rib-
Fig. 1 A series of diagrams to illustrate Goeppert’s interpretation of the
change in the vertebral attachment of the rib in urodeles. Black, rib-bearer;
stippled, parapophysis (basal stump of Goeppert); clear, rib; b, basal portion of
basal stump; b-d, distal portion of same; n.a., neural arch; n.c., notochord.
A, Necturus; B, Salamandra; C, Triton.
bearer’ (fig. 1A, black). This passes dorsally to the neural arch,
laterad of the vertebral artery, and then continues dorsocaudally
over the surface of the arch. The rib-bearer is separated from
the cartilage of the neural arch by a sheath of connective tissue
and later bone. Laterad of the rib-bearer the basal stump (fig.
1A, stippled) continues horizontally, and the rib (fig. 1A, clear)
forms as a mere prolongation of this element. Further laterad
still the rib develops a dorsal process which extends mesally
toward the vertebra and becomes the dorsal head of the rib.
In the larval Salamandra, according to Goeppert, the basal
portion of the basal stump has disappeared, and its place is taken
by a slender bar of bone (fig. 1B, 6). The distal end of the basal
stump (fig. 1B, b-d), that is, the portion which projects laterad
THE TRANSVERSE PROCESSES OF NECTURUS 539
of the rib-bearer in Necturus, is much shortened and is indis-
tinguishable from the rib-bearer. Now, the rib is attached
directly to the neural arch, although it has never actually lost its
connection with the distal end of the basal stump. According
to this interpretation of the dorsal shifting of the rib attachment,
the rib-bearer is substituted for the proximal portion of the basal
stump in attaching the rib to the vertebral column.
In view of these conflicting opinions regarding the independence
of the rib and the way in which its attachment changes, and
because of the possibility that the two stages (24 and 43 mm.)
studied by Goeppert failed to furnish all the facts, it was deemed
advisable to undertake a study of the development of the ribs
and transverse processes in a more complete series. Accord-
ingly, Necturus larvae of the following stages were studied:
20, 21, 22, 23, 24, 25, 26, 30, 40, 50, and 70 mm., respectively,
and in addition various stages of other species.
After a study of these stages, it is believed that the dorsal
shifting of the ribs in urodeles is not the complicated process that
Goeppert supposed and that all the steps in this change in attach-
ment can be illustrated in the single form, Necturus.
In discussing the development of these structures the follow-
ing order will be followed: 1) Fourth to antepenultimate trunk
vertebrae. 2) First three vertebrae. 3) Definitive estate of trunk
vertebrae. 4) Last trunk and first and second tail vertebrae.
DEVELOPMENT OF TRUNK VERTEBRAE (FOURTH TO
ANTEPENULTIMATE)
In the 20-mm. larva (fig. 2) the only cartilage present is that
of the neural arch which is not yet complete dorsally. The
neural arches of consecutive vertebrae are separated by quite
an interval. The sheath of the notochord is made up of an outer
skeletogenous layer, the elastica externa, within which is a thinner
layer, the elastica interna, while the outer cells of the notochord
are arranged in an epithelial-like layer, the notochordal epithe-
lium. In the middle of the future centrum the notochord sheath
is thin and all three layers are close together. Intervertebrally
the sheath is much thicker, due to the great increase in the num-
540 D. L. GAMBLE
THE TRANSVERSE PROCESSES OF NECTURUS 541
ber of cells between the elastica externa and interna. Between
the notochord and lateral muscle mass is found loose mesenchy-
mal tissue, in which at this stage a condensation is appearing
laterally, which extends outward into the horizontal septum
marking the position of the future rib (fig. 2, 7.p.) and also up-
ward to the neural arch along the inner edge of the transverse
septum in the position of the future rib-bearer (fig. 2, r.b.p.).
In tracing this condensation mesally toward the notochord, it
will be seen, in the greater number of vertebrae, to become much
weaker and gradually dissolve into what appears to be typical
mesenchymal tissue. In some vertebrae the basal stump is
indicated by a few slightly modified mesenchymal cells which
line up between the notochord and the rib proton? (fig. 2, p.a.p.).
In either case the greatest condensation of mesenchyme appears
laterally at the inner margin of the lateral muscles, and weakens
toward the notochord.
Goeppert’s belief in the rib as a lateral outgrowth of the basal
stump and the rib-bearer as a dorsal outgrowth of the same
element were based apparently upon the mistaken observation
that these structures are continuous in the proton stage. The
examination of a number of vertebrae at this stage of development
shows that there is a difference in the time of appearance of the
protons of these structures. The rib and rib-bearer protons
are well marked out in many of the vertebrae before the proton
Fig. 2 Transection through the fourth vertebra of a 20-mm. larva. n.a.,
neural arch; n.c., notochord; p.a.p., proton of parapophysis; r.b.p., rib-bearer
proton; r.p., rib eas v.d. steal artery.
Fig. 3 Transection aheaaee the fourth vertebra of a ee larva slightly
more advanced in development than that shown in figure 2. n.a., neural arch;
p.a., parapophysis; r.b.p., rib-bearer proton; r.p., rib proton.
Fig. 4 Transection through the fifth vertebra of a 2l-mm. larva. h.a.,
haemapophysis; n.a., neural arch; p.a., parapophysis; r.b.p., rib-bearer proton;
r.p., rib proton.
Fig. 5 Transection through the eighth vertebra of a 21-mm. larva. h.a.,
haemapopbysis; n.a., neural arch; p.a., parapophysis; r.b.p., rib-bearer proton;
r.p., rib proton.
2 The term ‘proton,’ introduced by Prof. B. G. Wilder, is used in the present
communication in preference to the word ‘anlage.’ It has the same meaning and
has the added advantage of being an English term.
542 D. L. GAMBLE
of the basal stump appears. When it does form it has a dis-
tinctly different appearance; that is, it is weaker and made up
of fewer cells and stains much less intensely with haematoxylin.
This difference between the proton of the rib and rib-bearer on
one hand and that of the basal stump on the other, points to the
former as elements formed in the muscle septa and seems to
indicate that the basal stump only is a lateral product of a verte-
bral element.
In a slightly more advanced larva which also measured 20 mm.
the basal stump makes its first appearance (fig. 3, p.a.). It is
seen here as a cartilaginous process projecting laterally from the
notochord. ‘This cartilage in the fourth trunk vertebra does not
lie in a horizontal plane, but projects slightly dorsad as well as
laterad.
At its tip the hyaline matrix disappears and it becomes con-
tinuous with a strand of procartilage cells (the proton of the rib-
bearer) which extends upward to the neural arch. At a level
with the base of the neural arch this strand of cells is also con-
tinuous with the rib proton (fig. 3, r.p.) which passes into the
horizontal septum. Farther back in the trunk region the prox-
imal end of the rib proton appears on a level with the middle
of the notochord and here the basal stump lies horizontally
(fig. 4, p.a.) and is directly continuous with the rib proton (fig. 4,
r.p.). When this is the case the distal end of the basal stump is
connected with the neural arch by a strand of procartilage cells
as in the anterior region (fig. 4, 7.b.p.).
In the 21-mm. larva the neural arch is completed dorsally,
but the basal stump appears no further advanced than in the
20-mm. larva. The first rib chondrification is to be noticed in
the anterior trunk region of the 21-mm. larva. This first cartilage
is developed distally, and between it and the cartilage of the
basal stump procartilage is found.
On the ventral side of the notochord just ventrad of the base
of the basal stump are found in many of the trunk vertebrae
small knobs of cartilage (fig. 4, h.a.). Occasionally two are found
in a Single vertebra, one lying on either side of the aorta. Gen-
erally, however, only one is present. The hyaline matrix of
THE TRANSVERSE PROCESSES OF NECTURUS 543
these ventral cartilages stains as deeply as that of the neural
arch, the lacunae are large and contain large cells. This is in
sharp contrast to the matrix of the lateral cartilage (‘basal stump’
of Goeppert) which stains very faintly and gives every indication
of having been formed more recently. .
In the 5th, 6th, 7th, 8th, 11th, and 12th vertebrae the ventral
cartilages are entirely independent of the lateral cartilages (fig. 4)
but in the 9th, 18th, 14th, 16th, and 17th they are joined with
the bases of the lateral cartilages, from the proximal ends of
which they appear as ventral outgrowths (fig. 5, h.a.). These
elements are absent in the remaining trunk vertebrae, but in
several of these there is to be noted a slight tendency of the base
of the lateral cartilage to bulge downward. This possibly
locates the position of the ventral cartilage. It is seen that
anteriorly the greater number of the vertebrae possess separate
ventral elements, but that posteriorly the ventral and lateral
cartilages are joined. In several the ventral elements have lost
their identity entirely.
These ventral knobs of cartilage were not observed by Goeppert
in Necturus, but he saw them in Salamandra (’96). Ventrad of
the slender bar of bone (fig. 1, 6), which he interpreted as the
reduced basal stump, ventral cartilages were located which dis-
appeared at the onset of bone formation. Goeppert observes:
‘“There occurs in Salamandra an unhitherto described peculiarity.
On the ventral side of the notochord, cartilage elements are found
belonging to the haemal arch system. ‘These are present in
the young animal. In the tail region they elongate to form the
haemal arches. There is no connection between the bony strand
of the rib-bearer and these elements. This separation must be
considered as a secondary condition.” The way in which
Goeppert believes this separation comes about will be described
when the vertebrae of the tail-trunk region of Necturus are
discussed.
In a 30-mm. Polypterus larva Budgett (’01) found a meta-
meric series of cartilages resting upon the notochordal sheath.
There was a dorsal row, the bases of the neural arch, a lateral
row, forming the foundation of the transverse processes and ribs
544 Dic Li GAMBEE
and a still smaller series of ventral cartilages forming the founda-
tions of the ventral ribs. He found all three series well developed
in the anterior region. The lateral series were developed out
into the horizontal septum as long processes, but at that stage
had no connection with the ribs which he saw forming inde-
pendently in the lateral portion of the horizontal septum, but
having no connection with the lateral cartilages. The ventral
series were also well developed and passed out between the ventro-
lateral muscles and the kidneys, never reaching, however, the
peritoneum at this stage. In the caudal region the lateral
series was not found and the ventral series had elongated to form
the haemal arches.
Budgett stated in conclusion: ‘‘In the possession of three
pairs of vertebrally placed cartilages resting upon the noto-
chordal sheath, before the commencement of bone formation,
Polypterus differs from all living vertebrates. ”’
It is interesting to note that in the possession of these cartilages
the 21-mm. Necturus larva at this stage corresponds very closely
to Polypterus. Furthermore, cartilage is appearing in the rib
distally, apparently as it does in Polypterus. The difference
between the two forms lies in the fact that in Necturus the ventral
cartilages, in some of the trunk vertebrae, are joined with the
bases of the lateral cartilage and in the posterior trunk region
become more or less indistinguishable from them. In the tail
region this basal portion which represents ventral cartilage grows
down to form the haemal arch while in Polypterus the haemal
arch is formed by the ventral cartilages which have remained in-
dependent of the lateral cartilages throughout the trunk region.
In Polypterus the ventral cartilages are present throughout the
life of the individual and elongate to form the ventral ribs, while
in Necturus they disappear when bone formation begins.
Salamandra, in so far as the ventral cartilages are concerned,
‘ also corresponds closely to Polypterus. Here at an early stage
they are found in the trunk region and in the tail form the haemal
arch. Salamandra differs from Polypterus, however, in the loss
of the lateral cartilage.
THE TRANSVERSE PROCESSES OF NECTURUS 545
The ventral cartilages were first seen in a 20-mm. Necturus
larva (fig. 6, h.a.). Here a lateral outgrowth of the ventral
cartilages is just beginning to appear (p.a.). Although these
two processes are continuous, it is very evident that the cartilage
of the ventral element is better developed and that the cells of
the lateral process have just begun to secrete a hyaline matrix.
This shows clearly that the primitive basal stump is growing in
two directions, ventrally giving rise to a haemapophysis and
laterally becoming the parapophysis. When bone forms in the
skeletogenous layer of the notochord the haemapophyses of the
trunk disappear. From this point on the terms of Owen will be
used while discussing these ‘derivatives’ of the basal stump.
The lateral cartilage will be called the parapophysis and the
ventral, the haemapophysis.
In the 22-mm. larva the cartilage of the rib has developed
mesally so that the area of procartilage between it and the para-
pophysis is much less extensive. Caudad of the third vertebra
the ventral cartilages or haemapophyses do not appear, the
reason for this being apparently that bone is beginning to make
its appearance in the outer layer of the notochordal sheath.
In this stage (fig. 7) in the fourth and fifth vertebrae the para-
pophysis makes a cartilaginous connection with the rib-bearer
for the first time. Because of the fact that in these vertebrae the
basal stump tends to project dorsolaterally, no distinction can
be made between it and the rib-bearer. In passing caudally,
however, it will be seen that as the parapophysis comes to lie in
the horizontal plane, the cartilage of the rib-bearer disappears
and in its place appears a strand of procartilage cells (fig. 8).
In the 24-mm. larva structures are considerably more advanced.
The dorsal portion of the neural arch has begun to develop
caudally into a median posterior articular process and cephalad
into two anterior articular processes, one on either side of the
middorsal line. The parapophysis in the midtrunk region
projects laterally and in the strand of cells which connects it
and the neural arch in the preceding stage, cartilage appears
(fig W720.)
546 D. L. GAMBLE
THE TRANSVERSE PROCESSES OF NECTURUS 547
Following the law of cephalocaudal growth and differentiation,
structures nearer the anterior end of the body have differentiated
further than those which are located more caudally, so that in
passing backward through the trunk region successively younger
stages in the formation of ribs and transverse processes are
encountered.
This law applies to all the trunk vertebrae except the first
three. Here, as will be discussed later, the development of the
various parts of the transverse process and ribs seems to lag
behind the trunk vertebrae immediately caudad. This condition
may be interpreted as the beginning of a process which in higher
forms leads to a reduction of ribs in the cervical region.
Beginning with the fourth vertebra, the rib-bearer is seen
connecting the neural arch and the distal end of the para-
pophysis, while farther caudad this connection has not yet been
made and the rib-bearer appears as a knob of cartilage attached
to the neural arch (fig. 10). Still farther caudad the cartilage
of the rib-bearer has not yet made its appearance.
In the 43-mm. larva studied by Goeppert this cartilage of the
rib-bearer next to the neural arch is found, but he concluded that
the lack of continuity between it and the basal stump was due
to a secondary degeneration of the cartilage. When intermediate
stages are studied, however, it is shown very clearly that cartilage
between the two may never have been formed. The study of
older stages shows that in the greater number of the trunk
vertebrae this connection is finally made, but that in a few in
which development is arrested the two are separated. Insuch an
event the result is that of the relations seen by Goeppert and
interpreted by him as a secondary degeneration.
Fig. 6 Transection through the second vertebra of a 20-mm. larva. n.a.,
neural arch; h.a., haemapophysis; ».a., parapophysis; 7.b.p., rib-bearer proton.
Fig. 7 Transection through the fourth vertebra of a 23-mm. larva. a.a.p.,
anterior articular processes; ”.a., neural arch; r.b., rib-bearer; p.a., parapophysis.
Fig. 8 Transection through a midtrunk vertebra of a 23-mm. larva. n.a.,
neural arch;7.b.p., rib-bearer proton; r.p., rib proton; p.a., parapophysis.
Fig.9 Transection through the sixth vertebra of a 24-mm. larva. n.a.,neural
arch; r.b., rib-bearer; r.p., rib proton; p.a., parapophysis.
