a Ri
ld
JOURNAL
MORPHOLOGY.
EDITED BY
C. O. WHITMAN,
Head Professor of Biology, Chicago University.
WITH THE CO-OPERATION OF
EDWARD PHELPS ALLIS.
Milwaukee.
OcToBER, 1898.
BOSTON, U.S.A.:
GINN & COMPANY.
AGENT FOR GREAT BRITAIN: AGENTS FOR GERMANY: AGENT FOR FRANCE;
EDWARD ARNOLD, R; FRIEDLANDER & SOHN, JULES PEELMAN,
EVA Bedford Street, Strand, Berlin, N.W., 2 rue Antoine-Dubois,
-» London, W.C, Caristrasse 14. Paris, France.
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CONTENTS OF No. 1, OCTOBER, 1808.
ees
PAGES
I. A New Peripatus from Mexico”. . . . . 1-8
WILLIAM MORTON WHEELER.
Il. The Germ-Ring in the Egg of the Toad-Fish
CUB AL GGIUS GUN is esas Giana Nicene g-16
LouIsE B. WALLACE.
Ill. The Metamerism of Nephelis. A Contribution
to the Morphology of the Nervous System,
with a Description of Nephelis Lateralis. 17-72
CHARLES L. BRISTOL.
IV. The -Growth of the Ovum, Formation of the
Polar Bodies, and the Fertilization in
POLY CROEKUS NCAUAALUS Ae pice on) ae gj LO
EDWARD G. GARDINER.
Che Atheneum Press.
GINN & COMPANY, BOSTON, U.S.A
JOURNAL
OF
MORPHOLOGY.
EDITED BY
C. O. WHITMAN,
With the Co-operation of
EDWARD PHELPS ALLIS,
MILWAUKEE.
Vou, XV
BOSTON, U.S.A.:
GINN & COMPANY
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CONTENTS” OF VOL. “XV.
No. 1.— October, 1898.
WittiamM Morton WHEELER.
A New Peripatus from Mexico .
LoutsE B. WALLACE.
The Germ-Ring in the Egg of the Toadfish
(Batrachus Tau) ae so at
CHARLES L. BriIsTOoL.
The Metamerism of Nephelis. A Contribution
to the Morphology of the Nervous System,
wth a Description of Nephelis Lateralis
EDWARD G. GARDINER.
The Growth of the Ovum, Formation of the
Polar Bodies, and the Fertilization in Poly-
choerus Caudatus .
No. 2.— November, 1898.
Joun P. Munson.
The Ovarian Egg of Limulus. A Contribution
to the Problem of the Centrosome and Yolk-
Nucleus
CornELia M. Crapp.
The Lateral Line System of Batrachus Tau
PAGEs
9-16
72
73=1O
- [11-220
. 223-264
iv CONTENTS.
PaGes
III. THos. H. Montcomery, Jr., Px.D.
Comparative Cytological Studies, with Especial
Regard to the Morphology of the Nucleolus . 265-582
No. 3.— February, 1899.
I. Brapney B. GRIFFIN.
Studies on the Maturation, Fertilization, and
Cleavage of Thalassema and Zirphaea . . 583-634
II. Gustav Etsen, Pu.D.
On the Blood-Plates of the Human Blood, with
Notes on the Erythrocytes of Amphiuma and
WN ECLUXUS Se te te ss) ee & @ a OZ 5—660
III. CHARLES WILSON GREENE.
The Phosphorescent Organs in the Toadfish,
Porichthys Notatus Girard. . . . . . 667-606
IV. W. G. MacCatium.
On the Species Clinostomum Heterostomum . 697-710
V. Gary N. CALkIns.
Mitosis in Noctiluca Miliaris and its Bearing
on the Nuclear Relations of the Protozoa and
Metazoa™. a ee ee a
Che Atheneum Press.
GINN & COMPANY, BOSTON, U.S.A.
Volume XIV, Number 3, of the Journal of Morphology
has been unavoidably delayed, but will be sent to all
subscribers as soon as completed.
GINN & Company, Pudlishers.
Volume XV. October, 1898. Number I.
JOURNAL
OF
MOR OLOGY,
A NEW PERIPATUS FROM MEXICO.
WILLIAM MORTON WHEELER.
Dr. Gustav EIsen, of San Francisco, has most generously
placed at my disposal all his material—some 87 specimens
—of a new species of Peripatus, with permission to describe
the animal and the development of its embryos. The speci-
mens were taken by Dr. Eisen during November, 1894, at
Tepic, Mexico, at an altitude of 4000 feet. He found them
under stones and pieces of wood in a shady spot along a ditch
of water flowing from some baths on the outskirts of the town.
The animals were killed in a fully extended condition by
drowning them in water, to which a few drops of ammonia had
been added. They were then carefully hardened in corrosive
sublimate.
The species which I take pleasure in naming Peripfatus
eisentt, after its discoverer, is a true neo-tropical Peripatus.
With the exception of P. edwardsii Sedgwick, from Venezuela,
the descriptions of the American species are not very satis-
factory. I shall, therefore, make most use of a comparison
with this species in the following description of the exterior
of P. etsenttz, Anatomical and embryological details are
reserved for future publication.
2 WHEELER. [VoL. XV.
The specimens vary considerably in length. The youngest
individuals, evidently just born, measure from 13 to 20 mm.,
the adults from 40 to 57 mm. One female only 30 mm. in
length contains full-grown pigmented embryos.
Dr. Eisen informs me that the preservation in alcohol has
not altered the colors of the animal. These are very variable,
the dorsal surface of the body and legs ranging through some
four or five shades, — from light pinkish brown in many of the
youngest specimens to a dark reddish chocolate color in some
of the oldest. Most of them show a distinct darker brown
median dorsal stripe, and some have the faint transverse inter-
segmental stripes indicated in the figure (Fig. 1). Along
either side of the mid-dorsal line are seen a number of white
dots, which are in reality the unusually large pale papillae seen
under a higher magnification in Fig. 8. The ventral surface,
too, is variable in its coloration. Some specimens are pale
yellow, like Figs. 2 and 5, others are white, still others flushed
with pink. The lips of the oral orifice, the oral papillae, and
the spinous creeping pads on the feet are paler than other por-
tions of the ventral surface.
The transverse ridges of the integument bear each a single
row of papillae. Some of these are enlarged, especially on
the legs, and consist of two segments, —a broad basal moiety
which is usually conical, although in some cases it approaches
the condition called “cylindrical” by Sedgwick ! in his descrip-
tion of P. edwardsit, and a more slender tapering apical
moiety, tipped with a spinule and unpigmented. It would
seem that the animals have the power of retracting the pale
distal moiety with its spinule, but this can only be determined
by a study of the living animal.
In the mid-dorsal line the transverse ridges of the integu-
ment are interrupted by a delicate but perfectly distinct im-
pressed white line (Fig. 8). The absence of this white line
is emphasized by Sedgwick as a diagnostic character of the
American, or neo-tropical, species of Peripatus. I find this
1 Adam Sedgwick, “A Monograph of the Species and Distribution of the
Genus Peripatus.” Quar. Journ. Micr. Sci., vol. xxviii, 1888. Also in Studies from
the Morph. Lab. Univ. Camb., vol. iv, pt. ii, 1888, pp. 147-212, Pls. XIV-XX.
No. I.] A NEW PERIPATUS FROM MEXICO. 3
line also in some specimens of P. trintdadensis Sedgwick,
although it is so much obscured by the shrinkage of the integu-
ment that I should have overlooked it had I not previously
found it in P. ezsenzz. The impressed line may be somewhat
narrower and fainter than it is in the Cape species (P. capensis
Grube and P. moseleyi Wood Mason; see Sedgwick’s Pl. XVII,
Fig. 10), but its presence in at least two of the American
species is sufficient reason for excluding it in future from the
diagnosis of the South African division of the genus.
The antennae and oral papillae resemble the corresponding
organs of P. edwardsiz. The jaws, too, resemble the jaws of
the Venezuelan species, except that the inner blade bears three
teeth before the diastema instead of two. The additional
tooth is a smaller second “minor” tooth. The teeth of the
series beyond the diastema are less numerous and blunter
than they are in P. edwardsiz. The outer blade of the jaw in
P. etsenit bears only two teeth, like that of the other species
of the genus.
The papillae and folds surrounding the mouth differ from
those in the same position in P. edwardsiz. These differences
are best seen by comparing Sedgwick’s Pl. XVIII, Fig. 15,
with my Fig. 2.
P. ewseniz resembles the other American species in having
a variable number of legs. The legs in all the specimens, old
and young, were counted, and were found to vary from 23 to
29 pairs. The extremes were each represented by a single
specimen. Four had 26 pairs, 11 had 24, 17 had 25, 22 had 27,
and 30 had 28 pairs. The peculiar curve plotted from these
data has two summits as shown in the accompanying figure.
This peculiar variation in the number of pairs of legs is not
due to growth, as Sedgwick has shown in other neo-tropical
species. The animal is born with the definitive number of legs,
and no further pairs are added during post-embryonic life.
Several of what I take to be just-born young, only 11 to 13 mm.
long, have 27 or 28 pairs of legs, whereas several larger speci-
mens, 20 to 28 mm. long, have only 24 or 25 pairs.
According to Sedgwick, the greater numbers are found in
the females, the lesser in the males. I have been unable as
4 WHEELER. [VoL. XV.
yet to find any males in the material. Of 16 adult females
containing embryos, 6 had 27, 9 had 28, and 1 had 29 pairs
of legs. This would tend to corroborate Sedgwick’s statement
and to show that the second summit of the curve represents
the usual number in the female, the first the usual number for
the male of P. ezsenzz, This point can be determined only
after the smaller individuals have been sectioned and their sex
ascertained.
The following list gives the numbers of pairs of legs in
the better known species of Peripatus, from both hemispheres,
as recorded by Sedgwick :
Peripatus brevis Blainv. S. Africa. 14
novae zealandiae Hutton. New Zealand. 1S
leuckarti Saenger. Queensland. 15
capensts Grube. S. Africa. 17
balfourt Sedgw. S. Africa. 18
moseleyt Wood Mason. S. Africa. 21-24
chiliensis Sedgw. Chili. 19-2
eisenti 0. sp. Mexico. 23-29
peruanus Grube. Peru. 2
demeraranus Sedgw. Demerara. 27-31
No. 1.] A NEW PERIPATUS FROM MEXICO. 5
Peripatus trinidadensis Sedgw. Trinidad. 28-32
juliformis Gilding. St. Vincent. 33
edwardstt Sedgw. Venezuela. 29-34
guitensis Schmarda. Quito. 36
torqguatus Kennel. Trinidad. 41-42
It will be seen from this list, in which we have a gradu-
ated series of forms with legs varying from 14 to 42 pairs, that
the Mexican Peripatus has the lowest number of legs of any
American species with the exception of P. chzliensis.1
In the structure of its legs P. ezsenzz resembles the other
neo-tropical species. All the feet have four spiny creeping
pads, except the last two pairs, which have only three (Fig. 5).
The most proximal pad on the antepenultimate pair is very
small, so that this pair forms a transition between the feet
in front with four pads and the posterior feet with three.
According to Sedgwick, only the last pair of feet has three
spinous creeping pads in P. edwardsiz.
The opening of the nephridium on the fourth and fifth pairs
of legs in P. ezsendi differs from that observed in other Ameri-
can species. The position of this opening is shown in Fig. 7.
The second pad from the base of the appendage is broken in
two, -—a short posterior and a much longer anterior piece, —
and between them lies the papilla with the nephridial orifice.
In P. edwardsiz the second spinous creeping pad is entire,
though somewhat narrowed in the middle; and the nephridial
papilla lies between it and the most proximal pad in the middle
line of the appendage, according to Sedgwick (Pl. XVII, Fig.
11). In the old-world species figured by Sedgwick (P. dalfour?,
Pl. XVII, Fig. 9, and P. novae zealandiae, P|). XIX, Fig. 21)
the nephridial papilla lies in the middle of the proximal pad,
which corresponds to the second pad from the base of the
appendage in P. ezsentz and other new-world species.
The position of the nephridial papilla in the middle longitu-
dinal line of the appendage, as represented by Sedgwick for P.
edwardsit, will not hold good for at least one other American
species, vzz., P. trinidadensis. In the specimens of this species
1 The number of pairs of legs in this species has not been satisfactorily
reported. See Sedgwick, p. 197.
6 WHEELER.
which I have examined the nephridial papilla lies between the
_ most proximal and the succeeding pad, as in P. edwardsiz, but dis-
tinctly nearer the posterior than the anterior surface of the leg.
The pedal grooves are conspicuous on the inner ventral sur-
faces of all the legs in P. ezsenzz. They are slit-shaped, with
thickened, somewhat folded lips. None of the specimens show
conspicuous tubercles behind the grooves of some of the pos-
terior legs, like those which have been found in the males of
P. edwardst.
The anus is terminal; the reproductive orifice is in the mid-
ventral line, between the penultimate pair of legs, as in other
American species.
HuLL ZOOLOGICAL LABORATORY, UNIVERSITY OF CHICAGO,
February 28, 1898.
8 WHEELER.
EXPLANATION OF PLATE I.
Fic. 1.— Dorsal view of a full-grown, rather dark-colored female Peripatus
eisenii N. Sp., magnified about 3} diameters.
Fic. 2. — View of ventral surface of the head of same specimen enlarged.
Fic. 3.— Outer blade of jaw.
Fic. 4.— Inner blade of jaw.
Fic. 5. — View of ventral surface of posterior end of the specimen represented
in Fig. 1.
Fic. 6.— Lateral view of one of the feet.
Fic. 7. — Ventral view of the fifth leg, showing the position of the nephridial
papilla.
Fic. 8.— Piece of integument, showing the mid-dorsal “ white line” interrupt-
ing the papillated ridges of the integument.
Journal. of Morphology Vol.X\
PLT
om
THE GERM-RING IN THE EGG OF THE TOAD-
FISH (BATRACHUS TAU).
LOUISE B. WALLACE.
Sm1TH CoLLeGE, NoRTHAMPTON, Mass.
Since the Toad-fish has a number of characteristics peculiar
to itself, it is natural to expect that it would differ from the
ordinary teleost in its mode of development. The marked
resemblance of the egg to that of Elasmobranchs has already
been noted,! but the formation of the germ-ring has been left
an open question. My observations were made during the
summers of ’95 and ’96, under the direction of Dr. Whitman,
at the Marine Biological Laboratory, Woods Holl, Mass.
It gives me great pleasure to express my indebtedness also
to Dr. Cornelia M. Clapp for many helpful courtesies.
After the middle of June most of the material found in nests
in Buzzards Bay was in an advanced condition, and it was neces-
sary to resort to artificial fertilization. Eggs were fertilized
by hundreds, covered with sea-water in shallow dishes, and
studied from the earliest stages. The egg of Batrachus is 5
mm. in diameter, being much distended with yolk, and is, when
deposited, attached to some foreign body by means of an
adhesive disc. The blastoderm, in encompassing the yolk, is
spread out into a cap of extreme tenuity, requiring delicate
treatment. After repeated effort the paraffin method was
given up and good results were obtained by use of the cel-
loidin method with either Hermann’s fluid or Flemming’s fluid
as fixing reagents.
Not until the fourth or fifth day after fertilization does a
distinct axial thickening appear, with oftentimes a slight, mar-
ginal notch at the embryonic pole, and there is no marginal
thickening around most of the blastoderm (Pl. II, Fig. 6). A
median, longitudinal section is shown in PI. III, Fig. 1. In
1 Cornelia M. Clapp, “Some Points on the Development of the Toad-fish
(BSatrachus tau).” Journ. of Morph., vol. v, No. 3.
IO WALLACE. [VoL. XV.
the extra-embryonic region the ectoderm is two cells deep, with
no peripheral thickening ; while in the embryonic region there
is a centripetal growth of cells, thickest near the margin and
thinning out anteriorly until some of the cells appear to be lying
loose on the periblastic floor. A cross-section, passing through
the axial thickening, shows that this tongue of cells also thins
out laterally (Pl. III, Fig. 2). Very soon after the germ-ring
attains its maximum development (Pl. II, Fig. 7) it begins
to decrease. This decrease is shown in an enlarged view of a
later stage, Pl. III, Fig. 9, in which the ring is narrow at the
anterior pole, gradually broadening toward the posterior or
embryonic pole. Cross-sections of the rim at the cardinal
points reveal an interesting modification of the germ-ring in
the ordinary teleostean egg. Ina section at the anterior pole
no invagination obtains, but rather a centripetal proliferation of
cells from the ectoderm (PI. III, Fig. 3). In Professor Wilson’s
paper on the Sea Bass! he says: “The peripheral part of the
blastoderm, both where there is a large Randwulst and none at
all, is an undifferentiated area, and the germ-ring consequently
starts at some distance from the extreme edge of the blasto-
derm.”’ If we follow this interpretation of terms, we have in
Batrachus no germ-ring, as there is no under layer of cells dif-
ferentiated from the rest of the blastoderm. A section through
the lateral region more strongly expresses the fact that there
is no invagination, the blastoderm being actually thinner at the
periphery, as shown in Pl. III, Fig. 4. Here, also, we find no
distinct under layer, but a few loose cells which are budded off
centripetally from the slight peripheral thickening. As the
occurrence of these loose cells is constant, might they not rep-
resent the distinct layer found in other forms? Passing to the
posterior pole, a section is shown through the longitudinal axis
of the embryo (Pl. III, Fig. 5). Here is a decidedly invagi-
nated appearance, but no real invagination, so far as can be
judged from a study of successive stages. The appearance
may be due to a rapid proliferation of cells both centripetally
and dorso-ventrally (cf Pl. III, Fig. 1), and also to the growth
1 Henry V. Wilson, “The Embryology of the Sea Bass (Serranus atrarius).”
Bulletin of the U. S. F. C., vol. ix. For 1889.
No. 1.] THE EGG OF THE TOAD-FISH. II
of the ectoderm over the yolk. The ectoderm is sharply dif-
ferentiated from the ingrowing tongue of cells, especially at the
periphery. From this time the ring becomes less and less pro-
nounced. In surface views of the stage shown in Pl. II, Fig. 8,
a little irregular thickening can be seen at the anterior pole,
and in sections a few scattered cells are found lying beneath
the thin, flattened ectoderm (PI. III, Fig. 6). In some sections
not even this much of the thickening remains, as the cells occur
in patches. In the lateral region the reduction is not yet car-
ried so far (Pl. III, Fig. 7). Sections through the rim of the
stage shown in Pl. II, Fig. 9, have no thickening even in the
lateral region, while the tongue of cells at the embryonic pole
is steadily lengthening.
In Ctenolabrus Dr. Whitman finds that there is ‘a plain rolling
under or involution as an initiatory step in the formation of the
ring,’’ but believes that the process is more correctly described
as ‘an ingrowth due both to a rapid multiplication of the cells
and also to the centrifugal expansion of the ectoderm.” At
the posterior margin ‘the inrolling portion presents a strongly
voluted outline, while’ at the anterior border it is much more
feebly expressed.”’? In Batrachus, around most of the margin
there is found simply ‘the initiatory step,” and even that
lacks the voluted outline, except at the embryonic pole. The
loose cells budded off from this small peripheral thickening
represent, I believe, a true germ-ring. In the Sea Bass, Pro-
fessor Wilson finds, at the embryonic pole, an apparent invagi-
nation caused by a centripetal growth of cells, and forming a
Randwulst from which cells are proliferated centripetally to form
a germ-ring. “Round the rest of the edge the ingrowth is like-
wise, at least in most places, preceded by the formation of a
Randwulst, which, however, is inconspicuous.”
From the stage given in Pl. II, Fig. 9, down to the closure
of the blastopore at a distance behind the embryo, there is an
apparent marginal thickening visible even in the living egg (PI.
II, Figs. 3-5). In specimens killed in Perenyi’s fluid, a distinct
1 Alexander Agassiz and C. O. Whitman, “On the Development of Some
Pelagic Fish Eggs.” Preliminary notice. Proceedings of the American Academy
of Arts and Sciences, vol. xx.
12 WALLACE.
opaque rim is noticeable, while in preparations with osmic acid,
the rim becomes much darker than the rest of the blastoderm.
By the study of surface mounts and from sections, this thick-
ness was found to be due to the greater thickness of the peri-
blast in that region, and also to the accumulation of huge
periblastic nuclei. The presence of oil globules increases the
effect, especially in the living egg.
Pl. II, Fig. 10, is a reproduction of Dr. Clapp’s Fig. 1, d.
She says: ‘In Fig. 1, d, this notch is seen at a little dis-
tance behind the embryo; a shadowy connection may be traced
between the germ-ring and the embryo.’’ While at this time
only the appearance of a germ-ring exists, the “shadowy con-
nection” between the ‘“germ-ring’”’ and the embryo has a
more substantial basis. The same stage is given in Pl. II,
Fig. 5. A longitudinal section through the embryo and the
margin of the blastopore is shown in PI. III, Fig. 8. A few
cells, marked ‘‘m,” are seen lying beneath the ectoderm and
reaching from the posterior end of the embryo almost to the
lip of the blastopore. Sections all around the blastopore show
no thickening except of periblast.
Summary.—In the egg of Batrachus there is a centripetal
growth of cells at the embryonic pole, the ingrowth having a
voluted outline in sections. Around the remainder of the
blastoderm there is not even the appearance of an invagination,
but only a slight thickening due to an ingrowth of cells from
the ectoderm, and a few loose cells which may represent a true
germ-ring found as a layer in ordinary forms. The peripheral
thickening gradually fades out, first at the anterior pole, until
the last remnant is found in a few cells lying beneath the ecto-
derm, forming a linear streak from the posterior end of the
embryo to the lip of the closing blastopore. In the gradual
disappearance of the thickening, beginning at the anterior pole
and continuing on either side toward the posterior pole, accom-
panied by the lengthening of the embryo, we see a highly
modified form of concrescence.
Woops Ho.t, Mass.,
September, 1896.
14 WALLACE.
EXPLANATION OF PLATE II.
Fic. 1. Ovum with blastoderm covering + of yolk surface. X 9.
Fic. 2. Ovum with blastoderm covering 4 of yolk surface. X 9.
Fic. 3. Ovum with blastoderm covering over %4 of yolk surface. X 9,
Fic. 4. Ovum with blastoderm covering nearly 3¢ of yolk surface. X 9.
Fic. 5. Ovum near the time of the closure of the blastopore. X 9. a.d.=
adhesive disc; 7. lip of blastopore.
Fic. 6. Blastoderm of an earlier stage than Fig. 1.
Fic. 7. Blastoderm with maximum development of germ-ring. X 16. gr.
= germ-ring ; az¢. = anterior ; per.2. = periblast nuclei.
Fic. 8. Blastoderm of later stage than Pl. III, Fig. 9. X 16.
Fic. 9. Blastoderm of a slightly earlier stage than that of Fig. 3.
Fic. 10. Reproduction of Dr. Clapp’s figure showing “ shadowy connection,”
m., between “ germ-ring ” and embryo.
Journal of Morphology Vol xv. ;
Sik, Werner & Winter Frankfort 2a
16 WALLACE.
EXPLANATION OF PLATE IIL.
Fic. 1, Longitudinal median section of blastoderm shown in P\. II, Fig. 6.
X 100. per. = periblast.
Fic. 2. Cross-section of the same. X Ioo.
Fic. 3. Cross-section of rim at anterior pole of stage shown in PI. III, Fig. 9.
x 160.
Fic. 4. Cross-section of rim in lateral region of stage shown in PI. III, Fig. 9.
X 160.
Fic. 5. Longitudinal section through axial thickening of stage shown in
Pl. II, Fig. 9. X 160.
Fic. 6. Cross-section of rim at anterior pole of blastoderm shown in PI. II,
Fig. 8. X 160.
Fic. 7. Cross-section of rim in lateral region of blastoderm shown in PI. II,
Fig. 8. X 160.
Fic. 8. Longitudinal median section through embryo and lip of blastopore at
stage shown in Pl. II, Fig. 5. X 160. A .v.—=Kupffer’s vesicle; /.=lip of
blastopore.
Fic. 9. Enlarged view of blastoderm in which the germ-ring is beginning to
disappear at the anterior pole. X 45. (Drawn by Mr. Hayashi.)
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4
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Journal of Morphology Vol.Xxv. ; PL. It
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THE METAMERISM OF NEPHELIS.
A CONTRIBUTION TO THE MORPHOLOGY OF THE NER-
VOUS SYSTEM, WITH A DESCRIPTION OF
NEPHELIS LATERALIS.
CHARLES L. BRISTOL.
TABLE OF CONTENTS.
PAGE PAGE
INGER. © EU CLL Nias cen corer soreeceensncesenserecsasee 17 Wey dig?s| Cell). reccecseresearesccees-enee-s 39
HISTORICAL . Median Nerve Cells .................. 41
METHODS ....... The Fibrous Portion .................. 41
SYSTEMATIC ..........:ececescesseseeeceeeeseneeee> 21. | INNERVATION OF A Boby METAa-
ID ES GRUPII ON | sectesvesenecccseet-essecacsnacearecess 24. MERE oc.-cshsscsseescsetetecesssvessntetecses 43
General of WV. lateralis ........-..-+- 26 | INNERVATION OF THE TERMINAL
SOS OS cecenenecchecneceoeea enero 27 SOMITES s.c.---2--: . 44
Head Region. 28 The Anal Region.. ae CU
Anal Region. 28 MhevEleadsRe pony esses scarssscnenes 46
Summary... . 28 | THE INTERMUSCULAR NERVE
NEAIANBUTDAST yo ese ss ston dec cectstcccesatesezsecen aos 29 MRUTIN Gass cane set ncuteeessascveesceceneses 51
EVAR IMS SHOOD sETC. cecssesssersconeeceeasent 30 | THE “LARGE” NERVE CELLS ........ 57
INERVOUS! SYSDEM(.....cc-sc.s0cec-2cecsns-00 32 | THE SYMPATHETIC SYSTEM............ 55
Whitman’s Description of the SUMMARY ............ OE
Nervous System of Clepsine 33 | BIBLIOGRAPHY.............. ma 03
NERVOUS SYSTEM OF NEPHELIS .... 36 | DESCRIPTION OF PLATES....-..-..---+.--. 64
Central Nervous System .......... Ve PAB BRE VILAUE LON Site cccsersracecascassesasesnsane 72
A Typical Neuromere................ 38
INTRODUCTION.
Tue work which forms the basis of the present paper was
begun in 1891 at Clark University, Worcester, and continued
at the Marine Biological Laboratory at Woods Holl and Chicago
University, and I wish to acknowledge here my indebtedness to
the authorities of these institutions for the facilities and the
Fellowship privileges granted to me.
To- Professor Whitman,
at whose suggestion I began the investigation, I am deeply
indebted for aid and encouragement and for many courtesies
extended to me.
Iam under obligations to Dr. Wm. M. Wheeler
18 BRISTOL. [VoL. XV.
for aid and advice, and to Prof. S. A. Forbes, of the Uni-
versity of Illinois, for the privilege of examining the specimens
of Nephelis collected by him under the auspices of the U. S.
Fish Commission in the Yellowstone region in Wyoming.
HISTORICAL.
In1767 Linné enumerated nine species of leeches in one genus,
Hirudo. This classification was followed by most later authors,
for example, Cuvier, Blumenbach, Carena, and Dumeril, until
about 1817, when Savigny, in his Systeme des Annelides,
announced the separation of Linné’s genus into seven genera.
The name Nephelis appears in this work for the first time,
although Oken set this leech apart from Hirudo in 1815 under
the name Helluo, which genus was to include all fresh-water
leeches not provided with jaws. In 1818 Lamarck, at the
suggestion of Blainville, proposed the name Erpobdella, which
Blainville (1828) urged for acceptance because it contained the
descriptive part “bdella.” In 1826 Moquin-Tandon adopted
Savigny’s name Nephelis and continued it in the second edition
of his Monographie des Hirudinées (1). The name has since
become generally accepted, notwithstanding the fact that
Oken’s Helluo holds priority and Lamarck’s Erpobdella is
more descriptive.
The first description of Nephelis in America was made by
Thomas Say (2) in 1824 under the name of Hzrudo lateralis.
In 1872 Verrill (3) changed this to Wephelis lateralis, which, for
reasons given in another part of this paper, I have given to the
leech I have studied.
METHODS.
The leeches are easily kept in aquaria, for which I used the
low glass dishes known as crystallizing dishes, or white earthen-
1 A. Moquin-Tandon: Monographie de la famille des hirudinées. Paris. 1846.
2 T. Say: Major Long’s Second Expedition to the Source of St. Peter’s River,
vol. ii, Appendix to the Natural History. 1824. (Republished in Dyesing’s
Systime Helminthologique, vol. i.)
8 A. E. Verrill: “Synopsis of the North American Fresh-water Leeches.” U.S.
Fish Commissioner’s Report for 1872-74. (Refers to the American Journal of
Science, vol. iii, 1872.)
No. 1.] THE METAMERISM OF NEPHELIS. 19
ware cooking dishes, known to the trade as nappies. In some
instances I supplied the aquaria witha layer of mud and bottom
débris, together with a few plants such as Ceratophyllum or
Valisneria. When such an aquarium is covered with a glass
plate it will keep fresh and clean for a long time and will
furnish considerable food for the leeches. Generally, however,
I used the plain dish, cleaning out the dédrzs and slime and
changing the water when necessary. I fed chopped fresh-
water clams, but I do not doubt that salt-water clams will serve
as well. I have kept individuals for over a year in normal
condition and have raised many young under these conditions.
When they are first transferred to the aquarium it must be
covered for a day or two, to prevent escape. For superficial
examination the leeches were killed in very dilute chromic
acid, % to % per cent solutions. There is one period just
before the acid penetrates very deeply when the surface mark-
ings stand out very clearly. The leeches usually extend them-
selves very well, and if killed in a wax tray they may be guided
by pins. The best medium for histological details is a 4% to
¥% per cent solution of chromic acid, allowed to act for at least
24 hours. The stains used were borax carmine, Delafield’s
haematoxylin, and Bizzozero’s picro-carmine. The macroscopic
characters of the nerve chain were studied from maceration
preparations. The leech is killed in a 20 per cent solution of
nitric acid and left in it for from 24 to 36 hours, or until the
skin and muscles can be easily removed with a porcupine
bristle or a glass rod drawn out to a point. These were all
carefully dissected away, leaving the chain entire. After
thorough washing, the chain may be slightly stained in borax
carmine and mounted in glycerine.
A number of details were worked out by the use of Haller’s
fluid. For example, the head was cut off, slit open on the
ventral or dorsal side as wished, and killed in Haller’s fluid
while it was flattened under a piece of glass. After two days
the specimen was transferred to glycerine.
The method that has given me the best results for nerves
and sense organs is a gold chloride process kindly given me
by Miss Julia B. Platt. It is so simple, so sure, and so exqui-
20 BRISTOL. [Vov. XV.
sitely delicate in some of its effects that it deserves extended
use. It may be used with equal success on vertebrate or
invertebrate, adult or larval tissue. It must be adapted to the
tissue studied ; but this can easily be done after a few experi-
ments. The formic acid appears to be the variable factor, and
upon its strength and the time it acts depends the measure of
success. I give the procedure applicable to Nephelis.
The leech is killed in a 10 or 15 per cent solution of formic
acid, left from 5 to 10 minutes, and then put without washing
into a I per cent solution of gold chloride for 25 minutes. From
this it is transferred, without washing, into a large volume of
I per cent formic acid, and left for 12 or 18 hours, or until
reduction has taken place. It is next washed, passed through
the alcohols to chloroform, and then imbedded in paraffin.
The sections were cut 18 micra thick. The specimen will
appear a rich purple when the reduction has taken place under
the best conditions. The precautions are : to use small pieces
of material, not thicker than 5 mm., to avoid maceration by
reducing the strength of the formic acid and the time of action.
My solutions were all well sunned, but no especial precautions
were observed.
In tracing out the innervation of the somites it was neces-
sary to examine long, continuous series of sections, and some-
times it was necessary to check results found in one somite by
comparison with the next somite. The following method was
used which would apply to other purposes. An ordinary library
reference card, about 8 cm. by 10 cm., is ruled so as to include
as many small rectangles in the same number of rows as the
slide to be examined contains sections. The unused margin
serves for making notes. An ordinary check mark denotes
that the section occupying the same place on the slide that the
rectangle does on the card has been examined but does not
contain the element under examination. Initials, symbols,
different colored pencils, etc., may be used to indicate various
details, and each card is numbered the same as the slide. After
a number of slides have been carefully plotted in this manner,
the cards may be arranged in series and studied as a map. It
furnishes an excellent reference-card system for any set of
No. I.] THE METAMERISM OF NEPHELIS. 21
serial sections, and permits a rapid glance at the order of
sequence of any character in different somites or individuals.
SYSTEMATIC.
Nephelis differs from nearly all other leeches in the external
topography of the somite. While the somite in the Hirudinea,
as a group, is characterized by prominent sense organs on the
first ring, in Nephelis these are conspicuously absent, save on
a few segments near the anus and in rare instances on a few
rings near the mouth. The absence of these characters has
compelled investigators to resort to other criteria for the
determination of species, such as color markings, and the
occurrence of four stripes of pigment on the dorsal side is
sufficiently well marked to furnish a criterion of generic, if not
of specific, value. In Europe the only well-established species
is WV. octoculata Bergmann. Blanchard (4) says: “Jusqu’a
Savigny, la seule espece admise sans contest était la WV. octo-
culata Bergmann : Savigny a distingué plusieurs espéces basées
exclusivement sur les differences de coloration : mais aucune de
ces espéces nominale n’est representée et n’est surement recon-
naisible. En outre de la 1. atomaria, nous croyons pouvoir
séparer de l’ancienne JV. octoculata plusieurs autres formes
spécifiques bien distinctes.”
Moquin-Tandon in the first edition of his monograph accepts
the description given by Carena for 1. atomaria as a species,
but in the second edition lowers it to the rank of a variety of
LV. octoculata.
The first mention of the genus in this country that I have
found was made by Thomas Say (2) in 1824 under the name
of Hirudo lateralis, and this was changed by Verrill (3) in 1872
to WV. lateralis. Leidy (5) described a form (1870) under the
name JV. warmorata. Verrill describes four species of Nephelis
found in the United States and says concerning three of them :
4R. Blanchard: “Courtes notices sur les Hirudinées, III. Description de la
Nephelis atomaria Carena.” Bull. de la Soc. Zool. de France, tome xvii, p. 165,
1892.
5 Jos. Leidy: “ Description of Nephelis punctata.” Proc. Acad. Nat. Sci. of
Philadelphia, p. 89, 1870.
22 BRISTOL. [VoL. XV.
«When a larger series of living specimens from various locali-
ties can be studied, the three preceding forms (lV. /ateralis,
NV. quadristriata, N. marmorata), admitted here as distinct, may
prove to be mere varieties of one species, no less variable than
NV. vulgaris of Europe.” The fourth species, WV. fervida, is
described from specimens taken from Lake Superior and has
eight ocelli. I have not collected a Nephelis answering to this
description.
The genus is widely distributed in the United States. My
own collections have been made in Massachusetts, Connecticut,
Illinois, New York, and South Dakota. I have received speci-
mens from Mr. A. J. Hunter, of Toronto, collected near To-
ronto, Can., and Professor Forbes, of the University of Illinois,
kindly loaned me for examination the specimens of Nephelis
collected by him in the Yellowstone region in 1890. Verrill
records collections from Maine, Massachusetts, Connecticut,
New Jersey, Wisconsin, Nebraska, Colorado (at an elevation
of 9000 feet on Longs Peak), and from the waters of Lakes
Superior and Huron. The area included covers about 35
degrees of longitude and 10 degrees of latitude; it embraces
the Atlantic slope, the Great Lake Region, the Missouri Valley,
and the Rocky Mountains.
Investigations on my own collections lead me to agree with
Savigny, Moquin-Tandon, and Verrill that it is difficult to dis-
tinguish species by the criteria used by them, color and color
markings, and to disagree with the methods and results, pub-
lished by Lindenfeld and Pietruszynski (6), who rely on these
features exclusively. My first attempts to classify the specimens
which I collected were naturally based on the descriptions given
by previous investigators, but it proved so difficult a task to
determine what value to place on the various statements of color,
and so many of my specimens could with equal propriety be
placed in either of two or three categories, that it became
evident that some different method of diagnosis would be
necessary. The necessity of going beyond color markings was
plainly shown by the following experiments.
® Von Lindenfeld und Pietruszynski: “ Beitrage zur Hirudineen fauna Polens.”
Reviewed by Nusbaum. JSvo/. Centralblatt, Bd. xii, p. 55, 1892.
No. 1.] THE METAMERISM OF NEPHELIS. 23
I attempted to separate all the individuals collected from one
locality near Worcester, Mass., according to color and color
marks. I provided five aquaria and sorted each lot as I col-
lected them until the whole number of individuals exceeded one
hundred and twenty-five. It was very evident at a glance that
the leeches in the first aquarium were light colored, and that
those in the fifth were dark colored, but it was impossible to
divide them so that each aquarium should be free from tran-
sitional forms. I repeated the effort on my collections from
Wolf Lake, near Chicago, IIl., and with like results. The very
light and very dark individuals were about equally rare, while
the great bulk of each lot was made up of leeches varying in
shade but having the same stripes more or less distinctly
accented according to the amount of pigment present. These
trials led me to adopt the method proposed by Whitman (7) and
used by Blanchard (4). The method consists in determining
the number of rings in the entire body and the limits of each
somite. The first ring of each somite in the Hirudinea bears
eight sense organs on the dorsal side, as Whitman has shown,
four of which are serially homologous with the eyes.
The typical somite of Hirudo contains five rings. This
number holds good throughout the middle body region, but
falls to three towards the two ends, then to two, and finally to
one. The amount and the manner of reduction vary in dif-
ferent genera, but are constant in any given genus. In Nephe-
lis, also, the typical somite has five rings, but the limits of
the somites and the number of rings in the terminal ones are
not readily determined by the arrangement of the sensillae, for
with certain exceptions mentioned hereafter these appear about
equally prominent on every ring throughout the entire body.
My first attempts to determine these points by means of the
sensillae failed ; I succeeded later in the following way.
When Nephelis is thrown into weak chromic acid, — Y6 to
¥Y% per cent solution, —there soon comes a time when the
sensillae stand out with perfect distinctness ; later the contrast
in color between them and the surrounding surface becomes
7C. O. Whitman: “The External Morphology of the Leech.” Proc. Am.
Acad. of Science, vol. xx, p. 76, 1884.
24 BRISTOL. [VoL. XV.
less and less marked. At the time of greatest distinctness
one may see that the sensillae are rather more strongly marked
on the terminal somites, especially those at the hind end. It
‘was here that I was able to find a starting point for determin-
ing the external metamerization. The 97th ring (Pl. VI, Fig. 3)
was strongly marked with sensillae, and between the 96th and
g7th rings were found the pores of the 17th and last pair of
nephridia. These two conditions gave me a starting point
from which I could fix with certainty the limits of the somites
towards the anterior until the reduced somites of the eye region
were reached. The nephridial pores were used as the limiting
marks of the somite forward to the first pair of pores which lie
between the 16th and 17th rings. From this point forward
the sensillae aided somewhat, but the final results were based
on the distribution of the nerves.
A careful reéxamination of my material now showed that,
with one exception, I had collected or examined but one spe-
cies of Nephelis. The exception was found among the leeches
collected by Forbes in the Yellowstone region, and while the
differences are such that I feel warranted in suggesting that
proper study may show them to belong to another species, I
could not, from the specimens at hand, determine this point.
The common species of Nephelis found east of the Rocky
Mountains is the one that I have used in my investigations.
The names adopted by Verrill (3) must, as he prophesies, be
abandoned, and the name WVephelts lateralis be retained for this
species so widely distributed over the United States.
DESCRIPTION.
The size of the sexually mature adult varies from 4 cm. to
10 cm. at rest. Anterior to the sexual openings the body tapers
gradually to the mouth ; posterior to them the body continues
about the same size until a little in front of the anus, where
it narrows to the sucker. The transection of the body is
lenticular, though in the pre-clitellar region it approximates
acircle. The body flattens in swimming as it does in Macro-
bdella and Hirudo.
No. 1.] THE METAMERISM OF NEPHELIS. 25
The color of the adults varies from a light chocolate brown
free from any mark of pigmentation to almost a coal black free
from any light areas. Between these extremes of very light
and very dark all gradations of color and varieties of pigmenta-
tion may be found in individuals collected in the same pond or
stream. The very light adults are comparatively rare, while
among the young smaller individuals unpigmented specimens
are quite common. The very dark adults are about as
frequent as the very light adults, while a young dark individual
is very rare. Most of the individuals that I have collected
would fall into two sorts : those in which the pigmentation is
diffuse, varying only in intensity through many shades, and
those in which the pigmentation is arranged to a greater or
less degree in longitudinal stripes. I have collected three
individuals that showed definite pigmentation on the first ring
-of each somite, such as Blanchard (4) describes as constant for
NV. octoculata. Two were from Coonamasset pond near Woods
Holl, and one was from Wolf Lake near Chicago. Other speci-
mens with the diffuse type of pigmentation have shown a
slightly accented color on the first rings of some of the
somites, but not to the extent of defining all the somites.
The ground color is either a light mahogany brown or a pale
plumbeous gray. This may be observed on the ventral side,
which is usually free from pigment. The color of an indi-
vidual depends upon the amount of dark opaque pigment
present either as small granular particles or as highly branch-
ing pigment cells. If the view of Graf (8) is correct, that the
chloragogen cells wander into the epidermis and there break
up, leaving their remains as pigment particles, then the wide
variation in individuals taken from the same locality may be
explained as individual variations in the manner of excretion.
It is interesting to note that the stripes of color so common
in Nephelis lie in the lines of least resistance for wandering
chloragogen cells. A reference to Pl. VIII, Fig. 18, shows five
spaces between the bundles of long muscles on the dorsal side
through which pass the dorso-ventral muscles, nerves, and
8 Arnold Graf: “ Beitrige zur Kenntniss der Exkretionsorgane von Nephelis
vulgaris.” Jenaische Zeitschrift fiir Wissenschaft, N. ¥., Bd. xxi, p. 163, 1893.
26 BRISTOL. [Vou. XV.
blood vessels, and it is directly over these or some of them
that the pigment collects. I hope to make some further obser-
vations on this point by raising the progeny of one leech by
themselves until they attain the adult markings.
Description of Nephelis Lateralis.
Since the analysis of Clepsine by Whitman (9) has given
the prostomium the value of a somite consisting of one ring, I
have followed the notation used by him and have counted the
prostomium as ring No. 1 and somite No. 1.
Excepting the clitellum during its active phase, the body
is not divided into obvious regions. The oral sucker is not
prominent as in some species of Clepsine and the anal sucker
is small, exceeding the body but little in width. The male
orifice lies normally between rings 36 and 37. The female
orifice lies normally between rings 38 and 39 (Pl. VI, Fig. 3).
The first pair of nephridiopores lies between rings 16 and 17
at the posterior edge of the 7th somite. There are four pairs
of nephridia anterior to the male orifice, and these differ from
the succeeding nephridia by reduction of certain parts. The
pores of the first pair of nephridia behind the male pore lie
between rings 36 and 37, about midway between the median
plane and the margin, and these are followed in regular order,
at intervals of five rings, by the remaining pores. The last
pores lie between rings 96 and 97, and the whole number of
pairs of nephridia is seventeen. The anus is dorsal and lies
behind the ro4th ring.
The clitellum consists of fifteen rings—from 28 to 42
inclusive. It includes the last four rings of somite X and the
first of somite XIII. It is plainly visible only during sexual
activity ; at other times it can scarcely be distinguished from
the adjacent rings. In the active condition it is paler in color
and may be swollen so as to become larger than any other part
of the body.
9“ The Metamerism of Clepsine.” Festschrift fiir Leuckart, p. 395, 1895.
No. 1.] THE METAMERISM OF NEPHELIS. 27
Somttes.
The number of rings in the typical somite is five, but this
number is reduced at each extremity. Unlike Clepsine, Mac-
robdella, Hirudo, and some other leeches, Nephelis does not
have the first ring of each somite, except in the anal region,
marked by especially large sensillae, and the study of a large
number of individuals showed the arrangement of sensillae
to be constant in this region. The last nephridiopores lie
between rings 96 and 97, and 97 is well marked by sensillae
(see Pl. VI, Fig. 7). This, then, is the first ring of a somite.
The next four rings following have no prominent sensillae, but
ring 102 is again strongly marked with them ; 103 is a broad
double ring ; 104 is another double ring, the latter half of
which bears sensillae. The anus sometimes divides this part
of the ring and so comes to be bounded anteriorly by 103, but
generally a thin portion of 104 forms the anterior lip of the
anus. Rings 102, 103, and the anterior half of 104 make up a
pre-anal abbreviated somite, while rings 97 to 101 form a com-
plete post-nephridial somite. Now going forward as far as the
first pair of nephridiopores (Fig. 3) the somites may be readily
traced by the nephridial openings, and they consist of five rings
each. At this point another criterion enables us to determine
one complete pre-nephridial somite. The ganglion in each
typical somite lies almost wholly in the first ring. If we count
five rings forward from the first nephridiopores, we find the
first ganglion of the nerve cord lying in this ring, the 12th.
The innervation of these five rings also proves that they make
up a complete somite. To recapitulate: The somite anterior
to the anus is reduced to two and a half, morphologically four,
rings ; thence forward to the 12th ring inclusive we find eight-
een complete somites, innervated by the eighteen separate
ganglia of the nerve cord.
The reduced somites of the head region are innervated from
the “brain” and sub-oesophageal ganglia, while the reduced
somites of the anal region are innervated from the anal
ganglia.
