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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
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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-
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- Arch. d. Zool. Exp. et Gen., T. 2, ser. 4, pp. 257-335.
1906 Recherches sur la biologie et le developpement des hymenopters
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gelechiae. Biol. Bull., vol. 29, pp. 333-373.
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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
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1919 Polyembryony and sex. Jour. Heredity, vol. 10, pp. 344-352.
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lized eggs. Biol. Bull., vol. 33, pp. 38-51.
PerRRIER, E., pt GRAvieR C. 1902 La tachygenese ou acceleration embryo-
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Proc. of Ent. Sco. of Phila., vol. 1, pp. 368-370.
44
J.) T. PATTERSON
SaRRA, RAFFAELLE 1915 Osservazioni biologiche -sull’ Anarsia lineatella Z.
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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 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.
58 100.
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. ee eens
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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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Se RE 4
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.
Aettn
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Saeatrihaet,
\
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
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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.
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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. af } \
co. Ola,
prone! RCL) Aull i oti" DSi hy Ae? eee) BF
rid 4 )
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et yvIon ere peritnl Lette
1.494 tnaeilli-y/. jtoduy
BP odio nui fl } jp oe Lae e| Lf 2 i a ee AT ithe
Orman.
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4
ry
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ivy
'
.
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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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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1917 The production of light by animals—The fireflies or Lampyridae.
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266 WALTER N. HESS
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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
BIBLIOGRAPHY
Batrour, F. M. 1878 A monograph on the development of elasmobranch
fishes. London.
Bosanus 1819-21 Anatome Testudinis europeae. Vilna.
Born, G. 1883 Ueber die Derivate der embryonalen Schlundbogen und Schlund-
spalten bei Siugethieren. Arch. mikr. Anat., Bd. 22.
Donrn, A. 1886 Studien, usw. VIII. Die Thyroidea bei Petromyzon, Am-
phioxus und den Tunicaten. Mitth. Zool. Sta. Neapel, Bd. 6.
1886-7 Studien, usw. XI. Thyreoidea und MHypobranchialrinne.
Mitth. Z.S. Neapel, Bd. 7.
FiscHetis, Pu. 1885 Beitrige zur Kenntnis der Entwickelungsgeschichte der
Glandula thyreoidea und Gl. Thymus. Arch. mik. Anat., Bd. 25.
Gorrrn, A. 1867 Beitrige zur Entwickelungsgeschichte des Darmkanals des
Hiinchens. Tiibingen.
1875 Die Entwickelungsgeschichte der Unke. Leipzig.
His, W. 1868 Untersuchungen iiber die erste Anlage des Wirbelthierleibes.
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1880-85 Anatomie menschlicher Embryonen. Leipzig.
1889 Schlundspalten und Thymusanlage. Arch. Anat. u. Physiol.,
Anat. Abth.
1891 Der Tractus thyreoglossus und seine Beziehung zur Zungenbein.
Arch. Anat. u. phys., Anat. Abth.
Huscukn, E. 1826 Ueber die Umbildung des Darmcanals und der Kiemen der
Froschquappen. Oken’s Isis, Bd. 1.
Jacopy,M. 1894 Ueber die mediane Schilddriisenanlage bei Saugern (Schwein).
Anat. Anz. Bd., 10.
1896 Ueber die Entwicklung der Nebendriisen, der Schilddriisen
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
Saugethieren Arch. f. mikr. Anat.,, Bd. 30.
1887 Das Schlundspaltengebiet des Hiinchens. Arch. Anat. u. Entw.,
Anat. Abth.
K6iircer, A. 1879 Entwickelungsgeschichte des Menschen und der héheren
Thiere. Leipzig.
1902 Handbuch der Gewebelehre des Menschen. Leipzig.
Livint, F. 1902 Organi del sistema timotiroideo nella Salamandrina perspicil-
lata. Arch. Ital. Anat. e Embriol., T. 1
Mavrer, F. 1885 Schilddriise und Thymus der. Teleostier. Morph. Jahrb.,
Bd. 11.
1888 Schilddriise, Thymus und Kiemenreste der Amphibien. Morph.
Jahrb., Bd. 18.
1898 Der Derivate der Schlundspalten bei der Eidechsen. Verh.
Anat. Gesellsch., Bd. 12.
1899 Schlundspalten Derivate von Echidna. Verh. Anat. Gesellsch..,
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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Mixiier, W. 1871 Ueber die Entwickelung der Schilddriise. Jena. Zeitsch.,
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Penpge, N. 1918 Endocrinologia. Milano.
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Wiirzburg.
Piatt, J. 1896 The development of the thyroid gland and of the supraperi-
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Prenant, A. 1894 Contribution a l’étude du développement organique et
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tidienne. La Cellule, T. 10.
1896 Elements d’embryologie de l’homme et des vertebrés. Paris.
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Van BEeMMELN, J. F. 1885 Ueber vermuthliche rudimentire Kiemenspalten
bei Elasmobranchiern. Mitth. Zool. Sta. Neapel., Bd. 6.
1893 Ueber die Entwickelung der Kiementaschen und der Aortabogen
bei den Seeschildkréten untersucht an Embryonen von Chelonia
viridis. Anat. Anz., Bd. 11.
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Wourter, A. 1880 Ueber die Entwicklung und Bau der Schilddriise. Berlin.
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)
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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. —$—. nee Se =
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— 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
= a meee a
—————— 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
LA ENE,
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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. '’ Yin >
cy a ae a ae
a>) yt oe
A, tar v" ats i ig! we .
was (aa
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