Fig. 10 Transection through a trunk vertebra of a 25-mm. larva. r., rib;
n.a., neural arch; 7.b., rib-bearer; p.a., parapophysis. ,
548 D. L. GAMBLE
The two processes which were seen just beginning to develop
forward from the dorsal portion of the neural arch in the 24-mm.
stage in the 26-mm. larva have continued their cephalic growth
until they meet the process developing caudad from the pre-
ceding vertebra. These will form the articular processes of the
definitive vertebra. The transverse process is in about the same
condition except that the rib-bearer has developed dorsocaudally
over the outer surface of the neural arch. In other words,
cartilage is forming in the inner edge of the transverse septum
along the line of its attachment to the neural arch.
The cartilage of the rib now extends inward to the distal end
of the parapophysis, but procartilage still persists between these
two elements. After the fusion of the rib-bearer and the para-
pophysis, a lateral extension of the transverse process takes place
distad of this point. As the animal grows the rib attachment
must be shifted laterally to bring it into its final position. This
means that procartilage cells must persist between the trans-
verse process and the rib, so that proliferation of cartilage cells
may take place and the parapophysis grow laterally. It was in
a larva of about this age that Goeppert saw the rib-bearer ap-
pearing as a dorsal outgrowth from about the middle of the basal
stump. The study of younger stages shows that this is not the
case, but that the rib-bearer grows downward and fuses with the
distal end of the parapophysis and that the ventral head of the
rib is borne upon an extension of the parapophysis which develops
laterally after the rib-bearer has united with it.
The rib gives off a dorsal process which becomes the tubercular
head. This extends dorsomesally toward the neural arch. In
the trunk region it never connects with a corresponding process
of the rib-bearer, but in the anterior region this does occur.
This will be discussed more in detail later.
DEVELOPMENT OF ANTERIOR TRUNK VERTEBRAE
As before stated, Goeppert’s conclusions concerning the dorsal
shifting of the rib attachment in urodeles were based upon a
comparison of the conditions found in Necturus, Salamandra, and
Triton. After the study of a complete series of Necturus larvae,
THE TRANSVERSE PROCESSES OF NECTURUS 549
it is believed by the writer that all of the steps in this shifting of
attachment can be illustrated by this one form alone. In
Necturus the horizontal septum is relatively high in the extreme
anterior region of the trunk and is lower posteriorly, and there-
fore might be looked to with a reasonable amount of assurance,
to show how the rib has become attached to the neural arch.
This has already been suggested by Wilder (’03) in his memoir
on the skeletal system of Necturus. Wilder makes the following
statement in this connection: ‘‘It would thus seem, judging from
the purely anatomical evidence, that the condition described by
Goeppert as characteristic of Necturus, is not a universal one
applicable to all the vertebrae, but is restricted to a certain
region approximately that of vertebrae 8 to 18.”
“Tt would seem important to investigate the development of
the transverse process and rib in certain of the other vertebrae,
for example the second and the fourth.”
It was found in the present study that Wilder’s suggestion
was a good one and that the second, third, and fourth vertebrae
are important in making intelligible the morphology of these
structures.
The first vertebra never bears a definitive rib, although a
cartilaginous rib rudiment was found in this vertebra in a 23-mm.
larva (fig. 14). The first vertebra has one peculiarity, however,
which distinguishes it from all the other vertebrae. The bases
of the neural arch of the first vertebra are enlarged and extend
much farther ventrad than in others. These enlargements seem
to represent basal stumps which have fused with the bases of the
neural arch (fig. 11). Continuity of neural arch and _ basal
stump was seen in none of the other vertebrae with a single excep-
tion (fig. 12). In a 21-mm. larva in the second vertebra the
neural arch extended ventrally exactly as in the first. In follow-
ing back through the sections, however, it was seen that the
ventrally projecting end of the neural arch extended caudad so
that a section which passed through the vertebral column just
caudad of the neural arch would cut through these caudal pro-
jections. Here, in cross-section, they appear as typical basal
stumps before the appearance of any lateral cartilage (fig. 13, b.s.).
550 D. L. GAMBLE
THE TRANSVERSE PROCESSES OF NECTURUS 5a
Extending downward and inward from the neural arch is the
strand of procartilage cells in which will later form the cartilage
of the rib-bearer. It is very noticeable here that this condensa-
tion of cells weakens markedly and finally disappears in passing
toward the notochord. It is further seen that a mesenchymal
condensation has appeared all along the inner edge of the trans-
verse septum where it attaches to the neural arch. This
represents the dorsal continuation of the rib-bearer proton.
Cartilage appears at this stage in the extreme distal portion of
the rib.
In the third vertebra a broad cartilage appears on the ventral
side of the notochord on one side only, while on neither side is
there any suggestion of a lateral cartilage. This cartilage is to
be interpreted as basal stump before the development of the
lateral cartilage (parapophysis). As in the preceding vertebra,
the procartilage cells are seen in the inner edge of the transverse
septum, where it is attached to the neural arch and also between
the neural arch and notochord. Here again is noticed the decided
weakening of this proton mesally. The beginning of the rib pro-
ton is on a level with the base of the neural arch.
In the second vertebra of a 22-mm. larva (fig. 15) the basal
stump is very broad at the base. The direction of growth is
dorsolateral (p.a.), this process dominating the development of
the ventral projection of the basal stump (haemapophysis) which
appears as a single cartilage cell much more advanced than the
Fig. 11 Transection through the first vertebra of a 20-mm. larva. b.s.,
basal stump; 7.a., neural arch.
Fig. 12 Transection through the anterior part of the second vertebra of a
21-mm. larva. n.a., neural arch; b.s., basal stump.
Fig. 13 Transection through the posterior part of same vertebra. n.a.,
neural arch; b.s., basal stump.
Fig. 14 Transection through the vertebral column between the first and
second vertebrae. e.m., epaxial muscle mass; e.7., elastica interna; h.m., hy-
paxial muscle mass; 7.r., intervertebral ring; /.l.a., lateral-line artery; 1.l.n , lat-
eral-line nerve; .c., notochord; r., rib; r.a., right aorta.
Fig. 15 Transection through the second vertebra of a 22-mm. larva. n.a.,
neural arch; p.a., parapophysis; h.a., haemapophysis; b.s., basal stump.
Fig. 16 A more caudal transection of the same vertebra. n.a., neural arch;
r.b., rib-bearer; p.a., parapophysis.
GAMBLE
Darke
552
“fy
\28
Oran,
THE TRANSVERSE PROCESSES OF NECTURUS ada
surrounding cartilage (fig. 15, h.a.). Cartilage has developed
in the inner edge of the transverse septum along its line of attach-
ment to the neural arch, and this extends downward and inward
toward the upwardly developing parapophysis, from which it is
separated by mesenchymal cells (fig. 16, 7.b.).
In the next (third) vertebra, cartilage has not formed all along
the line of attachment of the transverse septum to the neural
arch, but is found only at its ventral end (fig. 17, 7.b.). In other
words, the rib-bearer appears as a knob of cartilage projecting
ventrolaterally from the neural arch, while the parapophysis
extends dorsolaterally toward it, the two being separated by a
considerable interval. The proton of the rib is continuous with
the cartilage attached to the neural arch (rib-bearer) and has
no connection with the lateral cartilage (parapophysis) (fig. 17).
It will have been noted by this time that the second and third
vertebrae differ in several ways from those farther back in the
trunk. These differences can be summed up as follows:
1. Developmental processes seem to be retarded in this
region. The parapophyses and the rib-bearer unite much later,
the cartilage of the rib develops mesally more slowly, and the
haemapophyses persist longer than in the midtrunk region.
As before mentioned, this relative slowing up of the development
of these structures may be an expression of the reduction of ribs
in the cervical region of higher vertebrates.
2. The parapophyses do not lie in a horizontal plane, but
project dorsolaterally.
Fig. 17. Transection through the third vertebra of a 22-mm, larva. n.a.,
neural arch; r.b., rib-bearer; p.a., parapophysis.
Fig. 18 Transection through the second vertebra of a 24-mm. larva. n.a.,
neural arch; r.b., rib-bearer; p.a., parapophysis; h.a., haemapophysis; b.s.,
basal stump.
Fig. 19 Transection through the third vertebra of a 24-mm. larva. n.c.,
notochord; n.a., neural arch; 7.b., rib-bearer; p.a., parapophysis.
Fig. 20 Transection through the fourth vertebra of a 24-mm. larva. n.a.,
neural arch; r.b., rib-bearer; r., rib; p.a., parapophysis.
Fig. 21 Transection through the second vertebra of a 25-mm. larva. n.a.,
neural arch; r.b., rib-bearer; r., rib; p.a., parapophysis.
Fig. 22 Transection through a trunk vertebra of Amblystoma. 1.4., neural
arch; r., rib; 7r.b., rib-bearer,
554 D. L. GAMBLE
3. The rib-bearer and the parapophysis approach end to end,
while in the trunk region they meet at an angle of 90°.
4. The rib attaches to the rib-bearer and has no connnection
with the parapophysis.
On one side of the second vertebra of the 24-mm. larva ventral
and lateral cartilages are both present and continuous proximally
(fig. 18, p.a., h.a.). On the other side lateral cartilage only is
present, the haemapophysis (h.a.) having disappeared at the
onset of bone formation. On the side where both elements are
present, the basal stump appears as a forked structure in which
growth is taking place in two directions dorsolaterally and
ventrally. The lateral outgrowth does not quite meet the carti-
lage of the rib-bearer, the two being separated by mesenchyme.
The rib proton is continuous with the rib-bearer. On the other
side the connection between the parapophysis and rib-bearer is
almost made, the two being separated by a very narrow zone of
procartilage.
In the third vertebra the parapophysis is still unconnected
with the rib-bearer, and the rib proton extends laterally from it
(fig. 19, p.a.). The cartilage of the rib has developed mesally
and is separated from the rib-bearer by a limited zone of procar-
tilage cells.
The fourth vertebra of this larva is very important in eluci-
dating the morphology of the rib-bearer, basal stump, and rib
(fig. 20). On the left side the basal stump projects laterally from
the notochord and is continuous distally with the rib-bearer, as
in the stages previously described in the trunk region. The rib
is borne, also as in the trunk region, at the point of union of
these two elements (r). On the right side the parapophysis was
tardy in development as compared with the left, and is seen pro-
jecting laterally a very short distance and making no connection
with the rib-bearer whatever. The rib is borne by the rib-bearer
and has no connection with the basal stump.
This condition cannot be explained on the basis of Goeppert’s
interpretation of the morphology of these structures (fig. 1).
Here the development of the parapophysis has lagged behind
that of the rib-bearer and the rib has become attached to the
THE TRANSVERSE PROCESSES OF NECTURUS 955
latter before there is the slightest connection between the para-
pophysis and the rib-bearer. Three points are thereby made
clear:
1. The rib-bearer is not an upgrowth of the parapophysis.
2. The distal end of the parapophysis (basal stump of Goep-
pert) does lose its attachment with the rib and does not become
attached to the rib-bearer until after the rib has connected with
the same structure.
3. The rib simply shifts its attachment from the basal stump
to the rib-bearer as the horizontal septum moves upward.
After the attachment of the rib to the rib-bearer the para-
pophysis may develop dorsolaterally and fuse with the rib-bearer
secondarily. This connection may again be lost after ossification
sets in. This will be discussed more fully later.
Another possibility is that the parapophysis may be suppressed |
altogether. This is seen to be the case in the third vertebra of
a 25-mm. larva (fig. 21). Here haemapophyses do not appear
at all and the presence of one or two cartilage cells next to the
notochordal sheath is all that marks the position of the para-
pophysis. The ventral end of the rib-bearer projects downward
and inward toward the notochord, terminating in a weak strand
of cells which extends mesally to the vestige of the parapophysis.
The rib is directly continuous with the ventral end of the rib-
bearer (fig. 21, r.).
If this vertebra be compared with that of a similar stage in
Amblystoma (fig. 22), it will be seen to be strikingly similar. The
chief difference is that the rib-bearer of the latter is continuous
with the cartilage of the neural arch. However, many instances
of a direct connection between these two elements were found in
Necturus, although they are usually separated. (Compare rib-
bearer of opposite sides in fig. 19.) Furthermore, in place of the
weak strand of cells between the rib-bearer and notochord found
in Necturus, in Amblystoma there is a thin bar of bone.
556 D. L. GAMBLE
DEFINITIVE ESTATE
While the body of the animal is growing the ribs must shift
laterally to come into their final position, thus necessitating the
elongation of the transverse processes. This is interfered with
by the formation of bone around these structures. Hard skeletal
parts always interfere with the growth of softer parts, and provi- -
sion must always be made for it. In higher animals bone growth
and absorption go hand in hand. In Necturus, however, no
resorption of bone deposited around the transverse process and
rib was observed. Growth is provided for by the failure of bone
to form over the point of union between the proximal end of
the rib and the distal end of the parapophysis. Furthermore,
between these two elements is found procartilage cells which by
proliferation bring about a lengthening of the parapophysis.
In principle it is similar to the lengthening of the long bones
through the proliferation of cells in the diapophysial plate. This
point of growth together with the one at the distal end of the rib
makes possible the accommodation of these structures to the
growth of the body.
As before mentioned, the dorsal head of the rib develops as a
dorsal outgrowth from the rib itself. In the trunk this cartilag-
inous process does not reach the vertebra, connection being
made by a strand of connective tissue. In the second, third, and
fourth vertebrae the tubercular head of the rib meets a corre-
sponding outgrowth from the rib-bearer. Between the two, pro-
cartilage is found and no bone develops over the articulation.
This again must be interpreted as a provision for growth, for as the
parapophysis grows outward the dorsal part of the transverse
process must keep pace with it. The transverse processes of the
second, third and fourth vertebrae therefore possess both a ventral
and a dorsal cartilaginous rod surrounded by a sheath of bone.
Between the dorsal and the ventral rod a thin layer of bone
develops which Wilder (’03) calls the vertical lamina.
Goeppert, in describing the transverse process of Necturus,
states that the dorsal part does not appear to be so clearly a rod
as it does in Salamandra, and that it lacks the distal excavation
seen in Salamandra, the tubercular head of the rib not attaching
directly to it.
THE TRANSVERSE PROCESSES OF NECTURUS 55d
As Wilder pointed out, the distal cavity which contains the
dorsal cartilaginous process of the rib-bearer in the living state is
present in the second, third, and fourth vertebrae in Necturus,
and this connects with the dorsal head of the rib.
In studying cross-sections of the second vertebrae of a 70-mm.
larva, it was observed that the ventral cartilaginous rod of the
transverse process is located dorsad of the vertebral artery. In
the trunk vertebrae this element is ventrad of the same artery.
Figures 23 and 24 illustrate the morphology of this vertebrae.
The parapophysis (fig. 23, p.a.) extends a short distance laterad
from the notochord, but makes no connection with the rib-
bearer (r.b.)
Two processes are seen developing caudolaterally from the rib-
bearer (figs. 23 and 24, d.p., v.p.), and these connect with the
heads of the rib. This means that the capitular and tubercular
heads of the rib of the second vertebra are attached to out-
growths of the rib-bearer and the parapophysis takes no part in
this connection.
This failure of the parapophyses to connect with the rib-bearer
may or may not be secondary. As was seen by the study of
younger stages, the parapophysis may be entirely suppressed.
In other cases it may be poorly developed and make no connec-
tion with the rib-bearer or it may connect only to be again sepa-
rated when bone begins to form. However, whether or not the
connection between these two elements is made, the rib is borne
upon outgrowths of the rib-bearer.
If the macerated vertebral column of an adult Necturus be
examined (fig. 31), it will be observed that the transverse proc-
esses of the second and third vertebrae are higher than those
farther caudad. In tracing the ventral rod of the transverse
process of the second vertebra (v.p.) proximad, it will be seen to
extend dorsad of the foramen for the vertebral artery. In the
third vertebra it appears to be on the same level with this fora-
men, while in the fourth it extends below it.