28 BRISTOL. [VoL. XV.
Hlead Region.
The innervation of the rings of the head region shows, as
will be demonstrated later, the limits of the reduced somites
to be as follows : (Fig. 3) I consists of the prostomium ; II of
rings No. 2 and 3; III of a single broad ring, No. 4; IV of a
single ring, No. 5 (this ring lies in the plane of flexion of the
body on the oral sucker and is very narrow) ; V consists of
three rings, Nos. 6, 7, and 8; VI consists also of three rings,
Nos. 9, 10, and 11.
Anal Region.
In the anal region (Pl. VI, Figs. 3 and 7) the innervation shows
the limits as follows : XXV consists of rings 102, 103, and the
anterior half of 104. XXVI consists of the posterior half of
104 and 105. 105 isa broad ring which in some individuals
shows a tendency to divide into two, sometimes three, rings.
It lies in the plane of flexion between the body and the anal
sucker. XXVII consists of 106, the last ring of the body and
the dorsal area of the sucker. XXVIII to XXXIV consist of
the sucker disc. External evidence of this is found in the six
radial lines of sensillae on either side of the median plane.
Summary.
The number of rings is 106 from prostomium to sucker.
The first pair of eyes (Fig. 4) lies in the 2d ring; the second
pair lies wholly in the 4th, while the third pair lies usually
between the 4th and 5th rings. The clitellum consists of
fifteen rings, Nos. 28 to 42 inclusive, or the 2d to 5th ring
of somite X, the ten rings of XI and XII, and the Ist ring of
somite XIII. The male orifice lies, usually, between the 36th
and 37th rings. The female orifice between the 38th and 39th
rings.
The anus opens in the hinder portion of the 104th ring, or
between the 104th and r1osth rings.
The first nephridiopore lies between the 16th and 17th
rings ; the last and 17th nephridiopore lies between the 96th
and 97th rings.
No. 1.] THE METAMERISM OF NEPHELIS. 29
The head region consists of the first six somites, comprising
the first eleven rings. The first body ganglion lies in the 12th
ring, the Ist ring of somite VII.
The 18th and last body ganglion lies in the 97th ring, the
Ist ring of somite XXIV.
The body region extends from ring 12 to ring 101, somites
VII to XXIV inclusive.
The anal region extends from ring 102 to the disc of the
sucker; somites XXV, XXVI, and XXVII.
The sucker contains seven somites, XXVIII to XXXIV.
HaBITAT.
Like other leeches, Nephelis keeps its body for the most
part in the dark, and must be sought for according to the con- .
ditions of the bottom of the pond or stream. Ina stony brook
or pond beach they may be found adhering to the underside
of the stones ; ona sand beach unshaded from the sun they
bury themselves almost completely in the sand, projecting
their heads at short intervals in search of food. Where the
overhanging trees have dropped their leaves into the water
they will be found on the underside of the leaves. They may
be found on the underside of the water-lily leaves, on floating
pieces of wood, and between the bark and the wood of rotting,
water-logged branches of trees.
They thrive under widely different conditions of water, soil,
and temperature, so long as food is obtainable. I have collected
them in the Charles River at Cambridge, Mass., during low
water in midsummer, when the river was reeking with sewage
and the chemical wastes from paper mills, while the tempera-
ture was but a few degrees lower than that of the air. Yet,
within a stone’s throw of the river bank I have collected them
quite as readily in a clear spring-water brook, in which the
water was so cold that collecting in it was almost painful.
The abundant food supply appeared to be the only feature
common to the two places.
The character of the bottom of a pond seems to be an
indifferent factor, for in the same pond they may be as numer-
30 BRISTOL. [VoOL. XV.
ous on a mud bottom as on a sand bottom. This was a matter
of surprise to me until I found the explanation. I noticed
that I invariably made the best collections on the shore that
looks towards the prevailing summer winds ; that is, the shore
towards which the surface current flows, bringing with it
crustaceans, dead fish, and various other food materials. The
windward shore is almost always barren of Nephelis, for the
water on that shore is the cool water of the deeper parts and
is poor in food for Nephelis. That this food supplying current
is the important factor in influencing the distribution of
Nephelis is beautifully demonstrated in the small fresh-water
ponds near Woods Holl.
These ponds lie in basins scooped out by glacial action, and
many of them have no outlet. Some are nearly circular,
others are elliptical or long and narrow. The surrounding
hills are comparatively high, and the direction of the prevailing
wind over the pond is frequently determined by the trend of
the lower land or valley near the pond. This exposure to
wind varies in different ponds lying near together, and Nephelis
are always more abundant on the lee shore. In brooks they
are usually more abundant near the mouth of the stream,
whether it flows into another stream or into a pond. This is
explained in the same way. Food brought down by the brook
is more plentiful at that point than at any other.
Hapsits, Foon, ETc.
Nephelis, like Aulostoma, is non-parasitic and differs from
the parasitic leeches in many of its habits. It does not readily
leave the water like Hirudo or Macrobdella, and in confinement
it seldom attempts to leave the aquarium after the first twenty-
four hours, if there be plenty of food. It swims freely and
rapidly with the same undulating movement that Hirudo
employs. In creeping it never brings the anal sucker up to
the oral sucker as Clepsine, Hirudo, and Macrobdella, but
usually attaches it about halfway between the two in the out-
stretched body. In common with other leeches, Nephelis has
the habit of fixing itself by the anal sucker and then undulating
No. 1.] THE METAMERISM OF NEPHELIS. 31
its body as in swimming. In repose it commonly seeks shelter
under a stone, a leaf, a clump of weeds, or in the upper layer
of mud or sand at the bottom, exposing only the anterior third
of the body. It rests in this position for comparatively long
periods and seems, at times, to be sleeping, or at least so slug-
gish as to require considerable stimulating before it responds.
Sometimes it rests curled up in a spiral with its head in the
center and attached by its anal sucker. When undisturbed
and active it creeps in search of food and stopping now and
then it attaches itself by the anal sucker and explores an arc
of the circle of which its body is the radius. The head sways
from right to left, up and down, while the body is extended
gradually to full length ; then the body is shortened and moved
through a small angle and the first process is repeated.
When hungry, either at rest or creeping in search of food,
Nephelis is quick to perceive its presence ; but while swim-
ming it seems to be less attracted, although it may swim nearly
in contact with the foodstuff. My experiments were made on
leeches in aquaria, purposely left without food for some days.
Leeches fresh from the pond gave practically the same results
as leeches that had been kept in confinement for long periods.
When the water is about 3 cm. deep an individual at rest on the
bottom will perceive a portion of food let down gently over-
head almost as soon as it touches the surface ; after a short
interval, fifteen or twenty seconds, the leeches lying from 4 to
6 cm. away will give evidence of perception and they will set
out to find it. If at another time, when the leeches are bunched
together in a mass, the food be placed about 10 cm. away, a
minute or a minute and a half may elapse before one shows
any sign of awakening and starting in search of the food.
Others follow more or less rapidly at intervals. Under these
conditions there seems to be some evidence of a sense of
direction, but it is vague, if not mere chance. Some, not
always the first ones, will start off in the proper direction;
others will stray afar; some will come within 1 cm. of the
food and pass on without noticing it ; others will start to swim
briskly in irregular paths as if to trace the scent, and when
near the food will suddenly settle down, fix the anal sucker,
32 BRISTOL. [VoL. XV.
and explore. They areas likely to explore away from the food
as towards it. If, while swimming, any portion of the body
touches the food, a leech will often perceive it, stop short, and
feed on it.
If a leech is feeding, any other leech that comes in contact
with it perceives instantly what the other is doing and rapidly
creeps along its body to partake of the meal. I have frequently
started up every individual in a bunch of a dozen or more, by
gently pushing a morsel to one which projected its head a little
beyond the others. The first motion of seizure would be enough
to set the whole bunch in commotion. If a bit of food be
gently placed on the back of an individual at rest, it will often
whirl rapidly about and seize it, though it remains indifferent
to another leech creeping over the same place. If, in a clear
aquarium containing some hungry Nephelis, the finger be rubbed
over the bottom and continued up the side out of the water, the
leeches as they creep along the bottom will perceive the scent
and follow the trail, even to some distance out of water.
These experiments indicate the same general conditions of
perception as Professor Whitman has found in Macrobdella (10).
In the summer time Nephelis lives in the shallow waters of
the pond, but in winter it goes down to the deeper parts or into
the mud of the edges if there is a good food supply. I have
found them in midwinter in seven feet of water when the ice
was 25 cm. thick, and in another place in the mud near the
edge when the ice was 50 cm. thick, leaving only 8 or 10 cm.
of water over the mud. In both cases the individuals were as
active as in summer, and some of them laid eggs after being a
few weeks in the aquaria. These developed and produced nor-
mal individuals which in one instance gave me a supply of
small individuals very opportunely.
Nervous SYSTEM.
When this work on the nervous system of Nephelis was
begun, the chief object in view was to determine the innerva-
tion of the somites as a means of elucidating the metamerism
of Nephelis.
10 “ The Leeches of Japan.” Quar. Journ. Micr. Soc. vol. xxvi, p. 317, 1886.
No. 1.] THE METAMERISM OF NEPHELIS. 33
Professor Whitman was making a study of the nervous sys-
tem of Clepsine (9) for this purpose, and suggested that the
same be done with Nephelis, in order to bring the two genera
into comparison. In order that the relations between the two
may be clear, I present the following summary of his paper, so
far as it bears on this question.
He presents some considerations drawn from embryological
evidence to show that the head includes a number of true
metameres. ‘Does it include anything more?”
“Tn the adult head we find the segments fairly well defined
behind the eyes, but how far the metameric division extends
into the prae-ocular region remains to be determined. With
reference to the origin of the head, we are compelled to take
one of two views. The head consists either (1) of a non-
metameric lobe plus a number of metameres originally belong-
ing to the trunk, or (2) of such metameres only, the non-
metameric head element of the ancestral form having been lost
or incorporated in the first metamere.”
Each body neuromere in Clepsine, disregarding the longi-
tudinal nerve cords which fuse regularly at the level of each
metameric center, comprises three pairs of nerves and _ six
ganglionic masses, each mass being contained in its own cap-
sule. Two of these are always ventral and median, the remain-
ing four are arranged in pairs, two on either side above the
nerve roots. The sub-oesophageal ganglia readily show their
metameric origin; the ventral capsules of the body neuromeres
persist, arranged in a median row with only the two anterior
capsules crowded into bilateral positions. The corresponding
lateral capsules are readily identified in the 6th, 5th, and
4th segments, while the others in the 3d and 2d have been
crowded out of the places they would naturally occupy. The
nerves from this region are also identified as containing the
elements of the single neuromere. VI, V, and IV have three
roots each; III shows only two roots, and II issues as a single
root, which soon divides into two branches. Sections show
very plainly the presence of five nerve roots, each with its pair
of median nerve cells. Thus the evidence is conclusive that the
sub-oesophageal region consists of five metameres (II to VI).
34 BRISTOL. - [Von. XV.
“ The surprising thing is that we have left what seems to be
the exact equivalent of a trunk neuromere ; one pair of nerves
(1) and six ganglionic sacs, of which two are median and four
are lateral. Whether there is a pair of ‘median nerve cells’
connected with this part of the nervous system, I cannot say.
I have not found them, but my search has not been exhaustive.
The equivalence in other respects is so complete that there
seems to be no escape from the conclusion that the ganglionic
centers of the ventral cord are simple repetitions, element for
element, of the ‘brain. The nervous system is made up of seg-
ments of equal morphological value throughout. It must be
regarded then either as a series of ‘brains’ or as a series of
ventral neuromeres, one or more of which have been carried
secondarily to the dorsal side, and which here take the place of
a brain that has been lost or confounded with the metameric
system. That a portion of neuromere II has suffered trans-
portation from the ventral to the dorsal side is certain; but
the development of the supra-oesophageal system does not
permit us to believe that neuromere I was ever post-oral in
position. Allowing that it represents genetically the annelid
brain, as it certainly seems to do, the ventral cord must be
regarded as a chain of brains. The dorsa/ position of the brain
signifies nothing more than that the anterior end of the double
nerve cord has been bent upward from its prae-oral and ventral
position and slipped backward over the oesophagus.”
In the caudal region, although the concentration is quite as
great as in the head region, the elements of the neuromeres
are plainly resolvable. Each neuromere is complete in the
number of capsules and the nerve roots, which, however, are
here reduced to two. The whole nerve chain is divisible into
three portions: the head with six neuromeres, the trunk with
twenty-one, and the caudal disc with seven, making a total of
thirty-four neuromeres. Referring to Pl. I (Pl. IV here), the
innervation of a typical body somite is made clear. We find the
nerve divided into ¢#ree distinct parts which we may designate
as anterior, middle, and posterior nerve, respectively. “A
glance will make clear one very interesting feature in the dis-
tribution of these nerves. They ¢xnervate three successive rings,
No. 1.] THE METAMERISM OF NEPHELIS. 35
the first and second of their own segment, and the third of the pre-
ceding segment. The distribution is thus triannulate and dimeric.”
Passing to the head region, we find a number of interesting
modifications of the plan found in the body somites. Nerve
VI has three parts, ‘but they are no longer the precise equiva-
lents of ‘anterior,’ ‘middle,’ and ‘posterior’ nerves. What
before appeared as the dorsal branch of the posterior nerve
now appears as the middle nerve, supplying the same sense
organs as before and, in addition, the inner lateral sense organ
of segment V. The third nerve has no dorsal branch except
the short one to the outer lateral sense organ. It has two
main branches, however, one of which takes the place of the
‘middle’ nerve, the other that of the ‘posterior’ nerve. The
first nerve alone remains the unchanged ‘anterior’ nerve.
The branch running to the inner lateral sense organ (z./.) of
segment V belongs, according to what we saw in typical seg-
ments, not to segment VI, but to segment V.
“In segment V we find three nerves, but their composition and
distribution depart still further from the typical arrangement.
This nerve, as shown in Pl. I [Pl. IV here], gives off a number
of motor branches, and then passes to the outer lateral and
marginal sense organs and the labial organs of four rings (8-12).
It innervates then the first and second rings of its own segment,
and two rings (9-10) of segment IV. It corresponds then to
the ‘middle’ nerve in the trunk region, but contains also fibers
belonging to three other nerves, namely, the ‘ posterior’ nerve
of the preceding segment, and the ‘anterior’ and ‘posterior’
of its own segment. Just above and a little in advance of this
root appears another quite strong nerve, which rises and passes
forward over the lateral angle of the supra-oesophageal ganglia.
This nerve divides just in front of the head ganglia, sending
one branch to the inner lateral organ of segment IV, and the
other to the median organs of segments IV and V._ This nerve
then corresponds to the dorsal sensory branch of a ‘posterior’
nerve, and includes so far as it goes the fibers of two such
branches, for segments IV and V.
“In segment IV we find only two nerves, one small motor,
corresponding to the ‘anterior’ nerve, and one large nerve
36 BRISTOL. [Vot. XV.
which, after giving off several motor nerves, runs to the labial
sense organs of three rings (6-8) and to the outer lateral organ
of ring 8 in its own segment. This nerve corresponds in the
main to a ‘middle’ nerve. The sensory ‘dorsal branch’ of the
‘posterior’ nerve of this segment, as we have seen, is united
with the corresponding nerve of segment V.
“In segment III we find only two nerves, corresponding with
the two seen in segment IV. Where is the sensory ‘dorsal
branch’? On examining nerve II, we find it contains the
missing nerve united with the corresponding nerve of segment
II. Nerve II supplies not only the rudimentary eyes (median
sense organs) of its own segment, but also the pair of large
eyes and the inner lateral organ of segment III. One of its
two main branches supplies the outer lateral organ and the
labial organs of segment II.
“Nerve I innervates the median, the inner lateral, and labial
sense organs of the most anterior division of the head.”
This species shows also very plainly that some of the meta-
meric sense organs acquire eye-like properties in the head
region which gradually increase towards the anterior somites.
“Tn no other species hitherto described do we find the sensillae
passing by such gradations into the eyes. The serial homology
of these organs with the eyes is then a fact demonstrated not only
by the embryonic development, but also by the structural grada-
tions in the adult animal.”
In the concluding portion Professor Whitman reviews the
evidence derived from the innervation of the head region and
says: “The morphological equivalence of segment I with the
following segments is evident to a degree that is really aston-
ishing. It makes no departure from the typical trunk segment
which is not led up to through gradations represented in the
segments immediately following it.”
Tue Nervous System oF NEPHELIS.
The nervous system of Nephelis may for convenience be
divided into two parts: that portion which responds to external
stimuli and codrdinates the muscles of locomotion, the central
No. 1.] THE METAMERISM OF NEPHELITS. 37
nervous system; and that portion intimately connected with
the control of the organs of internal life which I shall call the
sympathetic system. These two parts differ widely in certain
characteristics of structure as well as of function. The central
nervous system is strongly metameric throughout its length.
Its cells are relatively larger and are referable to the unipolar
and bipolar types for the most part. The fibers of these cells
always tend to run in bundles and never to form plexuses. The
sympathetic, on the other hand, is free from any discoverable
trace of metamerism; its cells are small and frequently multi-
polar, and the fibers always tend to form plexuses (Pl. VIII,
Fig. 19).
The Central Nervous System.
The entire ganglionic chain in Nephelis, as in other leeches,
is contained in the ventral blood sinus, which, according to
Bourne and others, is one of the vestiges of the original coelomic
cavity. This sinus runs directly under the alimentary canal
and is readily distinguished by its dark pigmentation and the
swellings within which lie the ganglia.
The anterior end of the chain, called the sub-oesophageal
ganglia and the brain, consists of a mass of neuromeres more
or less completely fused together, and forming a collar about
the oesophagus. The posterior end, called the “anal ganglia,”
consists likewise of a number of neuromeres more or less com-
pletely fused. Between these terminal portions lie eighteen
neuromeres joined each to the next by two connectives. Between
these, and dorsal to the axis of the chain, lies a small bundle of
fibers known as the median nerve, or Faivre’s nerve. These
connectives are longest in the mid-body region and decrease in
length towards either end of the body, becoming almost nil in
the most fused parts at both extremities. Within each connective
lies a “colossal axial” cell, the nucleus lying about midway be-
tween the neuromeres, as has been described for other leeches.
At the points of junction between the connectives and the
neuromeres the fibers of the connectives do not separate into
small bundles as they do in Hirudo and Macrobdella, but each
continues into the body of the neuromere as a single bundle.
38 BRISTOL. [VoL. XV.
A Typical Neuromere.
In order to analyze the “brain” and the “anal ganglia”’
it is necessary to know the component parts of a typical neu-
romere and to grasp their relations to each other under normal
conditions.
The general shape of a ganglion is that of a flattened ellip-
soid, the long axis of which is parallel to that of the body; the
ventral surface being slightly more convex than the dorsal (PI.
VI, Fig. 9). Each ganglion gives rise to two nerves on each
side which leave the ganglion and proceed for a short distance
in a horizontal plane, and then branching, go to the dorsal or
ventral side.
The anterior nerve, however, is not a single nerve. It
results from the fusion of a ventral and a dorsal root, the
fusion taking place almost immediately after their departure
from the body of the ganglion (Pl. VI, Fig. 9). This fact
enables us to homologize the two lateral nerves of Nephelis
with the three of Clepsine as follows : I and II in Clepsine are
represented by I in Nephelis. III in Clepsine is II in Nephe-
lis. This homology is also shown by the correspondence of
the areas innervated by I in Nephelis and I and II in Clepsine
(Pll. 1V and V). I have not been able to find evidence of the
similar origin of the anterior nerve in Hirudo or Macrobdella,
and this fact suggests that Nephelis is an intermediate form
between the Clepsinidae and the five-ring leeches.
Between each pair of the lateral nerves, andnear the ganglion,
lies a bipolar cell, the principal prolongations of which pass
outward along the trunks of the lateral nerves for a short
distance and then fuse with them so as to be indistinguish-
able from them. This cell is found in other Hirudinea and
has been called from its discoverer “ Leydig’s cell.’’ Its pres-
ence throughout the entire ganglion chain, its variation under
different conditions, and its possible relations to other extra-
ganglionic cells are of sufficient interest to demand for it
separate consideration.
The nerve cells of the ganglion, with the exception of
“ Leydig’s cell,” are gathered into six groups or clusters lying
No. 1.] THE METAMERISM OF NEPHELIS. 39
outside of the central fibrous portion in capsules as in the other
Hirudinea. Pl. VI, Fig 9, shows the general arrangement.
Two clusters lie on the ventral surface in the median line (one
anterior and the other posterior) and two clusters on each
lateral face. The anterior lateral clusters are anterior to the
anterior nerves, and the posterior lateral clusters lie between
the two nerves. These lateral clusters rise slightly above the
dorsal surface of the body of the ganglion, and their posterior
edges are notched by the lateral nerves as they pass out from
the ganglion (Pl. VI, Fig. 12). The number six is constant in
the whole chain and the position is also constant except in the
supra-oesophageal ganglia or “brain.” In the “anal ganglia”
the lateral clusters tend to become dorsal towards the posterior
portion owing to compression, but they are perfectly recogniz-
able and referable to their proper neuromeres (PI. VI, Figs.
14 and I5).
Leydig's Cell.
Lying between the two nerve trunks of either side of the
ganglion, and nearly in contact with the posterior lateral
capsule, lies a large bipolar cell whose prolongations follow
along the lateral nerves as they pass outward, and finally fuse
with them (Pl. VI, Fig. 9). This cell was described first by
Leydig in Hzrudo medicinalis ; it was called “ Leydig’s cell”
by Hermann (11), and is found in all the Gnathobdellidae in
the same relation to the ganglion. I have not been able to
find any trace of fibers going to the ganglion; so far as I
have traced them they all pass outward along the nerves. This
cell, the significance of which is as yet entirely unknown, is
constant throughout the central nervous system. I have found
it in every neuromere of the body. In the first four neuromeres
it lies upon the fused nerves at some distance anterior to the
“brain” (Pl. V, Fig. 2). In the fifth neuromere (PI. VI, Fig. 11)
the cell is found at the angle formed by the separation of the
hitherto fused portions of the nerve trunks. In the sixth
neuromere, the last nerve of the sub-oesophageal mass, the cell
11 Emst Hermann: Das Central Nervensystem von Hirudo medicinalis.
Miinchen. 1875.
40 BRISTOL. [VoL. XV.
lies much closer to the mass and exhibits more of the characters
of the normal cells found in the body ganglia.
In the “anal ganglia” (Pl. VI, Figs. 14 and 15, and Fig. 1 in
the text), the first neuromere, XXV, the cell is normal. In
the second, XXVI, the two nerves partly fuse at a little
distance from the margin,
and continue thus for a
short distance, when they
become fully separate.
Within this region of par-
tial fusion “ Leydig’s cell”
is found lying between the
two trunks compressed and
changed into a_ spindle-
shaped cell; the prolonga-
tions extending median
and lateral as in the cells
of the first four neuro-
meres. In the third “anal”
neuromere, XXVII, the
cell appears in the same
relative position, but is
more compressed and elon-
gated. Good histological
Text-FiG. 1.— The four anterior neuromeres of the :
‘anal ganglia ’’ seen from the ventral side, showing preparations of these cells
the stages of compression of “‘ Leydig’s cell” tillit show that the size and
appears outside of the fused trunk in XXVIII (1st
anal in Clepsine) and the succeeding nerves. (From structure of the nucleus
sc are te orem ed ete nel con avec
from sections.) tical with those of a cell
from a mid-body region.
The fourth, XXVIII, and succeeding neuromeres, XXIX
to XXXIV, innervate the sucker. The fusion here is as com-
plete as in the first four neuromeres, and the “ Leydig’s cell”
has been pushed out, in the more complete fusion of the nerve
trunks, until it lies completely outside of and upon the nerve,
about midway from the anal ganglion to the edge of the
sucker ; the prolongations extending, as before, median and
lateral. In these most posterior neuromeres the size and
No. 1.] THE METAMERISM OF NEPHELIS. 41
structure of the characteristic features of the cell remain
unchanged from those of the normal mid-body cell.
Median Nerve Cells.
Within the fibrous body portion of the normal ganglion, near
the median plane, lie two “median nerve cells,” one slightly
anterior, the other posterior, tothe center. They are found in
all the Hirudinea and have been described by several authors.
Retzius (12) and Biedermann (13) show them in their figures of
Hirudo obtained by methylen blue, and they continue to appear
forward in the sub-oesophageal ganglia. In my analysis of the
“brain” I shall speak of these in detail. I have also found
them in the anterior neuromeres of the “anal ganglia,” but I
am not able to say whether they are present in the posterior
neuromeres or not.
The Fibrous Portion.
The fibrous part of the ganglion occupies the axial portion
and, macroscopically, appears to consist of thickenings of the
two connectives that afterwards fuse. It is perforated by two
small holes which lie close together on either side of the median
plane at the level of the anterior nerves. These perforations
persist in the fused portions of the nerve chain and afford good
evidence of the fusion of originally separate neuromeres.
According to Biedermann (/.c.) and Retzius (/. c.), this fibrous
part is made up of fibers from three different sources : (1) from
the connectives, part of which continue through the ganglion ;
(2) the efferent fibers from the neurones, which fill the six cap-
sules of the ganglion ; and (3) the afferent fibers, which are
the central termini of neurones whose trophic centers lie out-
side of the ganglia.
The first two sources are readily demonstrated, but Retzius
failed to find the source of all the fibers in the third set. His
12 G. Retzius: Biologische Untersuchungen. Neue Folge, 2. Stockholm. 18go.
18 W. Biedermann : “ Ueber den Ursprung und die Endigungsweise der Nerven
in den Ganglien wirbelloser Thiere.” /enaische Zeitschrift fiir Naturwiss., Bd. xxv,
1891.
42 BRISTOL. [VOL. XV.
figures are very clear and show the structure of the fibrous
portion of the ganglion with great detail.
He traces out the course of the axis cylinders from the cells
of each capsule, and of those fibers whose trophic centers lie
outside of the ganglion. He separates these fibers into six
groups, five of which come into the ganglion by way of the
connectives and one by way of the lateral nerves. This last
group, the sixth of Retzius, is of peculiar interest, because
while Retzius was drawn by his examination farther and farther
from the ganglion to search for the cell-bodies of this class of
fibers, until he reached the epidermis, he did not succeed in
finding them.
He says (N. F., ii, p. 21) : “Was stellen nun diese Fasern
dar? Sie sind offenbar Nervenfasern, welche peripherisch
verlaufen. Wo sind aber ihre Ganglienzellen? Da ich bei
den Crustaceen ahnliche, durch die peripheren Nervenzweige
aus den Ganglien des Bauchstrangs, austretende Nervenfasern
mit grossen in den Ganglien befindlichen Ganglienzellen in
Verbindung gefunden hatte, so schien es mir auch bei den
Hirudineen moglich zu sein, dass die fraglichen Fasern von
intraganglionaren Zellen entspringen konnten. Es erwies aber
durch zahlreiche Versuche, dass dieses nicht der Fall war ;
keine Ganglienzellen konnten mit ihnen in Verbindung ange-
troffen werden.
“Die fraglichen Fasern treten offenbar von der Peripherie
her in die Ganglien hinein, um hinter der geschilderten
Verastelung sich in ihre Punktsubstanz aufzuldsen.
“ Der von Hermann u. a. gemachte Befund grosser Gang-
lienzellen im Verlauf der peripheren Nervenzweige erklart aber
in sehr plausibler Weise ihre morphologische Bedeutung ;
sie miissen eine Art von Nebenfortsatzen dieser peripheren
Ganglienzellen darstellen, welche durch sie die contactartige
Verbindung mit den Elementen der Ganglien, d. h. dem cen-
tralen Nervensystem, aufrecht erhalten. Ich versuchte nun,
durch Methylenblaulosung die fraglichen Ganglienzellen und
ihre Fortzatze zu farben, aber der Pigmentreichthum und die
Scheidenbildungen verhinderten leider bei Aulastoma und
Hirudo die endgiiltige Lésung dieses interessanten Problems.”
No. 1.] THE METAMERISM OF NEPHELIS. 43
I believe that I have found the source of these fibers in
the bipolar cells that lie in the intermuscular nerve ring, the
description of which will be given later.
INNERVATION OF A Bopy METAMERE.
As I have said before, the ganglion lies in the first ring of
the somite and the two lateral nerves pass out, for a little
distance, in a horizontal plane and at right angles to the long
axis of the body. Then they divide into dorsal and ventral
branches, and again divide and subdivide to innervate the
various organs, as described in detail below. The first and
most striking fact is that the distribution is morphologically
identical with that of Clepsine, and the second that it confirms
Professor Whitman’s explanation of the derivation of the five-
ring metamere from a three-ring type: e,g., Clepsine.
A glance at Pl. V, metamere VIII, will show that the
anterior nerve innervates the 4th and 5th annuli of the pre-
ceding metamere on the ventral side and the extreme lateral
sensillae of the Ist annulus of its own metamere, and sends
fibers to the intermuscular nerve ring in the 5th annulus.
The posterior nerve sends one ventral branch to the sense
organs on the ventral side of the 3d annulus and two ventral
branches to the intermuscular nerve ring in the 2d annulus,
one of which by subdivision makes two connections with the
nerve ring. The principal branch of the posterior nerve is
dorsal, and this branch innervates, first, the few dorsal sensillae
on the 4th annulus of the preceding metamere; second, the
dorsal side of the nerve ring in the 5th annulus; third, the
large sensillae in the Ist ring of its somite; fourth, the dorsal
side of the nerve ring in the 2d annulus; and fifth, a few
dorsal sensillae in the 3d annulus. A comparison now with
Professor Whitman’s work on Clepsine will show how com-
pletely identical the distribution is (Pll. [Vand V). The 4th
and 5th annuli are morphologically the 3d annulus of Clepsine;
they are innervated by ventral portions of the anterior nerve,
as in Clepsine. The branch of the anterior nerve in Nephelis
that represents the middle nerve in Clepsine innervates exactly
44 BRISTOL. [Vot. XV.
the corresponding area in Nephelis, the ventral side of the Ist
annulus together with the outer lateral sensillae. The posterior
nerve in Nephelis, as in Clepsine, is the principal sensory
nerve, innervates dorsal sensillae in all five rings, and ventral
organs in annuli 2 and 3 (Clepsine 2).
Remembering, then, that annuli 4 and 5 represent annulus
3 in Clepsine, that 2 and 3 represent annulus 2 in Clepsine,
together with the homology of the nerves, the anterior nerve
of Nephelis representing the anterior and middle nerves of
Clepsine, we may use Professor Whitman’s words (9, p. 388) to
describe the distribution of the nerves of Clepsine for Nephelis
as well: “They innervate three successive rings, the Ist and
2d of their own segment and the 3d of the preceding segment.
The distribution is thus triannulate and dimeric.”
THE INNERVATION OF THE TERMINAL SOMITES.
We are now prepared to understand the modifications of the
plan of innervation found in a body somite as found in the
somites innervated by the more or less completely fused
terminal neuromeres in the head region, somites I to V, and
those in the anal region, somites XXV to XXXIV. These I
shall call terminal somites for convenience. Of these two
groups, those of the anal region present less departure from
the normal and hence will be described first.
The Anal Region.
As I have stated briefly elsewhere, the posterior portion of
the nerve chain is sometimes called the anal ganglia. As
Whitman and others have shown in other leeches, so in Nephe-
lis the nerve chain in this region consists of neuromeres more
or less fused together but retaining their fundamental charac-
teristics to such an extent that they can be easily identified.
Pl. VI, Figs. 14 and 15, show the dorsal and ventral views. The
number of neuromeres is ten, —the three anterior being less
modified by the fusion than the remaining seven. Koehler (14)
14 R. Koehler: Recherches sur la structure du systeme nerveux de la Nephelis.
8°. Nancy. 1882.
No. 1.] THE METAMERISM OF NEPHELIS. 45
is quoted by Francois (15) as assigning nine neuromeres to the
anal ganglia. The 1st anal neuromere, XXV, innervates the
first of the posterior terminal somites (Pl. V, Fig. 7), consisting
of annulus 102, the somewhat double annulus 103, and the ante-
rior half of 104. The 2d anal, XXVI, innervates the anus-
bearing somite, the posterior half of annulus 104, and the broad
annulus 105, which often shows traces of doubling. The 3d
anal, X XVII, innervates the last somite preceding the sucker,
which consists of annulus 106 and the “acetabulum,” the area
lying between the last annulus and the sucker. This area lies
in the plane of flexion of the body on the sucker and has lost
all trace of annulation. The succeeding seven neuromeres
innervate the sucker. The 4th anal, XXVIII, innervates
the anterior part of the sucker, immediately on either side of
the median plane. The last anal, XXXIV, innervates the
posterior part of the sucker in the same way, while the inter-
mediate neuromeres supply the rest of the sucker radially
between these parts. The XXVth or Ist anal somite resem-
bles in every particular a normal body somite. The connec-
tives to the XXVIth are very short and broad, and they
become shorter and more nearly uniform in breadth with the
body of the ganglion as we continue backward. They preserve
their characters as connectives, however, as is shown by the
oblong slits in the central fibrous portion, until the XXXIId
neuromere is reached, when the slits cease. The nerves of the
XXVIth and succeeding neuromeres are single, and the details
of their fusion have already been described under the “ Leydig’s
cell.” The arrangement of the capsules is interesting, for they
help to give evidence of the relations between Clepsine and
Nephelis (Fig. 1 in the text). The ventral capsules of a normal
neuromere lie one in front of the other, so that the line separat-
ing them is transverse. This obtains in XXV, XXVI, and
XXVII, the first three neuromeres of the “anal ganglia.”’
But in XXVIII and the succeeding neuromeres the ventral
capsules lie side by side, the line separating them being
longitudinal.
16 Ph. Frangois: Contribution a l’étude du systéme nerveux central des hiru-
dinges. Poitiers. 1885.
46 BRISTOL. [VoL. XV.
In discussing the anal region of Clepsine, in which the same
condition obtains, Whitman says (9, p. 388): “This arrange-
ment, evidently one of mechanical adjustment necessitated
by the shortening and crowding of the segments, prevails
throughout the caudal region with the exception of the first
segment (XXVIII) in which the sacs are placed one behind
the other as in typical trunk segments.”
Again, in his description of Clepsine plana (16, p. 413), he
says : ‘Reduction, as I have before pointed out, seems to have
begun at both extremities, and to have advanced from these
points towards the middle of the body. Its advance shows
how far a form has departed from the ancestral condition of
uniform somites. It is here that we discover a very important
guide to the systematic rank and relationship of different
forms.” These seven neuromeres, then, correspond to the
entire anal ganglia of Clepsine, as is shown by the degree of
fusion in the lateral nerves and in the arrangement of the ven-
tral capsules. The process of reduction in the anal region has
gone on further in Nephelis by three metameres than in Clep-
sine, while in the head region the number remains the same in
both forms.
The Head Region.
In order to make an analysis of the terminal somites of the
head region we must keep in mind that the external criteria of
a neuromere are six capsules, two being ventral, two pairs of
nerves, and a pair of ‘“ Leydig’s cells.” Beginning at the pos-
terior end and working forwards we shall have little, if any, dif-
ficulty in finding six neuromeres.
The nerves of the last neuromeres, VI (Pl. VI, Figs. 10-13),
arise as single trunks, but divide very near the body of the
ganglion, and in the angle of separation of each trunk lies a
“ Leydig’s cell.” Two pairs of lateral capsules, 6.6., separated
from the others, are easily identified as belonging to this nerve,
so that with the two end capsules of the two ventral series
we find all the elements of the typical body neuromeres. The
next nerve, V, arises as a single trunk and proceeds forwards as
16 “ Description of Clepsine plana.” Journ. of Morph., vol. iv, 1891.
No. 1.] THE METAMERISM OF NEPHELIS. 47
such until it passes the collar, when it divides into a ventral
and dorsal branch, and at this point of separation, as in VI,
lies a “ Leydig’s cell” (Pl. VII, Fig. 16). Two pairs of lateral
capsules lie well separated from the others, just anterior to
those belonging to VI, and two more, 5.5., of the ventral series
furnish the elements of this neuromere, V. The next nerve, IV,
arises as a single trunk, proceeds for a much longer distance
as a single trunk, sending off to the 5th annulus a dorsal branch
which quickly divides (Pl. V). This annulus is a very narrow
ring lying in the plane of flexion of the oral sucker and the
body. The “ Leydig’s cell” of this neuromere lies completely
outside of the nerve trunk, just as it does in XXVIII or the
Ath anal neuromere, and sends one fiber forward and one
backward (Pl. VII, Fig. 16). The two pairs of lateral capsules
belonging to the neuromere lie just anterior to those of neuro-
mere V close to the angle made by the collar. The third cap-
sule of the cluster at this point, lying close to, and anterior to,
these two, belongs to the next neuromere, III (Pl. VI, Figs. 11
and 13). These two lateral pairs of capsules, together with
two, 4.4., of the ventral capsules, complete the elements of
neuromere IV. The next nerve, III, arises just anterior to
IV and proceeds in much the same manner, dividing near
annulus 5 into dorsal and ventral branches. The “ Leydig’s
cell” lies alongside the trunk, as in IV (Pl. VII, Fig. 16). The
lateral capsules belonging to this neuromere show the same
peculiarity that Whitman found in Clepsine and that I have
seen in Macrobdella,—one pair lying close to the capsules
belonging to neuromere IV, while the other pair lies close to
the capsules of II, being separated by a wide space. The two
ventral capsules, 3.3., complete the elements of this neuromere
CEVA hioser tanden3):
The next nerve trunk arises from the collar as a single large
trunk and proceeds some little distance before it shows evidence
of separation, and just after separating a “ Leydig’s cell” appears
on each trunk as in III and IV (PI. VII, Fig. 16). We have
here, then, nerves II and I as their distribution also shows.
The lateral capsules of II are situated on the posterior side of
the collar, while the most anterior, 2.2., of the ventral capsules
48 BRISTOL. [Vou. XV.
complete the elements of this neuromere. The capsular ele-
ments of neuromere I differ from all the others, in that the
whole six are carried on the dorsal part of the collar (Pl. VI,
Figs. 11 and 13). Excepting the position of the ventral cap-
sules, the supra-oesophageal ganglion does not differ from the
typical neuromere, and the argument made by Whitman (9)
for Clepsine applies with equal force to Nephelis. Not only do
these nerve trunks, “ Leydig’s cells” and capsules, show by
their analysis the presence of six, and only six, neuromeres in
the head region, but the distribution in the peripheral parts
confirms it and sets the limits to the terminal somites in the
most conclusive manner.
The gold chloride stain was peculiarly valuable in this work,
and gave me sections with which it was only a question of
patience to follow out the well-defined nerve branches to their
peripheral parts. The fibers stand out distinct in form and
color, not to be confused with any other element in the head.
The spherical cysts of a parasitic nematode often furnished
excellent data for the perfect superposition of the drawings of
a series of sections and made it possible to follow out every
fiber represented in my drawings through its subdivisions to
the sense organs.
Beginning, as before, at the 11th annulus (Pl. V, Fig. 2)
I find the distribution from behind forward as follows: the
1Ith annulus contains an intermuscular nerve ring, and
receives its innervation from the succeeding neuromere, VII.
The posterior trunk nerve of VI sends a ventral branch to the
roth annulus and a dorsal branch which innervates dorsal
sensillae on the roth and oth annuli, as well as sending a
branch forward to the intermuscular nerve ring of annulus 8.
The anterior branch is wholly ventral and lateral, innervat-
ing the intermuscular nerve ring in the 8th annulus and a
few ventral sensillae. The gth, roth, and 11th annuli, there-
fore, make up metamere VI, the innervation of which is
strictly comparable to that of a body metamere, being dimeric
and triannulate. The most striking departure from the five-
ring metamere lies in the absence of the intermuscular nerve
ring from annulus 10, morphologically the 2d annulus of the
4, I.] THE METAMERISM OF NEPHELIS. 49
body metamere. Proceeding forwards, the 8th annulus has an
intermuscular nerve ring, innervated as has just been described
from the succeeding metamere, VI.
The inner, or median, branch of nerve V corresponds to the
anterior lateral nerve of a body somite and innervates a few
ventral sensillae on the 7th annulus, the outer lateral sensillae
of annulus 6, the ventral portion of the intermuscular nerve
ring in annulus 5, and thence passing forwards innervates the
labial sense organs on the ventral margin of the oral sucker.
The outer branch, corresponding to the posterior lateral nerve,
rises sharply to the dorsal side (Pl. VII, Fig. 16), innervates the
sensillae in the 7th and 6th annuli, and sends a branch to the
intermuscular nerve ring in the 5th annulus. The 6th, 7th,
and 8th annuli, then, form metamere V, and again we find the
dimeric and triannulate distribution found in Clepsine and in
the normal body metamere of Nephelis. From this metamere
forward the distribution is simpler but readily referable to the
body metamere. Annulus 5 is, as has been described, very
narrow and situated in the plane of flexion, yet it represents
metamere IV, for it contains an intermuscular nerve ring
innervated from the succeeding somite, and nerve IV gives off
a dorsal branch which, quickly dividing, innervates dorsal sen-
sillae and the third pair of eyes in this annulus, while the ven-
tral branch goes forward to innervate some of the lateral labial
sense organs. The persistence of this annulus in the plane of
flexion is a striking instance of the stability of the 1st annulus
of the metamere. Reduced, by its position, to the narrowest
annulus in the animal, so narrow that the eye belonging to
it has been forced partly outside of it into the broad 4th
annulus, it retains not only the characteristic features of the
Ist annulus, but also the intermuscular nerve ring belonging
to the posterior annulus of the normal body metamere.
Annulus 4 is broad and bears two rows of large sensillae
on its dorsal surface. It represents metamere III. Nerve III
divides as it enters the annulus, sending off a dorsal branch,
which soon divides, one branch going to the sensillae of the
annulus, the other innervating the second pair of eyes. The
ventral branch goes to labial organs on the dorso-lateral margin.
50 BRISTOL. [Vot. XV.
I have found evidences of the intermuscular nerve ring in
this annulus, but I have not been able to trace them com-
pletely enough to describe the relations which the ring bears
to the other parts of the nervous system.
The annuli lying in front of annulus 4 are incomplete, as
shown in Pl. VI, Figs. 5 and 6. Annulus 3 is well marked off
on the dorsal side, and the groove separating it from 2 is clear
and sharp in outline, while 1 is separated from 2 by a partial
groove extending about two-thirds of the way across the dorsal
surface.
Nerve II innervates the numerous, large, dorsal sensillae of
annulus 2, a few small ones on annulus 3, and the large first
pair of eyes in annulus 2. The ventral branch of this nerve is
reduced to a small branch that traverses the long axis of the
first eye and proceeds to a few of the dorsal labial organs.
Annuli 3 and 2, therefore, make up metamere II, and the
nerves of this metamere, like those described, innervate the
preceding metamere. Nerve I innervates annulus 1, supply-
ing the numerous large sensillae and the numerous mid-dorsal
labial organs. This fact raises the prostomium to the rank of
a metamere, and it must be counted as one. It has been cus-
tomary to disregard this reduced portion in numbering the
metameres and annuli, but hereafter it must be reckoned in
the count of metameres and annuli, as Whitman has done in
Clepsine (Z. c.).
Thus far the external features of the sub-oesophageal ganglia
and the “brain”’ and the distribution of the nerves have been
analyzed with concordant results ; there remains an internal
factor that adds still further proof for Professor Whitman’s
proposition. In Nephelis, as in Clepsine and other Hirudinea,
each body neuromere contains, as I have said, two “ median
nerve cells.” In Nephelis, as in Clepsine, they are found in
the sub-oesophageal portion, but arranged numerically, four
pairs appearing instead of five, as Whitman finds in Clepsine.
Careful examination of excellent sections reveals further that
in each side of the collar, near the capsules, 2.2., ascribed to
metamere II is a “median nerve cell” somewhat irregularly
compressed. The volume of the cell is still large, and the
No. 1.] THE METAMERISM OF NEPHELIS. 5I
nucleus has the same size and characteristics of the typical
“median nerve cell.” Still further dorsally, lying between
the capsules ascribed to metamere I, I find the pair of remain-
ing “median nerve cells” here compressed into a spindle
ANT. CAPSULES.
TExt-FiG. 2.— The ‘‘ median ”’ cell in neuromere I as seen in a horizontal section through the
dorsal part of the collar. The section passes through the capsules, 1.1.1., of Fig. 13, Pl. VI.
Camera outlines. ys immersion oc. 3. Reduced one-half.
form (Fig. 2 in the text). This discovery enables us now to
say, without reserve, that every element recognized in the body
neuromere is found in the supra-oesophageal ganglia, and,
therefore, that the supra-oesophageal ganglion or “brain” is
homologous with a body neuromere.
Tue INTERMUSCULAR NERVE RING.
Intimately connected with the central nervous system and
probably closely related to it in origin is a remarkable periph-
eral system of nerves hitherto, so far as I have been able to
learn, wholly unknown and unsuspected by all investigators
who have worked especially on the details of the nervous sys-
tem of leeches. For the discovery, I must again thank the
gold-chloride method of staining, for the only elements of it
52 BRISTOL. [VoL. XV.
that show in control preparations are the large bipolar cells,
and these are constant whatever method is employed.
Traces of this system are found in the most anterior portions
of the head in the form of large bipolar cells, whose connec-
tions I have not yet determined. The Ist ring occurs in
metamere IV, annulus 5; the 2d in metamere V, annulus 8;
the 3d in metamere VI, annulus 11, the last annulus of the
head region. From this point onward two rings are found in
each full metamere, in the 2d and 5th annuli respectively. I
have not found the ring behind metamere XXIV, or any well-
defined traces of it.