Although in the third vertebra the parapophysis in a greater
number of cases connects with the rib-bearer, here also the rib is
attached to outgrowths of the rib-bearer. In the fourth vertebra
JOURNAL OF MORPHOLOGY, VOL. 36, No. 4
D.
L.
GAMBLE
GaN.
v.a.
p
THE TRANSVERSE PROCESSES OF NECTURUS 559
the capitular head is borne upon an extension of the parapophysis,
while the tubercular head is attached to a process of the rib-bearer.
It is of interest to note that the higher position of the first three
ribs is in some way correlated with the formation of a direct
connection between the tubercular head of the rib and the dorsal
process of the rib-bearer. It is also important to note that the
relation of the rib to the rib-bearer in these anterior vertebrae is
almost exactly similar to the relations of the same structures
throughout the trunk region of Amblystoma and Salamandra.
The one minor difference between them is that in Necturus the
rib-bearer is generally separated from the cartilage of the neural
arch by connective tissue, while in Amblystoma and Salamandra
the two are continuous. As shown in figure 19, however, this
continuity often obtains in Necturus.
DEVELOPMENT OF VERTEBRAE IN THE TAIL-TRUNK
TRANSITIONAL REGION
In the trunk region of the younger larvae (21 to 22 mm.) it
has been seen that distinct ventral cartilages or haemapophyses
often appear. In some vertebrae these are independent elements
and in others are fused with the bases of the parapophyses and
appear as ventral projections of the same. In several cases the
basal stump appears to be growing in two directions, laterally and
ventrally, although the lateral element becomes dominant and
the ventral element in time disappears.
Fig. 23 Transection through a second vertebra of a 70-mm. larva. e.c.,
chordal cartilage; d.p., dorsal process of rib-bearer; v.p., ventral process of rib-
bearer; n.a., neural arch; v.a.,vertebral artery; 7r.b., rib-bearer; p.a., parapophysis.
Fig. 24 More caudal transection of the same vertebra. d.p., dorsal process
of rib-bearer; c.c., chordal cartilage; v.p., ventral process of rib-bearer; v.a.,
vertebral artery; p.a., parapophysis.
Fig. 25 Transection through the last trunk vertebra of a 25-mm. larva. n.a.,
neural arch; r.b.p., rib-bearer proton; p.a., parapophysis’, h.a., haemapophysis.
Fig. 26 Transection through the first tail vertebra of a 25-mm. larva. n.a.,
neural arch; r.b.p., rib-bearer proton; p.a., parapophysis; h.a., haemapophysis.
Fig. 27 Transection through the second tail vertebra of a 26-mm. larva.
t.v.r., intervertebral ring; n.c., notochord; h.a., haemapophysis; p.a., para-
pophysis; n.a., neural arch.
560 D. L. GAMBLE
In the transitional region between the trunk and tail no sepa-
rate haemapophyses were found in any of the larvae studied.
The haemal arch is formed by the downgrowth of the bases of the
parapophysis and not by the elongation of distinct haemapophyses,
as in Salamandra and Amblystoma.
In the 25-mm. larva (fig. 25) in the last trunk vertebra the
proximal portion of the basal stump shows a tendency to bulge
downward. On one side this is very noticeable, while on the
other the process is in an incipient stage. On the left the proxi-
mal portion of the basal stump bends downward considerably
below the horizontal septum. A short distance from the noto-
chord, growth apparently is taking place in two directions,
laterally to form the lateral process and ventrally to form the
haemal-arch element. The more ventral outgrowth becomes
dominant over the lateral one and leaves it behind, so that it
appears as a lateral process of the haemapophysis. On the other
side the ventral outgrowth has not yet become dominant over the
lateral and appears here only as a rounded knob projecting slightly
ventrad from the base of the parapophysis (fig. 25).
In the first tail vertebra of the same larva (fig. 26) both haema-
pophyses have developed ventrally and completed the haemal
arch below. ‘The parapophysis appears as a small lateral process
of the haemal arch. In the second tail vertebra this lateral
process has lost its connection with the haemal arch and exists
as an independent element (fig. 27, h.a.).
It is seen, therefore, that the basal stump of Necturus shows a
tendency to fork not only in this region, but also farther forward
in the trunk of younger larvae. Anteriorly the lateral division
becomes dominant as the parapophysis and the lateral disappears,
while posteriorly the ventral one becomes dominant as the haema-
pophysis and the ventral disappears.
These two divisions of the basal stump, however, tend to sepa-
rate secondarily not only in the anterior tail region, but also in
the trunk. This separation is permanent in other salamanders
and in Polypterus, but, as Goeppert states, such cases in all
probability represent a secondary separation.
THE TRANSVERSE PROCESSES OF NECTURUS 561
SUMMARY
1. The protons of the rib, rib-bearer, and parapophysis are
all condensations in the muscle septa, and are therefore continuous
at this stage. The proton of the rib and rib-bearer is made up
of a greater number of cells, which is in sharp contrast to the
proton of the parapophysis in this respect. The parapophysis is
made up of a few cells more or less regularly lined up between the
notochord and the lateral muscle mass (fig. 2).
Fororer
0. O20
Fig. 28 Series of diagrams to illustrate the development of the parapophysis
and haemapophysis in Necturus. A, trunk region; B, tail-trunk transection
region.
2. The first cartilage to form is that of the basal stump which
appears as a knob projecting from the notochordal sheath in a
lateroventral direction (fig. 28A, b.s.). Later a laterodorsal
outgrowth from this becomes the parapophysis (fig. 28A, p.a.).
3. The first cartilage of the rib appears distally, and this later
develops mesally (figs. 29 and 30, A and B).
4. The first cartilage of the rib-bearer appears next to the
neural arch (fig. 29A). Later this develops ventrally and fuses
with the distal end of the parapophysis, and also develops dorso-
caudally over the outer surface of the neural arch (fig. 29B).
5. The proximal end of the rib is relatively high in the second
and third vertebrae; i.e., it is on a level with the base of the neural
562 D. L. GAMBLE
arch (fig. 30A). Posteriorly the rib is on a level with the middle
of the centrum (fig. 29A).
6. The parapophyses of the vertebrae in which the ribs are
high do not lie in a horizontal plane, as they do farther back in
the trunk, but extend dorsolaterally and approach the rib-bearer
Fig. 29 Series of diagrams to illustrate the morphology of the ribs and trans-
verse processes in the trunk region of Necturus. ¢.h., capitular head of rib;
t.h., tubercular head of rib; black, rib-bearer; stippled, parapophysis and
haemapophysis; white, rib. A, B, and C represent successive stages in the devel-
opment of these structures. D, E, and F show the successively less differentiated
condition encountered in passing toward the tail! in a larva in which the transverse
processes of the anterior vertebrae are at a stage similar to that represented by B.
end to end (fig. 830A). In this anterior region the rib-bearer and
parapophysis do not fuse until relatively late, while the rib
becomes attached to the rib-bearer before rib-bearer and para-
pophysis come together (fig. 30 A, B, C).
7. In the second and third vertebrae the capitular as well as
the tubercular heads of the rib attach to corresponding proc-
esses of the rib-bearer. In these vertebrae the parapophysis
THE TRANSVERSE PROCESSES OF NECTURUS 563
takes no direct part in the formation of the rib-attachment ap-
paratus (fig. 30 E and F).
Fig. 30 A series of diagrams to illustrate the development of the transverse
process in the anterior trunk region. Development may proceed in either of the
following ways: ABC ForABDE.
Fig. 31 Drawing of the lateral aspect of the first five vertebrae of an adult
Necturus, f.v.a., foramen for vertebral artery; d.p., dorsal process of rib-bearer;
v.p., ventral process of rib-bearer; p.a., parapophysis.
8. In the trunk region the capitular head of the rib attaches to
the parapophysis and the tubercular head makes no connection
with a process of the rib-bearer (fig. 29 B and C).
9. In the trunk the rib-bearer fuses with the distal end of
the parapophysis, and as growth takes place the distal end of
564 D. L. GAMBLE
the parapophysis extends laterally past this point of union
(fig. 29 A, B, C).
10. Between the dorsal and ventral cartilaginous rods of the
transverse processes and the dorsal and ventral rib heads in the
second and third vertebrae, procartilage cells persist which by
proliferation bring about the elongation of the transverse process.
In the trunk the tubercular head of the rib has no cartilaginous
connection with the rib-bearer, so this provision is necessary only
in the case of the parapophysis. ;
11. The haemal-arch element (haemapophysis) appears as a
downgrowth from the base of the parapophysis. This growth
becomes dominant over the parapophysis which in the first tail
vertebra appears as a lateral process of the haemal arch (hae-
mapophysis). It loses its connection with the haemal arch in the
second tail vertebra and appears as a lateral projection from the
notochord (fig. 28B).
CONCLUSIONS
1. The dorsal shifting of the horizontal septum is the direct
cause of the change in rib attachment from the centrum to the
neural arch.
2. That the rib is an independent element is indicated by:
a. The apparent ease with which it loses its connection with the
parapophysis and becomes attached to the rib-bearer.
b. In the very earliest stages the rib proton may appear in-
dependent of any vertebral element.
3. Goeppert’s belief that the rib never actually loses its at-
tachment to the basal stump is incorrect. In the second and
third vertebrae it has been seen that the rib connects with the
rib-bearer before this structure and the parapophysis (Goeppert’s
basal stump) unite.
4. The rib-bearer is not a dorsal upgrowth from the middle of
the basal stump, as Goeppert maintains, but is a chondrification
in the inner edge of the transverse septum which fuses with the
distal end of the parapophysis. As growth of the body takes
place, the distal end of the parapophysis elongates laterally
past this point of union.
THE TRANSVERSE PROCESSES OF NECTURUS 565
5. The distal excavation which Goeppert did not observe in
the dorsal part of the transverse process of Necturus is present
in the second, third, and fourth vertebrae. This has already been
pointed out by Wilder. Therefore, in these vertebrae the carti-
laginous tubercular head of the rib is attached to an outgrowth
of the rib-bearer. In the remaining trunk vertebrae this con-
nection is not made.
6. The ventral cartilaginous rods of the transverse processes
in the second and third vertebrae are not homologous with those
of the other trunk vertebrae. This ventral rod in these two verte-
brae is an outgrowth of the rib-bearer, dorsad of the vertebral
artery and bears the capitular head of the rib. The ventral rod
of the transverse process in the remaining trunk vertebrae repre-
sents an elongation of the parapophysis which passes ventrad
of the vertebral artery.
7. The relation of the transverse process and rib heads of the
second and third trunk vertebrae in Necturus is exactly similar
to the condition throughout the trunk in Amblystoma and
Salamandra.
8. The primitive basal stump (Gadow’s basiventral element)
develops a lateral process, the parapophysis, and a ventral, the
haemapophysis. In the trunk region of a younger stage, both
are present. In some vertebrae these are joined and in others
are separated. This separation is secondary. In the trunk the
parapophyses persist and are connected with the ventral head of
the rib. The haemapophyses here disappear, while in the tail
region they elongate to form the haemal arch ‘and the para-
pophyses disappear.
9. In conclusion, therefore, it may be said that the change in
attachment of the rib, from the centrum to the neural arch in
urodeles, is not the complicated process that Goeppert thought.
The capitular head of the rib does not remain attached to the
basal stump or vestige of it, but loses its connection with the
parapophysis and joins with the rib-bearer (compare figs. 29 and
30). This change in rib attachment is correlated with the dorsal
shifting of the horizontal septum.
566 D. L. GAMBLE
LITERATURE CITED
BupGETT, JoHN SAMUEL 1901 On the structure of the larval Polypterus. Trans.
of Zo61]. Soc. London, vol. 16, part 7, Oct., 1902; or, Budgett Memorial
Volume, 1907, pp. 154.
Gapow, H., anp Miss E. C. Assotr 1896 On the evolution of the vertebral
column in fishes. Phil. Trans. Roy. Soc. London, vol. 186.
1896 On the evolution of the vertebral column in Amphibia and
Amniota. Phil. Trans. Roy. Soc. London, vol. 187.
GorrprerT, E. 1895 Zur Kenntnis der Amphibienrippen. Morph. Jahrb.,
Bd. 22.
1895 Untersuchungen zur Morphologie der Fischrippen. Morph.
Jahrb., Bd. 23.
1896 Die Morphologie der Amphibienrippen. Festschrift fiir Gegen-
baur, 1.
1897 Bemerkungen zur Auffassung der Morphologie der Rippen in
Rabl’s Theorie des Mesoderms. Morph. Jahrb., Bd. 25.
1898 Erliuternde Bemerkungen zur Demonstration von Praparaten
iiber die Amphibienrippen. Verhandl. Deutsch. Zoél. Gesellsch.
Leipzig.
Goerrtr, A. 1878 Beitraige zur vergleichenden Morphologie des Skelettsystems
der Wirbelthiere. lI. Die Wirbelsiule und ihre Anhinge. Arch. f.
mikro. Anat., Bd. 15.
1879 Same. Bd. 16.
Rasu, C. 1892 TheoriedesMesoderms. Morph. Jahrb., Bd. 19.
1897 TheoriedesMesoderms. 1. Teil, Vorwort. Leipzig.
ScHAUINSLAND, H. 1901-06 Die Entwickelung der Wirbelsiule nebst Rippen
und Brustbein. In Hertwig’s Handbook. 3.II.III. Bibliography.
Wiper, Harris HAwTHoRNE 1903 Theskeletal system of Necturus maculatus,
Rafinesque. Memoirs of the Boston Society of Natural History,
vol. 5, no. 9.
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Resumen por el autor, G. H. Bishop.
El metabolismo celular en el cuerpo adiposo de los insectos.
I. Los cambios citolégicos que acompafian al crecimiento y la
histolisis del cuerpo adiposo de Apis mellifica.
En las células del tejido adiposo de la larva de la abeja al
principio de la ninfosis, algunos de los granulos nucleares estan
dispersos por el citoplasma, para funcionar como cromidios en
el desarrollo de los glébulos de reservas albuminoides. Las
vacuolas grasas que pasan hacia el centro indentan al nitcleo
alargado; la pared nuclear desaparece, el nucleoplasma y
el citoplasma se mezclan parcialmente y este material se extiende
periféricamente como trabéculas desde la regién nuclear hasta
cerca de la membrana celular. Los granulos nucleares pasan
depués 4 través de los intersticios de las vacuolas, y pasan hacia
el exterior en las trabéculas. La membrana nuclear se regenera
alrededor de la vesicula muy deformada, las trabéculas que se
extienden desde el nticleo se desintegran, y los granulos cromidiales
se desarrollan 4 expensas del citoplasma y las vacuolas grasas
que contiene en glébulos que acaban por llenar la célula. Estos
gl6bulos parecen poseer al principio una estructura uniforme, pero
mds tarde adquieren vacuolas centrales, con una corteza peri-
férica de material basdéfilo, que finalmente se divide en finas
particulas en la superficie de los glébulos. Al disolverse la
membrana celular los glébulos quedan libres, disolviéndose en
la sangre para ser utilizados como alimentos para el crecimiento
del tejido imaginal. Si se considera al crecimiento del tejido
adiposo como un proceso anabdlico de acumulacién de alimentos
para el desarrollo imaginal y la disolucién de los glébulos albumi-
noides como el correspondiente proceso catabdélico, la transforma-
cidn de las substancias celulares—la grasa de las vacuolas,
la matriz citoplasmica y los granulos nucleares—en la forma co-
mun de glébulos albuminoides tiene lugar como un éstado inter-
mediario de metabolismo intracelular, siendo fisiol6gicamente
cada célula en cierto grado un ‘sistema cerrado.’
Translation by José F. Nonidez
Cornell Medical College, New York
AUTHOR’S ABSTRACT OF THIS PAPER ISSUED
BY THE BIBLIOGRAPHIC SERVICE, JULY 17
CELL METABOLISM IN THE INSECT FAT-BODY
I. CYTOLOGICAL CHANGES ACCOMPANYING GROWTH AND HISTOLYSIS
OF THE FAT-BODY OF APIS MELLIFICA
GEORGE H. BISHOP
Zoological Laboratories of the University of Wisconsin
SIX TEXT FIGURES AND THREE PLATES (THIRTY-SIX FIGURES)
CONTENTS
GAP TC WCUCIRG a6, 6S:6 CARRE OOS GUM o5 Gilde RAE RE Onan SUIS oc ciaicmindes ret denen 568
The role’of the fat-body in larval metabolism.........,20842242. 4.529 a.: 569
denlarvaledevelopment: scy4 tre bnew’ 4 otis, cists Ses tes eae and Serer Bere, bre? 569
TAO TE Sista nfiaietoe or Youn Fs 95) 0 1000 9 gaa eke eT ene ae Se ee 570
FS UOR CMC RESPESIN. <3, ha tee ree oe, A), oearmudmerhietsreete es ae oes 570
Ditterermtaaeiainee <2. sek ee eam A) ead Res ILE 571
Ana Long AEC MUG FON 4 terpyae ae eh ves feces Cpa eee Beies BE Sys. ot cua 571
Saelvelatronmtrous cry lie a Chlvdlityae cit Ui oy eet ees clooc, o eacatuccasie a os koe 573
AE RURRGH AMIS UAT OLPAOSIS 4 ic 5,6 gee, <4 osc ape, wn d's yes ats Ee, a a cio oo aye 573
Development and transformations in structure of the fat-body cell of the bee
Stitt be. «id UN ERLE Ae bC) Sg OMe: aie CRSA Ct) Sea ROR MMNOY Ce REC 6.024 oP 574
LUEN Caen II, ae: ne 8 ee ed a ee a ee nS | eee ee 574
a. ls Gaeease to imaginal fat-body. . : ay . 574
b. Destruction of larval fat-body; patoiyee sal nineeeytosin ere 574
Guietabolismoanythe:lanvalstat-oOdiye. jstere ache eae fo eicine ea ctl 575
2. Anatomical data. Structural changes accompanying cell metabolism
in he rat=MOty OF Ge DEG 1AYV A. werd ox 5a2 see che god oehn ant oe we ogc 3 577
(a ANA Dae ree Bat nae a at oe a a ried Dae le ah 577
[Foal EKG. A TING [es etna a des PALE ie i Me Lr. lal need ee 578
(By AMOK SCORCH ORCA ac bab He oo ido ob do Oko btRId OG DAC OAC ESO 579
SURG SEM SEG Ghats Ny CUTS) Ages Gene ae ee. ieee ge ee ae oe 580
hemnuel ears ern WLAN ere erie ee aes 5 Hors oa 44 vss eke 586
Gd: VALEEMIGRSUIOUL LYSE fracas eee ee tees Se cls es ee clo tie dle 589
Qneentandiworker cells sepa sete tee tere ole has (cle. d.cs itis eke tale 590
Oucenwandecroneycell sya ere, Beye sts ner oieeais erie oarete 592
Varia ti Onsmilln DO Givate IOUS Sw pea ne veteeas = 1+. 1s ckavors) gyersr ss aye ctalave 592
3. The metabolic significance of changes in structure................ 593
567
568 GEORGE H. BISHOP
INTRODUCTORY
The honey-bee larva, just at the beginning of pupation, ex-
hibits in the cell of its fat-tissue an abrupt and striking cytological
transformation. The nuclear wall disappears, and the peripheral
fat-vacuoles of the cell approach the nucleus; through the
interstices between these, granules of basophile material, pre-
sumably of the nature of nucleoli, pass from the nuclear area
and invade the cytoplasm as chondriosomes. By progressive
absorption, both of the surrounding cytoplasmic matrix and
of its fat-vacuoles, these granules finally develop into globules
containing albuminoid material, which are discharged into the
blood on dissolution of the cell wall. The nuclear membrane is
meanwhile reformed, without nuclear division, and may persist
until after disintegration of the cell wall and discharge of its
contained globules.
The cytological structure of these fat-body cells, or trophocytes,
will be described in the following account, and especially the
cytolytic changes they undergo during larval pupation. It has
seemed advisable to enter in the second part of this paper, into
certain rather speculative considerations which, based on the
work so far accomplished, form the tentative framework for
further research; but no attempt is made at this stage of the
investigation to propound the ultimate analysis of cell metabolism
in the insect fat-body.
The study of somatic cell metabolism has led to a wide diversity
of interpretation, depending on the point of view of the special
research dealing with it. The result has been a ‘chemical’
theory of function, or a ‘physical theory,’ ora ‘genetical’ theory,
when an adequate biological interpretation must involve all
of these. Especially in general physiology and physiological
chemistry does a large and rapidly augmenting body of data,
specifically applicable to the functioning of the somatic cell,
invite a reconsideration of normal ‘resting’ cell structure; it
demands, in fact, a more energetic name for the ‘resting’ cell
itself. As a method of coordinating these specialized studies,
it should be profitable to subject some one tissue to as many
different techniques as conditions admit of, and the bee fat-body
HISTOLYSIS OF FAT-BODY OF APIS 569
tissue seems well adapted for this. The present paper aims to
lay down the anatomical basis for such a series of experiments.
In part I the réle of the fat-body is defined in relation to general
larval activity, and the structure of its cells as functional ele-
ments is described and in part II is discussed the functional sig-
nificance of these cell elements in metabolism.
Acknowledgments are due especially to Dr. M. F. Guyer,
of the University of Wisconsin zoological laboratories, for much
valuable advice, and to Dr. Wm. 8. Marshall, of the same
institution, for literature references and suggestions as to insect
metabolism.
THE ROLE OF THE FAT-BODY IN LARVAL METABOLISM
1. Larval development
The larvae of the honey-bee are available in quantities and
lend themselves readily to investigation. From the laying of
the egg to emergence of the imago, a larva passes its existence
in a cell approximately 4 mm. across by 9 deep. For the first
part of its life it is half immersed in the partially digested food
administered to it, the food being later disgorged by the worker
bees directly into its mouth. It lies in the position in which it
was first hatched until the confines of the cell containing it
compress its flabby bulk into a flattened ring, whereupon it
straightens itself out and spinsa cocoon. The larva then pupates
and is quiescent until it quickens as a mature insect.
From the standpoint of metabolism, the bee larva approaches
what might be called a closed system: During a developmental
period of twenty-one days it passes no excreta. The content
of the malpighian tubules and a few faeces collect in the hind
intestine, which, however, does not make functional connection
with the stomach until late in development; the stomach contents
are regurgitated at the beginning of pupation. The food taken
in is of determinable and relatively unvarying composition. The
developmental period is definite in time, and the temperature of
the bee cluster is maintained with remarkable constancy. Thus
the natural environment of the larva furnishes controlled experi-
570 GEORGE H. BISHOP
mental conditions difficult to duplicate for animals undergoing
a less monotonous adolescence.
Internally the bee is no less adapted to experimental investiga-
tion. Its response to a life of sequestered inaction has been a
repression or a rudimentary development of many of the larval
organs that would be required to adapt a larva to an active and
independent mode of life. Locomotor muscles, elaborate modi-
fications of the digestive system, complications of chitinous
hypodermis for protection or aggression, are little developed or
are lacking entirely. The larval life is given over to one function
predominantly—the storage of nutriment—and this stored-up
nutriment, asthe fat-body, comprises at the time of metamorpho-
sis three-fourths of the body tissue (blood excepted).
After hatching, for five and a half days the chief activity of the
bee larva consists in the storage of food as fat-body tissue.
During the first four days of pupal existence this fat-body is
almost entirely histolyzed to furnish nutriment for the growth
of imaginal tissues. Hence, in the building up of this food into
fat-body cells, and the breaking down of these cells into tissue
nutriment again, one is justified in looking for a highly specialized
and, in a sense, an isolated process of nutritive metabolism.
2. Histology of the fat-body
Histogenesis. The fat-body in insects is a tissue of mesodermal
origin, the cells of which originate in the embryo by division and
segregation from the inner surface of the mesodermal tubes
along what is destined to be the ventrolateral aspect of the body-
space. These cells form a mass of tissue running along the body
between the intestine and the ventral body wall, attached to
each. Typically, during embryonic or early larval life, the cells
multiply to the full number to which the larval fat-body will
attain. Thereafter, the growth of this tissue consists in enlarge-
ment of its cells, and not in their numerical increase. The bulk
of the tissue relative to the size of the body varies in larvae of
different insects from a narrow band on either side of the nerve
chain in the embryonic position to a massive growth which
comprises the greater part of the bulk of the larva. In the bee
HISTOLYSIS OF FAT-BODY OF APIS 571
embryo (Nelson, 712) this tissue develops chiefly from the
splanchnic, instead of the parietai mesoderm as is usual in insects.
This gives rise to two groups of cells on each side of the nerve
chain, to which are added third groups from the parietal layer
near the heart. In the larva it comes to extend dorsalward in
two layers—splanchnic, enveloping the intestine, and parietal,
lining the body wall. At metamorphosis, this tissue is dis-
integrated with the other larval tissues, and is replaced by an
imaginal tissue, generally of much smaller bulk and more periph-
eral location.
Differentiation. Starting as a tissue of homogeneous compo-
sition in the bee, the fat-body attains complexity by reason of
two processes. Certain cells become differentiated from the
fat-tissue itself, and other elements invade it from the outside.
During larval life, scattering cells have been described that either
lose their fat-globule content or never acquire any, and, as larval
life proceeds, develop characteristic granules of sodium urate.
These have been termed excretory cells. From outside the fat-
body altogether the tissue is invaded by large amoeboid cells
originating segmentally from the margins of the spiracles, which
from their wine-red color in certain insects are termed oenocytes,
and to which are ascribed a secretory, and by some, a respiratory
function. Leucocytes are also found wandering through the
fat-tissue, presumably from the blood. After metamorphosis,
pericardial cells grow down in strings from the heart region and
become associated with the fat-body, as do also the imaginal
oenocytes. The fat-cells themselves acquire during imaginal
life granules of sodium urate. The functions of these various
elements are even more obscure in the imaginal than in the larval
economy.
Anatomy and function. Ignoring for the time being the con-
flicting interpretations that have been placed upon the fat-
body function, we note that the mass of this tissue in the bee,
aside from any other consideration, suggests a food reservoir.
In larvae a few days old the fat-body envelops most of the other
organs. It extends from the head region through the extreme
caudal segments, and, except for blood spaces, fills all the region
572 GEORGE H. BISHOP
from the intestine to the nerve cord. From here it extends
dorsally in two chief layers, the intestinal. layer just failing to
close over the gut dorsally, and the margins of the parietal layer
adjoining on either side the dorsal blood vessel. Through the
transparent chitin of the grown larvae may thus be seen a clear
Text fig. A One half of a cross-section of a mature bee larva. At., heart;
h.s., haemal space; int., mid-intestine; »., ventral nerve cords; sp., spinning
glands; m.t., malpighian tubules; m., muscles; erc., excretory cells of the fat-
tissue; 0e., oenocytes. The remainder of the cells, not shaded, are the fat-cells
or trophocytes. The cells of this tissue are arranged in folds or laminae, each
_two to five cells thick; the lamina run in general longitudinally in the body and
are separated by blood spaces, which also surround all the organs. The oenocytes
and excretory cells, both amoeboid, lie sometimes completely embedded in the
fat-tissue, sometimes along the margins of the blood spaces. Roughly, three
regions may be distinguished: a visceral or splanchnic layer of the tissue ventral
and lateral to the intestine; a parietal layer along the body wall, ventral and
lateral; and a cardiac layer, extending from the heart region lateroventrally
on either side the dorsal haemal space. These layers correspond to the three
mesoblastic anlages of this tissue in the embryo. Camera-lucida drawing.
blood space below the dorsal heart and pericardium, and through
this again is visible the yellowish content of the mid-intestine,
flanked on either side by white fat-tissue (text fig. A).
In cross-section the fat-body is seen to be longitudinally lami-
nated, each lamina two to five cells thick, and extending from the
central or peripheral layer into the blood space. A large pro-
portion of the cells thus lie in immediate contact with the blood.
HISTOLYSIS OF FAT-BODY OF APIS 573
There are no trachea extending into the laminae. In the deeper
layers are embedded the malpighian tubules and the salivary
and spinning glands. The hind-intestine, unlike the mid-
intestine, is entirely enveloped.
3. Relation to larval activity
This tissue occupies a space so prominent and so extensive in
the larval anatomy and forms so large a proportion of its mass
that the larva seems to be merely a mechanism for nourishing
the fat-body, rather than the fat-body an organ of the larva.
This illusion is supported by the low degree of development of
any organs not concerned with the assimilation of food; i.e., the
fat-body itself, the digestive apparatus, including the salivary and
spinning glands, and the excretory system. The physiological
assimilation of most of the food the larva takes goes on in the
fat-body, the functional unit of which is the individual fat-cell.
As in all the Hymenoptera, the metamorphosis is complete.
It involves the destruction of all the typically larval organs and
the formation of a full set of imaginal tissues. This degenerate
and highly specialized larva, consisting of a living sac full of
stored-up nutriment, thus transforms into one,of the most elabo- _
rately and highly specialized of mature insects. The transfor-
mation is so extreme and so abrupt that it demands the inter-
ruption of even such activity as the bee larva exhibits, and the
pupal stage is entirely inert.
4. Nutrition and metamorphosis
The fat-body of the bee, compensating as it does for the large
masses of muscle, etc., developed by more active larvae, is also
presumably a more efficient mechanism for food storage, by
reason of its high fat content, than the usual larval tissue. The
histolysis during larval metamorphosis, therefore, is to be con-
sidered as more than a mere removal of a no longer useful larval
organ; an intracellular digestion goes on by which a large amount
of reserve tissue nutriment is elaborated with very little waste.
Its cells may be interpreted as having two methods of functioning,
574 GEORGE H. BISHOP
on the one hand, storage of food materials especially adapted to
tissue growth and, on the other, a mechanism by which, during
pupation, this reserve is further modified into food constituents
suitable for immediate utilization by imaginal tissues. With a
study of these two phases of the activity of the cells the following
discussion concerns itself.
DEVELOPMENT AND TRANSFORMATIONS IN STRUCTURE OF THE
FAT BODY CELL OF THE BEE LARVA
1. Historical
Except for quite recent papers, the literature on the insect fat-
body has been reviewed so thoroughly that repetition is super-
fluous (Anglas, ’00; Perez, ’02, ’11). In general, three lines of
attack have been made on the question; Ist, the investigation of
the larval fat-body as the precursor of the imaginal fat-body;
2nd, the study of the mechanism by which the larval fat-tissue
is histolyzed (autolysis of phagocytosis), and, 3rd, consideration
of the fat-body as a larval food storage reservoir. The ele-
ments concerned are the fat-tissue cells with their fat-globules
and albuminoid granules, the oenocytes, the phagocytes, and
leucocytes.
a. Metamorphosis to imaginal fat-body. Four methods of
origin have been assigned to the imaginal fat-body: 1) from em-
bryonic cells developed from fragments of the fat-cells (Auerbach,
°74); 2) from embryonic cells developed from degenerating muscle
cells (Anglas, ’00); 3) by reformation of dispersed fat-cell frag-
ments about the old cell nucleus (Koschevnikow, ’00); 4) by
persistence of certain larval fat-cells to form the imaginal tissue
(Ganin, ’%75; de Bruyne, ’98; Berleze, ’01; Perez, ’02,’11). The
last interpretation seems best established.
b. Destruction of larval fat-body; autolysis and phagocytosis.