Beginning with metamere VII, I find eight bipolar cells con-
necting each ring with the succeeding one, as shown in Pl. V
and as will be described below. Each ring receives fibers from,
and sends fibers to, the central nervous system, and fibers
from the ring run to sensillae and muscles.
Figure 18, Pl. VII, is a diagram of one-half of a transection
made in the mid-body region, designed to show the arrange-
ment and relations of this nerve ring. It is a projection con-
structed from camera drawings of the neryous elements in
annuli 5 and 1 of adjoining somites upon a single plane,
viewed from anterior to posterior. The anterior nerve (com-
pare Pl. V, Fig. 2) runs laterally in the Ist annulus, sending
forward two ventral branches, between the 3d and 4th, and
the 4th and 5th bundles of long muscles, one to the nerve
ring in the 5th annulus, the other, the cut end of which is
shown in this figure, to a few ventral sensillae in the 4th
annulus. Passing out to the edge, it innervates three large
sensillae in annulus 1, as shown. The posterior nerve rises
dorsally and innervates three large sensillae on the dorsal sur-
face of annulus 1, and sends forward a branch which goes to
the nerve ring between the 4th and 5th bundles of dorsal
long muscles and on to a few dorsal sensillae in the 4th
annulus. The nerve ring here represented lies in the 5th
annulus. It consists of a complete ring of fibers which lie
between the layers of long muscles and the circular-oblique
muscle layers, whence the name I have given to it. Around
this ring at definite points are ten groups of bipolar cells, six
No. I.] THE METAMERISM OF NEPHELIS. 53
on the dorsal side, four on the ventral side. These cells
resemble in size and character of nucleus the ‘“ Leydig’s cell”
of the body neuromere. They all send fibers in both directions
into the ring, but I have been so far unable to follow them
to their terminations. The groups differ in characteristic
features, and for the purpose of description I have named them
as shown in the figure. They are constant in their position
and character throughout the leech, and this I determined by
comparing the six rings in three successive mid-body metameres
minutely, detail with detail, by the method described in the
early part of this paper.
The inner dorsal group (zz. d. dc.) consists of one bipolar
cell with a short pedicel, lying in the 3d muscle bundle
almost sessile on the ring. The outer dorsal group (0. d. dfc.)
consists of a cell with a very long pedicel, lying in the 4th
muscle bundle. The lateral dorsal group (/a¢. d. dpc.) consists
of six or eight cells, four to six of which are small, lying in the
outer or 6th muscle bundle.
The outer ventral group (0. v. dpc.) consists of one cell with
a short pedicel, lying in the 5th muscle bundle, and like the
inner ventral group, very near the point of junction with the
nerve trunk from the central system. The inner ventral group
(ix. v. dpc.) consists of two or three cells with pedicels of
medium length, lying near the edge of the 4th muscle bun-
dle. The connection with the central system at this point is
different from the others. The trunk divides just before reach-
ing the ring, and as I have traced fibers from this inner ventral
group into that branch nearest the group, it is evident that it
carries fibers from these cells to the ganglion, the sensory
fibers, while the other branch of the nerve trunk carries motor
fibers from the ganglion into the ring. If my data as just
given are sufficient, then we have the same morphological con-
ditions that obtain in the spinal ganglion of vertebrates. The
one piece of evidence wanting is the termination of the other
pole of the cell. These cells are without doubt those which
Retzius (2. c.) sought, as quoted above (page 42), to find in
the epidermis. These cells, together, possibly, with the outer
ventral cell, answer the conditions called for by Retzius for
54
TEXT-FIG. 3. — A very narrow
tangential section showing the
long bipolar cells running from
junction to junction through
two metameres and five rings.
(Camera drawing from a gold
chloride preparation.)
BRISTOL. : [Vou. XV.
those central endings which enter by
the anterior nerve. I have not been
able to determine which of the dorsal
cells send in fibers by way of the pos-
terior nerve. The ring itself, as shown
in the gold-chloride preparations, con-
sists of numerous fine fibers, which
come in part from the bipolar cells, in
part from the central system, and in
part from the diffuse sensillae in the
epidermis. It gives off fibrillae which,
ramifying the bundles of long muscles,
innervate the cells.
As I have said above, these rings
are connected, one with another, by
long bipolar cells lying between the
same muscle layers, as shown in Pl. V.
These connecting cells form, when taken
together, eight longitudinal paths, reach-
ing, according to my present investiga-
tions, from metamere VII through the
body region to metamere XXV._ I
have no doubt that they may be found
in the regions of the terminal somites,
but I have not yet been able to do so.
These long connective cells join the
nerve rings at the points where the
branches from the central system come
into the ring (Fig. 3 in the text).
Every point of connection of the ring
with the central system is also a point
of junction with two long bipolar cells.
I regret exceedingly that the histologi-
cal character of this junction is wholly
obscured by the swelling of the tissues
by the formic acid, and I hope to study
this detail by the use of Golgi’s method
of silver impregnation or methylen
No. 1.] THE METAMERISM OF NEPHELIS. 55
blue. The bodies of the cells lie about midway between
the nerve rings (Pl. V, long 4fc.), and present the same
general appearance in size and nucleus as the bipolar and
“ Leydig’s cells.”
The physiological réle played by this highly specialized
peripheral system is, doubtless, of the utmost importance, and
anything like a discussion of its functions can be made only
after the details of its constitution have been more fully worked
out, and something is known of the comparative anatomy of
the structure in other worms. It plainly offers a method for
short reflexes, such, for instance, as those controlling the
rhythmic undulating motions of the leech when at rest, and
supposed to be respiratory ; or for the successive stimulation
of the muscles in voluntary motions. The presence of this
system may throw some new light on the phenomena that go
under the name of ‘skin tension.”
Leaving its physiological functions for another investigation,
its presence may be brought to bear testimony on the question
of the derivation of the five-ring form of metamere. In his
Metamerism of Clepsine, Whitman says (p. 392): “In my
description of Clepszne plana (1891) the following note may be
found (p. 414): ‘I am reminded of an error into which I fell
in my paper on Japanese leeches. The error was the assump-
tion that all somites having less than five rings were abbre-
viated. The assumption should have been, as I now feel
convinced, that all somites with less than three rings are
abbreviated, and all with more than three have been increased
by the division of one or two of the three primary rings. I
have collected considerable evidence, which cannot be given
here, to show that in the evolution of Hirudo it was the 2d
and 3a rings that underwent division, while the 1st remained
undivided.’’’ On page 393 he continues, under the head of
“ Multiplication of annuli’’: “It is a fact of some importance,
in estimating the morphological value of the metamere, that
the multiplication of annuli seems to follow the same general
law as the multiplication of metameres in the embryo ; that is
to say, the posterior end of the metamere, like that of the
embryonic trunk, is the region of most rapid growth and elon-
56 BRISTOL. [VoL. XV.
gation, and the new rings are added by the division of the
ultimate (3d) ring alone, or by the division of both the ulti-
mate and the penultimate, somewhat as new metameres are
added by the division of the part lying behind the last one
formed. There is not then a uniform growth throughout the
trunk, but a curve for each metamere.”’
When I first found the intermuscular nerve ring I made a
very careful search in the 3d and 4th annuli for every trace
of nerves, and found that they were very weak in those struc-
tures, and when I came to plot down the nerve ring as it occurs
in the successive metameres, it became evident that the 5th
and 2d annuli of each somite (see Pl. V) were in strong
and equal connection with the Ist annulus which carries the
ganglion.
The absence of anything like a proportional division of
nerves between the several annuli shows, I believe, that the
annuli weak in nervous elements are the younger and second-
ary annuli, formed by the division of the 2d and 3d rings
as follows: the posterior half of Clepsine 2 becomes 3 in
Nephelis, and the anterior half of Clepsine 3 becomes 4 in
Nephelis. This mode of formation of the five-ring type of
somite does not involve any shifting of the nephridiopore, as
would happen if the posterior half of primitive 3 became 5 in
Nephelis.
This explanation assumes, of course, that the intermuscular
nerve ring is a constant feature in the leeches, and I feel
confident in predicting its discovery not only in the leeches,
but either that or its homologue in other annelids as soon as
they are studied with good nerve methods. There are many
evidences in the structure of the ring that it is an old and
very stable structure. The constancy through successive
metameres of such features as a long pedicelled cell always
in the same position on the ring ; the group of sensory fibers
separated from motor fibers near the inner ventral group of
bipolar cells ; the strong innervation from the central system,
and the definite longitudinal connectives, all point strongly to
the nerve ring, as we find it in Nephelis, as being very highly
specialized and the resultant of two originally distinct systems.
No. 1.] THE METAMERISM OF NEPHELIS. 57
In Lumbricus, for instance, both the afferent and efferent
fibers of the central system, though somewhat more diffuse than
in Nephelis, run in well-defined bundles, and Miss Langdon (17)
has found numerous bipolar cells along these nerves. These
two elements, fibers and cells, necessary to form the inter-
muscular nerve ring of Nephelis are present in Lumbricus,
and when one takes into consideration the vast differences
between the life habits of the sluggish, mainly herbivorous,
earthworm and the active, free-swimming, carnivorous Nephe-
lis, it does not seem difficult to believe that specialization, so
far advanced among the leeches in other particulars, may so
combine these factors as to produce the result found in Nephe-
lis. This suggestion by no means excludes any other explana-
tion; it is the one nearest at hand in the light of our present
knowledge. Recent investigations, with methylen blue espe-
cially, show that the peripheral bipolar ganglion cells connected
with the central system play an important part in the neural
system of some of the flat worms, and the whole matter of
peripheral nerve systems in this group, as well as that of the
annelids, is now in such a promising condition of investigation
that much light will, doubtless, soon be thrown upon it.
Tue “LARGE” NERVE CELLs.
Investigators of annelid and arthropod nervous systems have
been familiar for a long time with certain nerve cells so large
in comparison with the ordinary motor cells of a ganglion that
they have often designated them by such words as “ giant,”
“colossal,” and the like, and have described their location,
character, etc., without, so far as I know, bringing them into
any relation with each other. Such a relation exists in Nephe-
lis, though what its full significance may be I do not yet know.
I find the large cells in a body somite arranged as follows : in
the ventral chain two “median” or “ giant” cells wzthzn the
ganglion; in each connective detween the ganglia lies a ‘“colos-
sal”’ axial cell, which sends processes before and behind into
17 Fanny E. Langdon: “The Sense Organs of Lumbricus agricola Hoffm.”
Journ. of Morph., vol. xi, 1895.
58 BRISTOL. [Vou. XV.
the ganglion; therefore, zear the “median” cells. In each
intermuscular nerve ring are about twenty-two “large bipolar”
cells, some of which send processes zz¢o the ganglion in the
neighborhood of the “median” cells, while the eight “connec-
tive”’ bipolar cells joining the two rings terminate in some
manner in close proximity to the fibers of the nerve ring at
their junction with the ring. This leaves but one “ large”’ cell
yet to be accounted for, the “ Leydig’s cell” near the ganglion.
This is a bipolar cell, and the processes may be readily
distinguished as they pass outward on each lateral nerve toward
the periphery. These processes, however, soon fuse with the
nerve trunks, and I have not been able to follow them to any
considerable distance. It is quite significant that these same
trunks, or branches from them, send fibers into the intermuscu-
lar nerve ring, and furnish a path by which the processes of
this cell may reach to the other large cells. If it does not come
into proximity with the others, it forms an exception to all the
other “large” cells in the somite.
Another fact to be noted of all these cells, numbering nearly
fifty in each somite, is that the nuclei are practically the same
in size and character, and the volume of the different cells is,
so far as sections show, practically the same, whatever the
shape may be. Such a definite arrangement cannot be without
a purpose, the significance of which may in some measure be
revealed by their development, and this I hope to determine soon.
In brief, if by some means we could remove all other cells
and tissues in a somite excepting the “large” nerve cells and
leave them in their normal relations, we should find them all
joined together, forming a closed system capable, on the one
hand, of receiving impressions and stimulating muscles inde-
pendently, and, on the other hand, so related to the central
nervous system through the cells in the ganglia and connectives
as to make it completely adjunct to it.
Tue SYMPATHETIC SYSTEM.
Leydig (18) and others have found evidences of a sympathetic
system arising from the collar in certain leeches and other
18 F, Leydig: Tafeln zur vergleichenden Anatomie. Tiibingen. 1864.
INH Aisi] THE METAMERISM OF NEPHELIS. 59
annelids. In Nephelis, I have found it to be much more
extensive than has been figured in any annelid that has come
to my notice.
It arises in Nephelis very similar to the method figured by
Leydig (/. c.) for Haemopis vorax Brandt (Pl. IV, Fig. 5). In
this latter leech Leydig shows the sympathetic system lying on
the walls of the “crop,” but not connected with the part arising
from the collar. In Nephelis (Pl. VII, Fig. 17) the junction
between the two systems is on the median side of the collar
near the nerve root I-II. A fibrous projection from the anterior
side of the collar on each side gives rise to three branches
which run over the wall of the oesophagus; the dorsal and
ventral roots pass off in a 4 fashion, while the lateral root
comes off from the median side. Six capsules, three on either
side, contain nerve cells whose processes run into these
branches. One pair (Pl. VI, Figs. 11 and 13, symp.), the larger
of these capsules, is on the collar, the other two on the posterior
side of the dorsal branch. The ventral branches retain their
individuality for some distance as they approach the mid-
ventral line, but they soon become lost in a system of closed
meshes. The lateral branches continue as such, plainly taking
part in forming the meshes, but preserving their identity
throughout (Pl. VII, Fig. 17; Pl. VIII, Fig. 19).
The dorsal branch of each side rises parallel to the collar,
just in front of it. Professor Patten has called my attention to
the fact that this structure in this position is comparable to a
similar structure in Limulus. A narrower band connects them
in the mid-dorsal region so that they form together a half circle.
Two branches pass backward near the median plane into two
large ganglionic masses lying just under the collar (Pl. VIII,
Fig. 20). All these branches give off bundles of fibers that
run forward to the buccal cavity, and these bundles differ in
two ways from the plexuses behind the collar. They contain
but few, if any, cell bodies, being processes of the cells that lie
clustered together in ganglionic masses between the fibers and
the meshwork of the plexuses on the oesophageal walls. Fig.
20 shows this as it occurs on the dorsal side. It is a dorsal
view drawn from a Haller preparation and, hence, shows no
60 BRISTOL. [Vou. XV.
cells. The same characteristic difference between the fiber
bundles which run forward and the plexuses is found at each of
the other branches, and similar but smaller ganglionic masses
are present. Back of the collar, the muscular wall of the
alimentary canal is covered with a complicated meshwork, as
shown in Pl. VII, Fig. 17, and shown in greater detail in Pl.
VIII, Fig. 20. The cells are multipolar and send processes in
various directions, forming meshes. The processes ramify the
wall and innervate the muscle cells. The preparations made
with formic acid are not satisfactory for histological detail, and
Fig. 20 is introduced to show the distribution, not the structure.
This system continues over the whole alimentary tract in
substantially the same manner as shown on the oesophagus,
and though from theoretical considerations I expected and
sought diligently for metameric connections from the central
system, I am confident that none exist.
In a few very favorable sections I have seen what I believe
are traces of the sympathetic system in the post-anal region,
extending in the axial line of the acetabulum and the sucker.
The musculature in this region is so complicated that I cannot
determine this point to my complete satisfaction. Nerve cells
and fibers are certainly there and show plainly. There is no
theoretical objection to their being a part of the sympathetic,
for if in the ancestral form the anus was terminal and the
sympathetic system was present to the anus, then in the leech
the formation of the sucker undoubtedly made demands upon
the muscles of the alimentary tract that may have continued
after the anus moved forward and the sucker became imperforate.
Again, while feeding, the leeches always hold themselves fast
by the sucker, and the stronger stimuli to the muscles excited
by food in the alimentary tract, during a meal, may by this
same system be communicated to part of the muscles of the
sucker and may help to make the adhesion more effective.
No. 1.] THE METAMERISM OF NEPHELIS. 61
SUMMARY.
1. Nephelis differs from nearly all other leeches in the
external topography of the somite. The prominent sense
organs present in most genera are not easily visible in Nephelis,
excepting a few somites in the anal region.
2. Color and color markings do not afford criteria for the
determination of specific characters.
3. The body somites consist of five annuli as determined by
the nephridiopores. The somites of the terminal regions, contain-
ing less than five annuli were determined by the innervation.
4. But one species of Nephelis came under my observation
though the collections were made over a wide area of country.
5. The food supply is the controlling factor in the choice of
location in a pond or brook. ;
6. The head region contains six somites; the body region,
eighteen ; the anal, ten, making thirty-four in all.
7. The anterior nerve of a body neuromere arises from two
roots which fuse quickly ; the anterior nerve is, therefore, the
morphological equivalent of the first and second nerves of
Clepsine, while the posterior nerve in Nephelis is the equiva-
lent of the posterior nerve in Clepsine.
8. “Leydig’s cell” is present in every neuromere from one
to thirty-four.
g. The innervation of a body metamere in Nephelis is mor-
phologically identical with that of Clepsine.
10. The three anterior neuromeres of the anal ganglia show
evidences that reduction in Nephelis has progressed by so
much more than Clepsine, whose anal ganglia are represented
by the succeeding seven neuromeres. In the head region the
number of neuromeres is the same in both.
11. The distribution of the nerves in the somites of the ter-
minal regions is precisely referable to that of a body somite.
12. In both terminal regions each neuromere contains every
element found in a body neuromere.
13. A peripheral system of nerves composed of large bipolar
cells, which I have called intermuscular nerve rings, is in inti-
mate connection with the central system.
62 BRISTOL. [Vou. XV.
14. Some of these cells supply the fibers which Retzius and
Biedermann describe as ending in the ganglion.
15. These rings are connected longitudinally by other bi-
polar cells which thus form direct axial paths for nervous
impulses in addition to those furnished by the central system.
16. The relation of these rings to the distribution of nerves
in a body metamere affords striking proof in favor of Whitman’s
theory of the formation of a metamere with five annuli from
one of three annuli, that is, the posterior half of annulus 2
in Clepsine becomes annulus 3 in Nephelis; the anterior half
of annulus 3 in Clepsine becomes annulus 4 in Nephelis, while
the posterior half becomes annulus 5.
17. There is some evidence that this peripheral system of
nerves is an old and very stable structure, and will be found in
the other leeches. There is already some evidence that it or
its forerunner is present in other annelids.
18. The “giant” nerve cells in the two systems, central
and peripheral, are in close relation to each other, and are
strikingly alike in cytological characters.
19. The sympathetic nervous system is well developed, and
is connected with the central nervous system at the “collar.”
20. The branches at this connection form a nerve circle in
front of the “collar,” such as is found in the arthropoda.
21. The nerve cells in the sympathetic system are multipolar,
the processes forming meshes over the wall of the alimentary
tract.
22. There is some evidence that the sympathetic system
persists in the post-anal region, extending in the axial line to
the muscles of the concave side of the sucker.
No. 1.] THE METAMERISM OF NEPHELIS. 63
Il.
12.
BIBLIOGRAPHY.
Moguin-Tanpon, A. Monographie de la famille des hirudinées.
Paris. 1846.
Say, T. Major Long’s Second Expedition to the Source of St.
Peter’s River, vol. ii, Appendix to the Natural History. 1824.
(Republished in Diesing’s Systeme Helminthologique. Vol. i.)
VERRILL, A. E. Synopsis of the North American Fresh-water
Leeches. U. S. Fish Commissioner’s Report for 1872-74.
(Refers to the American Journal of Science. Vol. iii. 1872.)
BLANCHARD, R. Courtes notices sur les hirudinées. III. Descrip-
tion de la Nephelis atomaria Carena. Bxll. de la Soc. Zool. de
france. Tome xvii, p. 165. 1892.
LEIDy, Jos. Description of Nephelis punctata. Pyvoc. Acad. Nat.
Sct. of Philadelphia. P. 89. 1870.
Von LINDENFELD und PIETRUSZYNSKI. Beitrage zur Hirudineen
fauna Polens. Reviewed by Nusbaum. Biol. Centralblatt. Bd.
xii, p. 55. 1892.
WHITMAN, C. O. The External Morphology of the Leech. Proc.
Am. Acad. of Science. Vol. xx, p. 76. 1884.
GRAF, ARNOLD. Beitrage zur Kenntniss der Exkretionsorgane von
Nephelis vulgaris. /enatsche Zeitschrift fiir Wissenschaft. N.F.,
Bd. xxi, p. 163. 1893.
WHITMAN, C. O. The Metamerism of Clepsine. Festschrift fiir
Leuckart. P.395. 1895.
WHITMAN, C.O. The Leeches of Japan. Quar. Journ. Micr. Soc.
Vol. xxvi, p. 317. 1886.
HERMANN, Ernst. Das Central Nervensystem von Hirudo medici-
nalis. Miinchen. 1875.
ReEtzius, G. Biologische Untersuchungen. Neue Folge, 2. Stock-
holm. 1890.
BIEDERMANN, W. Ueber den Ursprung und die Endigungsweise der
Nerven in den Ganglien wirbelloser Thiere. /enaische Zeitschrift
Sir Naturwiss. Bd. xxv. 1891.
KOEHLER, R. Recherches sur la structure du systéme nerveux de la
Nephelis. 8°. Nancy. 1882.
FRANCOIS, PH. Contribution a l’étude du systéme nerveux central des
hirudinées. Poitiers. 1885.
WuitMAN, C. O. Description of Clepsine plana. Journ. of Morph.
Vol. iv. 1891.
LANGDON, Fanny E. The Sense Organs of Lumbricus agricola
Hoffm. Journ. of Morph. Vol. xi. 1895.
LeypiG, F. Tafeln zur vergleichenden Anatomie. Tiibingen. 1864.
64 BRISTOL.
DESCRIPTION OF PLATE Iv.
\
Reproduced through the kindness of Professor Whitman from the “ Fest-
schrift zum siebenzigsten Geburtstage Rudolph Leuckart’s,” Leipzig, 1892, being
Pl. XX XIX of that volume and appended to Professor Whitman’s article, “ The
Metamerism of Clepsine.”
Representing the first eight segments as reconstructed from sections and
surface views. X 50. The segments and nerves are numbered with Roman
characters, the annuli with Arabic numerals. The metameric and smaller
accessory sensillae of the dorsal side are represented in black, on the ventral side
by circles.
REFERENCE LETTERS.
Alphabetically arranged.
antm., anterior nerve. —é., buccal annulus.—d.é., dorsal branch of post.
nerve. —d.4. JV a. V, dorsal branch of nerves IV a. V.— £', rudimentary eyes.
— £%, principal pair of eyes. —/ 7 and 2, nephridial funnels. —g.d., bundles of
gland ducts. —z./., inner lateral sensillae. —/aé.g/., labial glands. — Z.s., longitu-
dinal septa. —., median sensillae.—g., marginal sensillae. —mg.s.. marginal
sinus. — m.s., median sinus. — miéd.z., middle nerve. — z.c., nerve cord. —meph.
z2, 1st and 2d nephridia. — zeph.f., nephridial pores. —o./., outer lateral sen-
sillae. —7.d., post-buccal annuli. — f/.g/., pharyngeal glands. — Zos¢.x., posterior
nerve. —s. 7-g, septa. —s.2., septal nerve branch. —¢y.s., transverse sinus. —
V 1-2, funnel vesicles. — v.d., ventral branch of posterior nerve.
atY
PL IV.
Morphology Vol. Xv.
Journal of
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66 BRISTOL.
DESCRIPTION OF PLATE V.
Dorsal view of the central nervous system of Nephelis in the first seven and
part of the eighth metameres as reconstructed from surface views and sections.
X 30 (circa). The metameres are indicated on the left of the drawing by Roman
numerals, the annuli are numbered by Arabic figures on the right. The nerves
are also numbered with Roman numerals. Dorsal sensillae appear as black
circles; ventral sensillae as light circles, excepting in the first five annuli, where
they are drawn as they appear in a surface view of a specimen killed and examined
in Haller’s fluid. The details of distribution are shown so plainly as to do away
with the need of explanatory references. On the left side, omitted from the right
for clearness, are seen the long bipolar cells (long 4./.c.) that connect the inter-
muscular nerve rings. They are found at the junctions (Junctions) of the nerve
branches with the rings, and the cut end of these branches are represented on that
side. Faivre’s nerve is indicated at #4. The first pair of nephridiopores (zs¢
uph.p.) lie between the 16th and 17th annuli.
Journal of Morphology. Vol.xv.
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THE METAMERISM OF NEPHELIS. 67
DESCRIPTION OF PLATE VI.
FIG. 3 is a diagram of Wephelis lateralis showing the boundaries of the somites
in Roman numerals, the annuli in Arabic figures, the nephridiopores (7f/.f.), the
boundaries of the clitellum (c/.), and the sexual openings (male ¢, female 9°).
The “brain” and the first two body neuromeres are sketched in to show their
relative positions.
Fic. 4 is a dorsal view of WV. dateralis from Wolf Lake near Chicago, IIL.,
showing the relative size of the annuli, and the number of sensillae as seen in a
specimen freshly killed in weak chromic acid. The eyes are represented by cres-
centic black areas, as seen in a specimen killed in Haller’s fluid. They represent
the pigmented part only, the large, clear visual cells extending in a cone-shaped
cluster from the concave side. The first pair look forward and outward, the
second and third pairs backward and outward. Camera drawing: Zeiss, comp.
oc. 1, obj. AA.
Fic. 5. Ventral view of the same showing the oral sucker as it appears in
a freshly killed and well-extended specimen. Annulus 3 is the first complete
annulus.
Fic. 6. Lateral views of the same showing the incomplete separation of annuli
1 and 2 and the doubling often seen in annuli 4 and 6.
Fic. 7. Anal region, dorsal view. Annuli 97, 102, 104, and 106 show well-
marked sensillae in specimens freshly killed in weak chromic acid, though the
number is not constant. Annulus 104 is double, the posterior half bearing the
sensillae. The semicircular area on top of the sucker, the “acetabulum,” is finely
wrinkled, all traces of annulation being lost.
Fic. 8. Lateral views of an unusual specimen showing the partial fusion of
the narrow annulus 5 with the broad annulus 4. Wolf Lake.
Fic. 9. Dorsal view of a normal body neuromere, showing the elliptical
fibrous part in the middle, the two perforations in it near the median line, the
slight groove in the median line passing between them. Faivre’s nerve is shown
as a narrow line between the two connectives. Two pairs of lateral capsules
containing nerve cells lie on either side of the fibrous portion, one lying anterior
to each of the lateral nerves. Two ventral capsules are shown under the fibrous
portion. The dorsal and ventral roots of the anterior lateral nerve are shown
together with the “ Leydig’s cell’*lying between the two lateral nerves. (Nitric
acid maceration ; slightly stained with borax carmine.) Camera: Zeiss, obj. A,
OC. 3.
Fic. 10. Dorsal view of the “brain,” the sub-oesophageal ganglia, and first
body neuromere, VII. (Nitric acid maceration.) Camera: Zeiss, obj. AA, oc. 2.
Fic. 11. Tracing of same. The capsules are numbered to correspond with
the neuromeres to which they belong. The capsule designated “Symph” con-
tributes its fibers to the sympathetic system only. It is not metameric.
Fic. 12. Side view of “brain” of same specimen, with same magnification.
Fic. 13. Tracing of same; capsules numbered as before. This view shows
the relations of the lateral capsules to the roots of their respective nerves in the
“brain,” and the same relations as they exist in the body neuromere, VII,
The separation of the lateral capsules, 3. 3., of neuromere III is also shown. The
position of the junction between the “brain” and the sympathetic is indicated on
68 BRISTOL.
the median side of the collar at the level of the root of the nerves I, II. One
large and two small capsules are connected with the sympathetic at each junction,
which sends a dorsal and a ventral nerve bundle, shown here, and a lateral
bundle not shown here, to the muscular wall of the alimentary canal. See
Pl. VI, Fig. 17.
Fic. 14. ‘Anal ganglia,” dorsal view.
Fic. 15. Ventral view of same. Metameres in Roman numerals. Capsules
in Arabic figures. In Fig. 14 the crowding of the lateral capsules to the dorsal
side begins with neuromere XXVIII, while the ventral capsules, Fig. 15, of the
same neuromere are also crowded out of the normal. The seven neuromeres,
XXVIII to XXXIV, constitute the entire “anal ganglia” of Clepsine. XXV is
nearly normal, lateral nerves not fused; XXVI is slightly compressed, lateral
nerves fused, but the two roots are plainly visible; XXVII is still more com-
pressed. These three neuromeres indicate the extent to which abbreviation has
proceeded farther in Nephelis than in Clepsine in this region.
Journal of Morphology Vol.xV
Sith. Warner 8 Wintes Franafoney,
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70 BRISTOL.
DESCRIPTION OF PLATE VII.
Fic. 16. Lateral view of the nervous system of the head region, reconstructed
from sections, showing the distribution and general paths of the principal nerve
trunks, the intermuscular nerve rings in annuli 5, 8, and r1, together with their
junctions with the central system, and other details. The principal eyes lie in
annulus 2, the small ventral branch of nerve II passing through it axially to the
labial sense organs. The second and third eyes look to the rear. On nerves I,
II, III, and IV the “ Leydig’s cells ” of those neuromeres lie outside of the nerve
trunks which are the fused anterior and posterior lateral nerves of their respective
neuromeres. In nerves V and VI the same cell is seen near the angle of separa-
tion of the fused parts.
Fic. 17. A reconstruction showing the general arrangement of the sympathetic
system. Combined from sections and specimens killed in Haller’s fluid. The
body outline is drawn from a median section, the same as used in Fig. 16, and
the parts of the central nervous system are traced in outline from Fig. 16. The
right half of the oesophagus is shown covered with the closed meshes of the
plexuses formed by the fibers from the multipolar cells. The cells are inadequately
shown ; see Fig. 20, Pl. VIII. Standing out from the median side of each half
collar, median to the fused roots of nerves I, II, is a fibrous projection which
gives rise to three trunks: (1) one going ventral and median, meeting the similar
trunk of the other side in the mid-ventral line; they form anastamoses and
gradually lose their outlines in the general plexus; (2) a lateral trunk that persists’
to the rear as shown; (3) a dorsal branch which runs parallel with the collar to
the median dorsal line; the details of this arrangement are shown in Fig. 20.
The dorsal trunk of each side carries two small capsules containing nerve cells,
while a third capsule, whose fibers go into the sympathetic system, is located on
the collar, not far from the origin of the connection between the central system
and the sympathetic. Anterior to the collar the sympathetic system runs to the
buccal cavity in fibrous bundles, apparently free from cell bodies.
a _
Journal of Morphology. Vol.Xxv:
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72 BRISTOL.
DESCRIPTION OF PLATE VIII.
Fic. 18. A projection of the nerves of the fifth and following first annuli
showing especially the intermuscular nerve ring and its relations to the central
nervous system. On the ring are ten (five in the half section) groups of bipolar
cells designated as below. The trunks from the central system join the ring at
the points marked 1 to 4 on the margin, which points also mark the points of junc-
tions of the long bipolar cells with the ring. The numerals in the figure designate
the bundles of longitudinal muscles, here seen in section, and drawn only in three
of the ventral bundles. In these are shown fibrillae which leave the ring to
innervate the muscle cells. The layers of circular and oblique muscles between
the epidermis and the long muscles are omitted for clearness, in order to show
the fibers from the sensillae to the ring.
ABBREVIATIONS.
al.
br. to rst ann. et seq.
intestine.
branches of nerves to the respective annuli.
cpt. epidermis.
gn. ganglion.
2u. lateral blood vessel.
med.bdl.l.m. median bundle of long muscles.
m.circ. and ob.
SENS LALA LLL
layer of circular and oblique muscles.
three types of sensillae.
in.vbp.C. inner ventral group of bipolar cells.
0.v.bf.C. outer “ “ “ “
in.d.b.p.c. inner dorsal “ “ “
0.a.b.p.c. outer ‘ “ “ “
lat.d.b.p.c. lateral “ “ “ “
I-4, marginal. junctions of ring with central system and the long con-
nective bipolar cells. See Pl. II, Junctions.
1-6, within. ends of the bundles of long muscles.
Outlines taken from camera drawings.
Fic. 19. Camera drawings of a section showing details of the sympathetic
system. The faint circles are the cut ends of muscle cells. In many of them
may be seen the nerve end plates. The wide bundle of fibers is part of the lateral
bundle. See Pl. VII, Fig. 17. Reichert, ;4; immersion, oc. 3.
Fic. 20. Dorsal view of part of the collar and sympathetic system. From a
flattened head killed in Perenyi’s fluid and viewed as a transparent object in
Haller’s fluid. It shows the continuity of the dorsal branches of the sympathetic,
the ganglionic masses under the collar (PI. VII, Fig. 17), and the different character
of the fibrous bundles running to the buccal cavity and the plexuses posterior to
the collar. Zeiss, c. oc. 2, camera drawing, reduced nearly one-half.
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THE GROWTH OF THE OVUM, FORMATION OF
THE POLAR BODIES, AND THE FPERTILIZA-
TION IN POLYCHOERUS CAUDATUS.
EDWARD G. GARDINER.
WHILE studying the segmentation of the ova of Polychoerus
caudatus, large numbers of these little animals were kept alive
in small aquaria under constant observation, and their habits
were carefully studied. When a number of them are placed
together in a small dish, after a short time most of them settle
on the bottom of the dish and remain quiet. There are gen-
erally, however, a noticeable few who keep on the move,
endeavoring to find a lodgment on the back of some other indi-
vidual. Sometimes one of the latter may remain motionless
and allow a pursuer to creep onto its back, but not infrequently
it moves off a short distance and then comes to rest again, in
which case the disturber follows and again endeavors to gain a
resting-place on its back. I have seen one of these worms
thus pursue others for upward of half an hour, and each time
just as it gained a lodgment on the back of the one sought,
the latter would move off. Finally, as if to get rid of further
annoyance, the pursued one came to the surface of the water,
where it swam with its ventral side up, thus preventing its
pursuer from accomplishing its purpose. After these observa-
tions had been repeated many times, it occurred to me that
the pursuer was endeavoring to fertilize the pursued by hypo-
dermic impregnation. To confirm this I have taken one of
these restless individuals and put it, with several others which
were at rest, in a shallow watch glass, where they could be
observed more closely. I found that after the restless one
gained a lodgment on another both were quiet for a short time
and then separated.
More frequently the under one moved first, and then in such
a manner as to suggest that it had been suddenly disturbed by
its burden. Several times I have seen the under one suddenly
74 GARDINER. [Vor. XV.
give convulsive struggles as if to rid itself of its annoyer and
swim rapidly away. Such individuals, when killed immediately,
showed in sections spermatozoa adhering to the back and
penetrating the tissues. In several of these specimens the
surface of the back where the spermatozoa were ez masse was
slightly abraded. Whether or not this abrasion was caused
by the action of another individual or was due to accident is
uncertain. It seems probable that the numerous chitinous
“mouth pieces,’ which are one of the characters on which
Mark (1) has founded the genus Polychoerus, may be used by
the animal to pierce the skin of other individuals so that sper-
matozoa deposited thereon may penetrate. The penis of this
form is unarmed, and is situated a short distance behind these
“mouth pieces,’ to which no definite function has hitherto
been attributed. That this is the normal method by which fer-
tilization is effected in this species I have no doubt whatever,
and believe it is but another case of hypodermic impregnation
to be added to the long list cited by Whitman (2) in his paper
on this subject. In no case, except when intertwined and
dying in stale water, have I seen two individuals bring their
ventral sides in contact as would be necessary in copulation.
In this group fertilization always takes place before the ova
are laid, and Dr. Sophie Pereyaslawzewa (3) states that in all
cases the polar bodies are formed while the ova are still in the
parent. That this is normally the rule in this species will be
shown later.
In specimens examined under the slight pressure of a cover
slip it is easy to determine whether or not the eggs have been
laid, for when present they can be distinctly seen each with
its large round germinal vesicle. Very frequently, however,
instead of the germinal vesicle, a clear, translucent, dumb-bell-
shaped structure, which occupies the greater portion of the
ovum, may be seen. This is the amphiaster of the first seg-
mentation spindle, which in this species is usually formed
before oviposition. This is, however, not an invariable rule,
for I have found, in normal egg capsules, ova with round, intact,
germinal vesicles. In such cases, when the polar bodies are
formed they are always extruded from the egg, while when
No. 1.] POLYCHOERUS CAUDATUS. 75
they are formed before the egg is laid they are always retained
within it. Further, it was noted that, when first captured,
animals are more apt to lay than when long in captivity, even
under the most favorable conditions of fresh sea water, etc.,
that I could devise. When, however, they are kept long
under rather unfavorable conditions, such as slightly stale or
too warm water, the dumb-bell-shaped structure disappears
by the drawing together of its extremities, and the nucleus
appears to return to its resting-stage.
Sections show, however, that the true resting-stage is never
attained. The centrospheres still exist fairly distinctly, each
containing in its center a faint centrosome. The cytoplasmic
network, which, when the spindle is fully formed, is so startlingly
conspicuous, has disappeared, and the achromatic spindle fibers
have become very indistinct and shortened. The chromo-
somes have lost their cheveron shape, and in some cases seem
to have melted so as to form round bodies, and the outline of
the whole structure is but slightly oval, and occupies very
much less space than does the fully formed amphiaster. In
this stage, when examined in the living specimen, this retro-
grade amphiaster may readily be mistaken for an intact
nucleus. Sections show that it is but poorly defined in outline,
and apparently the greater portion of the material which
formed the amphiaster has changed its chemistry, so that it no
longer differentiates by stains as formerly. To what extent
this degeneration can take place, without the power of recovery
being destroyed, it is impossible to say, but the ova of worms
which have been kept for a long time under such abnormal
conditions sometimes fail to develop.
This very unusual action of a spindle once fully formed sug-
gested immediately the “eigenthiimliche Art der Kernmeta-
morphose,” noted first by Selenka (4) in the ova of Thysano-
zoon, and afterwards in other forms by Lang (5) and Wheeler
(6). Selenka describes in the uterine ova of Thysanozoon
diesingii a spindle which must strongly resemble in its action
that just described in Polychoerus caudatus. To quote him
directly, he says: ‘‘Nachdem das Ei seine definitive Grésse
erreicht hat, beginnt das Keimblaschen sich in typischer
76 GARDINER. [VoL. XV.
Weise zur Theilung anzuschicken: die chromatischen Kern-
faden (ich gebrauche hier und in der Folge die Bezeich-
nungen welche Flemming eingefiihrt hat) ordnen sich zur
Knauelform, die achromatische Fadenspindel mit ihren Polar-
korpern, die zwei Radiensysteme der Eikorperstrahlung treten
auf u.s. w. Sobald aber die Fadenschleifen des Kernes die
«Sternform’ oder die Form der sog. Aequatorialplatte erlangt
haben, sistirt die begonnene Kerntheilung, und indem die
vorher weit auseinander geriickten Polarkérper sich langsam
wieder nahern, verschmelzen auch die Fadenschleifen wieder
zur ‘Knauelform,’ die Dotterstrahlung verschwindet nahezu
ganzlich und der Kern kehrt zur Ruheform zuriick. Der letztere
unterscheidet sich von dem friiheren Keimblaschen durch die
centrale Lage im Ei und den Mangel eines grossen Keimfleckes.
Der ganze Prozess kann also mit einer auf halbem Wege stehen
gebliebenen und wieder riickschreitenden indirecten Kern- und
Zelltheilung verglichen werden. Ein Resultat dieses Vorganges
ist leicht zu erkennen: namlich die Umgruppirung der Dotter-
kérnchen. Wahrend diese Dotterkornchen anfanglich gleich-
massig im Dotter zerstreut lagen, werden sie durch die erwahn-
ten Vorgange um die Centren der beiden Astera geschaart und
durch Annaherung der letzteren endlich in die Mitte des Eies
geschafft.”
In discussing this phenomenon, Lang says (pp. 295, 296):
“Die im Uterus enthaltenen Eier aller von mir untersuchten
Cotyleen und viele Acotyleen zeigen eigenthiimliche Veran-
derungen ihres Kernes, die vollstandig mit denen tibereinstim-
men, welche der Kern erleidet wenn sich die Zelle zur Theilung
anschickt. Ich kenne diese Veranderungen schon seit vielen
Jahren, vermochte aber nie fiir dieselben eine befriedigende
Erklarung zu finden.” Further, while Lang has no serviceable
hypothesis for the vazson d’étre of this phenomenon, he utterly
declines to accept that offered by Selenka, vzz., that it is a
normal action of the nucleus to bring about a redistribution of
the “ Dottermaterial’’ in the ovum. Still, Lang regards it asa
normal process of the nucleus, and does not connect it with the
conditions under which the animals bearing the ova may have
been brought just before being killed, as I think should be done.
No. 1.] POLYCHOERUS CAUDATUS. 77
While seeking to compare the above described retrograde
growth of the amphiaster in the ova of an Acoela with the
“disappearing spindle”’ in the uterine ova of a Polyclad, the fol-
lowing facts should be borne in mind. In both of these groups
the spermatozoa to be used in fertilizing the ova are contained
within the egg-bearing animal, introduced, probably, in most if
not in all cases, by hypodermic impregnation. (See Lang,
p. 636.) In the Polyclads the normal process is that, when the
eggs are laid, one or two spermatozoa pass with each ovum into
the egg capsule, where fertilization takes place later ; hence, if
for any reason oviposition is delayed or prevented, it would
seem perfectly possible that fertilization might be effected zx
utero. That such is the case under some circumstances the
following experiments show conclusively.
In the summer of 1895 and again in 1896 I obtained quite
a number of Leptoplana variabilis (Verrill), which laid quite
freely in captivity. In each egg capsule examined, from
one to three spermatozoa were found. In less than three
hours after oviposition the polar bodies were extruded, and
in from one to three hours after this the line of the first
cleavage plane was evident, and the two-celled stage was soon
after reached.
These facts being established, I brought six specimens which
appeared to contain ripe ova into a dish of warm, somewhat
stale sea water, in which were a number of Polychoerus caudatus
on which I was experimenting. Within an hour two laid ova
in which the development proceeded as above described. At
the end of eight hours the ova in the other four had, as far as
I could observe, undergone no change. The next morning
(twenty hours) the worms were almost dead; nor did they
appear to revive on being placed in fresh sea water. They were
then killed and sectioned. In two ova amphiasters were fully
formed, which from their small size appeared to be destined for
polar body formation rather than for the first segmentation
cleavage. Into one of these ova the sperm had penetrated.
In another ovum a polar body had been divided off, but not
extruded, and a sperm had entered the ovum; while in another,
two polar bodies were lying close to the egg membrane, and
78 GARDINER. [VoL. XV.
the first cleavage spindle was forming. These experiments
were repeated several times with about the same results.
The sections in no case gave very satisfactory preparations,
for frequently the tissues and the ova were abraded and injured
by the severity of the experiment. It was, however, clearly
demonstrated that if the animal bearing ripe ova were placed
under such conditions that it either could not, or would not, lay
its eggs, the development of the ova continued much as if
normal oviposition had taken place.
In several ova a larger spindle strongly resembling the disap-
pearing spindle as shown by Lang (Tafel 20, Fig. 4) was found.
I would suggest, therefore, that individuals of the Polyclads, in
which such structures are found, have before death been placed
under some abnormal conditions ; that the ovum has been ferti-
lized and the polar bodies formed ; that the first segmentation
spindle has been formed; and that the environment was such
that oviposition could not take place; consequently, that a retro-
grade development of this spindle has taken place exactly as
in Polychoerus. If this is so in one Polyclad, it may certainly
be so in others, and it seems much more logical to assume that
under fitting conditions the first segmentation amphiaster may be
formed in the uterine ovum and then undergo retrograde devel-
opment, as I have demonstrated to occur in P. caudatus, than
that an amphiaster should be formed with the express purpose
of disappearing again, as the observatiori above quoted would
indicate. The figures in von Graff's (9) great work show
in animals of several species ripe ova with large amphiasters
already formed within them. Von Graff, however, does not
discuss this matter in the text. It is interesting to note
the parallelism in the extrusion or non-extrusion of the polar
bodies in these two forms. In both, the polar bodies are
extruded only when the ovum has been laid before these
bodies are formed. If, for any reason, the polar bodies are
formed before the eggs are laid they are not extruded. It
may be that the sea water comes in more intimate contact
with the egg in the capsule than when within the parent, and
in some way stimulates the ovum to extrude the bodies.
No. 1.] POLYCHOERUS CAUDATUS. 79
Growth of the Ovum.
Several methods of killing and hardening were used, but by
far the best results were obtained after using Hermann’s fluid.
No other reagent seemed to preserve the nuclear structure so
satisfactorily. Pretty good results were also obtained with
Flemming’s fluid, and also with weak formaline, but corro-
sive sublimate solutions, either with or without the addition
of acetic acid, gave but poor results. As related in a former
paper (10) on the early development of this form, no satisfac-
tory method of killing the ova after they were laid in their
capsules was found. This is probably due to the impenetra-
bility of the capsule.
In his monograph on the Rhabdocoela, von Graff (8) states
that in the Acoela no vitellarium is present; and in this Dr.
Sophie Pereyaslawzewa (3) agrees. In a later work (9) he
describes with some detail the rapid growth of the ovum from
the small germ cell as being due to one germ cell absorbing
the substance of its immediate neighbors, which are thus con-
tinually reduced in size, while the only germ cells destined
to reach maturity grow at their expense. Thus, he says,
a struggle for existence and survival of the fittest occurs
among germ cells just as among individuals. That this
is the method of growth of the ova in many Acoela I have
satisfied myself by sectioning specimens of different spe-
cies ; but in P. caudatus it is quite different, for, when very
small, the ova pass from the ovary into an enlarged and dif-
ferentiated portion of the oviduct, which is charged with food
material, which the ova there absorb. Professor Mark (1)
speaks of this as a “differentiated portion of the ovary, where
the cells destined to reach maturity undergo . . . rapid increase
in size.” I think this should be described rather as a differen-
tiated portion of the oviduct ; for, as Pl. 1X, Fig. 1 (vz.), shows,
it lies between the ovary and the female genital pore. This
Fig. 1 is a diagram showing the condition of the female repro-
ductive organs at different seasons. The left half shows the
ovary (ov.) and enlarged oviduct filled with vitelline or food mate-
rial (v¢.), in the spring or early summer, before the near approach
80 GARDINER. [VoL. XV.
of the breeding season. The right half of the diagram shows
the same parts when the ova are rapidly approaching maturity.