The discussion of the release of the food materials of the larval
fat-body cells has taken to some extent the form of a debate as
to the respective merits and relative prevalance of autolysis, i.e.,
histolysis without attack by phagocytes, and phagocytosis or
destruction by leucocytes or other wandering cells. Five inter-
pretations have been placed upon the facts: 1) The development
HISTOLYSIS OF FAT-BODY OF APIS 575
of ‘Kérnchenkugeln’ from fat-cell débris, with aquisition of a
nucleus (Weissmann, 64). 2) Theattack of fat-cells by ‘excre-
tory-secretory cells’ (Anglas, ’00). 3) Lyocytosis, or dissolution
of the cells by enzymes from the imaginal tissue cells developing
(Anglas, 702). 4) Autolysis (Terre, ’99; Perez, ’02, ’11). 5)
Phagocytosis (Kowalewski, ’85; van Rees, ’87; deBruyne, ’98;
Karaweiew, 798; Anglas, ’00; Perez, ’02, ’11). The evidence
seems best for the processes of autolysis, with or without
leucocytic absorption of the débris, and phagocytosis by the
leucocytes themselves where a precocious breaking down of the
trophocytes is necessary.
c. Metabolism in the larval fat-body. After considerable
speculation on the possibility of excretory and other manners of
functioning of this tissue, attention has turned to the process by
which food material is released from the fat-cells and prepared
for tissue nourishment. Storage of nutriment in the fat-body
was first pointed out in 1875 by Kunckel d’ Herculais.
Berlese, ’99, (on the Diptera), believed that material from the
histolyzing intestine passed through the blood to the fat-cells,
forming the albuminoid granules. He named the fat-cells
‘trophocytes’ from this activity. Action on the granules by
nuclear enzymes was thought to render them basophilic, in digest-
ing them for tissue nourishment.
Anglas, 00, (wasp and bee), observed that in the wasp certain
cells undergo a marked transformation, assigned to attack by
excretory-secretory cells. The nuclear membrane disappears,
nuclear and cytoplasmic materials mingle, and the nucleus can
finally no longer be discerned. This process of phagocytosis was
not deseribed specifically, for the bee, though the author states
that the two forms are closely similar. The transformations
described were not noted in other than those cells attacked by the
phagocytes.
Perez, 02, (Formica), and ’11, (Polistes), describes the growth
of fine granules formed in the region around the nucleus, to
albuminoid globules, and a modification of the nucleus, without
attack of phagocytes. The fat-globules disappear presumably
by digestion, with the growth of the albuminoid globules. The
576 GEORGE H. BISHOP
nucleoli increase in number to this stage (beginning of pupation),
and the nucleus as a whole decreases in size with the formation
of the granules. The globules developed somewhat differently
in the different forms studied.
Hufnagel, ’11 (Hyponomeuta), described an ‘epuration’ of
chromatin from the nucleus of the cell. Thisprocesswasobserved
to commence in the larva, but persisted throughout pupation to
the formation of the imaginal fat-body. Granules of chromatic
material formed within the nucleus passed into the cytoplasm and
became enclosed by a chromatic portion of cytoplasm. Different
stages of ‘condensation’ of the chromatic substance were observed
in the same cells, due to the fact that the granules were developed,
not all at once, but in a successive order. The globules were
finally expelled from the cell and engulfed by phagocytes. The
fat-cells persisted after this expurgation of nuclear material to
form the imaginal fat-body.
Hollande, ’14 (Vanessa), conducted a chemical investigation
of the contents, especially of the albuminoid reserve globules, of
the fat-cells. He found that the globules in this form developed
as in other larvae from granules formed close about the nucleus;
that granules of sodium urate were also formed here, and he
concluded that the process represented an expulsion from the
nucleus, and consequent digestion, of nucleic acid containing
material, from which sodium urate was split off almost imme-
diately by enzyme action as waste material. This was demon-
strated to be formed endogenously, not acquired from without
in the functioning of the fat-body as an excretory organ. The
development of the granules from feebly basophile particles to
globules with acidophile margins, and finally to hyperacidophile
bodies, apparently by attack of enzymes, was checked by micro-
chemical tests which showed a transformation from nucleo-
proteids to albuminoids, and finally to biurette polypeptids and
erystalloids. The possibility is considered that part of the fat is
transferred to albuminoid (the reverse of the process reported by
Weinland (’08) of transformation of albuminoid to fat by fly
larvae), but no evidence was offered for such a process. Crystal-
loids were observed in the center of fat-globules.
HISTOLYSIS OF FAT-BODY OF APIS 577
Nakahara (17), in a research directed primarily to the func-
tioning of amitosis in the insect fat-body, reported incidentally
on the larval development of the fat-cell in Pieris. In ‘second-
stage’ larvae, apparently still very young, nuclear ramification
was observed, and in the ‘third’ stage spherical albuminoid glob-
ules in the cytoplasm; amitotic division of the nucleus occurred
with the production of as many as five nuclei to a cell. Ata
late larval stage some of the albuminoid globules ‘‘begin to show
dark dots, taking basic stains, indicating that the transformation
of albuminoid substance into urates is beginning to take place”’
(on the assumption that the basophile stain indicates the de-
generation of albuminous material to purine bodies, rather than
the acquisition of nuclear substance). ‘‘This possibly may be
regarded as one of the first signs of a histolytic process. Soon
afterwards, just before the larva enters the prepupal stage, the
nucleus loses its membrane and its structure becomes more or less
indistinct. This is, I believe, the sign of a karyolytic process,
which concludes the activity of the larval adipose cells.” The
further progress of these structures is not followed. Amitosis in
the fat-cells is inferred to result in the increase of surface ad-
vantageous to active nuclear functioning.
From these accounts may be derived a general description of
the fat-cell changes during growth and metamorphosis. The
conflicting details of various authors, while perhaps due in part
to faulty or incomplete observations of fact, are probably more
largely due to the actual differences in the details of the process
in the different orders of insects worked upon. It will be difficult
to compare these details critically until a more accurate knowledge
can be obtained of their physiological significance.
2. Anatomical data. Structural changes accompanying cell
metabolism in the fat-body of Apis mellifica
a. Material. Material was procured at closely timed stages
and examined consecutively with various stains. It was found
convenient to divide the larval and pupal development more or
less arbitrarily into periods according to the cell changes, as
follows:
578 GEORGE H. BISHOP
A. Embryonic development, in egg, two days; and multi-
plication stage in fat-body, egg, and young larva, one tot wo days.
B. Growth period. Characterized by one large fat-globule
in cell, and irregular cell shape. One to two days, text figure B
and plate 1, figure 1.
C. Fat storage. Development of peripheral ring of globules,
with further increase of size. Three days. Text figure C and
plate 1, figures 2 and 3.
D. Nuclear transformation. Cessation of larval feeding,
spinning of cocoon, quiescence. Few hours, of sixth day of
larval life. Text figure D and plate 1, figures 4, 5, 8.
E. Development of albuminoid globules. Head of imago
forms, prepupa stage. Twotothreedays. Text figures E and F
and plate 2, figures 11, 12, 13.
F. Globules released. Imaginal form assumed. About six
days. Plate 2, figures 14 and 16.
G. Imaginal fat-body formed, young bee emerges. Last two
days of pupation.
It will be noted that these periods represent not equal periods
of time, nor changes of larvae form, but are based on changes
within the cells themselves. From three to five substages were
examined in the critical periods, D, E, and F.
b. Technique. The technique employed in the microscopic
work was the conventional cytological technique for material to
be sectioned. The typical method was as follows:
A little fixative was injected into the blood space of a larva or
pupa with a capillary pipette, to harden the tissues sufficiently
for further cutting. The larva was then placed under fixative
and slit open along the back with fine curved-handled scissors,
since the fixatives would not readily penetrate the impervious
chitin. Under this treatment the larval stages showed slight.
distortion in the shape of the cells, due to contraction of body-
wall muscles which drew the slit-open larva into a bowed position,
with a consequent stretching of the intestinal layers of fat-body
cells. The intracellular structure, as checked by larvae killed
in hot water, was not materially distorted. Hot water, however,
seemed to leave the fat-globules less accurately defined.
HISTOLYSIS OF FAT-BODY OF APIS 579
The material when fixed was slit sagittally, and sections of
one half the body cut transversely, 3 or 4 mm. thick. These |
were carried up to 85 per cent alcohol by means of an apparatus
designed to produce a very gradual dehydration without shrinkage
(Bishop, 17). The pieces of tissue were then cleared in redistilled
anilin oil, passed through xylol and paraffin, changes being made
by small degrees, and infiltrated for two to four hours at 56 to
58°. Sections were cut 4 to 10 uw. The standard fixative used
was Allen’s B15 formula, with or without urea, although prep-
arations were made with picro-formol, formol-acetic, Flemming’s,
Gilson’s, hot water, etc. The stains used most successfully
were Heidenhain’s iron alum haematoxylin, safranin-gentian
violet mordanted with Gram’s solution, and Delafield’s haema-
toxylin, for chromatic structures; polychrome methylene blue
proved a very delicate stain for the nuclear membrane; for
cytoplasmic structures, the albuminoid globules, ete., eosin,
aurantia, acid fuchsin, licht griin, orange G, and Congo red.
Eosin and aurantia gave good differentiation oftheglobules. Fat
was stained with sudan III and osmic acid. An old much-used
bottle of Mallory’s phosphotungstic haematoxylin, diluted with
equal parts of distilled water, gave beautiful preparations showing
all structures, nuclear, cytoplasmic, and nuclear membrane,
but stained so generally that it masked all counterstains used for
qualitative differentiation in cytoplasmic structures, such as the
albuminoid globules.
c. Microscopic anatomy. The first stage to which close study
has been directed is that designated B, in which the fat-cells,
or trophocytes, having ceased to divide, are laying up appreciable
stores of globular fat. Before this stage, during what may be
designated rather loosely as a multiplication period, this tissue
is characterized rather by the irregular shape of the cells than
by their visible fat-content. From this period forward, the de-
posit and the metabolism of fat appear under the microscope as
the most striking and characteristic activities of the tissue. An
analysis of text figures B to F will comprise a presentation of
the chief anatomical findings of this paper.
580 GEORGE H. BISHOP
Stage B-C. In figure B, the distinctive elements brought out
_by staining are as follows: in the cytoplasm typically one large
fat-globule, and generally several smaller ones, showing after
alcoholic extraction as clear spaces in a homogeneous, non-
Text fig. B Larval fat-tissue cell, stage B, shortly after hatching, characterized
by one large fat-vacuole, which may distort the nucleus.
Text fig. C Larval fat-tissue cell, stage C—rapid cell-growth period, the usual
larval appearance—characterized by a peripheral ring of fat-vacuoles, central
densely staining cytoplasmic area, and densely granular nucleus.
Text fig. D Larval fat-tissue cell, stage D—larva just becoming quiescent,
dispersion of nuclear granules into the cytoplasm—characterized by a central
ring of fat-vacuoles indenting the nuclear vesicle, by loss of the nuclear membrane,
and by dispersal of the nuclear granules.
granular, or finely granular, rather heavily staining matrix; and in
the nucleus, many large chromatic granules in a clear, lightly
staining nuclear sap, with smaller granules and a very lightly
staining linin network between them, the whole enclosed by a
HISTOLYSIS OF FAT-BODY OF APIS 581
faintly basophilic nuclear membrane. The amount of chromatic
nuclear material and the density of its staining reaction suggest
pronounced nuclear participation in the cell’s metabolism.
The nucleus is as a rule pushed to one side and distorted, even
to the extent of being indented, by the one large fat-globule.
This stage of the fat-cell is not limited by definite or abrupt
changes either at its inception or at its conclusion. The fat-
globule increases in diameter, at first more rapidly than the cell
containing it, compressing the nucleus as it enlarges. Later it
grows more slowly (relatively) until the nucleus relaxes to the
smooth oval shape of the later stage.
Stage C. Text figure C presents a section of a larger fat-cell,
the difference in structure of which, from that of figure B, appears
to be due less to a difference of physiological functioning than to
mere increase in size and content. The cell comes to present an
appearance strikingly different from the first figure, by the mere
mechanical rearrangement of elements whose individual aspects
are precisely the same as in the former stage. The nucleus is
surrounded by a homogeneous mass of cytoplasm extending
uniformly from nuclear membrane to cell wall, except where dis-
placed by fat-globules. A peripheral ring of these globules, the
largest of which approximate the size of the prominent globule
of the former stage, and the smallest of which surpass the limits
of microscopic vision, displace most of the readily staining cyto-
plasm from among them, and give the appearance of lighter stain-
ing in this region. The globules are often so numerous and so
massed that they distort each other from the characteristic
spherical form. This accretion of peripheral fat continues with
a progressively increasing number of vacuoles, until just after the
sealing over of the larva and the cessation of the nutritive supply.!
1 Some light is thrown on the mechanics of the change in fat disposal from
stage B to C (text figs. B and C) by aconsideration of the surface: volume ratio.
The volume of a sphere increases as the cube of its radius; the larger the cell diame-
ter relative to the diameters of the contained fat-vacuoles, the larger will be the
radius of that portion of the cell’s volume into which the non-fatty material
gathers centrally. For instance, one-half the volume of a sphere whose radius
is 1 is contained in a sphere at its center whose radius is 0.79, the other half in a
peripheral shell of 0.21 thickness. If, say, 50 per cent of the cell were fat, it could
582 GEORGE H. BISHOP
Stage D. In figure D is pictured a stage which differs from the
two former stages figured not only in the mechanical arrangement
of the cytological elements, but evidently in the nature and con-
dition of the substances present. Both nucleus and cytoplasm
show marked changes from the previous stage. In the cytoplasm
the peripheral vacuoles have decreased in size and number, while
a layer of them has appeared centrally along the sides of the
oval nucleus. Early in this stage the nuclear membrane has
either disintegrated or lost its precise staining capacity, and the
nuclear granules have become more scattered than previously
throughout an area more elongated than the oval of the former
nucleus. Finally, in the cytoplasm, and especially out from the
be gathered into one layer at the cell’s periphery whose thickness would be about
one-fifth the radius of the cell. But gathered into one vacuole (as in fig. 1), the
globule’s diameter would be nearly four-fifths that of the cell, and must push the
nucleus to one side. With the fat in smaller vacuoles relative to the size of the
cell, the center of the cell may be free from fat (fig. C.)
The vacuoles of the larger cells are not much larger than the prominent ones of
the earlier stage. The reason for the limit to the size of the vacuoles, which seems
to be the factor causing the difference of appearance between them, may be
deduced tentatively from a consideration of the relation of the volume of asphere
to its absorptive surface. Considering the fat-cell as a chemical plant for the
metabolism of fat as well as a storehouse for the product, the chemical activity
must take place somewhere between the cell wall and the vacuole where it is
deposited. The fat is presumably condensed into vaculoes from the emulsoid
form, from the peripheral cytoplasm where it is elaborated. In a small cell the
distance is small through which material must be transported from any part of the
surface to the one large vacuole, but with growth of the cell the distance increases,
and, moreover, the volume of the cell and presumably, the rate of fat metabolism,
increase even faster—as the cube of the linear distance. A number of relatively
small vacuoles dispersed through the cell’s substance, and especially near its
surface, where the material for fat production must be received from the blood,
will furnish more surface and distribute this surface more effectively for the
accretion of fat than one large one, and as the rate of fat production is increased
small vacuoles will be condensed before the material can be transferred to the
original large one. Moreover, the ratio of surface to volume would be greater
in many small vacuoles then in one large one, and the small vacuoles would conse-
quently ‘grow’ faster, which would make for uniformity of size. The anatomical
developments satisfy this hypothesis, without demonstrating its finality. As
the cell diameter increases, the fat tends to be deposited in smaller and more
peripheral vaculoes, and especially in the queen larva, where the development is
most rapid and the cells are largest, the fat-vacuoles are both relatively and
actually smaller than in the worker. Differences in consistence of the cytoplasm
may also effect the size of vacuoles.
HISTOLYSIS OF FAT-BODY OF APIS 583
ends of this elongated nuclear area, appear dark staining granules
identical in size and shape, at first at least, with the nuclear
granules, and exhibiting certain of the staining reactions of these.
An interpretation may here be anticipated; that these bodies
are identical with the larger chromatic or basophile granules of
the nucleus; that they leave the nucleus and invade the cytoplasm
when the nuclear wall disintegrates, or becomes permeable to
them; that their subsequent activity may be in part at least an
enzymatic one, and is certainly concerned with the further me-
tabolism of the stored fat of the cell.
The detailed anatomy of this stage merits closer scrutiny
(pl. 1, figs. 5, 6, 7). Several facts are apparent. First, vacuoles
from the peripheral ring may be traced passing in toward the
nucleus, through the densely staining cytoplasm surrounding it;
and the appearance of basophile granules in the cytoplasm coin-
cides with the disappearance of the distinct outline of the nuclear
vesicle—may, in fact, shortly precede the indentation of the
nucleus by fat-vacuoles. This picture is so constant and so
characteristic of whole sections, when it occurs at all, that it
evidently signalizes an important crisis of the metabolic activity
of the larva itself. The series of changes follows the cessation
of feeding, and precedes the transformation of larva to pupa,
so immediately, that their interpretation must be correlated with
the process of metamorphosis as a whole. If other forms which
have been worked upon exhibit the same phenomena, the failure
of the workers handling them to demonstrate this change may be
accounted for by the abruptness and rapidity with which the
cell is transformed from one relatively permanentstatetoanother.
The first sign of the transformation is the diffusion of the nu-
clear wall and the elongation of the nucleus in the long axis of
the oval. The line of demarcation between nucleus and cyto-
plasm does not at once vanish, but gradually blurs, as if the
substance of the membrane were partially dissolved by the
material on either side of it, or as if a membrane, formed by sur-
face tension between two non-soluble substances were obliterated
by their becoming soluble. This blurring is most pronounced
at the ends of the nucleus, and here a little later the transition
584 GEORGE H. BISHOP
from nucleus to cytoplasm becomes least abrupt. The margins
of the central vacuoles take the stain more sharply than the ad-
jacent cytoplasm—a conditon not obtaining for the periphral
vacuoles—and they lie so close to one another as virtually to
form a reénforcement to the diffuse nuclear wall, which appears
to follow their contour and fill their interstices. The heavier
stain in their margins may be due to the presence of the sub-
stance of the nuclear vesicle in their surface films. They appear
to compress the nucleus, which elongates to two or three trans-
verse diameters. The cytoplasm of the central portion, sur-
rounding these central vacuoles, takes the stain more heavily
than that surrounding what peripheral vacuoles still persist.?
The cell is now in the condition represented by figure D.
Material at this stage shows the basophile granules not only just
outside the nucleus, but’precisely in the areas at the ends of the
nucleus from which the nuclear wall has disappeared (pl. 1, figs.
8, 9). The diffuse structure and pronounced staining reaction
of the region renders difficult the exact location of these granules
with respect to the blurred residuum of the nuclear wall. The
appearance occasionally is that of a gap pushed outward through
the wall between nucleus and cytoplasm, flanked on either side
by the fat-globules; the granules contained in the nucleus are
escaping through the opening.
The granules in the cytoplasm increase in number and become
evenly dispersed from nucleus to cell-wall. They are not con-
fined to the regions of the cells near the ends of the nucleus where
they first appear. They seem also to have passed out laterally
between the vacuoles in considerable number. These granules
enlarge to spherical globules, and at the first the largest lie well
2 What the difference is between the central and peripheral cytoplasm is not
clear, but the different aspect seems to be due to the. different distribution of
globular or finely emulsified fat. If it were demonstrated that the fat were synthe-
sized in this region next the cell wall, and deposited in the cytoplasm in the form
of an emulsion, from which it condensed as the fat-content increased to droplets
which grew by accretion into vacuoles, then the lighter staining reaction might
be assigned to the fine dispersal of fat in the cytoplasm peripherally. This may
be a partial explanation, but, as will appear from later consideration of nuclear
activity, this central cytoplasm seems to be influenced also by the nucleus more
pronouncedly than the peripheral.
=
HISTOLYSIS OF FAT-BODY OF APIS 585
toward the periphery of the cell, where one might expect to find
those which had earliest left the nuclear region. The central
fat-vacuoles also recede from the nucleus, and become dispersed
among the enlarging granules. The nuclear membrane gradually
reappears, as the vacuoles leave the region of the nucleus; but
the latter does not reassume its former oval shape; it becomes
even more attenuated, sometimes so extended that if straightened,
it might touch the opposite sides of the cell. The ends of the
nucleus are the last regions to be enclosed. Often these may be
seen open in a nucleus of a much later stage of the cell, with small
dark-staining granules near the aperture (text fig. E). Cells
may be observed in which, instead of two ends, three or more
areas of a nucleus appear to have opened out; and a plane section
would fail to reveal the extent of this radiate condition of the
nucleus in a large proportion of the times it might exist. In the
queen pupa, a multipolar extravasation of the nuclear granules
is the rule.
Stage E. The result of these changes is seen in figure 5. After
the previous stage the fat cell does not increase in size. Once
the nuclear wall is reformed there is no further visible evidence
of activity within the nucleus. The wall stains much more
sharply than before its dissolution. The large basophile granules
are still present in considerable numbers, but the extreme dis-
tortion of the nucleus makes difficult a comparison with previous
stages as to its size or content. The finer basophile granules are
more numerous; relatively to the number of the larger ones which
remain in the nucleus, than before the transformation.
Outside the nucleus, however, the granules undergo a definite
development. They enlarge, staining less deeply with basic
dyes as size increases, and finally taking an acid stain, until,
with the same staining technique as before, the cytoplasmic dye
absorbed is often more prominent than the nuclear. They
finally become (typically) vacuolated spheres, the granular
peripheral shells of which stain slightly darker than the cytoplasm
of cells in previous stages, and the centers of which often appear
to be dissolved out in preparation much as the fat-globules are.
In the meantime both the cytoplasm and most of the vacuoles
586 GEORGE H. BISHOP
contained in it disappear. Since the spheres which develop
from the basophile granules fill the cell, the unavoidable conclu-
sion is that both cytoplasm and cytoplasmic fat-vacuoles are ab-
sorbed as material for the growth of the spheres (pl. 2, figs. 14, 16).
Further evidence is adduced from the cytoplasmic staining
reaction of these spheres, and, finally, the peripheral shell, and
especially the inside margin of it, is blackened by osmic acid,
indicating the presence of fat.
Stage E late. Figure F shows the later stage of the process
which brought about the condition pictured in figure E. A few
fat-globules are still present, and a few basophile granules are stiil
in early stages of development. The nucleus has the same aspect
as before, except that while earlier stages often show the ends of
the nucleus open, in this later stage the nuclear membrane is
always intact. The rest of the cell is occupied by the spheres
developed from the basophile granules, the interstices of which
are filled by a very light-staining cytoplasmic matrix (pl. 2,
figs. 14 to 16). |
As the pupa takes on the form of the imago and its tissues
demand food material for imaginal development, the trophocytes
proceed to the final stages of development and disintegration.
The cytoplasmic matrix stains less and less densely, and is re-
placed by the growing spheres, until only a clear plasma remains
between the latter. The cells become loosened from each other,
and round up from a polyhedral to a spherical form, while the
interstices so formed fill with lymph from the body fluid. The
cells subsequently float free in the body cavity. Finally the cell
wall itself dissolves or disintegrates (pl. 2, fig. 14), and the
spherical globules are released, to dissolve eventually and lose
their integrity in the body fluid.
The nuclear membrane. The behavior of the nuclear mem-
brane in this process is particularly striking. The nuclear plasma
in these fat-body cells appears to become fixed into an exceedingly
fine coagulum, so fine that its aggregations cannot be distin-
guished under the microscope. It thus gives the appearance
of a lightly staining, but entirely homogeneous mass, which
tends to take a basic stain as the change in form comes on. The
HISTOLYSIS OF FAT-BODY OF APIS 587
peripheral cytoplasmic mass (in the interstices of the fat-vacuoles)
precipitates upon fixation to a finely reticulate network, the clear
interstices of which are large enough to be distinguished under
the high power (pl. 1, fig. 3). The central or perinuclear cyto-
plasm fixes to a finer granulation, not so fine as the nucleoplasm,
but more homogeneous than the peripheral, and considerably
Text fig. KE Fat-tissue cell, stage E—early pupa—characterized by the reforma-
tion of the nuclear membrane, growth of the nuclear granules to albuminoid glob-
ules, disappearance of the fat-vacuoles, and resolution of the cytoplasmic
matrix,
Text fig. F Fat-tissue cell, stage F—cells ready to disintegrate, medium-
stage pupa; albuminoid globules matured, centers of globules acidophile, peripheral
granules in their walls feebly basophile, most of the cell cytoplasm absorbed by
the globules.
more densely staining. Some of the peripheral cytoplasm may
be carried centrally as the fat-globules approach the nucleus,
especially in cells of the queen larva (pl. 1, figs. 4,8). With deli-
cate staining the membrane separating these two masses, of
nucleus and cytoplasm, has no discernible structure or organiza-
tion, but appears to be merely a phase border between two im-
miscible fluids. The nucleus maintains the globular shape
JOURNAL OF MORPHOLOGY, VOL. 36, No. 4
588 GEORGE H. BISHOP
characteristic of such a condition as long as this state of the
membrane obtains: The first evidence of a change in the nucleus
appears as a slight modification in the condition of this phase
differenecee—a modification which is noticeable microscopically
as a slight thickening, a more cloudy staining, and less sharp
definition. This takes place even before the fat-globules reach
the nuclear region (stage of fig. 3, pl. 1). As the process goes
further, the membrane may finally be completely dissipated, and
no residue left that can be distinguished as the material of which
it was composed. The border-ground between nucleus and cyto-
plasm then grades imperceptibly from one to the other, staining
only a little more densely where the two materials appear to
diffuse; as if each retained its intrinsic staining capacity, and
the resulting stain was an additive effect of the characterisic
staining of both. This border-ground now appears to offer no
resistance to the passage of the basophile granules, which are
found indiscriminately on either side and within the region
where the two materials are diffused (pl. 1, figs. 5, 6, 7).
Along the sides of the nucleus it is exceedingly difficult to
discern just what the state of the border is, for here the central
fat vacuoles which indent the nucleus complicate the picture.
The surfaces of these vacuoles (or more accurately, the surface
of the material surrounding the vacuoles) stain more sharply
and more densely than the surfaces of vacuoles situated more
peripherally in the cells; and since these surfaces lose their sharp
staining capacity as the vacuoles disperse throughout the cyto-
plasm, it may be inferred that they are enveloped by a film con-
sisting of a mixture of the nuclear sap and cytoplasm, and
that the staining of this film is in reality a stain of the same
nucleus-cytoplasm complex as was the staining of the nuclear
membrane itself. The conditions causing a phase membrane
to disintegrate from the nucleus-cytoplasm surface might affect
the surface of the fat-vacuoles more tardily, and these might still
8 This supposition is of course somewhat hypothetical. Different proteins, in
colloid form, are affected differently by changes of acidity, some becoming more
and some less hydrophilic. A change in acidity of one or another in turn would
cause a change in the respective viscosities, which might be conceived to result in
membrane formation and other complicated physical phenomena, even such as
HISTOLYSIS OF FAT-BODY OF APIS 589
give evidence of the membrane after the nuclear membrane in
their interstices had become pervious to the basophile granules.
But microscopically this condition cannot be demonstrated,
except by inference from the presence of granules immediately
outside the layer of fat-vacuoles.
Conditions in the metabolic activity of the cell may be pictured,
e.g., cutting off of the nutrient supply to the cell with cessation
of larval nutrition, or possibly some regulatory mechanism in
the cell itself, which would at this specific stage of development
cause the constituents of nucleus and cytoplasm to approach
such a degree of acidity, say, as would cause them to approach
each other in fluidity. A membrane formed due to their previous
difference in consistency would then be destroyed, and the two
colloids might diffuse in the region of this surface. The same
force which in the intact condition of the nuclear wall prevents
the nuclear granules from fusing or agglutinating—apparently
some repellent force, such as like electrical charge—would upon
equalization of the physical conditions within and without the
nucleus cause a dispersal of the granules throughout the cell.
This dispersal would go only so far as to render the granules
equally numerous per volume within and without the nuclear
area; that is, not all the granules would pass into the cytoplasm,
but the process would cease when the granules were about equally
dispersed throughout the cell. It is worth noting that
this is approximately the case; moreover, that the granules tend
to remain for a time within the more central densely staining
cytoplasm—although the immediate change in the character
of the granules as they leave the nuclear area and commence
to absorb cytoplasmic substances renders precarious too strict
an interpretation of the appearances.
d. Variations from type. The above description of the fat-
body-cell development is based upon a study of, and applies most
accurately to, the cells of the abdominal region of the worker
may be thought of as taking place between nucleus and cytoplasm. In the case
of the fat vacuoles this explanation does not call for a destruction of their surface
membranes, for the fat does not become more soluble in the cell protoplasm than
it was before. A condensation of material at the surface of the fat-vacuole might
account for its heavy staining capacity.
590 GEORGE H. BISHOP
larva and pupa. ‘This cell is chosen as a type because its develop-
ment not only includes all the fundamental phenomena of the
other forms, but also because most of these phenomena are here
displayed in an orderly fashion. The process may, in fact
however, be modified in three respects, depending apparently,
Ist) on the rapidity with which the change from larva to pupa
and from pupa to imago takes place; 2nd) on the sex of the larva,
and, 3rd) on the locus, in the body, of the cell under considera-
tion. Perhaps these divergencies from what have been described
as the typical process may be correlated to a considerable extent
with the nature of the food supply of the respective larva on
the one hand, and on the other, with the demand for tissue-
building materials, made by the imaginal tissues on the larval
fat-body. More specifically, the difference, 1st) in the rate of the
change which takes place in the fat-body cells, at the time of
pupation, of worker and queen larva, respectively, seems to be
correlated with the difference in the total time of development
required by these forms (seventeen days for the queen, twenty-
one for the worker), which is again usually assigned to a
difference in larval feeding; the difference, 2nd) in the aspects of
larval cells in the fat-bodies of the different sexes (male and
female), seems to be due chiefly to a difference in the proportion
of fat stored in them, which again is probably correlated with the
difference in the food supplied to the larvae of the different sexes;
while, 3rd), the ‘precocious degeneration,’ noted by Perez, of
the cells of the thoracic region, which go to pieces before those
of the abdominal region, and before all the albuminoid globules
contained in them are fully developed, might reasonably be
assigned, in the light of the theory of reversible enzyme activity
in cell metabolism, to the earlier and more rapid exhaustion of
the end-products of katabolic enzyme activity in the fat-cells,
by the earlier development of the bulky thoracic muscle masses.
(This conception approaches, but is not identical with the
‘lyocytosis’ of Anglas.) The nature of these three modifications
of the typical process will be described.
Difference between queen and worker. In the queen larva the
disintegration of the nucleus of the fat-body cell and the dispersal
HISTOLYSIS OF FAT-BODY OF APIS 591
of the basophile granules into the cytoplasm is considerably more
striking, more abrupt, and in a sense more violent than in the
worker. The nucleus, instead of being enclosed by fat-vacuoles
in all but two, or a few regions literally sprawls all over the cell,
and sends out what might be described as trabecular processes in
every direction even as far as the periphery. The fat-vacuoles
are also smaller, both relatively and actually, than in the worker.
The nuclear granules are carried with these trabeculae pretty
evenly throughout the cell, not only in a few directions or from
a few poles of the nucleus as in the cell of the worker larva.!