The ova (.) which are destined for fertilization are contained
within the vitellarian portion of the oviduct (v¢.). These are
all about the same size and appear equally sure of surviving,
for all the ova in this gland are laid at the same time. Extend-
ing forward and at the same time upward from this vitellarium
is the ovary.
With but slight magnification three distinct stages in the
breeding season may be distinguished among _ individuals.
Those approaching the breeding season have enlarged vitel-
laria which do not contain ova ; later the vitellaria are crowded
with enlarging ova, and again later, after the ova are deposited,
the vitellaria are completely collapsed. I have kept animals
for several weeks after they have laid to determine whether or
not they laid a second time during the same season, but have
found no evidence that they do so. The vitellarium did not
recover from its collapsed state.
Double staining with lithio-carmine, followed by Lyons blue,
after a method described by Miss Katharine Foot (11), shows
that the substance of the vitellarium is very different from that
of the ovarian ovum. In tthe latter, both nuclei and cyto-
plasm take the red stain with great avidity, but do not stain at
all with the Lyons blue. On the other hand, the greater part
of the vitellarium is indifferent to the lithio-carmine, but does
stain with the blue. Sections through the vitellarium (Pl. IX,
Fig. 3) show it to be composed of large cells, the nuclei of which
are closely pressed against the cell walls. The nuclei, cell walls,
and a few fine protoplasmic filaments within the cell stain red,
while the cell contents stain a strong blue. Fig. 2 shows a sec-
tion through a portion of the vitellarium with three ova imbedded
in it. The ova have evidently just passed in from the ovary, for
they do not take the blue stain. Fig. 4 shows an ovum which
is about mature, having absorbed the dotter material from the
surrounding gland cells to such an extent that the red staining
cytoplasm, of which it was composed when it first entered the
vitellarium, is completely obscured. Around it are the col-
lapsed and empty gland cells.
No. 1.] POLYVCHOERUS CAUDATUS. 81
In the ova of Allolobophora foetida Miss Foot found that
the blue stain attacked the archoplasm and the spindle fibers,
while in the ova of P. caudatus its greatest affinity seems
to be to the lifeless dotter material, both while in the vitella-
rium and later while in the ova. It is curious that these stains
should act so differently on the ova of different animals. It
is unfortunate that this lithio-carmine and Lyons blue are
most valuable when the material has been killed in corrosive
sublimate, and are almost worthless where Hermann or any osmic
acid reagents have been used. Sublimate is the killing reagent
employed by Miss Foot, and from ova so treated she obtained
the beautiful figures shown in her paper. As narrated above,
sublimate shrinks and distorts the spindles in Polychoerus ova to
such an extent that it has much less value as a reagent. Many
animals killed in this way to study the vitelline glands proved
to contain ova in which the first segmentation spindles were
formed. The spindle fibers were often bent or distorted look-
ing, but the general outline of the spindle was distinctly red and
the periphery of the ovum equally distinctly blue. In no case
have I found any substance taking the blue stain in the ovum
which appeared to be archoplasmic or nucleolar in its origin.
In every individual, whether killed before, during, or after
the breeding season, immature ova closely clustered near the
lateral margins, quite close to the ventral wall of the body, are
conspicuous in sections. The ovarian ova (Fig. 5) are, as a
rule, oval in form, but are often packed so closely together
that almost any outline is possible. The dividing cell walls
are exceedingly indistinct and only to be made out in very
well-stained sections. The ova are finely granular, and show so
much greater affinity for almost all stains than do other tissues,
that, in sections in which the ovarian ova are the special object
of study, the color must be drawn until the other tissues are
but slightly tinged.
Even the nuclei of mature ova stain less vividly than do those
of immature cells. The probable reason for this is that at this
stage the whole protoplasm which is to constitute a part of the
future ovum is concentrated within the unripe ovum, and is
undiluted by the food yolk, which stains less intensely, and
$2 GARDINER. [Vou. XV.
which has not yet been drawn from the vitelline gland. Dr.
Sophie Pereyaslawzewa (3), in describing the young ovum of
what she terms the “ Pseudoacoela,’’ — practically the equiva-
lent of von Graff’s Acoela, — speaks of fine grains of “ vitellus
nutritif’’ adhering to the surface of the nucleus. The fate of
these grains she gives on page 149 as follows: “Cette dis-
position de toutes les parties inévitables de l’ceuf reste telle
jusqu’au moment de la fécundation, quand tout change: la vési-
cule disparait et emporte avec elle la force de l’attraction qui
jusqu’a ce moment retenait les grains du jaune d’ceuf adherents
a la surface de le vésicule ; ils montent tous a la périphérie
de l’ceuf, y stationnent, participent dans cette position a tous
les fractionnements de l’ceuf et restent inséparables du vitellus
formatif durant le développement embryonnaire. Comme il
reste inséparable du vitellus formatif, il est difficile de préciser
son role dans le développement embryonnaire.”’ I have never
observed either in the ova of P. cawdatus or that of any other
Turbellarian which I have studied any such changes as are here
described.
The nucleus of the ovarian ovum is quite peculiar in its struc-
ture, and the change which it undergoes as the egg matures
is worthy of some attention. In the smallest germ cell it is a
clear structure with well-marked granular network extending
from the nucleolus to the nuclear wall (Fig. 6). Within
the nucleolus is a spherical spot which stains very deeply.
This spot I have not been able to find in any but the ovarian
ova. Immediately after the ovum passes into the vitellarium
the whole nucleus increases enormously in size, so that its
diameter is quite as great as that of the ovarian ovum. This
increase is exceedingly rapid, for the intermediate stages are
seldom found. Still more striking are the changes which the
nucleolus undergoes (Fig. 7). Instead of a sphere, it grows to
be an enormous coil of densely staining substance, which forms
the most conspicuous feature in the whole ovum. The net-
work of chromatin in the nucleus (Fig. 6) gives place (Fig. 7)
to an exceedingly fine granular substance which stains less
deeply, and in which but faint traces of the formerly well-
marked network are to be seen.
No. 1.] POLYCHOERUS CAUDATUS. 83
In the stage shown in Fig. 7 the nucleolus has reached
its maximum development, and from now on it decreases
rapidly in size. As it decreases the structure of the whole
nucleus changes. A network of granular substance (Fig. 8)
resembling the chromatin granules of a still later stage occupies
the whole nucleus, while occasionally fragments of this net-
work stain as deeply as the chromatin granules of a later
stage. For the most part, however, the affinity for color is
much less than in the riper nucleus. The nucleolus has rela-
tively diminished in size, but not in its staining qualities. It
is to be noted that this structure never lies in the center of the
nucleus, but always near the nucleolar wall. In Fig. 9, which
represents the nucleus when fully ripe and ready to take part
in the formation of the first maturation spindle, the nucleolus
may be seen lying in close contact with the nuclear wall, com-
pletely broken up into fragments which are but faintly tinted
by a stain for which in an earlier stage this structure exhibited
the greatest affinity. On the other hand, the substance of the
nucleus shows a distinct granular reticulum of chromatin par-
ticles which now for the first time stains deeply. In following
the successive stages of the nucleus of the ovarian ovum (Figs.
5 and 6) through Figs. 7, 8, and g, one cannot but be impressed
with the fact that as the nucleolus diminishes in size and inten-
sity of affinity for stain, the nucleus acquires these very quali-
ties. This would suggest that the chromatin is built up at the
expense of the nucleolus, rather than that the nucleolus is a
by-product of the nucleus.
Formation of the Polar Bodies.
Before entering on the description of the formation of these
bodies it is necessary to make clear in what sense certain terms,
which have been applied by different authors to different struc-
tures, are used in this paper. The organ which in this ovum
presides over karyokinesis is a clear vesicle in which a dark
staining spot is formed only after the amphiaster is well devel-
oped. In accordance with the terminology adopted by Wilson
(13), I shall call this vesicle the centrosphere, and the spot
84 GARDINER. [VOL. XV.
within it the centrosome. Also, a few words are necessary on
the methods employed. Although in the egg-bearing animal,
and without injury to the animal, it is easy to determine
whether the ova are immature, about ripe, or whether the
amphiaster of the first segmentation cleavage has already been
formed, nothing further can be decided except by sections.
That is, no information whatsoever in regard to the formation
of the polar bodies or fertilization of the ovum can be obtained
by examination of the ova within the living parent. It was,
therefore, found most convenient and time-saving to kill large
numbers of individuals which examination with a hand lens
showed to contain large ova. Several of these were imbedded
in one block and sectioned without attempting to orient the
position of the ova. It was found that all the ova in one
animal were in very nearly the same stage of development,
but that the position of the axes of different ova differed ;
hence a section through the long axis of one ovum might cut
the ova next to it at a very different angle.
In this manner many hundred animals were sectioned, each
containing on an average half a dozen to a dozen or more eggs.
The size of the ova allowed from six to eight sections through
each, and as the whole worm was sectioned, the successive sec-
tions of any one ovum might be quite a distance apart. This
involved the use of a low power in order to be sure that the sec-
tion under inspection was the next in the series, and also every
section had to be carefully examined with the highest power
before the exact stage of development could be determined.
Hence, the amount of labor necessary to obtain anything like
a continuous history of the changes which occur in the ova has
been very great.
It was found that by far the most frequent stages were
either the nucleus intact, or the complete amphiaster of the first
segmentation cleavage fully formed, showing that the changes
between these two stages took place with great rapidity.
There are, as will be pointed out later on, several short gaps
in the chain of events between these two stages, but to fill
these an indefinite amount of section cutting might be required,
and the prospects of success were not sufficient to encourage
No. 1.] POLYCHOERUS CAUDATUS. 85
the undertaking. Also, it would have been desirable to have
followed the history of the centrospheres and centrosomes into
the segmenting ovum, but as related elsewhere (10), great diffi-
culty was experienced in getting sections of ova after they had
been inclosed in their gelatinous capsules. In nuclei in which
the nucleolus had broken down and begun to disintegrate, I
have noticed in two different cases, out of the many hundred
examined, adhering to the inner side of the nuclear wall and in
close proximity to the fragments of the nucleolus, a small, clear
vesicle which strongly resembles the primitive archoplasmic
vesicle as it appears in Fig. 9 outside of the nucleus. This
would indicate an intra-nucleolar origin for this body. The
substance of the nucleus in which this body is imbedded is,
however, so dense and stains so deeply that it is impossible to
be certain of this observation. The structure which I describe
may or may not be the primitive centrosphere.
It is therefore possible that in this form, as in Asteris as
described by Mathews (14), the centrosphere is developed and
remains within the nucleus until the ovum is fully matured.
The fact that at this time the nucleolus disintegrates suggests
that the centrosphere may have been located within this dense
mass. Although I have devoted much study to this point I
have not been able to demonstrate whether the centrosphere
actually migrates from within the nucleus or whether it origi-
nates in the cytoplasm.
I have sought in vain in young and almost mature ova for
some trace of this body or of a centrosome. The most careful
examination of serial sections, double stained with iron alum
haematoxylin and with Bordeau red or orange G, fail to dif-
ferentiate any such structure. If it exists in the cytoplasm, it
must be exceedingly minute, or else does not at this stage
respond to the same stains as it does later, and therefore must
be of a different chemical structure.
When first seen the centrosphere is a small, dark-walled
vesicle with a few short rays projecting in all directions (Fig. 9).
The center of this vesicle is quite clear and stains red with
Bordeau red or yellow with orange G. At this stage there is
no centrosome or speck of any kind within the vesicle. Still
86 GARDINER. [Vou. XV.
later this vesicle becomes dumb-bell-shaped and breaks into
two bodies which (Fig. 10) for a time are connected by fila-
ments. Apparently one of these bodies remains stationary
while the other migrates through an arc of 180° to the other
side of the nucleus, for, as Fig. 11 shows, one of the poles of
the amphiaster is formed near the broken-down nucleolus, at
which point the centrosphere first appeared.
In regard to the rays which extend out from these vesicles,
it is difficult to say whether they are composed of the same
substance as the walls of the vesicle, and therefore a part of it,
or whether they are formed directly from the cytoplasm in
which the centrosphere is imbedded. As, however, they grow
in length as the vesicle increases in size, both the vesicle and
rays must draw sustenance from the cytoplasm. These rays
seem to grow at either outer end by a direct change of the
cytoplasm, as Wilson (14) has described as occurring in the
fibers about the sperm asters of Zoropeneutes.
As soon as the centrospheres have attained their positions
at opposite sides of the nucleus the rays become speckled
with numerous fine microsomes, and at the same time lengthen
out so as to come in actual contact with the surface of the
nucleus, which at those points becomes very irregular in con-
tour (Fig. 11). It appears as if the ends of the fibers were
exerting mechanical pressure on the surface of the nucleus
and bending it in, but at the same time it is very evident
that the nuclear membrane is being dissolved at these points.
In a stage somewhat later than that shown in Fig. I1
the membrane is distinctly thinner, while in still later stages
(Figs. 12 and 13) it has entirely disappeared, leaving no
fragments in these regions, though it long continues round
the rest of the nucleus. With the first disappearance of the
membrane in this region, the substance of which the achromatic
spindle fibers are to be formed appears. Apparently it is the
linin of the nucleus which on the dissolution of the nuclear
wall flows out toward the centrosphere (Figs. 12 and 13).
At this stage it is homogeneous in appearance and does not
show the fibrous structure which characterizes it later. It
seems to flow directly out of the nucleus, and while so doing
No. 1.] _ POLYCHOERUS CAUDATUS. 87
to shorten the speckled rays from the centrospheres with
which it comes in contact. In Fig. 11 it will be seen that
these rays extend directly to the nuclear membrane; in Fig. 12
they are much shorter, while in Fig. 13 they have disappeared
on the sides directed toward the nucleus.
As the achromatic fibers are thus being formed from the
linin, that portion of the nucleus which lies directly between
the two centrospheres undergoes differentiation. The par-
ticles of chromatin collect to form irregular clumps (Fig. 13)
imbedded in clear linin. It looks as if the linin in flowing
out toward the aster centers had brought the chromatin par-
ticles into contact with one another, and that these gradually
melt together to form solid masses.
At first these masses show distinctly that they are made up
of separate chromatin granules (PI. XI, Fig. 29), but as the
amphiaster develops the particles knit together, and as the
achromatic rays begin to differentiate out of the amorphous
linin, these clumps become elongated into irregular rod-shaped
structures (Pl. X, Fig. 14, Pl. XII, Fig. 36). The number of
clumps or rod-shaped masses formed is by no means constant.
The manner in which the chromosomes are afterwards formed
from them will be described later. As these rod-shaped masses
are formed the whole amphiaster moves away from the broken
nucleus which remains in the center of the ovum. Fig. 14
shows this process. From the various positions in which I
have seen this amphiaster relative to the remnant of the
nucleus, I am inclined to believe that one of the aster centers
remains stationary as a pivot and that the other aster swings
through a wide arc of nearly, if not quite, 180°, thus freeing
the whole structure from the nucleus, and leaving the spindle
free to move towards its destination, the surface of the ovum,
which it does by moving very nearly in a straight line. The
fact that one side of the nucleus is intact in outline (Fig. 14)
while the other is completely destroyed bears out this view.
Also, at this stage numerous fragments of the nucleus are
scattered throughout the cytoplasm, particularly in the neigh-
borhood of the broken side of the nucleus. When this is the
case the whole cytoplasm stains more deeply than otherwise,
oe)
8 GARDINER. [VoL. XV.
owing apparently to the stain-absorbing substance of the
chromatin having been leached out into the cytoplasm. It
frequently happens that sections through ova containing such
fragments of the nucleus are perfectly unfit for study, owing to
the deepness of the stain, while other ova in the same worm and
on the same slide, and which, therefore, have been subjected
to exactly the same treatment, are not at all overstained.
The larger portion of the nucleus as shown in Fig. 14
remains in about the center of the ovum and undergoes a rapid
disintegration. The granular structure, in which the chroma-
tin particles are so distinctly separated from the linin (Fig. 13),
has completely disappeared, giving place to an ill-defined,
muddy-looking mass, as if the stainable substance of the
chromatin had leaked out and contaminated the hitherto clear
linin as well as the cytoplasm. This must indicate a chemical
change in both chromatin and linin. Also it should be noted
that this muddy appearance extends out around the amphiaster,
as if this structure while moving away from the degenerating
nucleus had dragged with it some color-absorbing substance.
The only chromatin which at this stage retains its former
power of being sharply differentiated by stain are the clumps
within the amphiaster, from which material the chromosomes
are to be formed. While the amphiaster moves on its way
to the surface of the ovum, the shattered portion of the
nucleus gradually fades in distinctness, the cytoplasm around
it still staining very deeply. Gradually, however, the nuclear
substance is so completely assimilated by the cytoplasm that
no trace of it remains, and the cytoplasm stains no more deeply
than before the nuclear wall was ruptured. The relative quan-
tity of substance taken from the nucleus to form the amphi-
aster as compared with the quantity assimilated or digested
by the cytoplasm will be discussed later (p. 99).
While this amphiaster still adheres to the remnant of the
nucleus, its length from aster to aster is of course greater than
the diameter of the nucleus. When, however, it once breaks
away from it, it is noticeable that instead of the asters drawing
apart, they begin to draw toward one another. Thus the length
shown in Fig. 14 is somewhat less than the diameter of the
No. 1.] POLYCHOERUS CAUDATUS. 89
nucleus. In Figs. 16 and 22 the length has evidently lessened
considerably. It must either be that the substance of the
spindle has undergone a condensation, or else that certain
material has been dissolved into the cytoplasm. There is, how-
ever, no evidence as to how either of these changes may have
been brought about. I have endeavored to determine the rela-
tion which the axis of the first maturation spindle, when first
formed, bears to the axis of the first segmentation spindle, but
with no satisfactory result. Fig. 26 is a diagram showing the
dumb-bell-shaped form of the first segmentation spindle; AA
the plane of the first cleavage ; BB the plane of the second
cleavage; P the polar bodies near the surface of the ovum.
The plane AA divides the ovum into two equal macromeres.
The plane BB cuts off two small cells which lie on the surface
of the macromeres. The polar bodies always lie near the sur-
face of one of the macromeres and near a point where AA and
BB intersect. It is evident that if the first maturation spindle
swung through an arc of 180° to break free from the nucleus
remnant, and then traveled in a strazghz line to the egg surface,
a line drawn through this amphiaster in anaphase when in con-
tact with the egg membrane as shown in Figs. 16 and 21, and
through the center of the ovum where the nucleus was, would
give the axis on which the amphiaster was formed. Since,
however, these facts cannot be accurately ascertained, except
by studying whole transparent ova, the relationship of these
axes still remains uncertain.
As the amphiaster moves towards its destination a very
distinct and beautiful cytoplasmic network is formed, extending
out from the asters. This could of course only be accomplished
by the continual breaking down and building up of the cyto-
plasmic material; for while the whole structure is in motion
there is at no time a distortion of the network (Fig. 22), such
as would result by the movement of the structure through the
ovum, — unless, indeed, the cytoplasm were very fluid and the
network a rigid structure. That the amphiaster is much more
rigid than the surrounding cytoplasm is shown by two instruc-
tive preparations which were the result of accident. Ova con-
taining amphiasters in the stage now under discussion were
go GARDINER. [VoL. XV.
ruptured just before the worm containing them was placed in
Hermann’s fluid. The cytoplasm had flowed or been pressed
out of the ovum carrying with it the amphiaster. In both
cases the cytoplasmic network had been completely bent and
twisted into a confused snarl. The achromatic rays were some-
what, but not nearly so much, distorted, but the centrospheres
were almost unchanged. From this I infer that the amphiaster
and the rays are on the whole much more rigid than the cyto-
plasmic network or the cytoplasm from which they were formed.
As one end of the amphiaster approaches the wall of the
ovum the network is brought in contact with it, and as the
amphiaster continues to approach, the network intervening dis-
appears, being absorbed into the cytoplasm, although round
the other aster the network continues as distinct as ever
(Fig. 16). At no time does the network extend far into the
cytoplasm, only the immediate neighborhood of the spindle being
involved. The rest of the cytoplasm looks in no way different
from that of an ovum in which the nucleus is still intact.
Shortly after the amphiaster has broken away from the
remnant of the nucleus the centrospheres increase enormously
in size, having a fairly reticular structure, merging gradually
on the one hand into the chromatic rays which connect it with
the cytoplasmic network, and on the other hand with the achro-
matic spindle fibers. Before the amphiaster is fully formed,
indeed at about the time the linin of the nucleus is beginning
to flow out (Fig. 12), an exceedingly small centrosome appears
for the first time in each centrosphere, and as these structures
enlarge the centrosomes become more conspicuous. By the
time the amphiaster breaks away from the nucleus (Fig. 14),
the centrosomes have become very prominent. In the stage
shown in Fig. 22 I have been able to discern a small central
black body within a small vesicle which stains a light blue.
This, however, is difficult to demonstrate, for in order to dis-
tinguish these structures the stain must be of exactly the right
intensity. The number of sections which show this stage is
comparatively small, and,many of them have been ruined —
after being studied — in removing the cover slip and experi-
menting for the exact amount of stain necessary. I have not
No. 1.] POLYCHOERUS CAUDATUS. gI
been fortunate enough to find any stages intermediate between
those shown in Figs. 16 and 22, therefore cannot detail the
changes which the centrosphere may undergo; but as shown in
Fig. 16, the centrospheres have become very much reduced in
size, and the centrosomes have entirely disappeared. As shown
in Fig. 21, a little more advanced stage, not only do the centro-
somes disappear, but the centrospheres are finally reduced to
small discs lying as it were on the ring of chromosomes.
From this stage on, the cytoplasmic network fades until there
is no trace of it left. The chromatic rays which connected
the centrospheres with the cytoplasmic network are still left,
though much reduced in size, as is shown in this last-men-
tioned figure. Fig. 17 shows the first polar body immediately
after the division has taken place. A small cap of the sub-
stance which composed the central mass of the centrosphere
rests on the ring of chromosomes, and below this may be seen
the remnants of the achromatic fibers. At a later stage the
fibers lose their structure and melt together with the stuff
forming the cap, and in this mass the chromatin of the chromo-
somes collects in scattered particles (Figs. 19 @ and 27).
The other daughter nucleus of the division lies close beside
it (Fig. 19 6), and several times I have seen what I believe to be
the first step towards a reorganization. The chromatin in-
creases in quantity and breaks up into numerous fine grains,
and at the same time the archoplasmic cap divides into two
masses, Fig. 18, as if about to form the centrospheres of the
new spindle. Fig. 19 a shows a polar body just formed, and 6
the other daughter product of the first polar spindle. In Jé the
chromatin granules are gathered in what seems to be the
region of the future equatorial plate. The achromatic rays
are beginning to form, and at each pole clear but small cen-
trospheres are formed. Between this stage and one in which
there are two polar bodies side by side, and a third body,
which without doubt is the female pronucleus, I have found
nothing. The polar bodies remain side by side just within the
egg membrane (Fig. 27) during several generations of cleavage
cells and, as they become less conspicuous with time, probably
they are ultimately absorbed.
92 GARDINER. [VoL. XV.
Evidently the formation of the second body is accomplished
with much greater rapidity than the first. Nor does this seem
strange when it is borne in mind that the formation of the
first maturation spindle involved action on a great nucleus,
of which some parts are apparently selected and some rejected,
and then the journey across half the diameter of the ovum,
before the anaphase can be accomplished, while in the forma-
tion of the second polar body the substance involved is small and
compact and no journey is necessary. Naturally, the process
takes less time, and while I much regret that the full process
of the reorganization has not come under my observation, the
vast number of ova which one might have to section before
finding the stages sought, presents too discouraging and unin-
teresting a piece of work to be contemplated.
In sections which show the two polar bodies side by side,
and near them the female pronucleus, no trace of the archo-
plasmic cap is visible in the latter. The remnant of this
body and of the achromatic fibers has entirely vanished.
Instead we find a mulberry-like-looking object, composed of a
large number of separate vesicles, each containing a round
bullet-like granule of chromatin (Fig. 20). In this form it
begins to migrate toward the center of the ovum, toward which
the male pronucleus is also moving. During the passage it
grows enormously and completely changes its structure. The
collections of separate vesicles disappear and give place to a
mass of chromatin grains imbedded in a linin network, almost
indistinguishable except from its smaller size and absence of a
nucleolus from the egg nucleus from which the first matura-
tion spindle was formed. It is to be noted that the centro-
somes in this form disappear with the anaphase.
Fertilization.
In no section have I been so fortunate to find the sperm
head in the act of entering the ovum, though I have frequently
found it within the cytoplasm quite close to the surface. In
such cases the outline of the ovum at the point nearest to the
sperm head showed a marked protrusion. This presumably
No. 1.] POLYCHOERUS CAUDATUS. 93
indicates the existence of an ‘“‘attraction”’ or “‘entrance cone,”’
though the structure of this part of the ovum differs very
slightly from the rest of it, except that it stains a little less
readily. Hence the manner, as well as exact point of entrance
with reference to the future plane of all cell cleavage, remains
undetermined. The fact that, shortly after entrance has been
effected, the sperm always lies near the pole opposite to that
at which the polar globules are formed seems, however, to
indicate that it has entered on the lower side of the ovum.
At the time when the first maturation spindle begins to
form, the sperm may generally be found at a distance from
the surface of less than one-fourth the egg diameter, and the
entrance cone has entirely disappeared. By this time the
sperm has increased enormously in diameter and is surrounded
by a peculiar substance (Fig. 23), which suggests in its appearance
a ball or snarl of thread, and which stains but slightly. This
substance must have been differentiated from the surrounding
cytoplasm while the sperm is moving toward the egg center.
While this growth of material about the sperm to form the
complete male pronucleus takes place, that portion of the
pronucleus which entered as sperm head increases very
markedly in diameter (Figs. 24 and 25), and at the same time
decreases in length as if it were melting away in the substance
built up around it by itself.
As yet no rays or aster are formed in connection with it,
although it progresses steadily toward the center of the
ovum. This is of interest, for in some cases the movement
through the cytoplasm has been attributed to the action of
the aster and in others to the movement of the sperm itself.
Since in this case no aster exists and the sperm is completely
imbedded in a substance built up by itself from the cytoplasm
and which moves with it, as if it were an integral part
of it, the translation must be due to other forces. When
close to the center of the ovum the remnant of the sperm
head is represented by a crescent-shaped dark-staining mass
(Fig. 25), still surrounded by a fibrous snarl of thread-like
substance. Then the chromatin begins to increase by the
breaking up of this head, until the whole pronucleus is filled
94 GARDINER. [Vou. XV.
with it, and a more or less broken spireme is formed. The
origin of the linin is difficult to account for, unless it is formed
from the substance which has been drawn from the cyto-
plasm and which surrounds the sperm head (Figs. 23-25).
At about this stage, at a point between the two pronuclei, but
much nearer to the male than to the female, a small but
distinct aster appears in the cytoplasm. The rays are very
straight and clear, are not affected by the stain, and the central
point shows no trace of a vesicle or centrosome. It can be
described simply as the central starting point of these rays,
and shows no structure which in any way differs from these
rays. Subsequent stages show conclusively that this aster
gives rise to the centrospheres of the cleavage spindle in
which the large black-staining centrosomes lie.
It is, therefore, evident that here, as in the beginning of the
first maturation spindle, the substance forming the centro-
sphere does not preéxist as such (unless too small to be seen),
or that its chemical structure is so different that it will not
react under the same stains as later. In regard to the origin
of this aster it appears to be purely cytoplasmic. There is
no evidence that it is in any way connected with the sperm
nucleus. With the study of this point in view, hundreds of
sections have been most carefully examined, but in no one
could any distinct particle resembling a centrosome or vesicle
be detected. A cytoplasmic origin of the centrosomes and
centrosphere of the male pronucleus and consequently of the
first cleavage spindle is too important a variation from the
usually ascribed origin of these bodies to rest on anything but
the best of evidence, and in this case the evidence is more of
a negative than of a‘positive nature. It agrees, however, with
the origin of the male centrosome as described by Wheeler (7)
in Mysostoma and in Allolobophora by Miss Foot (12).
As this aster increases in size the chromatin in both pronuclei
increases also, and except for the absence of nucleoli and their
smaller size, either might be mistaken for the original germinal
vesicle. The nuclear wall is, however, absent or but little
developed. Soon the aster centers divide (Fig. 15), the rays
from each extending out to the surfaces of the approaching
No. 1.] POLYCHOERUS CAUDATUS. 95
pronuclei, which they penetrate in a manner very different
from that in which the aster rays of the first maturation
centrosphere attach the egg nucleus, as will be explained
presently.
Formation of the First Cleavage Spindle.
The formation of this amphiaster is from its very inception so
different from that of the first maturation spindle that the two
can never be mistaken the one for the other. When in the
first maturation spindle the two centrospheres begin to act on
the nucleus they are at the opposite poles of the nucleus, while
in the first segmentation spindle the centrospheres lie between
the pronuclei (Fig. 15). I have never seen a complete union
of the pronuclei until both have been deeply penetrated by the
rays from the centrospheres. The relation of the rays to the
chromatin granules is different in the two cases, as a comparison
of Figs. 13 and 35 will show. In the former the rays which
emanate from the centrosphere do not reach to the surface of
the nucleus, while in the latter they pierce deeply into the
chromatic substance, the granules of which seem attracted by
the rays. In the first maturation spindle the centrospheres
never draw widely apart so as to form a very large structure,
while in the segmentation spindle the whole structure grows very
rapidly and soon occupies the whole interior of the ovum. In
the former the clumps and rods of chromatin are formed before
the spindle breaks away from the nucleus, while in the latter
these rods and clumps are first formed when the spindle has
almost attained its full size (Figs. 36 and 37). It is evident,
however, in both cases that the bulk of the chromatin con-
tained in these clumps far exceeds the amount which at a
later stage is found in the chromosomes. It is clear, there-
fore, that either a condensation of particles takes place or else
that some of the material in the clumps is removed. That
this latter is the case is very clearly shown in a later stage
before the chromosomes are completely formed. But before
the description of this is given it will be well to account for
that portion of the pronuclei not drawn into the clumps.
96 GARDINER. [VoL. XV.
As the centrospheres draw apart, the nuclei break to pieces
and the whole substance, except what is contained in the
clumps, is scattered throughout the cytoplasm, which in con-
sequence stains much more deeply for a time. Meantime,
when this spindle has about attained its full size the rods of
chromatin do not continue to elongate, but on the contrary flow
together at about their middle points, thus forming a continuous
ring which lies in exactly the position afterwards occupied by
the equatorial plate. This ring even while forming has numer-
ous outward prolongations extending into the surrounding cyto-
plasm (Figs. 28 and 30). Sections showing this structure were
very carefully studied, for nothing similar to it has to my
knowledge been described in amphiaster formation. To guard
against the possibility that this structure might be an artifact,
ova killed in Hermann’s, corrosive, corrosive acetic, Flem-
ming’s, formaline, and picro formaline, were sectioned, and the
structure as here figured was found in ova killed with all these
different reagents; therefore it can be stated with confidence
that it occurs normally in the formation of the amphiaster in
this egg. As is shown in Fig. 28, the prolongations, or
equatorial rays as they might be called, are composed for the
most part of separate granules of chromatin, while the chro-
matin which occupies the position of the future chromosomes
is much more compact. This difference I believe to be due
to the absorbing action of the cytoplasm on these exposed rays.
It may indicate that as the granules aggregate to form the.
chromosomes, the surplus material flows away in the form of
these equatorial rays. The number of rays is apparently the
same as the number of chromosomes. At a little older stage,
sections through the equatorial plate (Fig. 34) show thirty-one
cheveron-shaped chromosomes, from the bases of which numer-
ous dark anastomosing lines radiate outward. These appar-
ently are in a measure the remnants of the chromatin pro-
longations and the whole area tinges somewhat more deeply
than the rest of the cytoplasm, exactly as is shown in Fig. 14,
when chromatin is dissolved by the cytoplasm. Later the
cheveron-shaped chromosomes break at the apexes to split into
two.
No.1] POLYCHOERUS CAUDATUS. 97
The manner in which the chromosomes were formed in the
first maturation spindle seems to be exactly the same as here
described, but the parts involved are so much smaller that it
is more difficult to make them out; also, since the duration of
time from the entrance of the sperm to the complete formation
of the first maturation spindle is very short, the number of sec-
tions obtained through the polar spindles are but few. In no
case did I obtain a section directly in the plane of the equatorial
plate during its formation, but several diagonal sections show
clearly that a structure similar to that here described is formed
in the maturation spindle. The true significance of this reduc-
tion of the quantity of the chromatin is difficult to explain.
Furthermore, the reduction occurs twice during the formation of
each spindle. First the clumps of chromatin are selected from
the nuclei and then but a portion of these clumps are taken to
form the chromosomes. There are very many cases recorded
in which but a small portion of the nucleus is utilized, and the
rest dissolved into the cytoplasm, but in this ovum the quantity
seems unusually great.
I have endeavored to form some estimate of the relative
quantity in the chromosomes as compared with the bulk of the
nucleus, by taking a small apple about the diameter of the mag-
nified nucleus as shown in Fig. 7, and dividing it in halves,
quarters, eighths, sixteenths, etc. The seventh division, which
would be =}; of the whole, would certainly afford many times
more material than is contained in all of the chromosomes in
the cleavage spindle, and a comparison of Figs. 22 and 34
shows that the material in the chromosomes of this spindle is
vastly more bulky than in the polar spindle. Since it is thus
clear that a very small fraction —possibly not more than ;4, —
of the chromatin substance of the germinal vesicle and later
of the pronuclei is preserved as chromosomes, while the rest
is dissolved, the conclusion is immediately forced on us either
that the chromatin of the chromosomes differs chemically
from the bulk of the chromatin or that the chromosomes are
protected mechanically from the dissolving properties in cyto-
plasm. When it is remembered that the chromatin of the
polar bodies (Fig. 27) remains intact for many cell generations
98 GARDINER. [VoL. XV.
in the very substance which so quickly dissolves and assimilates
the rest of the nucleus, there seems no escape from one or the
other of the above conclusions.
Again, in the cleavage spindle the chromosomes remain prac-
tically unaltered for days (p. 77), while the enormous nuclear
chromatin once scattered through the cytoplasm dissolves in
a very short time. We cannot, therefore, but conclude that
they must differ chemically, or else that the achromatic rays
or some other substance builds around the chromosomes a wall
protecting them from the attacks of the dissolving agents in the
cytoplasm. The greater portion of the clumps or rods of chro-
matin described above must have the same structure as the
bulk of the nucleus, for they also are dissolved, though more
slowly, and only the chromosomes are left. Since it thus
appears that the substance of the chromosomes differs from
that of the rest of the chromatin of the nucleus in being insol-
uble, there are three possibilities presented as to the manner
in which this difference may have arisen :— /zrst: that two
distinct chromatic substances have existed prior to the spindle
formation, one soluble and the other insoluble ; the former des-
tined to be formed into chromosomes bearing the hereditary
traits, and the other, food for the cytoplasm. Second: that
there is but one chromatic substance in the nucleus, and that
this is soluble in the cytoplasm ; as the spindle is formed cer-
tain particles are changed into insoluble stuff from which the
chromosomes are formed; with the breaking down of the
nuclear wall the rest of the chromatin is exposed to and dis-
solved by thecytoplasm. 7Z/zrd.: there is but one chromatic
substance in the nucleus and that this substance is insoluble
in the cytoplasm; of this substance the chromosomes are
formed ; the rest undergoes chemical degeneration and be-
comes soluble in the cytoplasm.
Now to consider these three propositions in the order stated
above. If we accept the first, we must assume that some
force causes a migration of the insoluble rather than the sol-
uble particles toward the center of the nucleus to be formed
into clumps. This migration, however, if it occur, does not
visibly disturb the structure of the rest of the nucleus. On
No. 1.] POLYCHOERUS CAUDATUS. 99
the other hand, the fact that the greater portion of these
clumps is dissolved when in the form of equatorial rays, might
be explained by supposing that the condensation of the already
formed chromosome particles drags with it granules of the less
differentiated, soluble chromatin which have in them nothing
which is to be transmitted to the next cell generation. This
theory in no way conflicts with the “reduction theory” or
with the existence and transmission of the ‘ determinants,”
“ids,” etc., of Weismann. It simply asserts the existence of
two chromatic substances in the nucleus, which, though micro-
scopically indistinguishable, differ in the fact that the one
which is retained contains all that is essential to heredity,
while the other contains substances which are not to be
transmitted to the daughter nuclei, but become a part of the
cytoplasm.
The absorption by the cytoplasm of some of the products of
the nucleus is not an uncommon phenomenon, and the connec-
tion between the nucleus and yolk nucleus (15) shows that
chromatin is not necessarily a substance transmittible to the
chromosomes alone. An objection to this theory is that it
assumes the existence of a force to hold together or to col-
lect the chromosome particles containing the “ determinants ”
and ‘‘ids”” near the center of the nucleus. If the second prop-
osition is accepted, we must assume that the insoluble particles
received from the parent odgonia have degenerated, for they
were at one time insoluble ; if the third is accepted we must
assume that the greater portion of the hereditary qualities
existing in the nucleus are dissolved up as food for the cyto-
plasm. The supposition then that there are two kinds of
chromatin stuff, the one insoluble and bearing the heredity
which is to be transmitted to the daughter cells, and the
other food for the cytoplasm, seems unavoidable.
The exact use of this food is of course pure conjecture, but
the following suggestion may be worthy of notice. When
the soluble chromatin is thrown out into the cytoplasm, it is
digested quite rapidly; and then, and not till then, do the cen-
trospheres (which up to this time have been very small) begin
to grow and attain their full size. May it not be that the
100 GARDINER. [VoL. XV.
soluble chromatin affords to the cytoplasm the material neces-
sary to supply this growth?
To return now to the growth of the first segmentation
spindle. By the time the chromatin has become condensed
into clumps the achromatic fibers have become much more dis-
tinct. As stated above, the rays from the aster centers seem
to grow down into the pronuclei (Fig. 35). This is a marked
contrast to what appears to take place in the formation of the
first maturation spindle. In this latter, when the nuclear wall
is dissolved the linin appears to flow out and form the
achromatic rays; while in development of the segmentation
spindle the rays from the aster centers appear to grow down
into the pronuclei. It is of course possible that the linin into
which they grow affords material for that growth, but since the
bulk of these achromatic fibers soon far exceeds that of the
linin, it is evident that material is being elaborated from other
sources to form these rays. In the fully formed spindle
(Fig. 37) these rays are exceedingly large and distinct.
As these fibers develop, an exceedingly strong cytoplasmic
network surrounds the whole spindle (Figs. 36 and 37). This
network is dotted with numerous microsomes which with iron
haematoxylin stain a deep blue, not unlike the centrosomes.
This network soon extends to the very uttermost limits of the
ovum, so that the first superficial section, which may cut off but
a very small portion of the ovum, reveals the network and fully
prepares the observer for the large spindle which deeper sec-
tions will disclose. As the network thus extends further into
the cytoplasm, the rays supporting it and connecting it with
the aster centers increase in size and length. In a former
paper (10) mention was made of the strange manner in which
certain pigment granules are moved about so as to lie in the
same plane as the equatorial plate, and reference (16) to simi-
lar observations in other ova is made. The size of the rays
and network in the ova is shown in Fig. 37 and also in Fig. 28,
where the polar globules are surrounded by the network.
At the time when (10) was written I had sectioned but few
well-preserved ova and was unacquainted with the remarkable
structure of this spindle, but while examining certain pigment
No. 1.] POLYCHOERUS CAUDATUS. IOI
granules which are very characteristic of these ova I was struck
with their peculiar movements on the surface of the egg. To
quote the description written at the time: ‘ Not infrequently
while examining the surface of a (living) ovum with an oil
immersion lens, I have seen one of these granules come up
from within the ovum and move across the field of vision... .
When the ovum is thus viewed it is clearly suggested that there
are wonderfully active forces at work within, for the surface
fairly scintillates with the movements of the protoplasm and
these pigment granules.” Had I at that time known the size
of the spindle rays and the extent of the cytoplasmic network,
I should have felt less wonderment at the strange movements
of the pigment granules. These always find final lodgment in
a ring over the equatorial plate when the cytoplasmic rays are
least developed. While watching the surface of an ovum in the
two-cell stage I have seen one of the polar bodies lying close
inside the egg membrane bulge out the surface almost to the
bursting of the egg membrane, as if by pressure from below.
In the paper (10) on the segmentation of this ovum the peculiar
distortions of the ovum in different stages are referred to and
figured. I cannot but believe that much of this is due to the
movements of cytoplasmic rays.
At the stage shown in Fig. 36, when this remarkable cyto-
plasmic network was beginning to form, the centrospheres can
hardly be said to exist as a distinct structure, for when the
cytoplasmic network ceases the achromatic fibers begin. There
are, however, small clear spaces at the points from which the
achromatic fibers radiate. These spaces are the beginnings of
the centrospheres. As, however, the spindle grows, the cen-
trospheres enlarge very rapidly and become most conspicuous
structures. They at first are clear, colorless structures, but as
maturity approaches they become somewhat granular, with
occasional dark specks scattered here and there. Soon in
the center of each appears a clear, translucent spot in which
there is a small but distinct black or dark blue staining cen-
trosome. If the section is at all over-stained, the centreole
will stain as deeply as the centrosome, so that the whole struc-
ture, both centreole and centrosome, appears like an enormous
102 GARDINER. [Vou. XV.
centrosome, while the other portions of the amphiaster are
not markedly over-stained. When, however, the color is prop-
erly drawn the centreole is tinged a faint blue, as is shown in
Fig. 36. There are at this stage a few faint, dark fibers which
radiate into the surrounding centrosphere from the centreole.
By the time the chromosomes are fully formed, these radiations
are exceedingly distinct (Fig. 31), but from this stage on they
begin to fade away. Also as the radiations fade the cen-
trosome enlarges so as almost to fill the centreole, and at
the same time it elongates in a direction at right angles to the
long axis of the spindle. This elongation occurs at about the
time the equatorial plate is completely formed, Figs. 31 and
34 being portions of the same amphiaster. I believe this is
the stage at which the ova are normally laid, for the anaphase
does not occur until after oviposition.
It is, however, not unusual to find a centrosome, as shown
in Fig. 32, apparently consisting of numerous fine granules
which stain very deeply ; also where it is exceedingly indis-
tinct, as in Fig. 33, although I have found no sections through
fully formed spindles when it is altogether absent. It is
noticeable that in these cases the whole spindle is somewhat
indistinct in outline, and I believe is undergoing the retro-
grade development described in p. 77. The large number of
cases cited by Wilson (13) and others, in which the centrosome
is shown to be a permanent organ of the cell, presents a curi-
ous contrast to its action in this ovum. The small body
shown in Fig. 9, which for want of a better name I have
called the archoplasmic vesicle, may be permanent, but the
spot within it, the centrosome, is not, unless its chemistry so
changes that it answers to a stain at one time and not at
another. It should be borne in mind that these studies are
made on different series hardened in sublimate, sublimo-acetic,
Hermann’s and Flemming’s fluids, and in no case have I found
any trace of a centrosome except when the spindle is well
advanced in its formation.
No. 1.] POLYCHOERUS CAUDATUS. 103
N
15.
16.
LITERATURE.
Mark, E. L. Polychoerus caudatus nov. gen. nov. spec. Fest
schrift zum siebenzigsten Geburtstage Rudolph Leuckarts.
WHITMAN, C.O. Spermatopheres asa Means of Hypodermic Impreg-
nation. /ourn. of Morph. Vol. iv, No. 3.
PEREYASLAWZEWA, DR. SOPHIE. Monographie des Turbellaries de
la Mer Noire.
SELENKA, E. Ueber eine eigenthiimliche Art der Kernmetamorphose.
Biol. Centralol. Bd. i.
LANG, ARNOLD. Die Polycladen des Golfes Neapel. Fauna und
flora des Golfes Neapel. Bd. xi. Monographie.
WHEELER, W. M. Planocera Inquilina, a Polyclad inhabiting the
Branchial Chamber of Sycotypus canaliculatus. Journ. of Morph.
Vol. ix, No. 2.
WHEELER, W.M. The Behavior of the Centrosomes in the Fertilized
Egg of Myzostoma glabrum (Leuckart). /ourn.of Morph. Vol. x,
No. I.
GRAFF, LuDWwiIG VON. Monographie der Turbellarien. I. Rhabdo-
coelida.
GraFF, LuDwic von. Die Organization der Turbellaria Acoela.
GARDINER, E. G. Early Development of Polychoerus caudatus
(Mark). Journ. of Morph. Vol. xi, No. t.
Foot, Miss KATHARINE. Yolk-Nucleus and Polar Rings. /Journ. of
Morph. Vol. xii, No. I.
Foot, Miss KATHARINE. The Origin of the Cleavage Centrosomes.
Journ. of Morph. Vol. xii, No. 3.
Witson, E. B. The Cell in Development and in Inheritance.
Witson, E. B., and MATHEWS, ALBERT P. Maturation, Fertiliza-
tion, and Polarity in the Echinoderm Egg. New light on the
“ Quadrille of the centers.” Journ. of Morph. Vol. x, No. t.