(pl. 3). The appearance is almost that of an explosive phenom-
enon, and the result is that the granules are not only more im-
mediately dispersed through the cytoplasm, but ali begin
their development at about the same time (pl. 1, fig. 8), and they
tend to remain more nearly the same size throughout the cell’s
existence. The diffusion of cytoplasmic and nuclear material is
also more extensive in the queen larva. Instead of three quite
clearly defined zones, consisting of peripheral cytoplasm lightly
staining and interspersed with fat-globules, central cytoplasm
more densely staining, and nuclear sap, there are now two regions
irregularly disposed, consisting, respectively, of the peripheral
cytoplasm and its fat-globules and the central cytoplasm and
nuclear sap interdiffused.* The nuclear granules do not remain
in the denser region; the two cytoplasmic areas gradually diffuse.
The nucleus reéstablishes itself out of the diffused mass into an
irregular many-processed body containing the typical chromatic
material in a clear lightly staining medium. ©The later stages
approach very closely the later stages of the worker pupae®
(ple Defies. 15, 16):
4 Plate 3 shows twenty cells from one-half of a single cross-section of a queen
larva, just in the stage of nuclear dispersion. The nuclear area, shaded black,
can here be distinguished by the methylene-blue stain, though eosin stains nucleus
and cytoplasm both.
5 The granules of the peripheral trabeculae, outside the nucleus proper, stain
less densely with nuclear stains (pl. 3).
§ The exact nature of the difference in food which might occasion this difference
in metabolic rate has not been investigated in detail. It is known that the queen
larva is fed during its whole feeding period upon the so-called ‘royal jelly,’ a
partially digested compound of fairly constant proportions of fat, carbohydrate,
592 GEORGE H. BISHOP
Difference in queen and drone. The difference in the aspects
of the fat-body of queen and drone is exhibited chiefly in a lesser
portion of visible fat-content in the male. This may be a result
of a difference in food. ‘The drone, like the worker larva, is fed
at first on partially digested food, but later receives considerable
crude pollen. A high percentage of protein, and particularly of
nuclein-forming materials, such as pollen yields, may be con-
sidered necessary in the drone’s diet to provide material for
building up the testes, which shortly before emergence of the
drone have displaced the fat-body, and nearly fill the large ab-
dominal cavity. At the termination of the larval ingestion of
food these organs are present, but slightly developed. Their
growth during pupation must be at the expense of the fat-body,
which tissue may be expected to have stored up the proper
nutrient elements in the proper proportions for that development.
The testes, large as they are, must demand a higher proportion
of nuclein-forming materials for the development of their sperms
than any comparable organs in the worker or queen pupae.
Difference in different body regions. No significant difference
in the development of the fat-body cells of thorax and abdomen
is discernible until after the disintegration and reforming of the
nucleus, and the partial development of the albuminoid globules.
At a relatively late period in this development, but before all the
globules of the cells concerned have attained the final structure
and staining capacity of the typical cell contents, scattering
and protein, prepared in the midintestine of the young workers or nurse bees.
The worker larvae are fed this material the first three days of their life, after
which considerable undigested pollen and honey is added. Investigators have
been unable so far to assign any other difference in treatment as a necessary
cause of the different development of the worker, and this cause seems to be a
sufficient one. The question remains whether this difference in the effect of the
different foods is due to partial digestion merely or to the extraction or modifica-
tion of some constituent of the crude pollen, or honey, which, when fed the worker
larva without modification, retards or modifies its development. Considering
the fact that pollen,the chief protein-containing constituent of the larval food,
contains a high percentage of nuclein, it seems possible that some constituent
of nucleic acid, such as purines, may be modified or extracted from the queen’s
food, and left in the larval metabolism of the worker to modify the development
of the imago.
HISTOLYSIS OF FAT-BODY OF APIS 993
cells, more numerous in the thoracic region where they are
destined to be replaced by the thoracic muscles, undergo a
‘precocious degeneration,’ as stated by Perez. This change com-
prises in the bee larva the dissolution of the cell wall and the
release of the cell contents into the blood space. Those globules
which appear not fully developed still stain with the nuclear
dyes, and especially with Heidenhain’s haematoxylin; they are
still small in size and compact in structure, without the vacuo-
lated center which seems to be characteristic of a late stage of the
normal development. This precocious change is apparently not
associated in the bee with the presence of any unusual cellular
element such as the leucocyte, nor of any condition other than
the early development of large masses of tissue in this region.
3. The metabolic significance of the changes in structure
Tracing the fate of the larval food through the nutritive
mechanism, the following résumé may serve to correlate the
nutritive process with the cellular metamorphosis.
The partially digested food of the early larva, the ‘royal
jelly’ elaborated by the worker bees, contains carbohydrate, fat,
and protein. This special food the queen larva receives all during
larval growth; after the third day the worker is fed considerable
amounts of honey and undigested pollen, and the male still
larger proportions of pollen. The fat and practically all of the
carbohydrates taken up by the fat-body are stored as fat-droplets?
until, at the beginning of metamorphosis, these droplets are
worked over into the so-called albuminoid globules developed
from granules arising from the nucleus.
Since there is very little protein in honey, the bulk of the
nitrogeneous food comes from pollen, chiefly in the form of nucleo-
proteids. These are presumably stored up as nucleoproteids in
nuclear chromatin and the chromatoid granules, and as more
simple proteins in the acidophile cytoplasmic matrix. In the
7 Nakahara reports glycogen in the developing fat-cells of Pieris demonstrated
by Gage’s methods. Glycogen could not be demonstrated in vitro in these tissues
by the ordinary chemical test of caustic hydrolysis and treatment with iodine.
594 GEORGE H. BISHOP
development of the albuminoid granules the chromatoid granules
of the nucleus, the protein of the cytoplasm, and the fat of the
vacuoles are all utilized, and merged into a form where the
different constituents are not only different from the former cell
constituents, but also can no longer be distinguished from each
other by staining reactions. The conclusion can be drawn,
however, that these albuminoid globules represent the cell element
in which the chemical transformations take place by which all
the cell constituents (except possibly the residual nucleus)
undergo chemical reorganization in preparation for tissue use.
In the following section of this paper an interpretation of the
changes in structure and in staining reaction of the cell elements
will be undertaken. A bibliography there included will also
cover the first section.
|
ab
PLATE 1
EXPLANATION OF FIGURES
Fat-tissue cells from larvae of the honey-bee. 330
Figures 7 and 9, X 1000
Figures 1, 2, 3, 6, and 7, iron-alum haematoxylin; figure 4, safranin-gentian
violet mordanted with Gram’s solution; figure 5, iron-alum haematoxylin and
eosin; figures 8 and 9, iron-alum haematoxylin and safranin.
1 Worker bee larva fat-tissue cell, early larval stage B, showing nucleus
pushed aside by one large fat-vacuole, with others forming.
2 Same as above, stage later, C, peripheral ring of fat-vacuoles forming,
nucleus in center undistorted.
3 Queen bee larva fat-tissue cell, late larva, stage C; pressed out of shape at
the edge of a mass of cells, cut a little at one side of center. A precocious scatter-
ing of nuclear granules is taking place, before the fat-vacuoles have reached the
central nuclear area.
4 An attenuated cell of early prepupal stage, D, queen larva, showing nuclear
dispersion of granules, and their developemnt into globules. In other sections
the granules may be seen in passage from the sides of the nucleus as well as from
the ends. The nuclear vesicle is apparently beginning to reform here along the
sides.
5 Slightly earlier phase than above of stage D, transforming queen larva,
nucleus actively dispersing granules of basophile material, ring of fat-vacuoles
pressing it centrally. A more spherical cell would show trabeculae of densely
staining material at other regions than the ends of the cell nuclei. Long and
narrow cells are chosen here for simplicity and definition of the cell conditions.
6 and 7 early prepupal stage D, worker larva, cross-section of a cell of the
shape shown in figure 5, cut through one end of the nucleus, and an enlarged drawing
of the central region of the same. Centrally, the nuclear area still contains large
granules, interspersed with many smaller ones, and the whole is surrounded by a
characteristic ring of fat-vacuoles, with very sharply defined walls. Through
the interstices of these vacuoles nuclear granules are still passing. Peripherally
to this again, the central cytoplasm extends out into many small trabeculae through
the peripheral lightly staining cytoplasm, to the marginal region of the cell.
8 Cell from a queen larva, same stage as above, longitudinal section, and
figure 9, a higher magnification of one end of its nuclear region. The typical
condition of smaller fat-vacuoles and a more violent dispersion of granules, with
more attenuated and numerous trabeculae, is characteristic of the queen larva
as compared with the worker. Most of the cells of the queen larva are even
more complex. Here again, as above, an elongated bi-polar cell was chosen,
for comparison with the typical bipolar cell of the worker. Extreme destaining
has obliterated the finer nuclear structures.
596
PLATE 1
S OF FAT-BODY OF APIS
GEORGE H. BISHOP
SI
HISTOLY
597
PLATE 2
EXPLANATION OF FIGURES
Fat-tissue cells from pupae of the honey-bee. > 330
Figures 11, 13, and 15, safranin-gentian violet with Gram/’s solution; figure
10, polychrome methylene blue and eosin; figures 12, 14, and 16, iron-alum haema-
toxylin and eosin.
10 Late stage D, worker prepupa, nuclear vesicle reforming. The nuclear
wall stains sharply with methylene blue; granules and globules of nuclear origin
in the cytoplasm fix eosin after they pass the nuclear border, and methylene blue
as long as they are within it. The two cells show the two most typical shapes of
cell and nucleus of the worker larvae, 1.e., bipolar and tripolar.
11 Stage E, early queén pupa, multipolar nucleus, with reformed wall,
albuminoid globules all in about the same stage of development though of different
sizes.
12 Intermediate stage of albuminoid globule formation, stage E, early worker
pupa. Granules of different sizes and different stages of development in the same
~ cell, presumably due to succesive emission from the nucleus.
13 Same as figure 12, different stain.
14 Unusually large cell from a worker pupa, stage E to F, undergoing dissolu-
tion of the cell wall—at upper left hand—before complete elaboration of its
albuminoid reserves.
15 Another cell from the same slide as figure 14, less than 2 mm. away, on
opposite side of the intestine; both are of the same size and the same distance
from the surface of the tissue as fixed, and both are in the layer of cells next the
intestine. The difference is a metabolic one, of unknown causation. In figure
15 may be seen two leucocytes, but no actual leucocytic adherence to or attack on
fat-cells could be discerned here. The fat-cells had become loosened from each
other and had rounded up, and were nearly surrounded by the body fluids.
16 One of the smaller cells from a queen pupa, showing conditions more typ-
ical of the worker pupa (figs. 12 and 13) in greater degree of diversity of develop-
ment of globules, fewer number of them, etc., than the larger cells of the worker
pupa tissue itself exhibits (figs. 14 and 15). The probable explanation of this is
that the difference in development depends on metabolic rate of nutrition, higher
in general in the queen larva, and that the smaller cells from the queen larva
or pupa developed more slowly, due to isolation from nutriment, etc., than the
best nourished of the worker larva’s cells.
PLATE 2
HISTOLYSIS OF FAT-BODY OF APIS
GEORGE H. BISHOP
599
PLATE 3
EXPLANATION OF FIGURES
Cell shape and nuclear transformations
Figures 17-36, X 165. Stain, methylene blue and eosin.
All these drawings were taken from one-half of one-cross section of a queen
larva whose fat-cell nuclei were just in the act of dispersing their nuclear granules
into the cytoplasm. The black areas are those in which the nuclear granules were
stained by methylene blue, the stippled regions are the areas of dense nucleo-
cytoplasmic trabeculae through which the granules are dispersing from the nuclei;
this stains deeply red with eosin. The clear areas are the regions of lighter
stained cytoplasm containing fat-vacuoles.
This plate is designed to show the relations of these three regions in the cells
of this stage; to demonstrate the relation between their disposition and the cells’
shape, and to present evidence bearing on the mechanics of the process of nuclear
extravasation and dispersal of basophile granules. Only those cells were drawn
which appeared to be cut almost exactly through the median plane of the nucleus,
and in the plane of greatest nuclear extravasation, through other trabeculae than
those figured of course extended above and below the plane of the section.
In figures 17-21 are indicated variations from the bipolar to the tripolar
type of cell; in figures 22-25 from bipolar to hexapolar; in figures 27-31, from
bipolar to asymmetrical tripolar, or possibly multipolar, and in figures 32-36
variations from bipolar to tetrapolar cells. Each cell exhibits that type of
nuclear distortion which will most effectively distribute the nuclear granules
throughout the cytoplasm of a cell of that particular shape.
Comparing figure 3, plate 1. which is of a cell before this stage, but shows the
beginnings of it, it is apparent that the central cytoplasm and nucleus both assume
approximately the shape of the cell outline even before the fat-vacuoles approach
the nucleus. This may be considered a predisposing factor in directing the tra-
beculae later, and may be taken to indicate incidentally that nucleoplasm and cyto-
plasm are so little different in density or consistency that the distortion by fat-
vacuoles affects the former against whatever surface tension the nuclear membrane
may exhibit, tending to form the nucleus into a sphere. When the fat-vacuoles
later move toward the nucleus, and the larger of these press in through the central
cytoplasm toward the vesicle, they may be the active agents in pressing the
nuclear mass further out of shape; their effect is presumably augmented, however,
by a centripetal tendency on the part of the larger nuclear granules. Both these
forces seem to be occasioned by the cessation of larval nutrition, and their nature
is not clear. It is fairly certain that the nucleoplasm itself does not disperse
with the granules, from evidence of the staining reaction of the granules them-
selves as they pass into the cytoplasm. The dispersing forces must lie in the
granules themselves, conceivably a like static charge, for instance.
HISTOLYSIS OF FAT-BODY OF APIS PLATE 3
GEORGE H. BISHOP
601
SUBJECT AND AUTHOR INDEX
MAROUCIUM constellatum (Verrill). IT.
The structure and organization of the
tadpolelarvyaveeeer: olsen s- sen. es 71
Amitosis in the ciliated cells of the gill fila-
WHEHtS Ol Cy Clagieeeer pss sence c was esses 103
Apis mellifica. Cell metabolism in the insect
fat-body. I. Cytological changes accom-
panying growth and histolysis of the fat-
Teli iG Gis Ae. ococite Os ods GSD Ue 567
Avanhnit: The circulatory system and seg-
USAIN Tene e Sooo On aoe ee 157
Bust Grrsta symmetry in the embryo
of Cryptobranchus allegheniensis. The
DANES Fie Hoeee se ene ie ao 357
BisHop, Groree H. Cell metabolism in the
insect fat-body. I. Cytological changes
accompanying growth and histolysis of the
fat-body of Apis mellifica................ 567
Block to normal development in cross-ferti-
lized eggs. I. Crosses with the egg of
Fundulus. II. Reciprocal crosses be-
tween Ctenolabrus and _ Prionotus.
JNEG Si tT) Pee Sao 5 are ere 0
Branchial derivatives in turtles............... 299
Ceres of Meemopsisn , Wher Sener. bce «. 495
Cell_metabolism in the insect fat-body. I.
Cytological changes accompanying growth
and histolysis of the parbody of Apis
mellifica. . 567
Cells of the gill filaments of Cy clas. Amitosis
PRAGHC\CUIAGE Es, tot wes eR, eaten: eet 103
Changes accompanying growth and histolysis
of the fat-body of Apis mellifica. Cell
metabolism in the insect fat-body. I.