CaLkins, G. N. Observations on the Yolk-Nucleus in the Eggs of
Lumbricus. Zvransactions of New York Academy of Science, June
18, 1895.
Nusspaum, J. Ueber die Theilung der Pigmentkérnchen bei Karyo-
kinese. Anat. Anz. Jahrg. viii, Nr. 20. 1893.
104
GARDINER.
DESCRIPTION OF PLATE Ix.
All figures on this plate except Figs. 1, 2, 4,and 5 are magnified about 1140
times.
FIG
. I.
Diagram showing relative position of ovary and vitellarium; 0., ripe
or nearly ripe ova in the vitellarium; ov., ovary ; v¢, vitellus.
Fic.
FIG
FIG.
FIG.
gland ;
FIG
FIG
archop
2.
5 Sh
4.
1 &
a0:
7s
Nl
. 8.
SO
lasm.
FIc.
Fic.
FIG.
FIG.
Io.
Il.
12.
13.
Immature ova in the vitelline gland ; 0., ova.
Vitelline gland cells; 2., nucleus.
Ripe ovum gorged with material from the vitellus.
Ovarian ova.
An ovarian ovum; z., nucleus; c., nucleolus.
The nucleus of an ovum after it has just passed into the vitelline
nucleolus.
The nucleus of a nearly ripe ovum; xc., nucleolus.
Nucleus with the archoplasmic body when it first appears; af.,
Division of the archoplasmic bodies.
Beginning of the first maturation spindle.
Same.
Same.
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Journal of Morphology Vol. xv.
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106 GARDINER.
DESCRIPTION OF PLATE xX.
All figures on this plate except Fig. 26 are enlarged 1140 diameters.
Fic. 14. First polar spindle leaving the nucleus.
Fic. 15. Sperm aster forming between the pronuclei.
Fic. 16. First polar spindle near the surface of the ovum.
Fic. 17. First polar body just formed.
Fic. 18. Regenerating nucleus after the first polar has been formed.
Fic. 19. (a) First polar body. (4) Regenerating nucleus.
Fic. 20. Female pronucleus regenerating after formation of the second polar
body.
Fic. 21. Anaphase of first polar spindle.
Fic. 22. First polar spindle showing centrosomes. (The boundaries of the
asters are drawn distinctly. No such lines exist in nature.)
Fic. 23. Spermatozoon shortly after it has entered the ovum.
Fic. 24. Spermatozoon somewhat nearer the center of the ovum.
Fic. 25. Spermatozoon still nearer the center of the ovum.
Fic. 26. Diagram of ovum in which the first cleavage spindle appears like
a translucent dumb-bell-shaped structure; /, the two polar bodies; AA, first
plane of cleavage; A, line of second plane of cleavage.
Fic. 27. Polar bodies in the cytoplasmic network of first cleavage spindle.
_ Sournal of Morphology Vol.xv,
.
4 26.
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108
GARDINER.
DESCRIPTION OF PLATE XI.
Fics. 28, 34, and 35 magnified 1140 times, all others 1500 times.
Fic. 28. Section through the region of the equatorial plate before the chro-
mosomes are formed.
Fic.
FIG.
FIG.
Fic.
Fic.
Fic.
FIG.
29.
30.
31.
32.
33:
34:
skp
Particles of chromatin consolidating in the nucleus.
A portion of Fig. 28 enlarged.
Centrosome.
Centrosome.
Centrosome.
The equatorial plate.
A portion of the sperm aster and the male pronucleus.
Journal of Morphology Vol.xv:
PUNT.
|
|
|
|
E.G. Gardiner
Bes,
cy
DESCRIPTION OF PLATE XII.
Both figures enlarged 1500 diameters.
Fic. 36. Beginning of the first cleavage spindle.
Fic. 37. One quarter of the first cleavage spindle.
a
es
Journal of ; Yorphology Vol. xv.
| ae : Biwi eee PUNI.
356.
= ———— Lith. Werner 4 Winter, Frankfort.
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£. 6. Gardiner
JOURNAL
DVORT EOP OC Y.
EDITED BY
Cc. O. WHITMAN,
Head Professor of Biology, Chicago University.
WITH THE CO-OPERATION OF
EDWARD PHELPS ALLIS.2-
Milwaukee. | 1c
Novemper, 1808.
BOSTON, U.S.A.:
GINN & COMPANY.
AGENT FOR GREAT BRITAIN: AGENTS FOR GERMANY: AGENT FOR FRANCE;
EDWARD ARNOLD, R. FRIEDLANDER & SOHN, JULES PEELMAN,
37, Bedford Street, Strand, Berlin, N.W., 2 rue Antoine-Dubois,
London, W.C. Carilstrasse 12. Paris, France.
RATA RIES
CONTENTS OF No. 2, NOVEMBER, 1898.
I. The Ovarian Egg of Limulus. A Contribution
to the Problem of the Centrosome and VYolk-
LAL EIS aS NONITE id COIN Rash ep NNCA WIO OT a ear coigh a
Joun P. Munson. ;
th TL. The Lateral Line System of Batrachus Tau. .
Hep ~ CorNELIA M. Cxapp.
‘ Ill. Comparative Cytological Studies, with Especial ;
4 — Regard to the Morphology of the Nucleolus . 265-58
Tuos. H. MonrTGOMERY, JR., PH.D. ae:
Che Atheneum Press,
GINN & COMPANY, BOSTON, U.S.A,
Volume XV. November, 189%. Number 2,
JOURNAL
OF
MORPHOLOGY.
THE OVARIAN EGG OF LIMULUS.
A CONTRIBUTION TO THE PROBLEM OF THE CENTRO-
SOME AND YOLK-NUCLEUS.
JOHN P. MUNSON.
TABLE OF CONTENTS.
PAGE PAGE
Introduction, Material, Methods, ihe wNwu cleolusiessssvsee ees 156
etc. . @. SSUMIMATY cesecccasecnccsecesesezeeee 162
Oecology ... : GempleMteLa tule eetereere eee tae 164
PUTO OM ai prsecresetes anc saccteceesevette Ponsa 122 Connection of the Egg with the
Position and General Appear- Ovarianyaubes: serene
ANCO le ecseene snosntenoneasenneseeensazeees, L 22, ibheiGytoplasmiiec-se.-c eos
MUS cles C Oats i erescsttsreenenessensesteses 12 ELIE N00) I eee ecco
Peritoneal! Coat... cec....csscssescsna-e 12 Polarity of the Egg
The Lining Epithelium................ 127 Peripheral Bodies and Yolk- Nt u-
Formation of Follicles ....... on Leto) CLOUT R ieee stearate avteccecetecen 171
Development of the Ovary.......... 132 The Egg Membrane .................... 172
PICT pire esters scrresatcteenesescrrnstescractear 135 | Zones and Yolk-Nucleus ............ 176
Relation of the Egg to the The Centrosome and Sphere (V el
Ovarys eee ceecrhe Hef} inte:DOdy) lessees er ee 183
tages Of Growth ........cccc--.ceescsccsseene 146 | Interpretation and Summary ......... 191
Sta pen tee see cress eieacerss ke ace Origin of the Sphere (Vitelline-
Stage a ieee etree OGY) \-::-scsccscsnncstascoascssserurecertes 192
Stage III ELOMOLOBY® 2-s2ceccesecenseccornctseecssactoree 195
Stage IV Rositioniess.-.- te ie
Degenerative Processes .............. 149 Nature of Metaniasca. meccvese 199
The Germinal Vesicle .................-... 152 Growth of Cytoplasm 200
Te MUNSON. [VoL. XV.
INTRODUCTION.
Tus work on the history and morphology of the ovarian egg
of the King Crab — Limulus polyphemus — has been done in the
Zoological Laboratory of the University of Chicago, and in the
Marine Biological Laboratory at Woods Holl, Mass., under
the direction of Professor Whitman.
The object of the work has been to determine, so far as
possible, the organization of the egg during its different stages
of growth, and to give a connected history of its phases. The
vitelline-body and centrosome have received special attention.
Much of the more valuable literature on this subject is of so
recent date that it has not seemed advisable to encumber the
paper with a historical compilation. Papers of special interest
will be referred to in connection with my own observations.
Historical. — The main facts concerning the position and
external form of the ovary have been known since 1828, when
Strauss Durckheim made known the internal anatomy of Limulus.
Somewhat later, 1838, J. Van Der Hoeven also published an
account of the ovary, its mode of branching, its ramifications
throughout the cephalothorax, and the astonishing number of
eggs produced.
Lockwood ('70) has treated in a popular, but exceedingly
interesting way the habits of Limulus, and mentions many
interesting facts in regard to its development.
Packard (’71) noted some points in the development of the
ovary, and among other things called attention to the laminated
structure of the egg membrane, which he calls a chorion.
Owen ('73) described and figured the ovary of Limulus, and
showed the relation of the ovarian tubes to the terminal ovi-
ducts, as well as the relation of the right and left ovary to each
other and to the underlying parts.
Among those who have devoted attention more directly to
the nature of the ovarian eggs may be mentioned Ludwig,
Gegenbaur, and Kingsley.
Ludwig ('74) called attention to the character of the germi-
nal epithelium, and especially to the cell nature of the egg.
He, however, based all of his conclusions on the observations
No. 2.] THE OVARIAN EGG OF LIMULUS. 113
of Gegenbaur ('58), who, he says, has had the good fortune of
having a living specimen for dissection.
Kingsley (92) has shown the relation of the egg to the ger-
minal epithelium, and, from the point of view of odgenesis, has
shown some of the similarities of Limulus to the spiders.
No one, however, so far as I know, has attempted to study
the ovarian egg of Limulus with the more fundamental prob-
lems in view. I have been compelled to go over the whole
ground and to reéxamine the observations previously recorded
concerning the ovary. Where my description agrees with
previous accounts, it has at least the value of a confirmation.
Material. — Material for the study of the mature eggs was
obtained, through the kindness of Professor Whitman, from
three female specimens that had been on exhibition in the
aquaria of the United States Fish Commission at the World’s
Columbian Exposition in Chicago. In the following June, July,
and August an ample supply of material was collected at
Woods Holl, Mass., consisting chiefly of material from
females having mature ovaries, and captured in the act of ovi-
positing. From some of this material, through natural and
artificial fertilization, a large number of embryos were pro-
duced and raised to the desired age and size. Young Limuli,
ranging from one-fourth inch to eight inches, were obtained in
abundance at North Falmouth, Mass.
The results here presented have been confirmed and ex-
tended on material ranging in size from eight inches to the
adult form, obtained in the latter part of October and the first
week in November, off New Haven, in Long Island Sound, in
water ranging from five to fifteen fathoms. The material has
been very abundant and the series complete. My thanks for
material are due to the following gentlemen: W. H. Munson,
W. H. Packard, Dr. Watasé, and Professor Whitman. I would
also acknowledge my obligations to Captain Barnes, of the
Oyster Steamer, Roe & Co., of New Haven, Conn.
My former teacher, Prof. Sidney I. Smith, has very kindly
enabled me to use the Yale Library; and Dr. Watasé has
given me much encouragement in my work. I desire to
express my appreciation of these favors.
114 MUNSON. [VoL. XV.
Methods. — The best preserved material of the young forms
was obtained by killing in (1) Kleinenberg’s picro-sulphuric,
(2) corrosive-acetic, and in (3) a mixture, in equal parts, of
a ten per cent solution of nitric acid and picro-sulphuric.
Material killed in this latter mixture was excellently preserved.
It has the advantages of staining readily, and is especially
suited to the double stain of Lyon’s blue and lithium-carmine.
By this means, the archoplasm and centrosomes are made very
distinct. It was not so favorable for the study of the various
phases of karyokinesis. For this, material preserved in Mer-
kel’s fluid was used.
The ovaries of the adult animal successfully preserved in (4)
Merkel’s fluid are excellent for the study of the centrosomes
and sphere. This fluid, however, does not always give equally
good results, even when most carefully applied. Slight irreg-
ularities in the preparation of the mixture, as well as differ-
ences in temperature, may account for some of the differences
in the effect; but physiological variations in the egg itself,
especially those changes arising from a constantly increasing
quantity of food material, are perhaps responsible for much of
the variation.
In that stage of the egg immediately preceding its escape
from the follicle, the following method was successfully em-
ployed: (5) one-fourth per cent aqueous solution of platinum
chloride applied for twenty-four to forty-eight hours, and the
eggs then passed through the various grades of alcohol. The
eggs may also first be killed by leaving them a few minutes
in Flemming’s fluid and then transferring them to platinum
chloride for forty-eight hours.
For the stages of the mature egg, after it gets into the ova-
rian tube, Kleinenberg’s picro-sulphuric has been found most
favorable, where attempts to imbed in paraffine have been made.
Owing to the difficulty of staining after Flemming’s and
Hermann’s fluid, these have not been extensively used,
although a number of the drawings have been made from
preparations of material preserved in this way, as well as
from material hardened in corrosive sublimate, corrosive-
acetic, and picro-sulphuric.
No. 2.] THE OVARIAN EGG OF LIMULUS. 115
Imbedding has been done in the usual way by means of par-
affine. The mature eggs of the ovarian tubes were imbedded
and sectioned in celloidin. To enable the imbedding medium
to penetrate, a slit was made in the chorion by means of a
sharp razor.
Previous to imbedding, the absolute alcohol was removed by
means of chloroform, saturated with dissolved paraffine. To
avoid the hardening effect of the chloroform on the yolk of
the larger eggs, xylol and turpentine were substituted for the
chloroform.
The paraffine sections, from five to ten m in thickness, were
fixed to the slide by means of water, Mayer’s albumen fixative,
or by the two combined.
Staining was done almost exclusively after the sections were
mounted on the slide. The sections of the larger eggs in cel-
loidin were stained with Delafield’s haematoxylin diluted ten
times with water and slightly acidulated with HCl. This
leaves the yolk spheres unstained and facilitates the search
for traces of the nucleus and the maturation spindle.
For the study of karyokinesis, Heidenhain’s iron-haematoxy-
lin was used. The archoplasm and centrosome in the younger
eggs were studied by means of Heidenhain’s iron-haematox-
ylin, either alone or followed with erythrosin, eosin, or acid
fuchsin. Erythrosin and cyanin have also been used to good
advantage ; also borax-carmine, followed with picric acid; Del-
afield’s haematoxylin, either alone or followed with picric acid ;
Weigert’s picro-carmine ; Ehrlich’s haematoxylin, either alone
or followed with erythrosin, eosin, and especially with acid
fuchsin; eosin and nigrosin to a limited extent; the Biondi-
Ehrlich mixture, and finally lithium-carmine and Lyon’s blue.
These stains all give good results, but they differ in the
extent to which they can be applied. The carmine stains
have not been found useful on material killed in Merkel’s or
Flemming’s fluids. To obtain the double stain with Lyon’s
blue, safranin has been substituted for the carmine on Mer-
kel’s material. In such cases the sections were first stained in
Lyon’s blue for twenty-four hours, after which they were
stained for twenty-four hours in safranin; and previous to
116 MUNSON. [VoL. XV.
clearing in xylol, the sections were dehydrated in absolute alco-
hol, containing powdered copper sulphate. This method was
reversed when lithium-carmine could be associated with the
Lyon’s blue. With the method above described, I have not
found it necessary to watch the stain under the microscope
with the care which Miss Foot ('96) seems to think is neces-
sary. On the whole, Ehrlich’s haematoxylin, followed with
acid fuchsin, has been the method that I have placed great-
est reliance on in the case of material killed in Merkel’s
fluid. Weigert’s picro-carmine has also been found very use-
ful. The specific effect of each is more profitably stated in
connection with the description which follows.
It has been found very profitable to verify as many of the
points as possible on the living material. Much uncertainty
has been removed in that way.
OECOLOGY.
Oviposition. —Many of the observations of Packard (’70),
Kingsley (92), Lockwood ('70), Agassiz ('78), and others have
been confirmed. Oviposition, at Woods Holl, takes place dur-
ing the months of May, June, and July. The females at this
season frequent a particular beach characterized by an abun-
dance of medium-sized sand and the entire absence of rocks.
They appear to come in with the tide. As stated by Kings-
ley and others, they are usually accompanied by one or more
males, one of which has attached himself to the posterior mar-
gin of the female carapace, the other males occupying a similar
position with reference to him and to each other.
If the male occupying the position described be seized and
raised out of the water, he does not let go his hold, but lifts
the much larger female out with him. If they are then
dropped into the water, they continue the same gait as if they
had never been molested.
The attachment of the male appears to take place in deeper
water; but frequently isolated males may be seen moving over
the shallow bottom off shore, apparently in search of females,
which, when they approach, they appear to recognize at consid-
No. 2.] THE OVARIAN EGG OF LIMULUS. D7
erable distances. Isolated females are also met with; but
oviposition in the absence of the male was not observed.
I can confirm Lockwood's observation to the effect that they
deposit their eggs at the point reached by the highest tides.
But Kingsley is also right when he affirms that there are
exceptions to this rule. Thus I have found them ovipositing
at a point where I was in some doubt as to whether the eggs
would ever be exposed to direct rays of the sun. On the
other hand, the nests can be found at the point reached by the
high tides, even where no superficial evidence of their presence
is visible, —an evidence that they are numerous at that point.
During oviposition the animals may be covered with as much
as a foot or more of water; but they usually approach so near
shore that their carapace is only partly covered.
The act of ovipositing is apparently accompanied with con-
siderable activity and excitement, which is indicated by an
accumulation of air bubbles on the surface of the water, form-
ing a distinct line, extending in the direction of movement of
the animals. By pursuing this line, they can be traced for
considerable distances. In ovipositing, the female is partly
buried in the sand, and only slight movements are visible from
above; but the appendages are evidently in rapid motion, exca-
vating a deep cavity from which the finer sand becomes sifted
out, and into which the eggs are discharged. The eggs thus
come to lie in the midst of sand peculiarly resembling the
eggs both in size and appearance.
Careful examination of these nests would seem to indicate
that the terminal oviducts are discharged into each nest.
It seems probable that most, but not all, of the eggs con-
tained in the ovarian tubes are laid during one season. The
females captured on June I usually show a turgid condition
of the ovary. At the end of the laying season, on the other
hand, the ovarian tubes are nearly collapsed.
On the other hand, there is much evidence to show that a
female Limulus does not oviposit every year, and that females
having the ovarian tubes filled with eggs may, even in a state
of nature, carry these over at least one season. On the
twenty-fifth of October, in Long Island Sound, females were
118 MUNSON. [Vou. XV.
taken by me whose ovaries were turgid with what appeared to.
be mature eggs, and which could not be distinguished from
those examined at Woods Holl at the beginning of the spawning
season. Others presented ovaries in which only comparatively
few eggs had arrived in the ovarian tubes. The condition of
ovaries taken from the females that had been on exhibition
at the World’s Fair was that of the former, z.e., the ovarian
tubes were filled with mature eggs. This being on November
I, it was concluded that the eggs had been retained because
of the confinement of the animals during the season when
oviposition takes place. The above observation shows that, in
their native element, females with ovaries in a similar con-.
dition, at the same season of the year, are abundant, and that
neither the fullness of the ovarian tubes, nor the apparent
maturity of the eggs, are sure indications of the time when
oviposition will take place. Yet, as Kingsley has observed,
there are reasons for believing that there is no oviposition
in confinement. The ovaries of the female Limuli kept in
the large floating aquaria of the Marine Biological Laboratory
at Woods Holl were always filled with mature eggs, even after
the spawning season was over.
The movement of Limulus is a uniform, gliding one.
Oblivious of everything except the business which occasions
its visit, it pursues a more or less direct path for the beach,
which is most favorable to the concealment and subsequent
development of its eggs. Any attempt at concealment or
betrayal of fear, by a hasty retreat, is not to be observed. If,
on its first arrival off the beach, it be disturbed, it cannot be
induced to deposit its eggs, but endeavors stubbornly to make
its way back into deep water. The male still clings to the
female, but I have in vain, for hours, endeavored to secure
freshly laid eggs by urging them towards the favorable point
of oviposition.
Moulting. — Lockwood found a soft-shelled specimen in the
month of February, and concluded that, while the young moult
four or five times the first year and adults usually only once, in
the month of August, there might be two moults a year even
in the case of the adult. As several soft-shelled ones were
No. 2.] THE OVARIAN EGG OF LIMULUS. 119
observed by me on the twenty-eighth day of October and the
first of November, it may be supposed that the moulting period
is not fixed to definite seasons, but that it may take place
at any time, according to the physiological condition of the
animal.
Although the soft-shelled females observed by me were of a
size not inferior to the largest hard-shelled specimens found to
possess an ovary filled with mature eggs, yet the eggs in these
soft-shelled ones were scarcely visible to the naked eye. The
larger eggs, however, when exposed to favorable light, showed
a decided reddish-pink coloration, indicating the second stage
of yolk formation. Numerous specimens, apparently fully
grown, appeared to have moulted at an earlier period. These
were distinguished from the hard-shelled ones, having mature
eggs, by the translucency of the carapace, and their consequent
brown appearance in contrast to the black appearance of the
hard-shelled ones. The carapace in the former was further
distinguished by many distinct internal markings not visible in
the hard shells; and, unlike the latter, the eggs had not yet
been discharged into the ovarian tubes, but many of them,
apparently, had reached follicular maturity. In no case did I
find one recently moulted containing mature eggs. All females
that were observed ovipositing at Woods Holl had hard shells.
Habitat. — Limuli, ranging from eight inches to the adult
forms, were found in abundance on the last of October and the
first of November, off New Haven, in Long Island Sound, in
water ranging from five to fifteen fathoms. It was the general
opinion among the oyster fishermen, who are engaged in dredg-
ing oysters during the greater part of the winter months, that
Limulus goes into deeper water later in the season. But very
little reliance can be placed on their observations; for, although
they had been engaged in dredging starfish previous to Novem-
ber 1, yet they seemed to be ignorant of the presence of
Limulus at that time, till they were asked to take notice
of them. 3
The earlier stages of Limulus, ranging from one-fourth inch
to eight inches, were abundant at North Falmouth, Mass., in
the month of August. The place where they are found is a
120 MUNSON. [VoL. XV.
large, level expanse of loose sand that is left entirely exposed
on the retreat of the tides, and receives fresh water from a stream
flowing into the estuary, as well as from several fresh-water
springs along the shore. The sand is rich in clam shells and
soft-bodied animals, and the abundance of organic material is
evidenced by the black coloration of the sand in which the
young Limuli live.
At low tide they lie quietly buried, just below the surface,
and no tracks or markings reveal their presence. As soon as
the incoming tide has covered the sand, however, the Limuli
begin to move about, not on the surface of the sand, but just
beneath the surface, being always covered with the uppermost
layer of sand. This upper layer has the usual color of sand,
while just below it is black. As the little Limuli plow their
way along, the upper layer is pushed aside and a black track
appears. Immediately after the incoming tide covers the sand,
these black lines appear running in every direction. The
beginning of the black line marks the resting place of the little
Limulus during the absence of the tide; the end marks the
distance which it has traveled. At this point it can always be
found. The search for these little creatures is, therefore, a
comparatively easy one, notwithstanding their protective color-
ing and their subterranean mode of locomotion. This mode of
locomotion is evidently useful to them as a means of protection
from the many enemies that infest the neighboring eelgrass.
Many little Limuli, departing from the path which nature has
marked out for them, can be seen to have fallen victims to these
enemies.
Food. — One large female Limulus was found nearly buried
in the mud in about three feet of water. An examination
showed that it was enjoying its dinner, which consisted of a
worm.
In Dr. Lockwood's vivid description of the habits of Limulus,
an instance is cited where it had been observed caught by a
clam, which, it was supposed, the Limulus had been trying to
consume. From the oyster growers at New Haven, and espe-
cially from Captain Barnes and his crew, it was learned that,
while the starfish is their dreaded foe, the horse-fish (which is
No. 2.] THE OVARIAN EGG OF LIMULUS. 121
the fisherman’s name for Limulus) is perfectly harmless so far
as oysters are concerned. I was delighted, however, after
traveling from Chicago to New Haven in order to secure
material late in the season, to find that they were abundant
on these oyster beds.
Vitality of the eggs.— At Woods Holl, in the summer of
1894, the eggs, laid while the animals were observed in the act,
were taken from the nest as soon as the animals were about to
leave. The animals were also taken. Owing to the secretion
of the oviduct covering the eggs, they adhered more or less
firmly to the sand, with which they were intimately mixed, and
formed balls. These eggs and sand were put into a dish, No.
I, containing sea-water. A considerable quantity of eggs were
taken from the ovary of the same female, and placed in a dish,
No. 2; and, after treating them with the contents of the male
genital ducts, the dish was rotated; the eggs arranged themselves
in a single layer, and adhered firmly to the wall of the dish. In
the bottom of another dish, No. 3, were placed a number of
glass slides, and eggs from the same source similarly treated.
They were fixed to the slides in the same way. In a dish, No.
4, eggs and sand from another nest were placed, and, like the
others, supplied with sea-water. Dish No. 1 was moved only
sufficiently to change the sea-water occasionally. Dish No. 2
was treated in the same way. The glass slides in No. 3, with
the eggs adhering, rested on supports, and were frequently
turned, so that the eggs were alternately above and below the
slide. The sand and eggs in dish No. 4 were vigorously stirred
several times a day. In all four dishes the eggs developed. At
the end of two months no perceptible difference, so far as the
development of the eggs was concerned, could be observed. By
means of the glass slides, the experiment of Patten (94) was
confirmed; but the changes in the first indications of cleavage
observed by him have no perceptible influence on the develop-
ment of the eggs.
The above experiments were begun on June 25. The eggs
were not exposed to sunlight, and the development was slow
and irregular. On the first of September many of the embryos
had hatched, while many of the eggs showed no sign of devel-
122 MUNSON. [VoL. XV.
opment, and indeed appeared as if they were more or less
decayed. Embryos, however, continued to be produced, and
the eggs and sand were removed to Chicago in jars, where the
development continued, the evaporated salt water being replaced
from time to time with ordinary fresh tap water. After pre-
serving as many of the different stages as seemed desirable
and no more embryos being hatched, the jars were put to one
side and neglected till a few days before Christmas, when a
hasty examination showed that the water had evaporated and all
signs of life had disappeared. In order to clean the jars they
were filled with tap water and allowed to soak over night. The
next day I could scarcely believe my own eyes when I found
the bottom of the jars swarming with little Limuli.
THE OVARY.
1. Position and general appearance. — The ovaries of
Limulus (Pl. XIII, Fig. 2, ov.) communicate with the exterior
by means of two horizontal slits, situated on the posterio-dorsal
side of the operculum, on each side of the median line, and ap-
proximated to within one-fourth of an inch of each other. Each
external meatus (g.o.) is guarded by an upper and lower thick-
ened lip, that is somewhat prominent externally ; and the orifice
is further closed by transverse ridges within.
From these two openings, the two terminal oviducts, lying
beneath the outer integument of the posterio-dorsal surface of
the operculum (of.), traverse the proximal part of the opercu-
lum, and, proceeding forward, upward and outward, enter the
cephalothorax. Here each duct soon divides into two large
branches, one of which takes a peripheral direction, while the
other takes a course toward the central axis of the body, where
it anastomoses with the corresponding branch of the opposite
side, in the median line directly above the alimentary canal (a/.c.).
The point of divergence of the central and peripheral branch of
each duct is entirely obliterated, giving the two branches the
appearance of one continuous tube, in the middle of the ven-
tral side of which is inserted the slightly larger terminal duct.
Between the point of insertion of the terminal oviduct (ov.d.)
No. 2.] THE OVARIAN EGG OF LIMULUS. 123
and the point of anastomosis of the right and left branch, each
of these branches gives off a large secondary branch (ov.2.),
which passes backward on the right and left side of the ali-
mentary tract to the anus (az.). These are not simple tubes,
however, as has been affirmed by Owen ; but rather a network
of anastomosing tubes which surrounds the alimentary tract,
nearly concealing it, except immediately over that part of the
operculum where the external openings are situated (¥-0.).
The lateral tubes of this system when filled with eggs are
larger than the tertiary branches above and below.
After anastomosing in the median line the two secondary
branches previously described again separate, and, retaining
approximately their original size, pass forward on each side of
the alimentary tract (a/.c.), along the adductor muscles (7.), in
the meantime sending smaller anastomosing branches over
the alimentary tract, and large branches between the muscles.
Finally they unite again in the anterior part of the cephalo-
thorax immediately over the oesophagus.
The right and left peripheral branches of the divided oviduct,
retaining for some distance the original dimensions, proceed
outward, slightly backward and downward, giving off, close to
the adductor muscles, a large branch. This passes along the
muscles to the right and left of these till they become united
in front of the adductor muscles to the tubes previously
described. They again give off branches passing between the
adductor muscles toward the central axis of the body; and
these unite with the corresponding tertiary branches proceed-
ing from those running parallel with the intestine previously
described.
This system of large tubes lying over the alimentary canal
and surrounding the adductor muscles has been described by
Owen ('73) as the ovary of Limulus.
In the further course of the peripheral branch of each of
the secondary branches numerous tertiary branches are given
off. The whole finally resolves itself into a number of small
tubes that anastomose with their neighbors and with tertiary
branches given off from the large system of tubes surrounding
the adductor muscles. Thus the whole ovary becomes a net-
124 MUNSON. [VoL. XV.
work of tubes covering the whole ventral and dorsal surface of
the animal, and, uniting at the extreme borders of the cephalo-
thorax, enclose the massive liver and the other internal organs.
The ovary, whose double nature is evident only from the two
terminal oviducts, has a bilateral symmetry ; but this is incom-
plete, because of the somewhat irregular anastomoses over the
median line.
The secondary branches, surrounding the adductor muscles
and running parallel with the alimentary tract, are compara-
tively large. Owing to their relatively thick walls, they
maintain more or less uniform dimensions, even when filled
with eggs. These being branches of the oviduct, they serve,
like the latter, as reservoirs and channels of transmission
for the vast number of eggs that are discharged into them by
the numerous tertiary branches. This network of tertiary
branches (ov.), covering the entire animal outside of the ad-
ductor muscles, is the real egg-producing portion of the ovary.
A portion of one of these tubes is represented in Pl. XIII,
Fig. 16, drawn with a camera from living material, taken from
a young animal thirteen inches long, including the tail.
In the adult animal these tubes are usually filled with eggs.
Owing to the feeble resistance offered by their thin walls, the
eggs in them are not evenly distributed along the lumen, but
are often massed together into large masses, causing irregular
swellings that obliterate the meshes between the tubes. This
gives the ovary a very irregular appearance, as if it were nothing
else than a huge mass of eggs covered by a thin membrane.
Over and between these large masses of eggs the various stages
of new generations of eggs can be seen.
After the discharge of the eggs this chaotic appearance of
the ovary, for the most part, disappears, and most of the irregular
sac-like swellings resume the normal dimensions of the ovarian
tubes. These now become conspicuous, not only from the
shining aspect of their walls, but from the fringes of the vari-
ous generations of new eggs that dot their surface. In the
adult animals, except such as have recently moulted, the tubes
(ov.7.) are never completely collapsed, but contain a larger or
smaller number of eggs. These, when not very numerous, are
No. 2.] THE OVARIAN EGG OF LIMULUS. 125
arranged in rows along the lumen, like so many intersecting
bead strings. The arrangement and appearance of the com-
pletely empty tubes can best be observed in a half-grown
animal, where no eggs have yet been discharged into the
tubes.
This network of tubes is suspended between the carapace
and the liver in a subcutaneous alveolar tissue, to be described
more minutely later. In the turgid condition of the ovary this
tissue becomes greatly flattened. The entire space between
the liver and carapace is occupied by the enormous mass of
eggs, the peritoneal tissue being reduced to a thin film. This
does not entirely obscure the eggs when the carapace (ca.) is
removed.
2. Muscle coats.— The terminal portion of the oviduct is
characterized by the firmness of its wall. This is due to a
highly developed muscle coat (Pl. XIII, Fig. 15, m.c.). This
coat consists of an outer tunic, underneath which is a thick
coat of muscle fibers woven together, apparently without much
order, into larger bundles that intersect and cross each other,
leaving vacant meshes between. These meshes, however, are
small as compared with the muscle bundles themselves. In
cross section the cut end of these muscle fibers and bundles
show various outlines, corresponding to the various degrees of
obliquity in which they have been cut ; but no trace of a differ-
entiation into a longitudinal and circular zone is to be recog-
nized. Sections of the larger branches of this duct, however,
show a distinct, rather thin inner, circular muscle coat, outside
of which is a thicker zone of intersecting and dividing muscle
fibers, with connective tissue, blood lacunae, blood vessels, and
capillaries. The outermost coat consists, for the most part, of
longitudinal muscle fibers, the whole forming a muscular wall
considerably less in thickness than that of the terminal duct.
This same muscle coat is continued over the ovarian tubes
proper, but in a very loose and attenuated form (Pl. XIII,
Fig. 15, w.c.). In both transverse and longitudinal sections
of the ovarian tubes the same features present themselves.
This is true chiefly in the mature ovary where follicles have
already been formed. The distinctive feature is that the fibers,
126 MUNSON. [VoL. XV.
both in longitudinal and in transverse section of the tube,
are cut transversely or obliquely, longitudinal sections of the
fibers rarely appearing. The cause of this becomes plain when
the tube is slit open, spread out on a slide, and stained 27 foto.
Those large sac-like swellings previously mentioned have been
selected for this purpose. Killed in picro-sulphuric, and hard-
ened in alcohol, the wall of the greatly expanded tube remains
sufficiently tough to allow its removal from the mass of con-
tained eggs; and its elasticity is sufficiently destroyed to pre-
vent its return to its normal contracted state after removal of
the eggs. Such a preparation, stained with the Biondi-Ehrlich
mixture, shows the following arrangement of the fibers. Very
symmetrically arranged oval areas are observed where no fibers
are present. Around these areas the fibers run parallel to one
another, constituting a sort of striated rim or border around the
area. Between these areas with the encircling muscle fibers
the fibers cross and intercross, some becoming continuous with
the encircling fibers of one area, others passing around another
area, and soon. The beginning and the end of these fibers
could not be made out. They seem to branch freely and have
nuclei imbedded in their substance.
The oval areas are due to the characteristic arrangement of
the muscle fibers; and this arrangement is the expression of
the regularly arranged follicles in the adult ovary of Limulus.
The oval areas themselves are the follicles which have become
obliterated through the stretching of the walls of the ova-
rian tubes. That the muscle fibers retain this characteristic
arrangement in the wall of the tube, when greatly extended, is
perhaps sufficient evidence that these are permanent features
of the muscle coat, and not, as might be supposed, transient
features due to displacement by developing eggs and likely to
occur at any point where an egg might chance to develop. In
the adult ovary it is this arrangement of the muscle fibers which
determines the position and makes possible the characteristic
follicles of Limulus. Something more concerning the origin of
this arrangement and the part which the growing egg may have
in its production is to be considered in connection with the
development of the ovary.
No. 2.] THE OVARIAN EGG OF LIMULUS. 127
3. The peritoneal coat. — As will appear more clearly in the
account of the developing ovary, the muscle coat of the ovarian
tube is surrounded by a loose coat or mantle, belonging to the
honeycombed peritoneal tissue. This has been mistaken for
the tunica propria by Kingsley ('92). It serves to suspend the
ovary between the carapace and the liver. This tissue is seen
to be laminated, and consists of greatly flattened cells, joined
edge to edge like the peritoneal linings of lymph spaces gen-
erally. The meshes of this laminated tissue appear to serve as
lymph sinuses, and are filled with granules and corpuscles of
various kinds. It is between two such lymph spaces that the
ovarian tube lies. The tube is organically connected with this
tissue along one of its sides, so as to hang suspended in a loose
tube, the walls of which constitute the walls of neighboring
lymph spaces. This peritoneal mantle or peritoneal coat of the
ovarian tube is comparatively loose; and it is the looseness of
this coat which permits the eggs, as they develop, to push out
through the follicular fenestrae of the muscle coat, and to
occupy the space between the ovarian tube and encircling peri-
toneal coat. Because of its looseness, also, the ovarian tube is
enabled to greatly enlarge when the eggs are discharged into
the tube. In dissecting out the ovarian tubes, the connection
of the tube with this peritoneal mantle is usually severed and
the mantle does not appear in connection with the ovarian tube.
In one sense, it belongs rather to the system of lymph sinuses
than to the ovarian tube; but its relation to the ovarian tube
is such that it serves a double purpose. The nuclei in the cells
of this coat are conspicuous. The corpuscles and granules, so
conspicuous in the lymph spaces, are not found within the
periovarian cavity bounded by the peritoneal coat.
4. The lining epithelium.— In the adult ovary, in its empty
and contracted state, the tunica propria becomes folded between
the follicles, owing to the tonicity of the muscle coat.
The folding is especially prominent around the borders of the
follicles, where rachis-like projections appear to extend into the
lumen of the follicle.
This folding causes a considerable lateral pressure on the epithe-
lial cells, which thus become greatly elongated and compressed.
128 MUNSON. [Vo. XV.
On the expansion of the ovarian tube, this lateral pressure is
relieved and the cells assume a more spherical form.
The size of the epithelial cells is subject to considerable
variation, and the thickness of the epithelium also varies within
considerable limits. This is due, in part at least, to the varying
lateral pressure on the cells, and on the amount of folding of
the tunica propria; for, where the tube is distended, the epithe-
lium may become flattened into what appears to be a mere thin
protoplasmic layer with scattered nuclei. In other portions of
the same tube, where the folding and lateral pressure still exists,
the epithelium appears to have considerable thickness. In all
cases, however, it consists of a single layer of cells.
This epithelium can always be distinguished from all other
tissues by its glassy transparency, except at certain stages,
when the cell protoplasm becomes filled with secretion granules.
The minute structure of the protoplasm assumes different ap-
pearances, as the cells are seen to be compressed or expanded.
The distinctness of cell boundaries also varies with the amount
of lateral pressure. Where the tube is expanded, and the cells
are more than usually flattened, cell boundaries are difficult
to make out. The cytoreticulum of one cell area seems to be
continuous with the cytoreticulum of neighboring cell areas, the
nuclei in each affording landmarks. Careful examination of this
protoplasm seems to indicate that the various nuclei are con-
nected by means of this system of cytoplasmic fibrils. These
fibrils can be seen to be massed into strands or bundles. The
bundles, however, can be analyzed into finer fibrils connecting
larger microsomes. An increase of the magnifying power shows
that the meshes of these fibrils are again traversed by still finer
fibrils. At each node there is always a stainable, spherical
enlargement, which usually diminishes with the fibril under con-
sideration. The whole has thus the appearance of a network
within a network, through a considerable series of gradations
in size, both of the fibrils and their nodal enlargements. There
appears to be no limit, except that which the microscope
imposes, to this continued decrease in the size of the fibrils,
the size of the stainable nodes, and the size of the enclosed
meshes.
No. 2.] THE OVARIAN EGG OF LIMULUS. 129
The terms fiber and fibrils have been here used as descriptive
terms for appearances as they present themselves under the
microscope; not in any way to designate the real character of
the appearance.
In the relaxed state of the ovarian tube, and in the folded
condition of the basement membrane, this appearance can no
longer be discerned. With the increase of lateral pressure the
epithelium becomes thicker, and faint indications of distinct
cell outlines present themselves. The fibrous network described
above is either very obscure or else not visible at all. In the
latter case the cell contents appear homogeneous, with granules
scattered throughout the protoplasm, and a distinct apparently
vesicular nucleus situated nearer the base of the cell. But this
is not a permanent condition. Cell outlines become more and
more distinct. The granules become larger, clearly defined,
uniform in size, and stain with some difficulty, except in such
powerful stains as acid fuchsin. They often have a more or
less bead-like arrangement. As the granules increase, however,
they form large aggregations, occupying nearly the entire cell,
and often obscuring the nucleus. At this stage the cell can
often be seen to be distinctly striated. The fibers to which
this striation is due run parallel with each other, and with the
long axis of the cell, perpendicular to the basement membrane.
The cells increase greatly in size, and, varying with the lateral
pressure, their long axis may be many times that of the short
or transverse axis. The form and apparent size of the cell vary
with the position which it occupies, and the consequent varia-
tion in pressure. Its short or transverse axis may be equal
throughout, or the cell may be greatly narrowed at the base and
expanded at the free end.
At this stage the free end of the cells has a regular, clean-cut
outline, and the fibers of the cell body appear to extend to the
very surface of the cell. This, however, is not a permanent
condition of the cell; for the free border gradually becomes
ragged; the fibers appear to be continued into the lumen of the
tube, in which a fibrous, deeply staining substance appears to
have been secreted. With this substance the cell becomes
apparently more and more continuous. The free border of the
130 MUNSON. [VoL. XV.
epithelium, previously distinctly and uniformly outlined, now
becomes obscure and ill-defined; the fibers of the cell body
become more distinct and fewer in number; cell boundaries
seem to become very indistinct, and large, open, non-stainable
spaces appear to occupy the larger portion of the former cell.
The remaining fibers appear packed along one side of the
former cell, and in this remnant of protoplasm the somewhat
obscured nucleus lies imbedded. The whole epithelium in sec-
tion now has a very irregular, ragged outline.
Owing to these changes in the epithelial cells, the same
method of preservation has different effects, not only on the
cytoplasm of the cell body, but also on the nuclei. In well-
preserved conditions of the cell, previous to the considerable
accumulation of granules, the chromatin consists of deeply
stainable spheres arranged in a circle around the periphery of
the spherical, vesicular nucleus, forming in haematoxylin stains
a dark beaded ring around a clear central area. At times, but
not always, a dark body more minute than the chromatin gran-
ules can be seen to occupy the center of this clear central
area.
After the accumulation of the granules in the cytoplasm,
however, this expanded spherical condition of the nucleus and
the regular peripheral arrangement of the chromatin is not to
be observed. The nucleus seems to collapse, become oval, and
the distribution of the chromatin becomes irregular. The chro-
matin bodies themselves, losing their uniformity in size and
their regular spherical form, appear as if broken up. They may
also become aggregated into a more or less homogeneous mass.
This latter condition of the nuclei is often to be observed in
those cells of the egg stalk which show evidence of gradual
disintegration. Since the other forms of the nucleus are found
in their immediate vicinity, this peculiarity cannot be attributed
to the reagents used. Certain methods of killing, however, as,
for example, Ehrlich’s bichromate, when allowed to act for
several days, give to the chromatin of all the nuclei an appear-
ance resembling this latter form.
5. Formation of follicles. —The pressure caused by the
tonicity of the muscle fibers is greatest between the follicular
No. 2.] THE OVARIAN EGG OF LIMULUS. 131
fenestrae. The epithelium over the area of the fenestrae, being
relieved of this pressure, protrudes through these as evaginated
pouches (Pl. XIII, Fig. 15).
The cells lining these follicles are spherical, as compared with
the greatly elongated, compressed cells between the follicles.
The cell outlines are not so distinct as in the elongated cells,
and the epithelial lining of the follicle often appears more like
a layer of protoplasm containing nuclei than like a well-marked
epithelium of columnar cells.
In very many cases these follicles are filled with a secretion
resembling yolk. This secretion is seen to arise in the cells of
the epithelium often in the form of granules, and frequently in
the form of a non-stainable mucus-like substance, which is
accumulated in large masses, occupying nearly the whole cell.
The secretion which is poured into the lumen of the follicle
may be discharged through the communication of the follicle
with the lumen of the ovarian tube into the latter, the fol-
licle in such a case serving the purpose of an ordinary gland.
There is no reason for supposing that these lining cells of the
follicle differ in any essential respect from those lining the
ovarian tube proper, since their difference in form can be
accounted for by their freedom from pressure. The epithelium
lining the follicles and that lining the tube proper are, in fact,
a continuous layer of similar secreting cells, the peculiar
arrangement which they have being due solely to the fenes-
trated nature of the muscle coat.
It frequently happens that, instead of being an open space as
it is often found to be, or else a space filled with secretion, the
lumen of the follicle is filled with large masses of protoplasm
in the form of large, ill-defined, irregular cells (Pl. XIV, Fig.
23). These cells, occupying the entire follicle, are also seen to
be secreting, inasmuch as they become filled with yolk-like
granules (s.g.), often to such an extent as to entirely obliterate
cell boundaries. In such cases the granular secretion appears
to arise and accumulate around the nucleus of each cell to such
an extent as to obscure the nucleus, and often to render its
detection difficult. The chromatin granules of the nuclei are
seen between the secretion granules, often apparently imbedded
1 Bh) MUNSON. [VOL. XV.
in their substance. The secretion takes a dark coloration in
osmic acid, and, like the secretion of the ordinary epithelial
cells of the ovarian tube, stains with difficulty, except with such
powerful stains as acid fuchsin. Occasionally such a follicle is
seen to have been transformed into a mass of non-stainable,
homogeneous substance, showing irregular dividing lines, per-
haps the original cell boundaries. These lines, probably the
last remnants of the protoplasm of the cells originally present
in the follicle, stain in haematoxylin. At their intersections
are observed deeper staining patches that might suggest the
presence of nuclei.
The large accumulation of secretion here described occurs
only in those follicles where an egg is absent.
Development of the ovary. —Ina young animal of one inch
or less there are indications of a subcutaneous alveolar tissue
between the liver and the carapace. At this stage numerous
deeply staining nuclei can be observed lying between the cara-
pace and the forming liver. The body of these cells can be
seen to be flattened, and the various cells bear a certain fixed
relation to one another. The meshes between the cells in-
crease, owing perhaps to accumulation of liquids. It then
appears that the cells, originally closely packed, now form the
thin walls of vesicles or cavities. The nuclei are still prom-
inent. Each of these vesicles has a wall of its own, composed
of flattened cells set edge to edge, very much as in the case
of lymph spaces in general. As these spaces increase in size
the cells of their walls become flatter and the nucleus less
distinct. Where two of these spaces are contiguous the wall
appears double, as if the two walls had become closely applied.
In sections of the young animal at this stage, and earlier,
there can sometimes be seen between these contiguous walls
isolated cells with a distinct, deeply staining nucleus, and a
well-defined cell body of protoplasm, having that characteristic
clearness which is peculiar to the germ cells during the period
of division, previous to the period of growth. The position
of these cells corresponds to the position of the ovarian tubes,
when they can first be definitely recognized as such.
In a series of transverse sections the cells are not to be
No. 2.] THE OVARIAN EGG OF LIMULUS. 133
seen in all sections, but, in favorable cases of tangential-longi-
tudinal sections, they appear to be arranged in a chain between
the lymph spaces. Sometimes it appears as if every other cell
belonged to opposite walls of the contiguous lymph spaces ;
and, being separated considerably, they alternate in such a way
as to appear to be asingle chain of cells. It seems highly prob-
able that these cells give rise to the future ovarian tubes.
I have not been able to trace the origin of the tubes, but
it seems to be somewhat as follows: by division these cells
give rise to chains of cells; which chains, consisting of per-
haps four cells in cross section (possibly enclosing a cavity or
lumen), take the direction of the lymph sinuses between whose
walls they lie. But the walls of these lymph spaces intersect
in various ways and at nearly all angles. The chains or tubes,
therefore, also intersect in the same way, and thus a network
is established. The cells continue to proliferate, and the num-
ber of cells in cross section increases. Such minute ovarian
tubes, from four to six cells in cross section, can be seen in
young animals from four to five inches.
With the increase in cross section the peritoneal walls
between which the tubes lie become more and more separated ;
and these walls, originally closely applied as the boundaries of
lymph spaces, become the peritoneal mantle, or loose perito-
neal coat of the ovarian tubes of the adult animal.
The cells of the rods early show an arrangement as far from
the common center of the long axis of the tube as space will
permit ; and thus a lumen early makes its appearance. Cells
can be seen to continue dividing by karyokinesis, and, becom-
ing pushed into the lumen, temporarily obliterate it. In
animals from five to seven inches the lumen of the tube is
already well established. The cross section of the tubes does
not increase equally in all tubes, and their development takes
place more rapidly on the dorsal than on the ventral side.
When the cells are sufficiently numerous in cross section to
constitute a tube, they are seen to be surrounded by a second
membrane, the tunica propria. At this early stage the tunica
propria is closely applied to the original peritoneal coat,
although they are easily seen to be entirely distinct, the peri-
134 MUNSON. [Vou. XV.
toneal coat being conspicuous by the distinct nuclei of its
cells, while in the second coat, or tunica propria, no nuclei can
be seen (Pl. XIV, Figs. 33-41, 27.).
Comparing the tube with its tunica propria, with the walls
of the enclosing lymph spaces, it is seen to differ from these
in this, that while in the latter the body of the cells is
greatly flattened (Pl. XIV, Figs. 37-41, f.c.), so that the
nucleus finally seems imbedded in the outer lamella, in the for-
mer the body of the cell remains conspicuous, and the nuclei
are accordingly considerably removed from the common invest-
ing membrane, the tunica propria. In the peritoneal cells the
intercellular or cementing substance is formed between the
cells, which thus become adherent at their circumference ;
while in the germ cells this intercellular substance appears at
the outer pole of the cell, which thus retains more of its
spherical form.
In some of the tubes of an embryo seven inches long,
traces of muscle cells can be seen between these two coats ;
and this is, of course, the future muscle coat of the ovarian
tube. As the animal increases in size the muscle coat
becomes more and more pronounced.
Even in an embryo five inches long it can be seen that some
of the germ cells have passed the period of multiplication, and
have entered on the period of growth (Pl. XIV, Fig. 38). As
they grow they push their way out, causing a separation of
the peritoneal membrane and the tunica propria on either side
(Pl. XIV, Figs. 40, 41, f.c., ¢.p.), and this circumstance seems
to determine the position of the follicular fenestrae of the
muscle coat (Pl. XIII, Fig. 15, .c., 2.f.). It might be asked
whether the increased pressure thus produced between the
egg and the peritoneal membrane, and the resulting diminution
of pressure on the other side of the egg, has anything to do
with the peculiar arrangement of the muscle fibers. It will
be seen later that these odcytes make their appearance at
regular intervals as the animal grows; and that this is inti-
mately connected with the increasing diameter of the ovarian
tube. But the further account relating to this will be intro-
duced with the history of the growing egg.
No. 2.] THE OVARIAN EGG OF LIMULUS. 135
THE Eae.
The germ cells lining the ovarian tubes of the young animal
when the lumen has appeared are spherical in form (Pl. XIV,
Fig. 38). The cytoplasm of the body is relatively abundant
in the resting state, and is comparatively free from granules.
The nucleus occupies approximately the center of the cell. It
is not relatively large, but it contains considerable chromatin,
which stains deeply.
As these cells prepare for division the nucleus becomes
greatly enlarged, and the chromatin assumes a distinct and
highly characteristic network (Pl. XIV, Fig. 33). Dark,
straight fibers are seen to intersect other straight fibers, at
various angles; and these, again, can be seen to unite with
similar fibers at the periphery of the nucleus. Here dark,
bead-like chains of chromatin granules appear to constitute
the only boundary of the nucleus. These bead-like bodies are
not always evenly distributed, but considerable spaces some-
times appear between them. By adjusting the focus, however,
similar granules appear in these spaces, indicating that we
have here to do with a network which lies at the boundary of
the nucleus. At the points of intersection of the chromatin
rods within the nucleus there is always a considerable enlarge-
ment. The karyolymph is hyaline, and the chromatin
element does not appear to be so abundant as to make the
nucleus as conspicuous as it becomes in the next stage (Fig.
35). Now the nucleus, retaining its former size or even
slightly increasing, is seen to be filled with a deeply stainable
thread which appears to have the form of a coil and which fills
the nuclear space. It could not be determined whether this
is a single thread or several threads. Appearances seem to
show that there are more than one thread ; at any rate, several
apparently free ends could be seen at all times. Careful
examination of this thread shows distinctly that it consists of
spherical bodies arranged in single rows closely applied so that
in places the rod seems continuous; but the granules are often
slightly separated one from the other. This, in all probability,
may be taken to be the preliminary phase, spireme, of karyo-
136 MUNSON. [Von XV.
kinesis, which follows in the ordinary way (Pl. XIV, Figs.
36, 37):
Unfortunately, notwithstanding the comparatively large size
of the nuclei in the earlier stages of this division process, the
spindles are small; and the hyaline nature of the cytoplasm
renders it difficult to determine the conditions at the poles of
the spindle. The difficulty is further increased by the rather
peculiar phenomena of several contiguous cells seeming to divide
at the same time. Occasionally, single cells in the various
stages can be seen; but, as a rule, a group of from four to
eight contiguous cells are in precisely the same phases of divi-
sion. The spindles seem to lie in all planes; and the close-
ness of the elements renders it difficult to observe an entire
spindle with its two poles. Sections of the equators of the
spindles showing the chromosomes are numerous; but the
confusion arising from the crowded condition of the elements
is such that any attempt at counting the chromosomes belong-
ing to a given spindle would not lead to reliable results. A
distinct centrosome imbedded in the accumulation of pro-
toplasm at one pole, however, has occasionally been observed.
The stage with the greatly enlarged nucleus and the char-
acteristic chromatin thread is the most conspicuous one in
the process. These have been observed in most of the
ovarian tubes of young animals from five to eight inches, and
in material preserved in the nitro-picro-sulphuric mixture (see
methods), as well as in material preserved in Merkel’s fluid.
In the latter material the thread is most beautifully preserved ;
while in the former the archoplasm and cytoplasm of the cell
are excellently preserved.
As stated above, these cells usually occur in groups ; but
single cells, in this phase, can also be observed. The most
natural conclusion to be drawn in regard to the cause of these
cells occurring together in this way is perhaps that they are
the sister cells of an original mother cell ; and, being of equal
ages, they pass through the same cycle of changes at the same
time.
In these groups of cells the large nuclei, occupying nearly
the whole cell, seem to lie closely applied to one another (PI.
No. 2.] THE OVARIAN EGG OF LIMULUS. 1heh/
XIV, Figs. 33, 35), and on first observing them one might
easily receive the impression that they are fusing. The dis-
tinctness of the outlines of the nuclei, however, renders it easy
to make out almost equally distinct cell boundaries. The pro-
toplasm of each cell is reduced to a thin rim, in which can be
seen the few fibers of the cytoreticulum, often not exceeding
three or four in number. Owing to the pressure the cell
body becomes pentagonal, each side being straight and
closely applied to a corresponding surface of a neighboring
cell. Yet the cell boundaries are distinct.
These groups of cells can be seen in both transverse and
longitudinal sections of the tube. In either case the general
appearance of the group is the same. In transverse sections
of the tubes only one group can be seen; while in longitudinal
sections several groups can be seen at regular intervals along
the tube.
In such sections the epithelium on the side opposite the
group has only about one-fourth of the thickness of what it has
on the side where the groups are situated. This same relation
holds also in transverse sections, and it is due to the fact that
each group pushes into the lumen of the tube, so as to obliter-
ate it at that point (Pl. XIV, Fig. 35). But in the larger
larvae, where the epithelial cells have become considerably
elongated in the direction of the lumen, these groups of cells
are more frequently enclosed by the protoplasm of neighbor-
ing cells as if they had originated beneath them, and pushed
their way partly up between them. The group, therefore, does
not project freely into the lumen.
In the larva these cells, when they assume the resting stage,
acquire the general appearance of the neighboring epithelial
cells. They, however, remain grouped together for some time.
After a certain number of divisions (the exact number can-
not be made out), one of these cells, usually one lying close to
the basement membrane, increases in size more rapidly than
the others. Without assuming the characteristic chromatin
network, and chromatin coil of the preceding stage, the nucleus
enlarges, the chromatin becomes divided into irregular gran-
ules that become distributed in an irregular fashion along a
138 MUNSON. [Von. XV.
system of netted fibers, but yet more abundant around the
periphery of the nucleus. Here some of the chromatin granules
become aggregated into a homogeneous mass, which is often
closely applied to the periphery of the nucleus, and flattened
on that side, but otherwise spherical. This being, no doubt,
the first appearance of the nucleolus, the nucleus has, at this
early stage, the main features of the germinal vesicle of later
stages. The cytoplasm is still rather limited in amount, but
a distinct cytoreticulum can be made out. This possesses
prominent cytomicrosomes that stain as readily in chromatin
stains, especially at the nodes, as the chromatin within the
nucleus.
In the cytoplasm, close to the nucleus, the archoplasm with
a distinct centrosome can be seen (Pl. XIV, Figs. 42, 43).
8. Relation of the egg to the ovary.—On the first appear-
ance of the growing odcyte, in animals from five to six inches,
the ovarian tubes are still round in section (Pl. XIV, Figs.
34, 37, 38), and no diverticula have appeared. As the odcyte
increases in size, however, it sinks more and more below the
neighboring epithelial cells, as stated by Kingsley (Pl. XIV,
Figs. 34, 38, 39). These, as has been observed, are the sister
cells of the same group, and for some time partly enclose the
growing odcyte as temporary follicle cells.
The basement membrane becomes gradually pushed out as
the egg grows, until it forms an investing membrane of the
egg, which, remaining organically connected with the follicle
cells only by a narrow isthmus, appears to lie wholly outside of
the tube, between the germinal epithelium and the peritoneal
coat (Pl. XIV, Fig. 40). Being still invested with the tunica
propria, however, it is still within the ovarian tube, and in fact
never leaves it (Pl. XIV, Fig. 19, 2p.). As the odcyte moves
outward the sister cells belonging to the group assume more
and more the appearance of epithelial cells (Pl. XIV, Fig. 34).
New oocytes within the tube begin a similar career of growth.
It thus happens that in an animal seven inches long where some
of the tubes have acquired a considerable lumen, two or three
stages of these young odcytes may be observed (Pl. XIV, Fig.
40). Only one diverticulum, in cross section, has yet been
No. 2.] THE OVARIAN EGG OF LIMULUS. 139
fully formed, and this is, as yet, to be observed only on the
tubes of the dorsal side of the animal.
In an animal eight inches long two diverticula in cross
section are fully formed.
In an animal thirteen inches long, being about half grown,
six diverticula are observed in a cross section of a tube.
Here also can be seen the relation of the ovarian tube to the
enclosing peritoneal coat or mantle (Pl. XIV, Fig. 41; Pl. XIII,
Fig. 15, f.c.). The germinal epithelium, with its basement
membrane and enclosing muscle coat, is in organic connection
with the peritoneal coat only along one of its sides (Fig. 41).
Here the various tissue elements become intimately blended,
and here, also, blood capillaries and blood vessels are to be
seen. At this point the tube increases in size, and it is here
that the earliest stages of the forming eggs are to be seen. The
epithelial cells are considerably elongated radially. At the base
of these cells at this point groups of closely packed, deeply stain-
ing nuclei can be seen. Gradually a large nucleus appears
surrounded by a definite cell body, which, unlike the cytoplasm
of the hyaline epithelial cells, is granular, and stains deeply
in the carmine and haematoxylin stains. No evagination of
the basement membrane at this point has yet appeared, but
the cells lying above the young egg cell seem often to be
bounded at their base by a definite membrane, which partly
encloses the space in which the young egg lies.
On either side of this point of attachment of the ovarian
tube where the first stage of the egg appears, the more advanced
stages, in regularly increasing series, are to be seen. Passing
around the tube the diverticula increase with the increasing
size of the egg, to that point of the tube opposite the point of
attachment, where the largest egg in the series is to be seen.
There is no connection between the peritoneal coat and the
ovarian tube except at the one point of attachment of the latter
(Pl. XIV, Fig. 41). The ovarian tube with its diverticula
hangs suspended from the inner wall of the enclosing peritoneal
coat, along one of its sides. In sections the peritoneal coat is
seen only when the ovary is sectioned zz sztu, which is the
more convenient in the young forms.
140 MUNSON. [VoL. XV.
In the adult the relation of the egg to the ovary and of the
various parts of the ovary to one another is essentially the same
as in the younger forms (Pl. XIII, Fig. 15). As compared
with the younger forms the ovarian tubes, in cross section, are
greatly enlarged, and the number of diverticula, in cross sec-
tion, are proportionately increased. But this is not necessa-
rily true of the number of eggs that may appear in cross
section, for after the discharge of the first set of eggs into the
ovarian tubes a number of empty pouches containing no eggs
may be seen (Pl. XIII, Fig. 15). These occupy the same posi-
tion which the discharged egg previously occupied. Originally
they arise, as has been seen, in the young animal by the push-
ing out of the tunica propria through the fenestrae of the
muscle coat as the egg grows. The sister cells of the egg
after division of the odgonia become the lining cells of the
stalk of the egg. The stalk of the egg, however, exists only
during the empty state of the tube, for when the tube becomes
stretched by mature eggs that have been discharged into it,
the stalk of the egg disappears and its epithelium constitutes
the lining epithelium of the ovarian tube.
While it is true that throughout the various stages of growth
of the animal the number of eggs in cross section of a tube
regularly increases, there appears to be a period beyond which
no new eggs are formed. In an animal eighteen inches long
eight eggs appear in a cross section of atube. Several animals,
among them a soft-shelled one, equaling in size some of the
females that were observed ovipositing at Woods Holl, had
discharged no eggs from the follicles, and yet the number of
eggs in cross sections had not increased.
Notwithstanding a most careful examination of ovaries from a
large number of adult females, collected both at Woods Holl
and at New Haven, and showing empty follicles, having evi-
dently reached the period of sexual maturity, I have in no case
been able to observe the first stages of the forming egg as it is
so easily done in the earlier stages of the growing animal. I
infer, therefore, that new eggs are not formed after a certain
period, and that this period is either earlier than the period of
the first discharge of the oldest egg into the ovarian tube or
No. 2.] THE OVARIAN EGG OF LIMULUS. 141
else coincides with it. So far as I have been able to determine
from an examination of many specimens throughout the grow-
ing series, the number of diverticula that appear in a cross
section of a tube is equal to, and does not exceed the num-
ber of odgonia in the cross section of a tube previous to the
formation of an oocyte.
Furthermore, in the adult animal having empty follicles,
the number of eggs in cross section of a tube decreases in
proportion as the empty follicles increase; and the size of the
smallest eggs is proportional to the number of empty follicles,
and inversely proportional to the number of eggs in cross
section.
In higher animals it is known that a period exists when eggs
for the first time are discharged. It is also known that a
period exists after which reproduction does not take place. It
is also known that in many higher animals it is impossible to
find the earliest stages of the egg in the adult animal, and it
frequently has been assumed, on this account, that not only
the origin, but the history of the egg in the adult differs radi-
cally from the history of the egg of the same animal in its
early stages. Thus Balfour ('78), in Elasmobranchs, describes
two methods by which the egg may arise: first, by a fusion of
a number of cells, which he thinks is the normal process; and,
second, by a gradual transformation of a primitive ovum into a
permanent ovum.
To enumerate, briefly, the observations: In the young
animal, up to five inches, the germ cells form the lining of the
ovarian tube. At this period growing odcytes make their
appearance as diverticula, and continue to be formed up to the
period of sexual maturity. After this period no new odcytes
are formed; but those already existing continue to grow as the
animal grows, until the period of sexual maturity, when the
eggs in the follicles first formed are discharged into the ovarian
tube. The first oviposition takes place considerably later, and
continues at intervals till all the eggs have been matured,
which may cover a period of at least eight years. With an
intermission of more than one year between the periods of
oviposition, as seems probable from the observations recorded
142 MUNSON. [Vou. XV.
in the chapter on natural history, this period may of course be
greatly extended. It is seen from those observations, also,
that after the period of sexual maturity, which may be reckoned
from the first discharge of eggs into the ovarian tubes, the
phenomena of moulting, if not entirely suspended, become at
least less frequent. It may be supposed that from the period
of sexual maturity the animal does not increase so rapidly in
size. That it does increase in size after the period of sexual
maturity seems probable from the fact that females that were
observed ovipositing differed considerably in size.
The original germ cells (odgonia), up to the time when the
embryo measures five inches, including the tail, multiply by an
equal division. At this stage they number about eight in cross
section. This marks the end of the period of multiplication.
At about the sixth-inch stage of the animal a new period in
their history begins — the fertod of growth. This is immedi-
ately preceded by a multiplication process differing from the
former in that the products of division are dissimilar. The
karyokinetic processes by which this takes place have previ-
ously been described. The result of this process is the forma-
tion of a group of cells, one of which becomes the growing
oocyte, while the others belonging to the same group become
temporarily the follicle which ultimately forms the permanent
epithelium of the ovarian tube. In this way the original germ
cell has acquired a new environment, inasmuch as it is hence-
forth destined to be removed farther from the lumen of the
ovarian tube, and is guarded by its daughter cells, which, as
follicle and epithelial cells, serve to nourish and protect it.
This transformation of the original odgonia into the pro-
tected, specially nourished, and consequently growing oécyte
does not take place simultaneously in all the original odgonia,
but it is first accomplished in that one farthest removed from
the point of attachment of the ovarian tube (Pl. XIV, Fig.
41). From now on, this first-formed odcyte continues to
grow as the animal grows, and is the first to arrive at that
stage of maturity which marks its discharge into the ovarian
tube when the period of sexual maturity of the animal is
reached.
No. 2.]} THE OVARIAN EGG OF LIMULUS. 143
At the seven-inch stage of the animal this first odcyte has
formed a complete diverticulum (Pl. XIV, Fig. 40). Longi-
tudinal sections of an ovarian tube, when it passes directly in
the plane connecting the point of attachment of the ovarian
tube and the diametrically opposite side, show a series of these
first odcytes all practically equal in size.
In transverse sections of the tube, in this stage, it is seen
that the immediate neighbor on the right is passing through
the same process (Pl. XIV, Fig. 34); and this being formed,
two diverticula of the ovarian tube may be seen in an animal
about eight inches long. Now a third on the left is forming a
follicle in the same way. Thus the forming odcytes with their
follicles and diverticula appear alternately on either side of the
one first formed. In the thirteen-inch animal five have formed
and a sixth is forming; while in the eighteen-inch stage eight
diverticula have been formed, the smallest being close to the
point of attachment of the ovarian tube.
As these oocytes increase in size uniformly from the time
of their first formation, the one first formed continues to be
the largest, the others on either side of this being smaller and
smaller, corresponding to the time of their appearance, as the
point of attachment of the tube is approached.
The regular sequence in which the odcytes make their
appearance gives to each a definite amount of space, which
relieves it from pressure during growth and preserves its
spherical form. It is readily seen, also, that this sequence
affords a compensation in the economizing of space in the
periovarian cavity; for when the first odcyte attains to a defi-
nite size it is discharged; and thus the amount of space by
successive discharges, as each in its turn grows, remains prac-
tically the same throughout. A portion of an ovarian tube,
taken from the living animal thirteen inches long, and exam-
ined under the microscope, presents the appearance of an
elongated cluster of grapes (Pl. XIII, Fig. 16). In this way,
also, it can be seen that the eggs decrease or increase uni-
formly in size as the tube is rotated on its longitudinal axis.
Up to this point it can be said that there exists a
correlation of growth between the parent organism, the
144 MUNSON. [Von. XV.
ovary, and the eggs, after they enter on the period of
growth.
It is known from the observations of Lockwood, which I can
fully confirm, that the young animal moults more frequently in
its earlier than in its later stages. This seems probable from
the observations related in the chapter on the natural history,
where it was stated that the apparently grown soft-shelled
specimens were found, on examination of the ovary, not to
have any mature eggs in the ovarian tubes; and that animals of
an equal size, having moulted earlier, were in a similar condition.
Histological examination of the ovaries of these animals showed
that no egg had been discharged from the follicles, this being
an easy matter to determine.
It was also stated that animals having mature eggs in the
ovarian tubes always had hard shells, which was also. true of
all those females observed at Woods Holl during the spawning
season.
These observations seem to show that the moulting is not a
phenomenon in any way connected with the season of the year,
but that it is intimately connected with the phenomena of
growth.
Now, it having been shown that the young of Limulus moult
much more frequently than the adult animals, and that with
each moult the young animal increases greatly in size, it is
extremely probable that the animal increases in size much more
rapidly in the earlier than in the later periods of existence.
From the comparative size of the first-formed diverticulum,
and its contained egg in the seven-inch animal and the later
stages, the same retardation of growth from earlier to later
periods, observed in the animal, seems to hold good also
in regard to the growth of the ovary and the eggs contained
In it.
This may explain the apparent contradiction in the correla-
tion of growth, which at first sight seems to present itself in
the case of those eggs that are still growing after the period of
sexual maturity is reached. For while the oocyte first formed
is discharged from the follicle at the first period of sexual
maturity, the one which is just formed in an animal eighteen
No. 2.] THE OVARIAN EGG OF LIMULUS. 145
inches long may not be discharged for many years thereafter,
even though the difference between the two in point of time
of first appearance may be much less.
It is known that in many higher animals, especially in the
human subject, precocious growth is often accompanied with
precocious sexual maturity, and that this marks an important
epoch in the life of the individual. It is known also that this
period is evidence of maturity of the sexual organs. In Limulus
it seems extremely probable that the discharge of the first egg
into the ovarian tube marks the period after which no new eggs
are formed. Those already formed continue to grow at the
decreasing rate at which the animal increases in size, after the
period of sexual maturity. They are discharged from the follicle
when they attain to the size which is normal to them; and,
continuing thus to be discharged and no new eggs being
formed, the time of sterility finally arrives.
On the discharge of the egg from the follicle into the ovarian
tube, it is severed from its organic connection with the parent
organism and acquires a new environment. Here the egg
increases to double its former size within a very short period
of time. As will appear later, this change in environment and
in the rate of growth is accompanied by marked internal changes
in the constitution of the yolk. The important fact to note
here is that with the severance of the egg from its organic con-
nection with the parent organism the correlation in growth no
longer exists ; and that the egg, having acquired an individual
existence, grows at a rate entirely out of proportion to the rate
of growth of the animal.
The egg now is surrounded on all sides by the secretion of
the epithelial cells; it no doubt utilizes this secretion as nutri-
ment. In studying the structure of the egg, it appears that the
egg membrane is radially striated, and that these radial striae
are due to protoplasmic fibers that extend out to the investing
tunica propria and are in some way connected with it. When
the egg is discharged it becomes separated from the tunica
propria. This remains behind as the only wall at that point of
the tube, and later becomes lined with a new epithelium, per-
haps regenerated from the surrounding epithelial cells. The
146 MUNSON. [VoL. XV.
cavity in which the egg lay becomes practically obliterated by
the stretching of the walls of the ovarian tube to accommodate
the eggs within it, and only later bulges out as an empty
follicle, after the tension within is relieved on the discharge of
the eggs in oviposition.
Concerning the réle which the radial protoplasmic fibers of
the chorion may have in the transfer of nourishing material
from without, I have nothing on which to base any positive
statements. Neither do I know whether these fibers are
retracted within the egg, thus leaving pores after the discharge
of the egg from the follicle. It may be supposed, perhaps, that
they serve somewhat as delicate pseudopodia in the transfer of
nutriment. Among others, Eimer (’72) has ascribed such a
function to them in the egg of reptiles.
However that may be, the fact remains that the eggs
increase greatly in size and become unfavorable for sectioning,
a feature that does not exist up to this time.
STAGES OF GROWTH.
The period of growth extends from the last division of the
odgonia to form follicles to a somewhat indefinite period after
the egg has entered the ovarian tube and has attained its full
size. By regularly recurring internal phenomena this period
divides itself into four stages. First, a stage extending from
the beginning of growth to the formation of the first layer of
the egg membrane. Second, a stage extending from the end
of the first to the time when the germinal vesicle begins to
move towards the periphery. Third, a stage beginning with
the gradual approach of the germinal vesicle to the periphery
of the egg and terminating with the discharge of the egg into
the ovarian tube. Fourth, a stage extending from the time of
entrance into the ovarian tube to the time of oviposition.
Each stage may be first briefly described, after which the
history of each part of the egg will be considered separately.
Stage I.— The most striking peculiarity of the growing egg
at the time when it can first be recognized as such is the deeply
stainable granular cytoplasm which, previous to growth, is char-
No. 2.] THE OVARIAN EGG OF LIMULUS. 147
acterized by a peculiar glassy translucency. The germinal
vesicle also, at first a nucleus not differing perceptibly from the
neighboring nuclei of the follicle and epithelial cells, increases
in size and becomes more conspicuous by the increase of stain-
able substance. Part of this becomes condensed, or separated
off and collected into a nucleolus, which previous to this time
could not be observed. At this time, also, the archoplasm,
centrosome, or vitelline-body, is more conspicuous in the
cytoplasm.
Perhaps the most conspicuous feature of the egg, as a whole,
in this early stage is the strong affinity of the cytoplasm and
germinal vesicle alike for carmine and haematoxylin stains.
This peculiarity becomes gradually lost after the first stage is
passed. Unlike the nuclei of the follicle and germinal epithelial
cells, as well as the nucleiof other tissue cells of the ovary, the
germinal vesicle cannot be made to show the green stain of the
Biondi-Ehrlich mixture. The loss of this property appears to
take place about the time when the nucleolus makes its appear-
ance. The germinal vesicle and cytoplasm stain deeply in
haematoxylin and carmine stains up to the time when the first
layer of the egg membrane is formed.
In the cytoplasm, during this stage, there is an area, usually
close to the germinal vesicle, which does not show this affinity
for carmine and haematoxylin stains, but which, on the other
hand, has a peculiar affinity for Lyon’s blue, picric acid, eosin,
acid fuchsin, and erythrosin. At this stage the germinal ves-
icle is regularly spherical, and its position is usually slightly,
but at times very excentric. The proportion between its size
and the amount of cytoplasm is perceptibly greater than it is
found to be in later stages.
Stage IT. —In this stage the amount of cytoplasm, as com-
pared with the size of the germinal vesicle, has increased. The
cytoplasm is surrounded by a thin layer of dense substance
immediately under the investing membrane. The germinal
vesicle, instead of being spherical as before, now shows sac-like
diverticula that appear like buds on its surface. The nucleolus
has increased proportionately in size, and shows changes that
are not to be observed in the previous stage. This stage as
148 MUNSON. » Pion. XV.
contrasted with the previous stage is marked by the consider-
able loss, by the cytoplasm, of that affinity for chromatin stains,
and by the greater size and clearness of the centrosphere. The
yolk granules are more abundant and many of them seem to
have increased perceptibly in size.
Stage I/I.—In this stage the germinal vesicle is relatively
more excentric in position, and subject to great variations in
form and size. Compared with the amount of cytoplasm and
yolk it is perceptibly smaller. The nucleolus is often very
large relatively, and shows many irregularities in form and
structure. Numerous “ Nebennucleoli” exist. The chorion
has increased greatly in thickness by the addition of new
layers. The cytoplasm is conspicuously marked by a polar
differentiation, one pole being rich in yolk granules and the
opposite pole comparatively free from these granules.
At the end of this period the germinal vesicle lies close to
the periphery, partly surrounded by a spongy, hyaline proto-
plasm that does not stain readily. The egg having attained
about half of its normal size, but as yet showing no true yolk
spheres, is at the end of this period discharged into the ovarian
tube. The manner in which this appears to be accomplished
has been described above. The egg has now entered on its
fourth and last stage.
Stage [V.— This stage is marked by a modification of the
cytoplasm that renders sectioning in paraffine extremely diffi-
cult. This appears to be due to marked changes in the yolk
granules. These assume regular spherical forms, and increase
very rapidly in size. Owing to the rapid increase of the yolk
spheres the egg increases proportionately in size, and this
increase in size appears to be a very rapid one. In the first
periods after its discharge from the follicle the egg can still be
sectioned in paraffine, but the yolk bodies can be seen to have
become vesicular and regular in outline, though still compara-
tively small. The yolk bodies, even now, adhere less firmly to
the slide, so that passing the slide through different grades of
alcohol or even dissolving the paraffine is liable to wash many
of them away. This was not the case in the previous stages.
All transition stages from these first definite yolk spheres to
No. 2.] THE OVARIAN EGG OF LIMULUS. 149
the fully grown yolk spheres can be observed, not in the same
egg, but in a series of eggs, according to the time which has
elapsed since their discharge.
In this stage the nucleolus has disappeared, and the greatly
increased yolk spheres often render it difficult to find any trace
of the germina] vesicle, except in the first part of the period,
when it still can be seen immediately under the egg membrane
or comparatively close to it.
Degenerative processes. —In the third stage it sometimes
happens that the egg, instead of being discharged into the
ovarian tube, undergoes degeneration. This has been observed
occasionally in material collected both at Woods Holl and at
New Haven; but it was most pronounced in the ovaries of
those animals which were obtained from the aquaria of the
United States Fish Commission at the World’s Columbian
Exposition in Chicago.
These animals had been kept in confinement for at least six
or seven months. It is probable that they had suffered from
lack of nourishment, as well as from other disturbing influ-
ences incident to a long confinement.
The ovarian tubes of these animals were filled with mature
eggs, and oviposition had probably been prevented by their
captivity. Many of the larger follicular eggs show the regres-
sive metamorphosis referred to.
The metamorphic process appears to take place in two ways:
first, by the gradual absorption of the egg without the inva-
sion of cells; second, by the appearance, within the egg, of
innumerable nuclei (Pl. XIV, Fig. 30).
In the latter case the germinal vesicle, so far as observed,
is in all cases absent. On their first appearance the nuclei
are found at the proximal pole, where, in this stage of the
egg, the germinal vesicle is normally found. With the increase
of these nuclei they spread throughout the central part of
the egg; and, without at first producing any abnormal appear-
ances of the yolk, gradually fill the entire egg (PI. XIV, Fig.
30). Simultaneously with this, one or several layers of well-
defined, polygonal cells surround the egg, between the outer
tunic and the egg membrane, in many cases giving the appear-
150 MUNSON. [VoL. XV.
ance of a true follicle epithelium. At times this layer of cells
may not extend to the distal pole; and in still other cases
several layers may appear at various points.
These enveloping cells appear to be continuous with the epi-
thelial cells of the stalk, but their boundaries are more sharply
defined. The nuclei of these cells resemble the nuclei of the
germinative epithelium, but their cytoplasm is always packed
with stainable granules resembling yolk granules. The nuclei
within the egg present every similarity to the nuclei of
these surrounding cells; and, like the latter, in advanced
stages of metamorphosis of the egg they are surrounded
by deeply staining granular areas of protoplasm, indicating
cell outlines. Often, however, the nuclei are seen imbedded
in interwoven strands of protoplasm, where no cell boundaries
are visible. This may occur in different portions of the same
egg. On their first appearance the nuclei are often uniformly
distributed throughout the yolk, in which cases the yolk may
be normal, or else slightly broken up into comparatively large
masses, giving a vague suggestion of cleavage.
In stages farther advanced the nuclei, which at first showed
no indication of cell boundaries, become more or less grouped
into patches. The yolk granules, previously evenly distributed
throughout the egg, evidently disappear in patches at different
times, till one pole of the egg may be nearly devoid of yolk
granules. It then shows only the strands of protoplasm with
scattered granules, and nuclei imbedded in them; while the
other pole may still have the normal appearance, with the
exception of here and there an isolated nucleus.
In section, except in the earliest phases of metamorphosis,
the outlines of these eggs become irregular (Pl. XIV, Fig. 31).
The egg membrane becomes indented, folded, and perforated
in various ways. The perforations may pass transversely or
obliquely, and in these perforations cells resembling the
granular cells surrounding the egg are often observed. These
perforations often communicate with spaces between the outer
tunic and the infolded egg membrane, which spaces may be
filled with granular cells resembling those observed in the
perforations.
No. 2.] THE OVARIAN EGG OF LIMULUS. {51
At the proximal pole the egg membrane is often partially
or completely destroyed; and a nucleated mass of proto-
plasm within the egg appears directly continuous with the
protoplasm of the cells lining the stalk (Pl. XIV, Fig. 31).
That these bodies in the egg are real nuclei there is no
reason to doubt. They differentiate very excellently with the
ordinary nuclear stains. Diluted Delafield’s haematoxylin,
slightly acidulated, makes them prominent ; and they show the
differential green stain of the Biondi-Ehrlich triple mixture.
From material collected at New Haven, where the animals
were in their normal habitat, preparations showing these
nuclei were obtained by means of the double stain of Lyon’s
blue and lithium-carmine, the nuclei alone taking the carmine
stain.
The final result of this process of absorption, both where
nuclei are present and where these are not to be observed,
seems to be the removal of the entire substance of the egg.
The last traces that are to be observed are those of the
egg membrane, which appears to persist for some time after
its contents have been absorbed.
The lymph spaces adjoining the ovary containing such eggs
are often seen to be crowded with granular cells resembling
very much the granular cells surrounding the egg.
Strahl (92) found that, in the mature follicles of Lacerta
agilis, when the animals are kept in confinement and separated
from the males, an atrophy takes place in the mature ovarian
egg. The first evidence of this is the disappearance of the
nucleus ; second, the segmentation of the yolk as in cleavage,
and finally the entrance of leucocytes. These at first appear
aggregated around the point where the nucleus was situated,
but later they distribute themselves throughout the egg.
The segmentation of the egg of the domestic fowl in an
unfertilized state has frequently been affirmed, among others,
by Oellacher (72). In these, as well as in the unfertilized
eggs of bony fishes, according to him, a division of the nu-
cleus and a real cleavage takes place. The same has been
described in the egg of the dove by Motta and Mayo.
Born claims to have observed a cleavage of the unfertilized
152 MUNSON. [VoL. XV.
egg of the frog. He was unable to state whether this was
accompanied by a division of the nucleus.
Balbiani ('93) found that the ovarian eggs of spiders also
degenerate ; and he figures follicles filled with cells.
Iam unable to make any positive statements in regard to
the immediate causes of metamorphosis. I believe, however,
that the following statement can be made: the cause of the
disturbance lies in the egg itself. In the present case there
is no true follicle epithelium surrounding the egg. The cells of
the stalk, which correspond to the follicle epithelium in other
eggs, and which appear to have a similar relation to the egg
so far as the function of nutrition is concerned, seem perfectly
normal. They often appear to be unusually active and evi-
dently enter the egg at the proximal pole (Pl. XIV, Fig. 31).
The conditions which make this possible, as it seems to me,
lie in the egg itself, and not in an abnormal condition of the
follicle cells, as has been supposed by Flemming ('95) in the
case of other eggs,
THE GERMINAL VESICLE.
A network can be distinguished quite early in the germinal
vesicle, and the stainable substance, losing more and more its
definite form, becomes distributed in irregular granules over
this network, and also between the meshes, being especially
abundant at the nodes. The stainable substance tends to
become massed at the periphery, and especially at one point,
where the nucleolus early makes its appearance. As the
nucleolus increases in size, the remainder of the germinal ves-
icle loses more and more its power of staining deeply in car-
mine and haematoxylin, and is no longer capable of being
differentiated, as ordinary nuclei are, by means of the green of
the Biondi-Ehrlich triple stain.
During the first stage of the egg the germinal vesicle is
spherical and occupies a slightly excentric position when
viewed in the plane passing through the centrosome and
sphere. Ina plane at right angles to this, its position is about
central (Pl. XIV, Figs. 34, 38-41).
No. 2.] THE OVARIAN EGG OF LIMULUS. 153
In the next stage the germinal vesicle shows a tendency to
become irregular, owing to the appearance on its surface of
numerous diverticula or pouches (Pl. XVI, Fig. 105; Pl. XIII,
Figs. 1, 6-8 ; Pl. XIV, Fig. 24). These are often of consider-
able size. They are, for the most part, spherical and remain
connected with the germinal vesicle by means of a narrow
neck or isthmus. The network and stainable granules of
the germinal vesicle extend into these, and they are fre-
quently observed to contain pale “ Nebennucleoli” (Pl. XIII,
Figs. 7, 8).
Very frequently there is an accumulation of stainable gran-
ules at one point near the periphery of the germinal vesicle,
and this is at times so prominent that it might be mistaken for
a second ‘“ Hauptnucleolus.” It, however, lacks the definite
form of the ‘“ Hauptnucleolus,” and consists of irregular bodies
of very different sizes that stain deeply. When this is formed
in the central part of the germinal vesicle, the strands of the
nuclear network appear to radiate from it as a center (Pl. XIII,
Fig.-10). Occasionally this is so marked that it assumes the
appearance of an aster. In some cases the granules are less
pronounced ; and it can then be seen to have all the features of
a centrosome and sphere —a deeply stainable central body,
surrounded by a clear zone, which in turn is again surrounded
by an outer ring, from which the larger strands of the nuclear
network radiate. A somewhat similar arrangement of the
nuclear network around the nucleolus is sometimes seen (PI.
XIII, Figs. 4, 8, 11). It is especially pronounced in material
hardened in Flemming’s fluid (Pl. XIII, Figs. 4, 8), but the
appearances are by no means confined to such material. The
chromatin network seems often to have a centralized arrange-
ment, and the center of radiation may coincide with the
nucleolus or be independent of it. When it is found near the
periphery of the nucleus, the wall of the latter often shows
an indentation in the form of an acute re-entrant angle at that
point (Pl. XIII, Fig. 15). In such cases, which are of frequent
occurrence, the principal strands of the network can be seen to
radiate from this point in a fan-shaped manner. It can be seen
that this point is connected with fibers proceeding directly from
154 MUNSON. [VoL. XV.
the centrosome and sphere in the cytoplasm. It often recalls
very forcibly the observations of Auerbach ('96), Leydig (gs,
'g8), and Rabl (’g9).
The nuclear network can also be distinctly seen in the living
egg, without the use of reagents, by causing the contents of
the egg to flow out. In such a preparation the nuclear network
is very distinct, and presents all of the principal features seen
in well-preserved material. It is clearer and better defined,
owing to the comparative absence of granules which in preserved
material obscure it. A germinal vesicle, removed in this way,
remains surrounded on its exterior by a delicate network of
fibers enclosing yolk granules. These seem to be intimately
connected with the germinal vesicle, and render it impossible
to obtain the latter entirely free from them. One might ask
whether the peculiarly close adherence of these fibers is not
due to a direct continuation with the nuclear network.
Everything seems to point to the conclusion that this stage
of the germinal vesicle is a period of great activity.
The germinal vesicle, containing a “ Hauptnucleolus”’ and
many “ Nebennucleoli,” and having attained its maximum size,
now begins to approach the periphery (Pl. XVI, Figs. 104, 114).
It varies much in form and size. At times long pseudopodia-
like processes extend radially far out into the body of the egg,
giving the germinal vesicle the appearance of a very irregular
amoeba. There may be one or several pseudopodia, and they
may thin out to such an extent that it is difficult to trace them.
The body of the germinal vesicle, in such a case, may be
reduced to a small central area, in which the often very large
nucleolus may be seen (Pl. XIII, Fig. 5). It may also be
greatly extended in one direction, so as to become flattened out
into the form of a fish or an arrowhead.
In all such cases the hyaline karyolymph appears to
be wanting, or nearly so. The chromatin granules lie closely
packed, and the peculiarly distorted body thus takes the stain
with avidity. The “ Hauptnucleolus”’ is always present. There
is often a strong temptation to regard these peculiar forms of
the germinal vesicle as shrunken conditions due to reagents.
As they occur, however, in the best preserved material, it is not
No. 2.] THE OVARIAN EGG OF LIMULUS. 155
easy to regard them as artifacts. Careful study of the living
egg reveals none of those movements of the germinal vesicle
and nucleolus frequently spoken of in other eggs as amoeboid.
The stage under consideration seems rather to be a period of
suspended activity on the part of the germinal vesicle, and the
processes extending out into the cytoplasm appear rather as
the expression of pressure to which the germinal vesicle is
being exposed owing to the increasing mass of yolk.
This period is followed later by one of renewed activity, in
which the germinal vesicle again becomes filled with the usual
hyaline karyolymph, and assumes a more definite spherical
form (Pl. XIII, Fig. 14). Having reached the periphery of the
egg, it is often comparatively large and is surrounded on all
sides, except that immediately in contact with the yolk, by a
hyaline, finely spongy protoplasm, which is comparatively free
from yolk granules (Pl. XIII, Figs. 11, 14). The contents of
the germinal vesicle in such cases show, especially around the
periphery, a finely spongy protoplasm, in every respect resem-
bling that surrounding it. It is still surrounded by an appar-
ently well-defined membrane, and contains still a large, deeply
staining nucleolus. This alone shows the characteristic stain
of chromatin.
The ultimate fate of the germinal vesicle appears to be that
its membrane disappears, the larger portion of its contents
becomes diffused through the spongy protoplasm. This may
be seen as a cap, covering perhaps half of the egg (PI. XIII,
Figs. 11, 14). It persists as such for a considerable period,
until the yolk spheres, now increasing very rapidly, occupy
practically all the space within the egg. They gradually
encroach on the protoplasmic cap till it is reduced to a thin
protoplasmic layer immediately under the egg membrane (PI.
XIII, Fig. 17).
The nucleolus having disappeared as such, the last remnant
of the germinal vesicle can be seen as a deeply stainable,
irregular, amoeboid body, lying in the yolk some little distance
below the egg membrane (Pl. XIII, Fig. 12). The yolk
surrounding this has a distinct radial arrangement, and this
radial arrangement can be traced as parallel striae to the periph-
156 MUNSON. [VoL. XV.
ery of the egg, where they are continuous with a small mass
of hyaline protoplasm. In one or two cases, stainable bodies
suggesting chromosomes have been seen in the midst of this
radial striation.
In one case, a mass of hyaline protoplasm, free from yolk
and having the form of a spindle, was observed imbedded in
the yolk some little distance below the egg membrane (PI. XIII,
Fig. 17). The latter showed a perforation running radially
through it at this point, suggesting a micropyle. As this is the
only trace of such a structure that has been observed, it cannot
be definitely stated to be a micropyle. The lumen of the
perforation was occupied by a number of small yolk granules.
In the case of other eggs, various causes have been assigned
for the movement of the germinal vesicle towards the periph-
ery.
The yolk accumulating at one pole continues to gradually
increase until that pole in which yolk does not accumulate and
with which the germinal vesicle is connected, becomes more
and more flattened out (Pl. XIII, Figs. 11, 14; Pl. XVI, Fig.
104), the hyaline spongy protoplasm, of which it is composed,
being forced more and more over the surface of the egg as the
vegetative pole increases. This process continuing, the germi-
nal vesicle soon comes to lie under the cap previously described.
Owing to the growth of the yolk at one pole, the germinal
vesicle and its surrounding hyaline protoplasm, originally near
the center of the egg (Pl. XIV, Figs. 20, 24), becomes more
and more displaced, the internal portion becoming turned out,
so to speak. It might perhaps be designated as an evagination,
somewhat like the finger of a glove when straightened out after
being turned in on itself.
The nucleolus. — In the stages of the odgonia preceding the
final division, resulting in the formation of a follicle, no trace
of a nucleolus can be discerned (Pl. XIV, Figs. 33, 35, 37). It
is first seen in the odcyte, at the time when the latter has
commenced to increase in size. As we have seen, the chromatin
at this time loses many of its previous characteristics, both with
regard to chemical reactions and general appearance. The
chromatin bodies, so far as they retain their regular form,
No. 2.] THE OVARIAN EGG OF LIMULUS. 157
become imbedded in a more or less viscid (or granular), stain-
able substance. In this substance the nucleolus makes its
appearance, usually close to the periphery of the nucleus (PI.