OutOIDRICAl sete ne > EET ae mya chae 567
Characters of elasmobranch fishes—the
claspers, clasper siphons, and clasper
glands. Memoir III. The comparative
morphology of the secondary sexual...... 191
Characters of Holocephali and elasmobranch
fishes—the claspers, clasper siphons, and
clasper glands. Memoir IV. The com-
parative morphology of the secondary
ROXANE <revck tte Paste: oc) on ayo se eased ses 199
Sees of Holocephali and elasmobranch
fishes—the claspers, clasper siphons, and
clasper glands. Memoir V. The com-
parative morphology of the secondary
BEXTIBI SE «20.3 ost id orate athe s Aotcl ps oe a eete 221
Ciliated cells of the gill filaments of Cyclas.
mitosis in thes 2085 Seabee cet e ei deco eee
Circulatory system and segmentation . in
Arachnida: “The! ..ietsccdnce ayn eee 157
Claspers, clasper siphons, and clasper glands.
Memoir III. The comparative morphol-
ogy of the secondary sexual characters of
elasmobranch fishes—the................. 191
Claspers, clasper siphons, and clasper glands.
Memoir IV. The comparative morphol-
ogy of the secondary sexual characters of
Holocephali and elasmobranch fishes—the 199
Claspers, clasper siphons, and clasper glands.
Memoir V. The comparative morphology
of the secondary sexual characters of
Holocephali and elasmobranch fishes—the 221
Cloaca and cloacal glands of the male Necturus.
fe
Cloacal glands of the male Necturus. The
UIT2 14706 A rT. ot ie EES Sar ae 447
Crosses with the egg of Fundulus. II. Recip-
rocal crosses between Ctenolabrus and
Prionotus. The initial block to normal
development in cross-fertilized eggs. I... 401
Cross-fertilized eggs. I. Crosses with the egg
of Fundulus. II. Reciprocal crosses be-
tween Ctenolabrus and Prionotus. The
initial block to normal developmentin.... 401
Cryptobranchus allegheniensis. The origin
of bilateral symmetry in the embryo of... 357
Ctenolabrusand Prionotus. The initial block
to normal delvelopment in cross-fertilized
eggs. I. Crosses with the egg of Fundulus.
II. Reciprocal crosses between........... 401
Cyclas. Amitosis in the ciliated cells of the
PalealeMents Of, ...%..uer. os eeedt as ee oe 103
Cytological changes accompanying growthand
histolysis of the fat-body of Apis mellifica.
Cell metabolism in the insect fat-body.
i
Cytoplasmic inclusions in the egg of Echina-
PACHBENG ET; 15... Seem rete IS). 8 way tau 3 467
AWSON, Aupen B. The cloaca and
cloacal glands of the male Necturus..... 447
Derivatives inturtles. Branchial............ 299
Dermochelys. On the phylogeny of the shell
of the Testudinata and the relationships
Development in_ cross-fertilized eggs. I.
Crosses with the egg of Fundulus. II.
Reciprocal crosses between Ctenolabrus
and Prionotus. The initial block to
normal .
Development of Paracopidosomopsis.
Development of the light-organs of Photurus
pennsylvanica De Geer. Originand..... 245
Division of Trichomonas muris (Hartmann).
eRMestructyre andes .cveces vais sss abies. « 119
CHINARACHNIUS parma. Cytoplas-
mic inclusions in theeggof.............. 467
Egg of Echinarachnius parma.
PM OMINONS My CHS, a erS clases Wapsh ois *
Eggs. I. Crosses with the egg of F undulus.
II. Reciprocal crosses between Cteno-
labrusand Prionotus. The initial block to
normal development in cross-fertilized.... 401
Elasmobranch fishes—the claspers, clasper
siphons, and clasper glands. Memoir III.
The comparative morphology of the
secondary sexual characters of............ 191
Elasmobranch fishes—the claspers siphons,
and clasper glands. Memoir IV. The
comparative morphology of the secondary
sexual characters of Holocephaliand...... 199
603
JOURNAL OF MORPHOLOGY, VOL. 36, NO. 4
Elasmobranch fishes—the claspers, clasper
siphons, and clasper glands. Memoir V.
The comparative morphology of the
secondary sexual characters of Holocephali
EUINCL va rey Eau eee wdca Rae aee ee ele oper 221
Embryo of Crytobranchus allegneniensis.
The origin of bilateralsymmetry inthe... 357
Emys europaea. Contribution to the mor-
phologie study of the thyreoid glandin... 279
AT-BODY. I. Cytological changes ac-
companying growth and histolysis of the
fat-body of Apis mellifica. Cell metabo-
lismamebpel sect, nh). cutie seca Pet moO)
Filaments of Cyclas. Amitosis in the ciliated
COs Ofphe CIE oo. jared ane tree ae 103
Fishes—the claspers, clasper siphons, and
clasper glands. Memoir III. The com-
parative morphology of the secondary sex-
ual characters of elasmobranch............ 191
———the claspers, clasper siphons, and clasper
glands. Memoir The comparative
morphology of the secondary sexual char-
acters of Holocephaliand elasmobranch. 199
——— the claspers, clasper siphons, and
clasper glands. Memoir V. The com-
parative morphology of the secondary
sexual characters of Holocephali and
elasmobranch 3s) seuss vice eech eaten e 221
Fundulus. II. Reciprocal crosses between
Ctenolabrus and Prionotus. The initial
block to normal development in cross-
fertilized eggs. I. Crosses with the egg of. 401
AMBLE, D. L. The morphology of the
ribs and transverse processes in Necturus
MAC UIAOUS shi: wee Sls oe ne ee 537
Gill filaments of Cyclas. Amitosis in the
ctlintedicellaofihes: sa sckesse renee. cee 103
Gland in Emys europaea. Contribution to
the morphologic study of the thyreoid.... 279
Glands. MemoirIII. The comparative mor-
phology of the secondary sexual characters
of elasmobranech fishes—the claspers,
clasper siphons, and clasper.............. 191
Memoir IV. The comparative mor-
phology of the secondary sexual characters
of Holocephali and elasmobranch fishes—
clasper siphons, and clasper.............. 199
———. Memoir V. The comparative mor-
phology of the secondary sexual characters
of Holocephali and elasmobranch fishes—
the claspers,clasper siphons, and clasper.. 221
Glands of the male Necturus. The cloaca and
Close gs om aks tecord Reena he Ree een 447
GRAVE, Caswetu. Amaroucium constella-
tum (Verrill). II. The structure and
organization of the tadpole larva......... 71
Growth and histolysis of the fat-body of Apis
mellifica. Cell metabolism in the insect
fat-body. I. Cytological changes accom-
paying). cea die eta cack oor tan knee 567
H*:: Oxtver P. On the phylogeny of the
shell of the Testudinata and the rela-
tionships of Dermochelys ............. 421
Head segmentation. Primary neuromeres
DAE EE Dee RE NE NSS Seen om 331
HELVESTINE, JR., FRANK. Amitosis in the
ciliated cells of the gill filaments of Cyclas. 103
Hess, Watrer N. Origin and development
of the light-organs of Photurus pennsyl-
Vanicea DeGeersl? ke Do ae meee 245
Hisparp, Hope. _ Cytoplasmic inclusions in
the egg of Echinarachnius parma........ 467
Histolysis of the fat-body of Apis mellifica.
Cell metabolism in theinsect fat-body. I.
Cyiclagiat changes accompanying growth
PD och ae Ree ae cece nee pie eae
INDEX
Holocephali and elasmobranch fishes—the
claspers, _clasper siphons, and clasper
glands. Memoir IV. The comparative
morphology of the secondary sexual
characters of ...... ore per tiiss7 oe 199
and elasmobranch fishes—the claspers,
clasper siphons, and clasper glands.
Memoir V. The comparative morphology
of the secondary sexual characters of...... 221
NCLUSIONS in the egg of Echinarachnius
parma Oy toplasmichr.tue. 1) eee eee 467
Insect fat-body. I. Cytological changes ac-
companying growth and histolysis of the
fat-body of Apis mellifica. Cell metabo-
lism in. chet. 2... seas soc sehee eee 567
OHNSON, Cuaries Evcene. Branchial
derivativesiniturtles). ‘fice yen 299
ARVA. Amaroucium constellatum (Ver-
rill). IL. The structure and organization
of. the-tadpole set. Aine a eee 71
LeIcH-SHARPE, W.Haroutp. The compara-
tive morphology of the secondary sexual
characters of elasmobranch fishes—the
claspers, clasper siphons, and clasper
Plands. Memormlitiha.:- ce. sane 191
The comparative morphology of the
secondary sexual characters of Holoceph-
ali and elasmobranch fishes—the claspers,
clasper siphons, and clasper glands. Me-
TOWLE V6 jasc on. o oo hacoRte ye afeeie ate koe AE 199
The comparative morphology of the
secondary sexual characters of Holocephali
and elasmobranch fishes—the claspers,
clasper siphons, and clasper glands.
Memoir, Vi. 25 cist: et cee eee 221
Light-organs of Photurus pennsylvanica
De Geer. Origin and development of the 245
ALE Necturus. The cloaca and cloacal
glands ofthe. 22 ...4 50h sehen ee aes 447
Memoir III. The comparative morphology
of the secondary sexual characters of
elasmobranch fishes—the claspers, clasper
siphons, and clasper glands............... 191
Memoir IV. The comparative morphology of
thesecondary sexual characters of Holo-
cephali and elasmobranch fishes—the
claspers, clasper siphons, and clasper
elamd ey! (cee yeh os ie ee 199
Memoir V. The comparative morphology of
secondary sexual characters of Holocephali
and elasmobranch fishes—the claspers,
clasper siphons, and clasper glands........ 221
Metabolism in the insect fat-body, I.
Cytological changes accompanying growth
and histolysis of the fat-body of Apis melli-
fies. @ellak 22S. oF. eR eet 567
Morphologic study of the thyreoid gland in
Emys europaea. Contribution to the.... 279
Morphology of the ribs and transverse proc-
esses in Necturus maculatus. The...... 537
Morphology of the secondary sexual characters
of elasmobranch fishes—the claspers,
clasper siphons, and clasper glands.
MemoirIII. The comparative........... 191
Morphology of the secondary sexual characters
of Holocephali and elasmobranch fishes—
the claspers, clasper siphons, and clasper
glands. Memoir IV. The comparative... 199
Morphology of the secondary sexual characters
of siphons, and clasper glands. Memoir
V2 ihe comparative see .ccween cree te 221
INDEX
Wea Sante. Contribution to the
morphologic study of the thyreoid gland
TEMS CULGPACAadas <= «ose. oes eee s 279
Necturus maculatus. The morphology of the
ribs and transverse processes in........... 537
Necturus. The cloaca and cloacal glands of
(UTES Pewee Om 8 oho ee 2 447
Neuromeres and head segmentation. Pri-
HG MRE eG Join So cao. IotOCGCU OCS TORE EEE 331
RGANIZATION of the tadpole larva.
Amaroucium constellatum (Verrill).
Ti Phe sirnetuneagid seer. cc ccsa sas 71
Origin and developmens of the light-organs
of Photurus pennsylvanica De Geer..... 245
2) Seren parte ance The develop-
PRET Gy OL pes eco RR se © eas S.0
Patterson, J. T. The development of
iPaALAcOpldGSOMIOPSISs2 ace kre awe: ea ne es. 1
PrerRUNKEVITCH, ALEXANDER. The circula-
tory system and segmentation in Arach-
BCL Cir Pe ET ey Ste ea Aes 2s 3 oo 157
Photurus pennsylvanica De Geer. Origin and
development of the light-organs of...... . 245
Phylogeny ofjthe shell of the Testudinata and
ihe elationahipe of Dermochelys. Onthe 421
Pinney, Epira. The initial block to normal
development in cross-fertilized eggs. I.
Crosses with the egg of Fundulus. II.
Reciprocal crosses between Ctenolabrus
and Pronotus.
Primary neuromeres and head segmentation. . 331
Prionotus. The initial block to normal
development in cross-fertilized eggs. I.
Crosses with the egg of Fundulus. II.
Reciprocal crosses between Ctenolabrus
BEST eaters Shei ces eck. acts Guest MAGIC IG aya alive 6,06 5.
Processes in Necturus maculatus. The
morphology of the ribs and transverse.... 537
ECIPROCA Lecrosses between Ctenolabrus
and Prionotus. The initial block to
normal development in cross-fertilized
eggs. I. Crosses with the egg of Fundu-
UVR di 9 = een oe 401
Ribs and transverse process in Necturus
maculatus. The morphology of the...... 537
ECONDARY sexual characters of elasmo-
branch fishes—the claspers, clasper
siphons, and clasper glands. Memoir III.
The comparative morphology of the:..... 191
Secondary sexual characters of Holocephali
and elasmobranch fishes--the claspers
clasper siphons, and clasper glands.
Memoir IV. The comparative morphol-
GP Va OlbNe enn ee cease 199
Seconday sexual characters of Holocephaliand
elasmobranch fishes—the claspers, clasper
piphony, and clasper glands. Memoir V.
Thecomparative morphology of the...... 221
Segmentation in Arachnida. The circulatory
PIV SLE FIP AIG ete Noe ek tee nhac fe cls a aicte eRe 157
Segmentation. Primar y neuromeres and
RERIGE”: =88 Son ccke OGRE eo AEE nE eee aer ne 331
605
Sexual characters of elasmobranch fishes—the
claspers, clasper siphons, and ciasper
glands. Memoir III. The comparative
morphology of the secondary............. 191
Sexual characters of Holocephali and elasmo-
branch fishes—the claspers, clasper
siphons, and clasper glands. Memoir IV.
The comparative morphology of the
SRCOMUAUM A ciicacs ay° ilalane ton: fod bane Sete 199
Sexual characters of Holocephali and elasmo-
branch fishes—the claspers, clasper
siphons, and clasper glands. Memoir V.
The comparative morphology of the
BECQMP AB Mcrae ts. Ween las oslo sacs cee 221
Shell of the A estudinata and the relationships
of Dermochelys. Onthephylogeny of the 421
Siphons, and clasper glands. Memoir III.
The comparative morphology of the
secondary sexual characters of elasmo-
branch fishes—the claspers, clasper....... 191
Siphons, and clasper glands. Memoir IV.
The comparative morphology of the
secondary sexual characters of Holocephali
and elasmobranch fishes—the celaspers,
(CLAS pre tamer eaves, cosas, Shee cittaala Sela 199
Siphons, and clasper glands. Memoir V.
The comparative morphology of the
secondary sexual characters of Holo-
cephali and elasmobranch fishes—the
GIRS HETSMOIASDCL. 2.50. . asks doce ucnaMaes 221
SmirH, Bertram G. The origin of bilateral
symmetry in the embryo of Crypto-
branchus allegheniensis................... 357
Structure and division of Trichomonas muris
(Barina: bhes. +c 88.8.0. 28 oe) oe 119
Structure and organization of the tadpole
larva. Amaroucium constellatum (Ver-
PULL) ULC eis las cw crac tee tate loon cutee 71
Study of the thyreoid gland in Emys europaea.
Contribution to the morphologic......... 279
StunKarp, Horace W. Primary neuromeres
and head segmentation................:.. 331
Symmetry in the embryo of Cryptobranchus
allegheniensis. The origin of bilateral.... 357
System and segmentation in Arachnida.
BeteImeMALOLY. occ ochre. eh cones 157
IADPOLE larva. Amaroucium constella-
tum (Verrill). IJ. The structure and
organization of the.
Mermop sis wue Castes Diy... eas wera: oot 495
Testudinata and the relationships of Dermo-
chelys. On the phylogeny of the shell of
the... 421
THOMPSON, ‘CAROLINE Burina. The castes
ObsWenmopnine ie iiss. oe ek eka oe 495
Thyreoid gland in Emys europaea. Contri-
bution to the morphologic study of the... 279
Transverse processes in Necturus maculatus.
The morphology of theribsand........... 537
Trichomonas muris (Hartmann). The struc-
ture and division of. . tReet Oe
Turtles. Branchial derivativesin............ 299
I Ya er teeiaee D. H. The structure and
division of Trichomonas muris (Hart-
TRLSULLY DO Fo cis &.o.0/e, 4) sla MAEM apa eee ares 119
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