XIV, Fig. 39). At first it is often flattened on the side next
to the wall of the nucleus, but elsewhere spherical, though at
times irregular in outline. In this early stage it often appears
to consist of granules. How much of this granular substance
may be due to the reagents cannot be definitely ascertained. It
soon becomes homogeneous and spherical, and takes up little
by little a more central position (Pl. XIV). At a very early
period in its history it can be seen to be differentiated into an
outer and an inner zone (Pl. XIV, Figs. 34, 44). The outer
zone seems more dense, and at first it seems like a compara-
tively thick investing coat of the internal central body. Occa-
sionally two nucleoli of essentially similar appearance can be
seen in this stage (Pl. XIV, Fig. 26). In both the investing,
homogeneous layer can be seen to be thinned off at one point,
so that the internal, central spherical body partly protrudes
through the homogeneous covering.
One of these nucleoli usually increases more rapidly in size,
and later becomes the only one visible. The growing nucleolus
becomes the future ‘‘ Hauptnucleolus,” of which there is usually
only one, but in some cases two.
As the nucleolus grows, it retains for a considerable time its
spherical form; and throughout the first period usually remains
more or less homogeneous, with now and then spherical vac-
uoles in its substance (Pl. XIV, Fig. 44; Pl. XV, Figs. 83, 87;
Pl. XVI, Figs. 99, 100). These vacuoles are not always mere
cavities or fluid particles; they may contain solid bodies that
stain a deep black in Heidenhain’s iron-haematoxylin.
In the second stage of the egg the nucleolus, although it
often seems homogeneous, and filled with vacuoles of different
sizes, is seen to possess, in a great many cases, a dense outer
layer enclosing a central, spherical mass (Pl. XV, Fig. 68; PI.
LOM HIS SO Ophea, Bigs. 10S, LIS, lz, to. eelne
central mass often has an excentric position, so that the outer
homogeneous part, in optical section, has the form of a cres-
cent. This can be distinctly seen in the living egg (Pl. XVI,
158 MUNSON. [Vou. XV.
Figs. 106, 115, 117, 118). It is one of the most pronounced
characteristics of the nucleolus at this stage. In the nucleolus
of the living egg the outer crescent-shaped zone appears to be
studded with spherical bodies imbedded in it (Pl. XVI, Fig.
108). Whether these are mere fluid vacuoles or solid bodies,
cannot be made out. The central body often appears like a
vacuole, but more frequently it is granular. The granules
vary in size, not only in the same nucleolus, but in different
nucleoli (Pl. XVI, Figs. 108, 115, 117).
In preserved material this central body is seen to be solid
or composed of granules, as was the case in the living egg.
The central body may at times be greatly enlarged. The
outer crescent-shaped body then appears as a cap at one pole
of the central body. The horns of the crescent, in sections,
becoming greatly thinned out, extend along the sides of the
central body. At other times the central body is not so
large, the outer zone being larger in proportion. The central
body may then be elongated into a cylinder-like body with
rounded ends, the outer end projecting through an opening in
the outer zone at the point where this is thinnest (Pl. XVI,
Figs. 111, 113). This reminds me strongly of the observations
of Aimé Schneider and Balbiani (83). The inner end of the
projecting body may be simply rounded, or it may be somewhat
enlarged. The whole body may be spherical in form (Fig. 112).
In these cases the outer zone stains more deeply than the
inner body, except in Heidenhain’s iron-haematoxylin, in which
the central body takes a dark stain.
The body can often be seen to have been extruded (Pl. XVI,
Fig. 110; Pl. XV, Fig. 79). In such cases a cavity, which
communicates with the exterior by means of a circular opening,
exists in the nucleolus. The extruded body can be seen in all
stages of extrusion. When this has occurred, it is sometimes
seen lying close to the opening (Pl. XVI, Fig. 110; Pl. XV,
Fig. 79).
The extruded body assumes a spherical form, and, except in
Heidenhain’s iron-haematoxylin, loses more and more its power
of staining. Finally, it resembles an ordinary yolk sphere of
the last stage of the egg.
No. 2.] THE OVARIAN EGG OF LIMULUS. 159
I see no reason why this may not be regarded as a so-called
«“ Nebennucleolus.”” The difficulty with which these bodies
stain seems to correspond to the condition of bodies described
under that name by various authors.
Besides this single body extruded in this way, it can some-
times be seen that the central cavity of the “ Hauptnucleolus ”’
is filled with a number of comparatively large spherical bodies
that behave toward stains similarly to the one just described
(Pl. XIII, Figs. 4, 8). In one case an opening in the thinnest
part of the ‘ Hauptnucleolus” was observed; and some of
these internal bodies appeared to be on the point of being
extruded (Pl. XIII, Fig. 4). One was lying at the opening
outside the ‘“ Hauptnucleolus,”’ and another just inside; the
rest of the internal cavity was occupied by several of these
bodies. They were surrounded by a finely granular substance
which was strongly contrasted with the outer zone, this being
very thin, but staining deeply.
I cannot say that all ‘“ Nebennucleoli” originate in this way.
Occasionally one may be seen partly imbedded in the outer
zone of the “Hauptnucleolus,’ and this may occur at any
point where the outer zone is thickest.
Similar bodies are found distributed throughout the germinal
vesicle (Pl. XIII, Fig. 15). In the living egg they appear as
shining vesicles, often occupying diverticula of the germinal
vesicle. They can also occasionally be observed in the cyto-
plasm of the living egg (Pl. XIII, Fig. 3; Pl. XVI, Fig. 112).
As I have never seen them actually pass out from the germinal
vesicle, I cannot say that they do so.
If the living egg is ruptured, and the contents made to flow
out, they can be seen still within the germinal vesicle, and also
in its neighborhood.
On a closer examination they are seen to be vesicles, con-
sisting of a delicate membrane, within which are a number of
granules, apparently suspended in a liquid. This can be made
to flow out when the membrane is ruptured.
The “ Hauptnucleolus”’ increases as the egg increases in
size, and, in the third stage of the egg, may often reach gigan-
tic proportions (Pl. XIII, Fig. 5; Pl. XVI, Figs. 104, 114). It
160 MUNSON. [VoL. XV.
is extremely variable. As a rule, it is spherical; but it may
be perforated with holes and cavities. The center is often
finely granular (Pl. XIII, Fig. 7). These granules may con-
stitute the entire nucleolus, except a thin outer homogeneous
membrane (Pl. XIII, Fig. 6).
Then again the center may be occupied by a relatively small,
spherical, strongly refractive body, the outer zone being rela-
tively uniform in thickness. Occasionally, this outer zone,
surrounding the central body, is seen to be radially striated.
The striae appear to be continuous with the network of the
germinal vesicle.
Instead of a central body, there may be a central cavity in
which nothing stainable appears to exist (Pl. XIII, Fig. 5).
More frequently, however, the central cavity is filled with a net-
work resembling the network of the germinal vesicle, except-
ing that the meshes are finer (Pl. XIII, Fig. 1; Pl. XVI,
Fig. 107). As in the latter case, the fibers of the network are
more or less covered with stainable granules, and the meshes
between these fibers remain unstained. This caving in of the
interior, so to speak, appears at times to continue till the
nucleolus is nothing but a thin hollow shell (Pl. XIII, Fig. 6).
This shell may be so large as to occupy nearly one-half of the
germinal vesicle. Such cases, however, are not frequent.
The interior of such a nucleolus is occupied by a chromatin
network which in every way resembles the chromatin network
of the germinal vesicle.
More frequently, in this stage of the egg, the nucleolus may
be seen to have preserved a solid constitution even to the time
when the germinal vesicle has reached the periphery. In most
cases it is comparatively large, and stains more intensely than
the rest of the germinal vesicle. Yet it is often completely
honeycombed with little vacuoles. It often appears as if these
vacuoles enlarge and flow together. The large nucleolus then
appears like a system of variously connected, stainable strands
of nucleolar substance, in appearance not unlike a coarse sponge.
In rare cases such a large, degenerated nucleolus is accom-
panied by another very much smaller, which does not show the
signs of degeneration so conspicuously.
Noy 25] THE OVARIAN EGG OF LIMULUS. 161
In eggs that have been discharged from the follicle into the
ovarian tube no trace of the nucleolus could be observed. The
last phases described seem to be stages of final dissolution, and
absorption of the nucleolus. It seems that the discharge of the
egg from the follicle marks its end, as the entrance of the egg
into the follicle marked its beginning. Its history coincides
with that period of growth of the egg in which the latter
remains in organic connection with the parent organism. This
would seem to associate it with the phenomena of nutrition and
growth of the egg.
There are cases also, in this period of growth, in which there
is no nucleolus in the germinal vesicle. Such cases occur when
the germinal vesicle is surrounded by a zone of deeply staining
granules, which resemble chromatin granules in their behavior
towards haematoxylin and carmine stains (Pl. XIV, Fig. 29).
Whether this is an abnormal condition, I cannot say. The
appearances will be discussed more fully in connection with the
cytoplasm. We have seen, also, that at the beginning there
may be two similar nucleoli (Pl. XIV, Fig. 26), while later one
of these has disappeared. In the second stage of the egg
two nucleoli are rarely observed. But we have seen that
towards the end, when the nucleolus has greatly degenerated,
there may be a second smaller one apparently having recently
arisen,
In view of these facts, together with its great variability, it
is safe to say that it is not a permanent organ.
The appearances described above seem to show that the
nucleolus is not simple, but composite. It consists of a
framework of linin similar to that of the germinal vesicle, and
a more or less homogeneous, semi-solid, stainable mass, which,
accumulating at the nodes of the linin network, flows together
into a spherical body, enclosing portions of the linin fibers.
Within this mass chemical changes appear to take place which
ultimately result in a substance resembling the yolk of the ma-
ture egg, and which, like it, assume the form of spherical refrac-
tive bodies. These when formed are extruded and give rise
to “ Nebennucleoli.” The chemical or other processes within
appear to continue; and the nucleolus, losing substance from
162 MUNSON. [Vou. XV.
within appears to receive additions from without. Thus a com-
paratively large hollow shell arises (Pl. XIII, Fig. 6). It would
appear almost as if the addition from without is in the form of
a precipitate, which becomes deposited on the surface of the
nucleolus.
Owing to its relation to the linin network, which is often to be
observed within it, the nucleolus may be considered as having
a fixed position. Its movements within the germinal vesicle
must necessarily be regulated by the linin fibers which consti-
tute its framework. The ‘ Nebennucleoli”’ appear to lie more
or less free in the meshes of the network.
The main feature of both the “Hauptnucleoli” and the
« Nebennucleoli’”’ can be seen in the living egg (Pl. XVI, Figs.
106, 108, 115-118).
a. Summary on the nucleolus. —1. The nucleolus appears
at the time when the egg begins to grow.
2. It arises as an irregular or spherical mass in an amorphous
stainable substance, surrounding the chromatin elements at the
time when the germinal vesicle assumes its specific character-
istics.
3. There is usually only one, but occasionally there are two
in this early stage.
4. As soon as it has assumed a definite spherical form,
it is differentiated into an outer zone enclosing a central
body.
5. At first the entire nucleolus stains as readily as the
chromatin.
6. The outer zone retains this power of staining, but the
inner body gradually loses it.
7. Thecentral body (endonucleolus or nucleololus) stains very
intensely in Heidenhain’s iron-haematoxylin. With double
staining of the latter stain, combined with eosin, the entire
nucleolus can be seen as a red outer zone and a black or blue
central sphere.
8. This central body may become elongated, so as to
protrude through the outer zone.
g. It is extruded from the nucleolus, which then appears as
a hollow sphere with an opening at one pole.
No. 2.] THE OVARIAN EGG OF LIMULUS. 163
10. Other similar bodies may form within, and these likewise
are extruded.
11. These extruded bodies are the so-called “ Nebennu-
cleoli.”’
12. In carmine and Delafield’s haematoxylin they stain
feebly.
13. In the living egg they appear as shining vesicles,
composed of a delicate membrane enclosing a fluid in which
granules are suspended.
14. They have the appearance of yolk spheres; but as they
arise at a time long before the yolk spheres are formed in the
cytoplasm, they are not yolk spheres. .
15. Similar bodies are seen in the cytoplasm at this stage,
but they are not permanent.
16. The part remaining after this extrusion retains its power
of staining in carmine stains, and may be designated the
nucleolus.
17. This often has the form of a crescent.
18. The interior of this, in rare cases, contains no stainable
substance, and appears as if it might be a fluid vacuole.
19. More frequently the interior is occupied by a linin
network like that of the germinal vesicle, and, like it, having
stainable granules imbedded in it or attached to it.
20. The crescent-shaped or circular nucleolus appears to lose
substance from within, and to receive substance from without.
21. It may thus become a large hollow shell of stainable
substance enclosing a reticulum.
22. The entire nucleolus may often appear as a spherical
mass of granules enclosed by a homogeneous membrane.
23. These granules may sometimes be scattered, and lie
imbedded in a homogeneous mass resembling the outer mem-
brane. The granules, when they become refractive and lose
their power of staining, may be mistaken for vacuoles. These,
however, can be stained intensely in Heidenhain’s iron-haema-
toxylin, so that a dark body appears to lie in an unstained
vacuole.
24. When only one of these granules exists, it may occupy
the center of the nucleolus.
164 MUNSON. [VoL. XV.
25. Occasionally the outer part can be seen to be radially
striated, so that when the central body is present the nucleolus
has the main features of a centrosome and a sphere.
26. Such a structure is sometimes to be observed in addition
to a second homogeneous nucleolus.
27. When the central body exists, there may be a network
surrounding it, which in turn is enclosed by the outer layer of
the nucleolus.
28. In such cases the nucleolus has all the features of a
germinal vesicle, with nucleolus network and nuclear membrane.
29. The nucleolus can be seen as long as the egg remains in
the follicle, but not after its discharge.
30. In the later stages it is often very large and stains deeply.
31. It may, however, become honeycombed with large
openings.
32. In such a condition it may be accompanied by a very
much smaller one, apparently more perfect, and, like it, staining
deeply. This distinguishes it from the somewhat numerous
«‘Nebennucleoli” that are spread throughout the germinal
vesicle, and in carmine stains have a yellow coloration.
33. The nucleolus may, therefore, consist of three different
constituents: (a) linin framework; (4) substance resembling
chromatin; (c) substance resembling mature yolk globules.
34. Movements of the nucleolus are regulated by the linin
framework which permeates it.
35. The nucleolus disappears as such when the egg is
discharged from the follicle, and when, as we shall see, an
entirely different process of growth of the egg takes place.
36. The history of the nucleolus coincides with the period of
growth of the egg, 7.e., while it remains in organic connection
with the parent organism.
b. Literature. — An extensive literature on the nucleolus
exists, from which many similar observations could be cited.
We are reminded at once of the observations of Balbiani ('ss)
and Aimé Schneider ('75).
I cannot accept Rhumbler’s ('93) mechanical explanation of
the radial feature of the nucleolus, nor his equally mechanical
explanation of the endonucleoli.
No. 2.] THE OVARIAN EGG OF LIMULUS. 165
Balbiani’s explanation of an extruded body in eggs of
Geophilus, as being a tube, would lose much of its incredible
features if the term tube had not been applied to it. The
observations themselves are no doubt correct, but his figures
are as diagrammatic as his language is colored by a vivid
imagination.
Space will not permit an extended consideration of the many
problems concerning the nature and function of the nucleolus.
Most of them are well known, and the bearings of these
observations will be readily perceived. I would refer the
reader to the following authors: Cramer (48), Ludwig (74),
v. Wittich (49), Leuckart ('53), Pfliiger ('63), Will (86), Rhumbler
(93), Balbiani ('83), Aimé Schneider ('75), Valentin Hacker ('95),
Gegenbaur ('61), Stuhlmann ('g6), Bumpus (91), Waldeyer ('gg),
Leydig ('55), Brandt ('78), Korschelt (89), La Valette St. George
(66), Gustav Mann (93), McFarlane (92), Goette (75), Balfour
(78), Henking (82), O. Hertwig (77, '92), Flemming (75), Auer-
bach (74), R. Zacharias ('87), Scharff ('a8), Mertens (93), Klein
(78), Holl (93), Jordan (93), G. R. Wagener (79), Platner (86),
Wielowiejski ('85).
CONNECTION OF THE EGG WITH THE OVARIAN TUBE.
Before the formation of the egg membrane the cytoplasm of
the egg is continuous with the cytoplasm of the epithelium of
the ovarian tube. At times the neck of the egg, by which it is
joined to the epithelium, is comparatively large, so that the
epithelium appears to lie in direct contact with the egg over a
considerable area; and its cytoplasm appears continuous with
several of the epithelial cells. In most cases the neck of the
egg is constricted to a narrow bridge of fibrous protoplasm,
proceeding from the epithelial cells and continuing into the egg
body as a polar mitosome. This polar mitosome can often be
seen to be continuous with a modified layer of protoplasm sur-
rounding. the egg immediately under the investing tunic; and
it can also be seen to spread out in a fan-shaped manner in that
part of the egg adjoining the stalk. In a few cases the parallel
fibers of which the mitosome is composed have been seen to
166 MUNSON. [Vou. XV.
be connected with a body lying close to the germinal vesicle,
the nature of which will be considered later. The polar mito-
some is perhaps a remnant of the spindle of the last division of
the odgonia, comparable to a similar structure observed by Plat-
ner (86) in the sperm cells of Helix, and by Bolles Lee (95), or
to the Zellkoppel of Zimmermann (91).
At the junction of the egg with the epithelial cells these
fibers are firmly bound together by a body which is prominent
in the younger eggs especially, and which stains deeply in acid
fuchsin and eosin. In Heidenhain’s iron-haematoxylin it stains
very deeply, somewhat like the peripheral bodies, which are to
be considered presently. If haematoxylin be followed with
eosin, it appears as a doubly convex, bright-red body. In many
cases sections through its center show the form of aring. In
the younger eggs it is large and conspicuous. It resembles
very closely the so-called ‘Zwischenkorper” of Flemming (91),
which by him was homologized with the cell plate in plants. A
similar body was observed by van Beneden in the egg of Ascaris,
by Hertwig in Spirochona, by Carnoy ('gs) in the spermatocytes
of Arthropods, and by Henking (91) in the spermatocytes of
insects. This body does not perhaps differ materially from the
numerous peripheral bodies which later make their appearance,
and which, as we shall see, give rise to the first layer of the
egg membrane. As the latter seem to be aggregations of little
spherical bodies, which are the first indications of a forming
egg membrane, so this polar body appears likewise to be a
concentration of such granules.
The cytoplasm.—The cytoplasm consists of at least two
distinct elements —a living formed element and a non-living
amorphous element (Watasé). The former has the form of a
reticulum of variously interwoven fibers, which show a central-
ized arrangement at the center of the egg. This will be dis-
cussed more fully in connection with the attraction sphere and
centrosome. The living substance appears to have many of the
characteristics of a sponge, in the lacunae, vacuoles, and meshes
of which the various amorphous elements are lodged.
The yolk. —The yolk lies either massed together at differ-
ent points, or else uniformly distributed throughout the egg.
No. 2.] THE OVARIAN EGG OF LIMULUS, 167
It occupies the meshes of the cytoreticulum, and appears to be
movable from one point to another, according to the condition
of the controlling living substance. It is, therefore, subject
to changes in mass, giving considerable variation to the appear-
ance of the cytoplasm. It must be pointed out here that the
unequal distribution of the yolk, as well as the variable conden-
sation of the living substance in different parts of the egg,
which is frequently to be observed, does not alter the spherical
form of the egg. It is hardly probable, therefore, that the
spherical form is due to surface tension, which implies an
equilibrium of similar molecules in all radii.
In the younger eggs the amount of yolk varies considerably.
It may be so abundant as to obscure the cytoreticulum, or it
may be very limited in amount. It has been shown that the
earliest formed eggs grow more rapidly than those formed in
the period preceding sexual maturity. In the latter the yolk
is sometimes relatively scarce, and the cytoreticulum is very
distinct. In such cases the nutriment of the egg is presumably
so limited that the surplus food material is used up in the
growth of the living substance. At any rate, it is certain that
the egg increases in size by the growth of the living substance,
and by mechanical expansion due to the accumulation of yolk.
The growth of the cytoreticulum predominates in the earlier
stages, while the accumulation of yolk is the chief cause of
increase in size after the eggs are discharged from the follicle.
It has been maintained that the yolk originates in all cases
within the egg, and it appears to be with considerable reluc-
tance that many, even now, admit the origin of yolk in any
other way. This reluctance seems to date back to the early
controversy regarding the cell nature of the egg. On the one
side it was claimed that the yolk spheres represent real cells ;
on the other, that the yolk originates within the egg.
An external origin of the yolk has frequently been main-
tained. I need only mention Ayers ('84), confirmed by L.
Will (84), in the egg of Oecanthus niveus.
There are reasons for believing that in Limulus a substance
having the essential characteristics of yolk is produced in
the epithelial cells; and that this, in the form of granules,
168 MUNSON. [Vou. XV.
suspended in a fluid, enters the vacuoles and meshes of the
living substance of the egg.
At the time when the first layer of the egg membrane is
formed it is sometimes seen that part of the yolk is not
included within the membrane, but is cut off and remains out-
side in that portion next to the stalk of the egg (Pl. XIV, Fig.
22, y.s.). The yolk lying outside of the egg in such cases is
often considerable in amount, and resembles in every particu-
lar the yolk inside of the egg. Two explanations of this
appearance suggest themselves, which, although appearing
different, may be essentially the same. In the first place, it is
safe to assume that the first layer of the egg membrane arises
at the extreme limits of the formed, living substance of the
egg. Now it may be suggested that in such cases as those
under consideration the amorphous elements of the egg extend
beyond the outer limits of the living protoplasm, and thus
become cut off when the membrane arises. Or it may be that
the yolk granules from the epithelial cells, being prevented
from entering by the membrane, accumulate outside, later per-
haps becoming dissolved and serving as food. It is suggestive
that at this stage in the history of the egg, when the yolk
granules from the outside are no longer capable of entering as
solid bodies, the cytoplasm of the egg undergoes that peculiar
change from an alkaline to an acid state of reaction.
The yolk spheres appear in their vesicular, clearly defined
form only after the egg has been discharged from the follicle.
Previous to this event several of the epithelial cells of the egg
stalk appear to degenerate and break up into granules that
have all the appearances of the yolk granules of the ovarian
egg. On the first appearance of the definite yolk bodies they
are small. Those first formed increase in size, and thus in
somewhat later stages the yolk bodies may show many different
sizes. Ultimately, however, they all attain to a considerable
size and fill the egg completely. There can be no doubt that
these yolk spheres originate within the egg. Their formation
appears to be in some way associated with the new mode of
nutrition of the egg after its arrival in the ovarian tube. As
previously stated, it is here bathed in the secretion of the cells
No. 2.] THE OVARIAN EGG OF LIMULUS. 169
lining the ovarian tubes. These contain considerable quanti-
ties of such secretions.
The disintegration of cells above referred to recalls forcibly
the condition in milk glands, as related by Foster ('93), and
also the account given by Nissen ('g6).
Polarity of the egg.— Adopting the terminology of Auerbach
(96), we have also here a “Kernpol”’ and a “Gegenpol.”’
The archoplasm and centrosome determine the position of the
“Gegenpol.”’ Apparently this is the vegetative pole, for in
later stages it becomes especially granular.
At the nuclear pole a hyaline area appears usually in the
third stage of the egg (Pl. XVI, Fig. 104). This is often irregu-
lar, at times crescent shaped, but not sharply defined from the
rest of the cytoplasm. It may partly enclose the germinal
vesicle, the horns of the crescent gradually merging into the
compact area at the vegetative pole. It often presents a
striking similarity to the hyaline area figured by Andrews (91)
in the egg of Diopatra, and recalls the polar rings in the eggs
of Clepsine and of Allolobophora observed by Professor
Whitman (78) and Miss Foot (96), respectively. I do not
know how far it could be compared to the polar differentia-
tion observed by Mark (go) in the ovarian egg of Lepidosteus,
which would seem more closely related to the observations of
Stauffacher (93) in the ovarian eggs of Cyclas. Possibly these
bodies are more closely related to the ‘“ Zwischenkoérper”’
described by me in connection with the egg membrane.
For reasons which are discussed in connection with the
cytoplasmic zones and yolk-nucleus, I consider this area due
to an infiltration of substance derived from the germinal ves-
icle. Its position is evidently determined by the relative
positions of the germinal vesicle and the sphere. It always
appears opposite the vegetative pole. A line might be
drawn through the vegetative pole, the germinal vesicle,
and the nuclear-pole area (Pl. XVI, Fig. 104). This
line, however, would not always pass through the point of
attachment of the egg where the “‘ Zwischenkérper’”’ is formed.
(See Plates.) The above statements hold true of all stages of
the egg from the beginning of growth. (See Pl. XIV.)
170 MUNSON. [VoL. XV.
As the cytoplasm increases at the vegetative pole, the
nuclear pole becomes more and more crowded, till it, with
the germinal vesicle, spreads out over the surface of the vege-
tative pole as previously described (Pl. XVI, Fig. 104; Pl.
XIII, Figs. 11, 14).
It is to be remembered that no true yolk spheres exist yet,
for these appear only after the discharge of the egg into the
ovarian tube (Pl. XIII, Figs. 12, 17).
This process, it seems to me, has some of the features of
gastrulation by invagination. The animal portion, less laden
with food material, finally comes to lie externally to the vege-
tative portion. The gastrulation, therefore, might be said to
take place previous to fertilization, or even to yolk forma-
tion, and the cleavage by delamination, described by Kingsley
and also suggested by Brooks (85) and Bruce ('85), might
be regarded as only a continuation of these early conditions.
The appearance after the discharge of the egg of a large accu-
mulation of yolk spheres obscures these relations. Yet it is
difficult to escape the conviction that a relation of some sort
exists between these later developmental processes and the
conditions that are found to exist even at the beginning
of the period of growth of the egg (Pl. XIV, Figs. 42-47).
According to both Kingsley and Brooks, the development of
the fertilized egg of Limulus is peculiar in that the first
evidence of cleavage appears only on the surface. From
what I am able to gather from the accounts of these writers,
this division into cells is a secondary matter, the whole egg
ultimately being converted into an embryo.
The polarity of the ovarian egg of Limulus is not a matter
of chance. It is not acquired during the growth of the egg,
but it dates from the beginning. (See Pll. XIV-XVI.) The
germinal vesicle alone does not constitute this polarity ; for, as
has been shown, the centrosome with its cytoplasm exists from
the very beginning.
In the history of the germinal vesicle, more particularly the
chromatin, I find nothing on which to base the assumption
that it is the ovigenic element, and that it is this which pre-
sides over all the formative processes. I find no evidence that
No. 2.] THE OVARIAN EGG OF LIMULUS. 171
the chromatin is the basis of the structure that underlies these
polar differentiations. As a matter of fact, the chromatin of the
germinal vesicle seems to vanish when the metabolic processes,
concerned with the elaboration of food, are at an end.
The peculiar arrangement of the chromatin in the spireme
stage, and the various phases of karyokinesis (Pl. XIV, Figs.
33-37) seen in the dividing odgonia do not appear to me to
be evidence of an organization existing in the chromatin, but
rather an orderly arrangement of an inert mass, brought about
by a structural basis which is common to the cytoplasm and
nucleus alike.
The division of the odgonia is manifestly a division of the
structural basis of both the nucleus and the cytoplasm, and
the orderly separation of the chromosomes is due to an orderly
separation of the spindle fibers. These spindle fibers I can
regard as nothing else than the reticulum of the cytoplasm
and the reticular basis of the nuclear network combined.
The vital manifestations reveal themselves, not in the passive
chromosomes, but in the centrosome, and in the network of
which it is a part. The entire history of the chromatin offers
nothing on which to base the assumption that it is the con-
trolling element.
The uniaxial feature, which is so prominent in the spindle
stage of the dividing odgonia (Pl. XIV, Figs. 36-38), continues
to exist throughout the history of the ovarian egg, and can
be accounted for only by the assumption of a continuity of
structure. It is inherent in the living matter of the egg.
Dr. Eycleshymer (95) has reviewed the literature on this sub-
ject, and has tested the relation of the polarity in the am-
phibian egg to cleavage and to the orientation of the embryo.
Peripheral bodies and yolk-nuclet.— On the same slide can
be seen the wide contrast between the eggs that are still in
the first period and those that have entered upon the second
period. In haematoxylin the former are dark blue, while the
latter are a very light blue. If the slide be dipped into picric
acid, previous to mounting in balsam, the former are not
affected, while the latter have yielded their former stain for
the new. The same result is obtained by means of borax-car-
172 MUNSON. [Vor. XV.
mine followed with picric acid. With the double stain of
erythrosin and cyanin the same difference can be observed,
the eggs in the first stage taking the blue, those in the second
taking the red stain. Lithium-carmine and Lyon’s blue show
the same peculiarity; the eggs in the first stage, in this case,
taking the red carmine stain, those in the second stage taking
the blue.
While this change in the cytoplasm is in progress, there is
a period when portions have undergone the change, while other
portions remain in the former condition. In such cases it fre-
quently happens that in haematoxylin stains, while most of the
cytoplasm takes a light-blue stain, round, dark-blue bodies
resembling nuclei are found scattered through the cytoplasm
(Pl. XIV, Fig. 27, y.z.). These may easily be taken for nuclei.
They, however, disappear as soon as the critical line dividing
the first and second period is reached.
I call these yolk-nuclei.
Another class of bodies, which I shall call peripheral bodies,
are scarcely less puzzling when first observed. In many cases
during the first period in the history of the egg, deeply stain-
able bodies resembling nuclei are found along the extreme
border of the egg (Pl. XIV, Figs. 25, 28; Pl. XV, Figs. 66,
67, 77). They are often regularly arranged at equal distances
from each other, and always immediately under the surround-
ing tunica propria or follicular membrane. They stain deeply
in Ehrlich’s, Delafield’s, and Heidenhain’s haematoxylin, as
well as in carmine and safranin. They do not, except in some
rare cases, have the clear outlines of nuclei, but seem rather
diffuse (Pl. XIV, Fig. 28). A comparison of a tangential and
transverse section shows them to be round discs with one
flat side and one convex side, turned inward (Pl. XV, Figs. 79,
go). They are studded with regularly arranged shining dots
(Pl. XIV, Fig. 28). At the end of this period the first layer
of the chorion is formed. The nature of these bodies becomes
evident when the formation of the chorion is observed (PI.
XIV, Fig. 25).
The egg membrane. — During the first stage of the egg, its
only covering is that formed by the tunica propria, which, as
No. 2.] THE OVARIAN EGG OF LIMULUS. 173
has been shown, is probably a product of the epithelial cells,
being a secretion of the protoplasm of the basal end of these
cells (Pl. XIV, Fig. 10, 2.p.).
The larger ovarian eggs offer excellent opportunities for the
study of this tunica propria. When removed from the egg,
and viewed in optical section under the microscope, it is seen
to be a homogeneous membrane, without cell boundaries and
without nuclei. It is, however, studded with closely-set shin-
ing dots, as if perforated with closely-set pin holes (Pl. XIII,
Fig. 18, f.c.). The nature of these becomes sufficiently evi-
dent when the formation of the egg membrane is examined.
There being no true follicle epithelium surrounding the egg,
the coverings, which in later stages make their appearance,
arise from the egg itself. On account of the considerable
development of this covering, I shall follow Packard and Kings-
ley and call it the chorion, being aware that, according to the
nomenclature adopted by Ludwig and van Beneden, we should
be obliged to call it the vitelline membrane.
The chorion is a product of the egg. In the living egg it
has a semi-solid consistency and offers considerable resistance
to pressure. It may be ruptured by inserting a needle and
severing it in that way. In so doing, it may be drawn out toa
sharp point somewhat like india-rubber; but, unlike rubber, it
does not return to its former position. Fresh eggs examined
in glycerine or normal salt solution often show the formation
of extraovates, without the rupture of the membrane.
Examined in the living state, the chorion is seen to consist
of one or several concentric layers according to the size of the
egg (Pl. XIII, Figs. 12, 13, 17, 18). Each layer is uniform in
thickness; but considerable variation may exist between the
different layers. The layers appear to consist of a dense sub-
stance, between every two layers of which there is a thin-
ner lamella of lighter, apparently less dense substance. These
layers of darker and lighter substance are not clearly separated,
but grade into each other. Preserved in most hardening rea-
gents, the chorion becomes hard and brittle, offering in the
mature egg considerable resistance to the entrance of paraffine
or celloidin. In such preparations the lamellae may also be seen ;
174 MUNSON. [Vo. XV.
but they are now separated by clear-cut lines, the less dense,
intermediate, lighter layer having apparently been converted
into very narrow crevices. In all cases the outer layer, which
is the first to originate, differs from the other layers (Pl. XIII,
Fig. 18).
Both in the living egg and in the preserved material studied
in sections the chorion is seen to be traversed by radial stria-
tions (Pl. XIII, Figs. 12, 17). These are closely set and per-
fectly parallel. In the different concentric layers the radial
striations coincide as if continuous one with the other. The
striations extend even into the outer or first layer of the
chorion (Pl. XIII, Fig. 18).
The first layer of the chorion arises at the end of the first
period, and seems to mark an important epoch in the history
of the egg, inasmuch as it is at this time that the cytoplasm
loses its affinity for haematoxylin, borax-carmine, and other
chromatin stains.
It arises in the form of peripheral bodies, which are scattered
at regular intervals over the surface of the egg, immediately
under the investing tunic (Pl. XV, Figs. 66, 67, 77, 79, 90).
These bodies appear at first as minute dots which increase in
size, and stain deeply in chromatin stains. They are often so
regularly arranged as to be easily mistaken for nuclei. As
they grow, two or three may coalesce, forming a conspicuous
body at the periphery of the egg (Pl. XV, Fig. 67). As they
increase in size by coalescence, they gradually lose their affinity
for chromatin stains, and eventually all blend into the first
layer of the chorion which, when formed, does not stain readily
(Pl. XIV, Fig. 25, cz.).
This first layer of the chorion sometimes appears to arise
as a continuous layer instead of in patches, as described above
(Pl. XIV, Fig. 20). The first indications of its appearance in
such cases are not the peripheral bodies, but a layer of deeply
staining dots just under the primary tunic. The granules are
not concentrated into larger isolated bodies, but are spread out
uniformly.
The history of the origin of the subsequent layers of the
chorion is a different one, although the process in itself may
No. 2.] THE OVARIAN EGG OF LIMULUS. 175
be essentially the same. Immediately beneath the first layer
of the chorion a homogeneous layer of protoplasm appears (PI.
XIII, Fig. 13), which does not stain so deeply as the more gran-
ular protoplasm which it surrounds. In favorable preparations
this layer appears to be composed of fibers which resemble
those of the cytoplasm except in the absence of the conspicu-
ous cyto-microsomes. The fibers are at first arranged more or
less perpendicularly to the surface of the egg (PI. XIII, Figs. 13,
18). At this stage they present the appearance of regularly
arranged cilia, covering the surface of the egg, and they are
imbedded in a transparent substance which solidifies into the
inner layer of the chorion.
The radial striations so conspicuous in the chorion of Limulus
eggs are therefore due to protoplasmic fibers. Originally, at
jeast, the radial striations are not due to radial pores, as is so
frequently asserted of other eggs. The process appears to be
practically similar to the formation of chiton and other cutic-
ular substances by the fusion of cilia. The shining pores
previously mentioned in connection with the primary egg cov-
ering and the peripheral bodies (Pl. XIII, Fig. 18; Pl. XIV,
Fig. 28) are either transverse sections of these fibers or their
points of insertion. As we have seen, the outer primordial
covering, — tunica propria, — which is the original basement
membrane of the germinal epithelium, arises in essentially the
same way as a cuticular hardening of the outer ends of the
epithelial cells.
The peripheral bodies, which in their earlier stages resemble
nuclei, call to mind the so-called “ Binnen”’ epithelium of
Eimer (72), which figured so prominently in the discussions
concerning the cell nature of meroblastic eggs, and may pos-
sibly explain the much disputed observations of Clark ('57) in
the ovarian egg of the turtle. They may possibly be compared
to the bodies observed by O. Schultze ('87) and Goette ('75) in
the peripheral layer of amphibian eggs, and I believe they
serve to explain the observation of Schiitz ('82) in the egg of
spiders. So far as I am aware, he is the only student of those
eggs who has claimed the existence of a follicular epithelium
surrounding them. Bruce (’85) and Brooks (86) have shown
176 MUNSON. [VoL. XV.
that the “inner egg membrane” mentioned by Packard (’71) is
the protoderm, the “rudely hexagonal cells” of Packard being
the casts of the ends of the blastoderm cells.
ZONES AND YOLK-NUCLEUS.
The cytoplasm is often divided into two distinct zones — an
outer and an inner zone (Pl. XIV, Figs. 19, 21, 24, 29). These
two zones are often separated by a distinct line suggesting the
presence of a membrane between them (Pl. XIII, Fig. 16). In
other cases the zones are separated by a line of large micro-
somes (Pl. XIV, Fig. 21). This line may run at a uniform
distance from the germinal vesicle (Pl. XIV, Fig. 24; Pl. XIII,
Fig. 16), or it may be extended at the proximal pole towards
the stalk (Pl. XIII, Fig. 16). Instead of this line of large
microsomes, the zones may be separated by parallel fibers
resembling those of the polar mitosome. In this line the
vitelline-body or sphere may sometimes be observed (Pl. XIV,
Fig. 21).
The inner zone at times appears less granular than the outer
zone, 7.¢., the granules appear smaller, making the inner zone
less stainable than the outer zone (Pl. XIV, Fig. 24). In other
cases this inner zone consists of large, irregular, closely packed
granules that stain intensely in haematoxylin and other chro-
matin stains, the outer zone staining less deeply (Pl. XIV, Figs.
19, 29). This form is of frequent occurrence, and is found in
the best preserved material, on the same slide with other eggs
showing no trace of it.
In the living egg of a half-grown specimen these two zones
can be distinctly recognized (Pl. XIII, Fig. 16). In this case
the outer zone is translucent, with the exception of a few scat-
tered granules. The inner zone, very sharply separated from
the outer zone, is opaque. The germinal vesicle in this case
contains a distinct nucleolus. The inner dark zone may form
a regular circle around the germinal vesicle (Pl. XIII, Fig. 16, 4),
or it may be extended on the proximal side towards the stalk
of the egg, where it seems to become continuous with the
oD)
protoplasm of the epithelium of the stalk (Pl. XIII, Fig. 16, ¢).
No. 2.] THE OVARIAN EGG OF LIMULUS. Wala)
As these eggs were taken from the ovary of the living
animal, and examined at once in the fluids of the ovary, the
effects of reagents cannot be considered responsible for these
appearances.
An examination of the same eggs in a living condition shows
also the vitelline-body, or sphere, and its intimate connection
with this peculiar inner zone of the cytoplasm (Pl. XIII, Fig.
16, a). Pl. XIV, Fig. 21, shows a similar egg in section. The
sphere is seen to bear a close relation to the dividing line
between the two zones, on the one hand, and to the polar
mitosome, on the other.
Eggs showing these zones were preserved and mounted
entire. The main features are excellently preserved.
General considerations. — The division of the body of the
egg into an outer and an inner zone has frequently been
observed in other eggs. Among others by Pfliiger (63), cat ;
Cohn (56), rotatorian ; O. Schultze (87), Bambeke (75, 'aa), frog ;
Waldeyer (70), bird; His (73), fish; Will (86), Korschelt ('89),
insects ; Holl (90), Leuckart ('53), chick ; Ludwig (74), echino-
derms; Lancaster ('75), molluscs; K. Schulin ('g1), bat and
human; Henneguy ('93), fish; Goette (75), bombinator; J. V.
Carus ('50), spiders; Leuckart (53), Scharff (ss), Eimer (72),
Ransome ('67), fish.
The zones in Limulus eggs present all the essential charac-
teristics of the figures given by the above observers, and may
also be seen to undergo many of the modifications that have
been observed in other eggs ; for example, by Pfliger.
A so-called free space around the nucleus of ordinary cells
has also been described; among others, by Leydig (gs) and
Brass (83). A zone around the nucleus of sperm cells has
recently been observed by Auerbach (96).
In the case of eggs the zones are usually considered as being
connected with the phenomena of growth and nutrition; but
the manner in which they arise is a disputed question.
Leydig and Auerbach take different views. Leydig inter-
prets the inner zone as a free cavity in the cell, into which the
nucleus has crowded by a process of budding from the cyto-
plasm, to which it remains intimately connected by means of a
I 78 MUNSON. [Vo.L. XV.
narrow bridge or stalk. Auerbach, on the other hand, regards
it as the expression of a condensation of the cytoplasm, which
takes place previous to the division of the cell, and which ulti-
mately results in the formation of a spherical body, the sphere,
or “ Nebenkern.”
Such an explanation of the zone around the germinal vesicle
in the egg of Limulus cannot be offered, inasmuch as it appears
during the period of growth of the egg, when it has ceased to
divide, and often after it has attained to a considerable size.
This appears to be true in the fish egg also, according to the
observations of Scharff. He considers the outer zone as cor-
responding to the “ Rindenschicht ” of Eimer, which the latter
believes to be identical with the “ Zonoid schicht”’ of His.
One objection to applying Auerbach’s interpretation to the
inner zone of the egg is the sharp line which in certain stages
separates the two zones (Pl. XIII, Fig. 16). This feature is
peculiarly striking in the living egg of Limulus, and is espe-
cially emphasized by Pfliiger as observed in the ege of the cat,
and appears to have attracted the attention of Schulin in the
egg of the bat and in the human ovum, and also of Will in
the egg of Colymbetes fuscus. It is, of course, difficult to say
what effect a condensation might have.
The changes of the inner zone from a hyaline to a granular
condition, observed by Pfliiger in the egg of the cat, and which
is so evident in the egg of Limulus (Pl. XIV, Figs. 19, 24),
appear to be evidence of chemical changes taking place in the
interfilar amorphous substances immediately surrounding the
germinal vesicle. This may, of course, be accompanied with
an increased condensation of the reticulum; but it would seem
that the latter would be more apparent than real, and due rather
to the increase in the amorphous granules. The sharp limita-
tion of the inner zone in certain phases of the contained gran-
ules, it seems to me, points to differences in composition of the
amorphous or granular matrix in which the cytoreticulum lies
(Pl. XIV, Fig. 48).
In endeavoring to account for the existence of this inner
zone, there are four important elements which demand atten-
tion,
No. 2.] THE OVARIAN EGG OF LIMULUS. 179
If we reject the explanation of Leydig, and also that of
Auerbach, how shall we account for the first feature of this
zone, 2.¢., the hyaline stage? Certain features that I have
noticed in connection with the germinal vesicle and nucleolus
appear to throw light on this question. It was seen that the
nucleolus extrudes bodies in the form of vesicles, consisting
of a membrane within which is a fluid containing granules
(Pl. XVI, Figs. 110-113). These vesicles, which I have called
«“ Nebennucleoli,” were seen to vary in number, and apparently
to appear and to disappear. There is reason for believing that
these vesicles lying in the meshes of the nuclear reticulum
finally dissolve, and add their contents to the nuclear sap, or
karyo-lymph. Where they happen to lie near the periphery of
the nucleus, the discharge of their contents extends the area
of the germinal vesicle in that direction, thus causing pouches
of the nuclear wall (Pl. XVI, Fig. 105; Pl. XIII, Figs. 3, 6-8).
The nucleus, to all appearances, is not bounded by a solid wall,
but by a special arrangement of the cytoreticulum.
It has been seen that the germinal vesicle, having become
greatly extended, more or less regular in outline, may in another
phase of activity become amoeboid, greatly contracted, and
apparently devoid of karyo-lymph (Pl. XIII, Fig. 5). In all
such cases a hyaline area is found to exist outside the germinal
vesicle, apparently caused by the entrance into the cytoplasm
of the karyo-lymph, which now becomes either a hyaline zone
around the germinal vesicle, or in later stages appears as
the polar area referred to in another place (Pl. XIV, Fig. 24;
Pl. XVI, Fig. 104).
It would appear that this hyaline zone may become lost in
the cytoplasm, in which it becomes diffused throughout the
interfilar spaces. We may now consider the question of the
origin of the internal granular zone of the egg.
The definite yolk spheres appear only after the egg is dis-
charged from the follicle into the ovarian tube. It is hardly
necessary, therefore, to consider the explanation of an internal-
zone in the egg of the fish offered by His, as his well-known
theory of migrating granular cells would not apply in this case,
and, so far as I know, has never been seriously considered in
180 MUNSON. [VoL. XV.
recent years. The theory of Waldeyer also offered in explana-
tion of two zones in the cytoplasm of the bird’s egg, and
extended also to the observation of Pfliiger in the egg of the
cat, namely, the direct apposition to the primitive egg cell of
the outer zone, conceived to be derived from the follicle epi-
thelium, does not apply in the present case, inasmuch as no
true follicle epithelium can be said to be present. Equally
inapplicable is the somewhat vague but decidedly radical
theory advanced by Leuckart (53) in regard to all eggs, and
the somewhat similar though more metaphysical theory
advanced by Balbiani (’83), according to which, in myriapods,
the egg consists of a “partie germinative fondamental”’
and of a “partie nutritive,” each of these parts being consti-
tuted “isolement et pour son propre compte.” The cbjection
to such an explanation would be that in the egg of Limulus the
division of the cytoplasm into an outer and inner more or less
granular zone is not a constant, but a periodical feature.
It remains to be considered how far the direct elimination of
chromatin from the germinal vesicle into the surrounding cyto-
plasm can explain the existence of the internal granular zone.
Such an explanation has been offered by Will in eggs of
amphibians and insects, by Bambeke (93) and by Calkins ('95)
in the egg of Lumbricus. Calkins’s observations receive in-
creased interest and importance from the evident acceptance
of his results by Professor Wilson ('96) in his new work on
the cell, and the evident stress which the latter author places
upon it in his theory of synthetic metabolism of the chromatin
of the germinal vesicle. In addition to the doubt cast upon Cal-
kins’s results by the observations of Miss Foot (96) on the
eggs of a closely allied species, Allolobophora, from which she
seems inclined to believe that Calkins’s results were obtained
from pathological material, the following considerations may
be urged against the sufficiency of the theory of elimination
of chromatin to explain the internal deeply staining zone:
1. It does not explain the existence of the hyaline zone, which
appears to precede or to follow the granular condition. The
existence of the hyaline zone proves that the zone is not due
to the extruded chromatin granules. 2. The elimination of
No. 2.] THE OVARIAN EGG OF LIMULUS. 181
chromatin granules from the germinal vesicle cannot explain
the extension of the inner granular zone towards the point of
attachment of the egg, as it can be seen both in sections and
in the living egg of Limulus (Pl. XIII, Fig. 16). 3. Contrary
to the results of Calkins, the internal granular zone does not
behave like ordinary nuclear chromatin towards the Biondi-
Ehrlich triple stain. In the degenerating eggs described in
another chapter, the chromatin that is found in considerable
quantities in the cytoplasm, and having the form of nuclei,
does take the green stain with the Biondi-Ehrlich mixture
(EE SXEV,) Pigss 30; 31):
I can find no reason, therefore, for concluding that the deeply
staining granules of the inner zone are eliminated chromatin
granules.
On the other hand, it has been shown that the epithelial
cells of the ovarian tube and of the egg stalk secrete a sub-
stance which, under the influence of reagents, becomes granu-
lar, and that this substance is seen at times to accumulate at
the point of attachment of the egg (Pl. XIV, Fig. 22, y.s.).
From a consideration of the foregoing objections and the facts
presented by such appearances as are represented in Pl. XIII,
Fig. 16, and Pl. XIV, Figs. 19, 20, 22, as well as from the fig-
ures of Korschelt (89) in the case of insect eggs, I conclude
that this secretion enters the egg and is carried along toward
the germinal vesicle, where, acted upon by the hyaline karyo-
lymph derived from the ‘ Nebennucleoli,” it becomes con-
verted into a stainable substance which I will call metaplasm.
While I have called the extension of the inner zone towards
the stalk of the egg a channel, I do not mean to imply
by that term that it is a tube in the sense in which Balbiani
(83) used the term in the case of the egg of Geophilus. It
appears to be rather the expression of the existence of an
interfilar fluid or substance, which, for the time being, does
-not mix with the surrounding hyaline cytoplasmic matrix, and
which is especially favorable for the entrance and chemical
modification of the crude food material which serves the egg
as nourishment. I would not therefore regard the inner
hyaline zone as an open space in the sense of Leydig, nor as
182 MUNSON. [Vou. XV.
a cytoplasmic condensation in the sense of Auerbach, nor as
a funnel-shaped tube in the sense of Balbiani, but rather as
an interfilar digestive fluid in the sense of Scharff, without the
bodily migration of nucleoli as held by him; the granular
phases being due to the entrance of food material, as held by
Korschelt in the insect egg, and being the result of a combina-
tion of a nucleolar product with the nutritive material.
The result of this combination is the metaplasm which later
becomes distributed throughout the body of the egg or else
collected around the centrosome and sphere.
It is evident, however, that the internal zone is closely
related to the centrosome and sphere; and our next problem
will be to consider what this relation may be. A relation of
some kind has been pointed out by Balbiani, Bambeke, Oscar
Schultze, Henneguy (93), and Auerbach, and is evident from
their figures as well as from Pl. XIII, Fig. 16; Pl. XIV, Figs.
19-22, 24, 48, in the egg of Limulus.
In the case of the sperm cell, Auerbach considers the spher-
ical «« Nebenkern”’ as a further condensation of the inner zone.
He, however, can give no reason why such a condensation
takes place at one place rather than at another; and if I
understand him rightly, he does not insist on a structural rela-
tion of the cytoreticulum that might serve as a basis for such
a condensation.
In the case of the other observers mentioned above, it
would seem that they, in a somewhat similar manner, regard
the body — vitelline-body of Balbiani—as a fortuitous aggre-
gation of the granules of the internal zone, the granules being
regarded either as extruded chromatin or as disintegrated
migrating nucleoli.
Against such a fortuitous aggregation can be urged the
observation of Balbiani himself, in the case of Geophilus, where
a structural body in the form of a centrosome, sphere and
aster, is clearly figured and described, in the midst of
amorphous granules supposed to be derived from the germinal
vesicle. Balbiani does not prove that this sphere and aster
originate from the amorphous granules. Furthermore, Mer-
tens (93) has conclusively shown that a centrosome and sphere
No. 2.] THE OVARIAN EGG OF LIMULUS. 183
exist, apparently independently of the granules derived from
migrating nucleoli.
In the egg of Limulus I have conclusive evidence of the exist-
ence, both in earlier and in later stages, of a centrosome and
sphere in the midst of the granules of the inner zones (Pl. XIV,
Figs. 21, 47, 48); and as will appear from the following con-
siderations of that body, I believe that this is the structural
basis around which the metaplasmic granules of the inner zone
collect and give rise to the conspicuous body known as the
vitelline-body of Balbiani. In that way I would account for
the appearances in the living egg represented in Pl. XIII,
Fig. 16, a.
THE CENTROSOME AND SPHERE (VITELLINE-Bopy).
As soon as the growing egg can be distinguished as such,
there may be seen in the cytoplasm, in the immediate neigh-
borhood of the germinal vesicle, a body which differs from all
other parts of the egg in its staining reactions (Pl. XIV, Figs.
34, 38, 40). It is brought prominently into view by means of
Heidenhain’s iron-haematoxylin; by the Biondi-Ehrlich mix-
ture; Weigert’s picro-carmine; Delafield’s haematoxylin, either
alone or followed with picric acid; by means of borax-carmine
and picric acid; Ehrlich’s haematoxylin and acid fuchsin or
eosin; by erythrosin, either alone or followed with cyanin ;
and finally by means of Lyon’s blue and lithium-carmine or
safranin.
When first observed, it has the form of a crescent closely
applied to the germinal vesicle. In the latter double stain it
may be made very conspicuous, being differentiated from all
other parts of the egg, both germinal vesicle and cytoplasm.
These stain red, while the crescent stains a bright blue. In the
widest portion of the crescent is a clear area containing central
granules (Fig. 42).
This central area, with its granules, appears to be the essen-
tial part of the body, inasmuch as it is this which continues to
exist in various forms as the egg continues to grow. The horns
of the crescent seem to disappear early (Figs. 42-48).
184 MUNSON. [VoL. XV.
A high magnifying power shows the body to consist of the
following parts: first, a round central body, surrounded at a
slight distance by a circle of microsomes (Fig. 45). From this
circle radial fibers pass out in all directions to another circle
of rather large microsomes (Fig. 47). Outside of this again a
dense layer exists, which can be seen to consist either of gran-
ules or of closely interwoven fibrils, which radiate out into the
cytoplasm, and send larger or smaller strands out along the
outer wall of the germinal vesicle. In Weigert’s picro-car-
mine, in which the circles of microsomes, as well as the dense
fibrous layer, are very distinct, the central granule is not vis-
ible. This, however, can be distinctly seen in the carmine and
Lyon’s blue (Figs. 42-44). After the disappearance of the horns
of the crescent, the body appears essentially as before. In
Lyon’s blue it is a large blue mass of fibers, with a somewhat
darker center, situated close to the germinal vesicle, and nearly
equal to it in size (Pl. XIV, Fig. 40). It is flattened or slightly
concave next to the germinal vesicle ; and it is surrounded by a
clearer zone, which is traversed by radial fibers that pass out
into the cytoplasm, where they become lost in the red cytoplas-
mic network. Stained with haematoxylin, followed with picric
acid, the cytoplasm and the contents of the germinal vesicle
stain a deep blue; the body, on the other hand, appears yellow-
ish (Pl. XIV, Figs. 45, 46). It is seen to consist of granules
somewhat closely packed at the center, but less closely packed
at the periphery, where the granules are seen to be connected
by the same substance as that of the cytoreticulum, into which
it passes by imperceptible gradations (Fig. 46). Eggs of the
same size as this, however, may show the typical form described
above. The indentation next to the germinal vesicle may, how-
ever, be so marked as to cause the central granule to lie close
to the germinal vesicle (Fig. 45), but yet distinct from the lat-
ter, as is clearly seen by the differential stain of Lyon’s blue
and carmine. At a somewhat more advanced stage, however,
the body may be seen about midway between the germinal ves-
icle and the periphery of the egg (Fig. 49). When stained in
Lyon’s blue and safranin, all parts of the egg, except this body,
take the red safranin stain. The body, however, appears as a
No. 2.] THE OVARIAN EGG OF LIMULUS. 185
blue or green sphere of interwoven fibrils, in the center of
which may be found one or several red granules arranged more
or less in a circle (Fig. 49). By means of this double stain,
the body can be seen in all stages up to the time of the forma-
tion of the egg membrane (Pl. XV, Fig. 79), when the changes
in the cytoplasm render the carmine and safranin ineffectual.
But even in this case it can often appear very distinctly on
account of its greater affinity for the blue, and its consequent
deeper stain. In most cases of this kind it appears as a defi-
nite, spherical, compact body, consisting of closely interwoven
fibers that may at times show a concentric arrangement about
a somewhat modified central body (Pl. XV, Fig. 75). There
may be two of these central bodies (Pl. XIV, Fig. 58). They
appear somewhat like nuclei, each being surrounded by a com-
pact layer of the outer mass of fibers.
In comparatively small eggs stained with various stains, such
as the Biondi-Ehrlich mixture or haematoxylin, either alone
or followed with acid fuchsin, the fibers of the cytoreticulum
can be seen to converge to a point near the center of the egg
(Figs. 50, 51, 54, 55). At the point where the fibers meet, a
highly refractive body staining deep red in acid fuchsin can be
seen. At times when the cytoreticulum is particularly dis-
tinct, the body is comparatively small and regular (Figs. 50,
51, 54). When the cytoplasm is more granular, the body may
be comparatively large, apparently composed of refractive
granules closely packed, and either regular in outline or ser-
rated by projecting processes comparable to the points of a
star, the body being very conspicuous because of its greater
affinity for the stain than the rest of the cytoplasm, the reticu-
lum of which can be seen to have a somewhat indistinct radial
arrangement with reference to it (Fig. 56).
In favorable cases, when the cytoreticulum is especially dis-
tinct, the body towards which the fibers of the cytoreticulum
converge appears as a ring of dense fibers, the reticular nature
of which can, however, be distinctly seen (Pl. XIV, Fig. 32).
Within this ring is a delicate network of fibers with distinct
microsomes at the nodes, and from the ring surrounded by the
cytoreticulum, comparatively straight isolated fibers radiate into
I 5 OL. 2 y
86 MUNSON. VoL. XV
the cytoreticulum, where they can be traced for a considerable
distance.
In cases where a distinct refractive body exists at the point
of convergence of the cytoplasmic fibrils, this point often lies
near the germinal vesicle; and being about in the center of
the egg, the latter occupies a somewhat excentric position
(Pl. XIV, Fig. 51). The fibers converging at this point, or, as
we may say, radiating from this point, are independent of one
another over a considerable area surrounding the point of con-
vergence; but they ultimately become continuous with the
more reticulated portions of the cytoplasm. An area thus
exists near the germinal vesicle, where the fibers radiate ; and
being less dense, this area, at times at least, appears lighter
(Pl. XIV, Figs. 50, 51, 54, 55). The area is usually round,
about equal to the germinal vesicle in size, and often flattened
or concave at that point where it is in contact with the germ1-
nal vesicle (Fig. 50). At times the radial fibers may be very
distinct and numerous (Fig. 52). The radial fibers are some-
times definitely limited; but at other times they can be traced
to the periphery of the egg, at that point opposite to the germi-
nal vesicle (Fig. 57), the central body being at times incon-
spicuous (Fig. 52), or at other times apparently consisting of
a relatively large granular body into which the radial fibers
can be seen to extend at variable distances (Fig. 57). The sys-
tem of radial fibers is not always so close to the germinal vesicle
as described above (Pl. XVI, Figs. 87, 92, 97). The condition
of the radial fibers seems to vary. At times they appear as
rows of microsomes (Pl. XIV, Fig. 57); while at other times
they appear as comparatively homogeneous silken fibers, in
which varicosities do not come prominently into view (Pl. XV,
Fig. 87). Some of this difference in appearance can no doubt
be ascribed in many cases to difference in the staining, but it
appears clear that the difference is often due to the condition
of the fibers themselves.
In somewhat larger eggs the body appears more complicated.
Stained in Weigert’s picro-carmine, it consists of a round, dark,
homogeneous central body surrounded by a clear zone which
again is surrounded by a somewhat lighter zone of granules (PI.
No. 2.] THE OVARIAN EGG OF LIMULUS. 187
XV, Fig. 89). This zone of granules is often not so definitely
outlined as the central body, but its outer border is often ser-
rated. It is, however, sharply differentiated from the surround-
ing cytoplasm. From this dark-brown body radial fibers extend
into the yellow cytoplasm surrounding it. In Ehrlich’s haema-
toxylin, followed with eosin, the body may show a blue center
composed of granules. This is then surrounded by a red zone
of concentrically wound fibers more or less interwoven; and
from this again red fibers can be seen to radiate through-
out the cytoplasm; and in some sections of the latter, where
granules are less numerous, the radial fibers can be traced to
the very periphery of the egg (Pl. XIV, Fig. 60 ; Pl. XV, Fig.
67). As the egg grows the body increases in size, and the
form described above may thus become relatively large and
conspicuous. Stained in erythrosin and cyanin, such a large
body may be seen to consist of a large central, spherical mass
of granules with a blue tint. This is again surrounded by a
comparatively thick zone of bright-red fibers arranged concen-
trically around the central granules. And around this again
can be seen a radial arrangement of the cytomicrosomes (PI.
XVI, Fig. 101).
In Ehrlich’s haematoxylin and acid fuchsin the body may
appear as a conspicuous deep red, rather small, definite, refrac-
tive body in the center of a zone of blue granules (Pl. XV,
Figs. 68, 82). The cytoplasm having a less intense red colora-
tion, this form of the body is often a conspicuous and beauti-
ful preparation. From this as a center, also, radial fibers can
often be distinctly traced to the periphery of the egg. Many
of the red radial fibers can be seen to penetrate the blue zone of
granules, and to proceed directly from the red central body
(Pl. XV, Fig. 68). At other times the blue granules of the
zone are so abundant that the radial fibers cannot be observed
in it, and they consequently appear to end at the periphery of
the blue zone (Fig. 67). The round, red central body is occa-
sionally seen to be surrounded by a clear space which again is
bound by a definite red staining wall, around which the blue
zone of granules is arranged (Figs. 67, 82). The central body
may be very minute; and radial fibers, and, as it often appears,
188 MUNSON. [Vou. XV.
radially arranged granules, immediately surround the small cen-
tral body. The radial structures are then often limited by a
broader or thinner zone of concentrically arranged fibers — the
whole being conspicuously differentiated from all the rest of
the cytoplasm (Pl. XIV, Fig. 53; Pl. XVI, Fig. 100). The
outer concentrically arranged fibers may be absent, and the
center may bea mass of granules surrounded by short, stiff,
radial rods that penetrate unequally into the central granular
mass. In the Biondi-Ehrlich stain, the central granules are
yellow, while the radial rods are conspicuously red, the whole
being definitely limited and sharply contrasted from the rest
of the cytoplasm (Pl. XV, Fig. 80). In this case three bodies,
connected by dense cytoplasmic fibers, are seen in the neigh-
borhood of the spherical body ; and from this the cytoplasmic
fibers, arranged radially, can be seen to extend to the periphery
of the egg, where they become continuous with the peripheral
layer of fibrous protoplasm, thus rendering this portion of
the egg peculiarly different from other portions of the same sec-
tion. Instead of the radial rods and the three neighboring
bodies, a comparatively large vesicular body, consisting of
granules surrounded by a very distinct thin wall or membrane,
has been observed. In this case only one body existed in its
vicinity ; and, as in the case above, the cytoplasmic fibers had
a similar arrangement.
Occasionally a number of refractive bodies are to be seen
at the apex of a cone of fibrous protoplasm, whose base is con-
tinuous with the peripheral protoplasm (Pl. XV, Fig. go). It
may also take the form of an oval sphere of interwoven fibers,
enclosing in its meshes refractive bodies, and joined to the
peripheral zone of fibrous protoplasm by a narrow stalk of
similar fibrous protoplasm (Pl. XV, Fig. 84). Occasionally two
refractive bodies removed from each other, but connected by a
band of fibrous protoplasm, can be seen. From each of these
refractive bodies, bundles of radial fibers extend far out into
the cytoplasm, which is sharply contrasted from it. The body,
when stained in Ehrlich’s haematoxylin and eosin, is also seen
to consist of a central sphere of yellow granules, which is
surrounded by a bright-red, homogeneous, fibrous, protoplasmic
No. 2.] THE QVARIAN EGG OF LIMULUS. 189
zone of considerable thickness, and having the form of a horse-
shoe (Pl. XV, Fig. 76). At the upper part of this fibrous
protoplasm are imbedded several aggregations of blue granules ;
and, surrounding the whole, and slightly removed from it, in
the red cytoplasm, is seen an irregular circle of these blue
granules like a wreath, which, at the opening of the horseshoe-
shaped protoplasm, forms an irregular mass of the blue
granules. When stained in Ehrlich’s haematoxylin alone, the
body appears as an unstained spherical mass of fibrous proto-
plasm of considerable size, and definitely limited, around which
are arranged, in a radial manner, numerous deep-blue granules
apparently associated witha radial system of fibers proceeding
from the body as a center (Pl. XVI, Fig. 96). Within such a
body similar blue granules are found distributed, but most
frequently aggregated into groups occupying vacuoles which
with the blue granules appear like nuclei. Again it may appear
as a conspicuous spherical body of the same fibrous protoplasm,
which stains a deep red in erythrosin or acid fuchsin. It may
enclose one or two nuclei-like bodies surrounded by a denser
zone of the same fibrous protoplasm (Pl. XV, Figs. 64, 86).
If the acid fuchsin be preceded with Ehrlich’s haematoxylin,
the central body (or bodies) is seen to contain the same blue
granules resembling chromatin granules of nuclei (Figs. 86, 88).
There may be several of these central bodies containing blue
granules scattered irregularly between the fibers of a large felt-
work of delicate protoplasmic fibers, as seen in Pl. XVI, Fig. 95.
Occasionally the blue granules appear as highly refractive
bodies lying between the fibers, that may have a comparatively
regularly concentric arrangement (Pl. XIV, Fig. 61; Pl. XV,
Fig. 72). Occasionally none of the granules can be observed
(Fig. 69), but this may be due to the staining. Erythrosin, for
instance, makes the whole body very conspicuous, but does not
always differentiate the internal structures. The whole body
may be relatively large, oval in form, and homogeneous in
structure, having in this case, as in most cases, a system of
surrounding radiations that extend far into the cytoplasm (PI.
XVI, Fig. 99). It may at times have the form of a spindle,
with its longitudinal axis not exceeding the transverse axis, the
190 MUNSON. [ VOL. XV.
spindle appearance being due to the rather definite arrangement
of the fibers with reference to two poles (Pl. XV, Fig. 65).
At one of these poles appears a structure consisting of con-
centrically arranged microsomes, from which radiate in a
somewhat irregular manner protoplasmic fibers.
In many cases the conspicuous, definitely limited spherical
body, enclosing a central structure or nucleus, is seen to be
surrounded by a definitely limited zone of protoplasm and gran-
ules that differ somewhat from the rest of the cytoplasm (PI.
XV, Fig. 71). The investing, interwoven, or concentrically
arranged fibrous protoplasm, which so frequently surrounds the
central nucleus or body, and which makes the whole so con-
spicuous when properly stained, is often limited in amount, or
even apparently absent. The body may then be a spherical
granular mass enclosed by a thin definite membrane. There
may be two of these situated close together. They are
made conspicuous by means of acid fuchsin or eosin (Pl. XV,
Fig. 66).
As the egg grows the cytoplasm becomes more and more
granular, and the body is less easily traced. The peculiar
fibrous protoplasm, which often renders it so conspicuous,
becomes less and less apparent. It often appears as a spherical
mass of granules surrounded by a zone, in which they are few
or entirely absent, and around which is another zone of similar
granules. These granules being larger than those of the cyto-
plasm, and reacting differently towards stains, the body is still
conspicuous (Pl. XVI, Fig. 102). With continued growth of
the egg the body often attains to considerable dimensions. It
may be a definitely spherical body surrounded by a clear ring,
which, in connection with its deeper stain, sets it off conspicu-
ously from the rest of the cytoplasm (Pl. XVI, Fig. 109). Or
it may be irregular in outline, rather star shaped, granular, and
may contain within it a spherical central body which alone often
comes prominently into view (Pl. XVI, Fig. 114). This may
be situated in the center of the egg and near to the germinal
vesicle (Figs. 103, 104, 114). It varies, however, considerably
in size ; and it is at this stage difficult to differentiate it from
the rest of the cytoplasm. Repeated attempts with a variety
No. 2.] THE OVARIAN EGG OF LIMULUS. IQI
of stains often bring it prominently into view even after all
hopes of observing it have been abandoned.
Occasionally it may be more excentric. It is often very
large, and consists of densely packed granules, which grade
gradually into the surrounding protoplasm in which more con-
spicuous traces of the reticulated arrangement of the granules
can be seen (Fig. 114). Here a zone of similar granules
extends around the periphery of the egg, under the perivitelline
layer of protoplasm. It is made conspicuous by means of the
Biondi-Ehrlich stain, in which it takes a darker tinge than the
rest of the cytoplasm. By means of Lyon’s blue it also appears
as a much darker body. By means of the double stain of
erythrosin and cyanin it can be differentiated as a blue body,
the rest of the cytoplasm being red (Fig. 104). The staining,
however, must be applied with the greatest of care, and ina
manner that can be learned only by repeated experiment.
Having once acquired the necessary skill, however, it is a
comparatively easy matter. From this it is not to be inferred
that it is all a matter of staining, for the preliminary method
of preserving is of even greater importance. Even with the
best method of staining, it is to be observed only in the most
perfectly preserved material. This is true not only of the later
stages just described, but it is true also of the earlier stages.
In this description no attempt has been made to exhaust the
subject, but merely to give the more prominent features of the
body as it appears in the various phases of the growing egg.
All the figures in the plates, beginning with Fig. 48, Pl. XIV,
are drawn with a Leitz camera, obj. 5, oc. 1-Leitz. Figs.
42-49 are drawn with a Leitz ;!, oil immersion.
Fig. 32 is one of the smallest eggs observed in the adult
ovary, drawn with a camera, Leitz ;!5 oil immersion.
INTERPRETATION AND SUMMARY.
After the last division of the odgonia to form a follicle, the
centrosome, with its surrounding structures, persists in the
cytoplasm. It has been observed in this case, not only during
karyokinesis, but after the last reconstruction of the nucleus,
192 MUUNSON. [VoL. XV.
when the increased size of the cell and its nucleus shows it to
be a growing egg (Pl. XIV, Figs. 34, 38-40). It consists, in
this early stage, of two concentric circles of microsomes —a
large outer circle and a smaller inner circle. In the center of
the smaller inner circle is a granule, which at first hardly
exceeds in size one of the microsomes of the surrounding cir-
cle. The microsomes of each circle appear to be connected by
a less conspicuous fibrous substance; and from the micro-
somes of the inner circle to the microsomes of the outer cir-
cle radial fibers connecting these can be observed (Pl. XIV,
Figs. 45-47). The minuteness of the inner circle and the
body contained in it does not permit a determination, at this
stage, of the presence or absence of radial fibers surrounding
the central granule. The central granule, however, is present,
though its minuteness often renders its detection difficult.
I do not hesitate to say that this is the centrosome of the
dividing odgonia, and that the central granule, with its sur-
rounding structure, corresponds very closely to that described
by van Beneden in the dividing egg of Ascaris.
The centrosome and surrounding circles of microsomes,
with their radial fibers, appear to be imbedded in a specially
modified, more or less amorphous substance which, through
the various effects of reagents and stains, renders the former
obscure or even invisible. For the present I will adopt the
term used by Boveri and call this imbedding substance archo-
plasm. This archoplasm is more conspicuous in some stains
than in others; and for that reason the centrosome and sur-
rounding structure may alone be distinctly visible ; while with
other stains the archoplasm appears prominent, often showing
no internal structure. The former is true of such stains as
picro-carmine (Fig. 47); the latter is often true of such stains as
Lyon’s blue and erythrosin and cyanin (Figs. 39, 40). A care-
ful comparison of these different effects shows that at other
stages both the radial system and circles of microsomes, as
well as the archoplasm, are present.
Being a direct continuation of the centrosome of the divid-
ing odgonia, it cannot be said to originate in the cytoplasm of
the growing egg.
No. 2.] THE OVARIAN EGG OF LIMULUS. 193
But is it derived from the young germinal vesicle? It exists
before a nucleolus has made its appearance in the germinal
vesicle. In this early stage the contents of the germinal ves-
icle and the granular cytoplasm stain a deep red in carmine or
safranin. If one of these stains be properly associated with
Lyon’s blue, the centrosphere with its archoplasm stains a deep
blue, the rest of the egg, both nucleus and cytoplasm, being
bright red. The blue body is then observed as a conspicuous
crescent-shaped structure partly enclosing the germinal vesicle.
In the broadest portion of this crescent-shaped body the
structure of the sphere and an enclosed centrosome, described
above, can be seen (Figs. 42-47). The horns of the blue
crescent appear to be due to an extension of the archoplasm of
the sphere and an aggregation along the sides of the young
germinal vesicle of the radial fibers belonging to the sphere.
The blue crescent, although lying close to the germinal vesicle,
is sharply differentiated from all parts of it, and is also at first
sharply differentiated from the cytoplasm (Figs. 34, 38),
although it later grades gradually into it. The red stain of the
contents of the germinal vesicle and cytoplasm is due to the
presence of granules of the nuclear network and the granules
of the cytoplasm that are strongly affected by the carmine or
safranin. The blue stain of the crescent-shaped sphere and
archoplasm I would interpret as indicating an absence of the
chromophilous granules. The nucleolus, when present, is also
strongly affected by the carmine and safranin stains. When
these stains alone are employed, the body remains obscure
because of the absence of staining in that region; and one
might easily, in such a case, be led to say that the body is not
present. If, however, the carmine be followed with picric acid,
the body comes into view as a yellowish body instead of the
blue of the former double staining. There being present none
of those granules which carmine so strongly affects, and which
are the distinguishing features of chromatin of the nucleus,
there is no ground on which to base the statement that this
body originates from the germinal vesicle. Its form also will
hardly admit the statement that it isa bud of the germinal
vesicle. Furthermore, such a bud would necessarily contain
194 MUNSON. [VoL. XV.
chromatin granules or else some substance capable of fixing
the Lyon’s blue more strongly than the carmine. The latter
substance is not to be observed in the germinal vesicle, for
no part of it takes the blue stain when carmine or safranin is
associated with the Lyon’s blue.
From these considerations, and others that will appear in
another connection, it may be said that the vztelline-body does
not arise in the cytoplasm of the growing egg, netther does tt
arise as a bud of the germinal vesicle ; nor as extruded chroma-
tin, nor as migrating nucleoli, It contains no nuclear chromatin.
In this connection, a few further considerations concerning
Lyon’s blue as a stain may be added. This stain not only dif-
ferentiates the body under consideration in its earliest stages ;
but, in material preserved in suitable hardening reagents, it
differentiates it conspicuously as long as safranin or carmine
can be associated with it. This, however, ceases when the
first period of growth is passed, since, after that period, these
stains do not affect the granules of the cytoplasm. Yet, even
after this, the body is made conspicuous by means of Lyon’s
blue used alone, because of its greater affinity for the stain and
consequently deeper blue coloration. It may thus profitably be
employed even in larger eggs of the second and third stages.
Even in those cases where nothing corresponding to the
archoplasm appears, where the fibers of the cytoreticulum con-
verge to a point as previously described, thus forming either a
real, conspicuous aster, or a more irregular area with radial
fibers, the center of this system is made conspicuous by the
Lyon’s blue, all the other parts of the egg being stained red
by means of safranin. Where the body assumes the form of
a large compact sphere of interwoven or concentric fibers, also,
it is made prominent as a blue or green sphere standing out
conspicuously from all the rest of the egg.
Although this stain, therefore, has a decided affinity for this
body, I cannot regard it as a specific stain, for it shows also a
decided affinity for the egg membrane after it has acquired
several layers. Its general nature as a stain is evident further
in those eggs belonging to the third stage, where it has been
pointed out that numerous nuclei are found within the egg.
No. 2.] THE OVARIAN EGG OF LIMULUS. 195
In such cases the carmine or safranin stains the chromatin
granules of the nuclei ; and when this is followed with Lyon’s
blue, everything in the cytoplasm, except these red nuclei,
stains a deep blue, making indeed handsome preparations. I
should certainly hesitate, therefore, to regard everything as
archoplasm in the sense in which Boveri used that term, which
stains blue with this when combined with carmine and safranin.
An examination of the plates of Miss Foot (96), where the
effect of this stain is extensively represented, would tend to
increase rather than diminish such a reluctance. When asso-
ciated with carmine or safranin, these are the specific stains.
The value of the Lyon’s blue lies in this, that it brings promi-
nently into view those areas containing no chromophilous
granules ; and for this purpose it is very convenient. In the
first period of growth of the egg of Limulus it has been
pointed out that both germinal vesicle and cytoplasm contain
these chromophilous granules, this body alone being devoid of
such granules. When eggs of Limulus are properly preserved,
there are none of those irregular areas in the cytoplasm which
Miss Foot has found in the egg of Allolobophora by means of
this stain.
The vitelline-body having been shown to possess, in its earli-
est stages, all of the features of the centrosome and sphere,
and to be, in fact, the centrosome of the dividing odgonia, it
remains to show that the body found in the cytoplasm in later
stages is the same centrosome. For this purpose the plates
will afford better evidence than a labored description. The
figures being drawn with a camera with the same magnifying
power, show the body in the various stages of the growing egg.
It can be seen that even in advanced stages of the egg the
body often presents the fundamental features seen in the earli-
est stage, and often very nearly the features of a typical cen-
trosome and sphere (Figs. 42-48, 67, 68, 82, 89,114). These
are characterized by the presence of a strongly refractive spher-
ical body often surrounded by a clear zone, which again is
surrounded by a zone of metaplasm (archoplasm Boveri) and
provided with a system of radial fibers which can be seen
to traverse the metaplasmic zone and to extend far out into
I 96 MUNSON. [VoL. XV.
the cytoplasm. As these appearances have been described
elsewhere, they need not be repeated here. The central re-
fringent granule, staining deep red in acid fuchsin and sur-
rounded by these radial fibers and metaplasmic zones containing
the blue granules, is undoubtedly the centrosome; and it, with
the surrounding structures, constitutes a real sphere. Accord-
ing to van Beneden ('87), the sphere consists of a central body
(centrosome) surrounded by a clear zone (medullary zone),
which again is surrounded by a granular zone (cortical zone).
All of these conditions can be seen in the vitelline-body, in the
egg of Limulus. According to Boveri (89, '95), the centrosome
is surrounded by a zone of archoplasm, which in some way
grows out into the cytoplasm in the form of astral rays, which
gradually replace the cytoreticulum. The vitelline-body pre-
sents the features of the sphere as defined by Boveri, and also
the characteristics of a real aster (Figs. 43-46, 50-52, 54, 55,
57, 60).
But it is the unusual features which this body assumes that
offer the greatest difficulties. Some of these are its excentric
position, its large size, and the fantastic appearance which it
often presents. The more common of these is the great
increase in size of the central body (Pl. XV, Figs. 71, 77;
Pl. XVI, Fig. 101), or the apparent absence of a definite central
structure; the concentric arrangement of the fibers; their
great increase or diminution; the often granular aspect of the
body; the vesicular form which it sometimes assumes; and
finally the combination in various ways of these different fea-
tures. An attempt to account for these features will be made
in the suggestions that are to be offered in the following
chapters on some of the physiological problems of growth and
metabolism.
These features are not foreign to the centrosome and sphere
as these are now understood. I will only invite a comparison
of some of the forms represented in the plates with the sphere
in sperm cells of the salamander as figured by Rawitz (95) and
Meves (94, '95), and in nerve cells as figured by Lenhossek
(95). Such a comparison will only serve to strengthen the
conviction that the vitelline-body is indeed a sphere which not
No. 2.] THE OVARIAN EGG OF LIMULUS. 197
only possesses the typical form of a centrosphere, the many
forms of the real aster found in the dividing cells, in leucocytes,
and in the fertilized egg of Ascaris megalocephala, but also the
less typical forms observed in sperm cells as ‘‘ Nebenkern,”
and in the resting ganglion cells.
The vitelline-body in the ovarian egg of Limulus is genet-
tically the centrosome and sphere of the dividing odgonia, and con-
tinues to be the centrosome and attraction sphere of the growing
ovarian egg.
That this centrosome and sphere may assume the form of
the vitelline-body as originally described, seems evident from
a comparison of Figs. 60-64, 70-72, 95 with the figures of
Balbiani ('64), ('79), ('82), (83), (93) ; of v. Wittich (49); Carus
('50) ; Schiitz (82) ; and Henking (87) ; and becomes very evi-
dent when preparations of the ovary of Limulus and of the
spider are directly compared.
The most important recent papers on the vitelline-body,
Julin (93), Mertens (93), Balbiani ('93), and Henneguy ('93),
also suggest strongly a probable relation of this body in other
eggs to the centrosome and sphere.
Position of the sphere. —In the youngest eggs the sphere
is always situated close to the germinal vesicle, as described
above. It may remain in this position or it may become
removed from the germinal vesicle so as to occupy a posi-
tion midway between it and the periphery of the egg (Pl. XIV,
Figs. 49, 56). At times it may even occupy a more excentric
position (Fig. 70).
There appear to be three causes that can be assigned for
this difference in position. First, a difference in the tension
or contraction of the radial fibers; second, a difference in
the local accumulation of the amorphous substances in the
interfilar spaces; third, differences in actual growth of the
cytoplasmic body.
The position of the metaplasm varies with reference to the
central structure. It may spread out on either side of it,
causing a density of the radial fibers lying close to the germi-
nal vesicle, and thus causing the horns of the crescent (Pl. XIV,
Fig. 47). From this position it may collect around the central
198 MUNSON. [Vou. XV.
structure, greatly obscuring the latter, and causing the whole
body to appear very conspicuous as a homogeneous solid body
(Fig. 40). On the other hand, it may spread out in a circle
surrounding the germinal vesicle (Fig. 48), and even become
extended towards the point of attachment of the egg (PI. XIII,
Fig. 16). In that way it appears to form a channel by which
food material is conveyed into the egg. In the vicinity of the
germinal vesicle the food material is acted upon, or at least
comes in contact with a clear fluid, perhaps karyolymph or
nuclear sap, and becomes converted into conspicuous stainable
granules. The metaplasm then, instead of collecting around
the central structure, may move out into the cytoplasm, caus-
ing the fibers of the crescent to expand into the general cyto-
plasm ; and the food granules surrounding the germinal vesicle
may likewise be variously distributed, causing the inner zone
surrounding the germinal vesicle either to entirely disappear or
else to become hyaline and devoid of granules (Pl. XIV, Figs.
19, 24).
The variable disposition of these three elements — the cyto-
lymph, as it may be called, the food granules, and the meta-
plasm — appears to be responsible for many of the variations,
not only in the position, but also in the form of the vitelline-
body. The position of the metaplasm with reference to this
body appears to determine the direction of growth. If the
metaplasm surrounds the central structure uniformly, the
cytoplasm increases uniformly, and the body thus becomes
gradually removed from the germinal vesicle. On the other
hand, if the central structure lies close to the germinal vesicle,
and the metaplasm on the distal side, the cytoplasm appears to
increase in the direction of greatest amount of metaplasm ;
and if the metaplasm is wholly absent from the structure, as
often appears to be the case (Figs. 50, 51, 55), the structure
most frequently is found, even in later stages of the egg, to
occupy the position which it formerly had (Pl. XVI, Fig. 104).
In the absence of the metaplasm from the vicinity of the cen-
tral structure, this latter does not appear as a conspicuous
massive body, but as a fibrous framework of radial fibers with
its refractive body in the center (Pl. XIV, Fig. 54).
No. 2.] THE OVARIAN EGG OF LIMULUS. 199
It has been pointed out that eggs showing this feature of
the body most conspicuously are chiefly those arising in later
stages of growth of the parent organism, and therefore grow-
ing more slowly. This appears to be owing to the absence of
nutritive material, which reveals itself in the very pronounced
appearance of the reticulum (Pl. XIV, Fig. 32), which in eggs
of the same size, arising earlier in the history of the animal, is
often greatly obscured by the presence of an abundance of
amorphous granules.
Nature of the metaplasm.—The yolk-nucleus (Pl. XIV, Fig.
27), I believe, can be considered as an early stage of the yolk,
through which all yolk material passes on its way to become
definite yolk spheres. It is often associated with the vitelline-
body. Such a case seems to present itself here in the form of
the metaplasm surrounding the centrosome and sphere.
The metaplasm consists of at least two kinds of granules.
Some of these granules possess an affinity for haematoxylin,
which is evident from their retaining this stain even when fol-
lowed with such a powerful stain as acid fuchsin. In picro-
carmine these granules show a reddish coloration in marked
contrast with other portions of the granular metaplasm.
Careful examination of a large quantity of material showing
these granules in connection with the vitelline-body and centro-
some appears to show conclusively that they issue as little drops
from the living substance of the cytoplasmic fibers and remain
closely adherent to them. This appears to take place most
freely when the fibrils are relaxed and in a state of rest. It
is evident that where the fibers converge and are most densely
packed, the number of these granules would be greatest. There
is reason to suspect that these granules may be converted into
yolk bodies, or be reabsorbed by the fiber so that no trace
of them remains.
It has been shown that the granules in the cytoplasm of the
first stages of the egg show an affinity for chromatin stains, and
that later this affinity disappears as the definite yolk is formed.
It has also been shown that these stainable granules are more
abundant in those eggs which increase rapidly in size, and
which may be supposed to be abundantly supplied with nutri-
200 MUNSON. [VOL. XV.
ment. It has also been shown that in those young eggs which
grow more slowly, being formed in a later period of the devel-
opment of the parent organism, when growth is consider-
ably retarded, and when therefore it may be supposed to be
scantily supplied with nutriment, these granules are often com-
paratively scarce, often almost absent (Pl. XIV, Fig. 32).
This fact suggests that the granules may have been used as
food for the living substance, and that this is analogous to the
appearance and disappearance of those granules which later
appear in connection with the vitelline-body.
GROWTH OF THE CYTOPLASM.
The vitelline-body, as we have seen, is at first a central gran-
ule situated close to the germinal vesicle, surrounded by circles
of large microsomes, probably connected by linin strands, and
a system of fibers connecting the microsomes radially (Pl. XIV,
Figs. 45, 46). In many cases this spherical structure is seen
to become more granular, the granules being arranged concen-
trically, but yet closely packed. It may thus increase greatly
in size (Pl. XIV, Fig. 56), while remaining refractive and appar-
ently homogeneous, staining a bright red in acid fuchsin, and
becoming surrounded by a zone of granules which retain the
blue haematoxylin stain (Pl. XV, Fig. 78). In later stages
this body may become still more enlarged, and consist of a
large number of granules staining like the microsomes of the
cytoreticulum and be surrounded by concentric or interwoven
fibers, which again are surrounded by still another zone of
granular substance (Pl. XV, Fig. 77). At the periphery of
the central granular body the meshes between the granules
may gradually increase so as to acquire the essential structure
of the cytoplasm (Pl. XIV, Figs. 32, 46); or else a number of
blue granules, like the chromatin of nuclei, are found within a
felted mass of fibers which at the periphery passes gradually
into the cytoreticulum (Pl. XV, Figs. 70, 76; Pl. XIV,
Fig. 53).
In the absence of the blue granules the vitelline-body re-
mains a compact mass consisting of closely packed microsomes
No. 2.] THE OVARIAN EGG OF LIMULUS. 201
or fibrillae (Pl. XV, Fig. 69). Ox the appearance of these gran-
ules, vacuoles arise; and the microsomes expand into the cytore-
ticulum (Pl. XIV, Figs. 46, 53; Pl. XV, Figs. 72, 86). The
formation of vacuoles and the resulting expansion may continue
until the entire vitelline-body is reduced to a network (Fig. 32),
and may seem to have entirely disappeared. The disappear-
ance, however, is manifestly an illusion (Pl. XIV, Figs. 50, 51,
54, 55).
It would seem that the attraction sphere, centrosome, and
vitelline-body are the primitive basis or center of growth of
the cytoplasm.
The growth of the cytoplasm is greatest in the direction in
which the blue granules are most abundant.
When the blue granules are unequally distributed around
the central body, growth takes place unequally, leaving the
body close to the germinal vesicle, or widely removed from it.
When the blue granules are equally distributed around the
central body, growth takes place equally in all directions, and
the body becomes the center of the cytoplasm.
202 MUNSON. [VoL. XV.
'78
Eh
"74
96
84
'83
'93
64,
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'B2
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88
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‘BS
LITERATURE REFERRED TO.
AGassiz, A. Note on the Habits of the Young Limulus. Amer.
Journ. Sct. Vol. iii. 1878.
ANDREWS, E. A. Reproductive Organs of Diopatra. Journ. of
Morph. Vol.v. 1891.
AUERBACH, L. Organologische Studien zur Charakteristik und Lebens-
geschichte der Zellkerne. Breslau. 1874.
AUERBACH, L. Untersuchungen iiber die Spermatogenese von Palu-
dina vivipara. Jena. Zeit, Bd. xxx. 1896.
Ayers, Howarp. On the Development of Oecanthus Niveus, etc.
Mem. Bost. Soc. Nat. Hist. Vol. iii, No. 8. 1884.
BALBIANI, E.G. Sur l’origin du follicle et du noyau vitellin de l’ceuf
chez les Geophiles. Zool. Anz. Nos. 155 et 156. 1883.
BALBIANI, E. G. Centrosome et Dotterkern. Journ. del’ Anat. et de
la Physiol. No.2. 1893.
'65 BALBIANI, E.G. Sur les mouvements qui se manifestent dans la
tache germinative chez quelques animaux. Compt. Rend. 1864 et
1865.
BaALBIANI, E. G. Sur la constitution du germe dans I’ceuf animal
avant la fécondation. Compt. Rend. Tome lviii, pp. 584-588 ;
zbid., pp. 621-625.
BALBIANI, E. G. Sur la signification des cellules polaires des insectes.
Compt. Rend. Tome xcv. 1882.
BALFour, F. M. On the Structure and Development of the Vertebrate
Ovary. Q./. M.S. No. 18. 1878.
BoverI, TH. Ueber das Verhalten der Centrosomen bei der Befruch-
tung des Seeigel-Eies, nebst allgemeinen Bemerkungen iiber Cen-
trosomen und Verwandtes. Verh. der phys.-med. Ges. Wirzburg.
Bd. xxix, N. F.. Nr. 1. 1895.
BoverI, Tu. Zellen-Studien. Jena. Zett. f. Naturwiss. N.S. 1889.
BovertI, TH. Ueber partielle Befruchtung. Sztzwngsber. Ges. Morph.
Phys. Miinchen. Bad. iv. 1888.
BRANDT, A. Ueber das Ei und seine Bildungsstatte. Leipzig. 1878.
Brass, ARNOLD. Zool. Anz. 1883.
Brooks, W. K. and Bruce, A. T. Abstract of Researches on the
Embryology of Limulus polyphemus. Johns Hopkins Univ. Cire.
No. 5. 1885.
Bumpus, H. C. The Embryology of the American Lobster. /ourn.
of Morph. Vol.v. 1891.
CaLxkins, G. N. Observations on the Yolk-Nucleus in the Eggs of
Lumbricus. Zyransactions N.Y. Acad. Sct. 1895.
Carnoy, J. B. La Cytodierese chez les Arthropodes. La Cellule.
1885.
No.
50
'S7
56
194
‘48
‘72
195
io
"75
B82
‘91
96
93
‘61
58
"75
pl
'94
95
"95
91
'87
'B2
2.] THE OVARIAN EGG OF LIMULUS. 203
Carus, J. V. Ueber die Entwicklung des Spinneneies. Zezt. f. wiss.
Zool, Bd.ii. 1850.
CLark, J. Embryology of the Turtle, in L. AGassiz, Contrib. to the
Nat. Hist. of the United States. Vol. ii. Boston. 1857.
Coun, F. Ueber die Fortpflanzung der Raderthiere. Zezt. f. wiss.
Zool. Bd. vii. 1856.
CONKLIN, E.G. The Fertilization of the Ovum. Bol. Lect. Marine
Biological Laboratory, Woods Holl. Boston. 1894.
CRAMER, H. Bemerkungen tber das Zellenleben in der Entwicklung
des Froscheies. J7iiller’s Archiv. 1848.
EImMer, TH. Untersuchungen tuber die Eier der Reptilien. Arch. f.
mtkr. Anat. Bad. viii. 1872.
EyCLESHYMER, A. C. The Early Development of Amblystoma.
Journ. of Morph. Vol. x, No. 2. 1895.
FLEMMING, W. Neue Beitrage zur Kenntniss der Zelle. II. Theil.
Arch. f. mikr. Anat. Bd. xxxvii. 1891.
FLEMMING, W. Studien in der Entwicklungsgeschichte der Najaden.
Sttzungsber. k. k. Akad. Wiss., Abtheil. 3. Bd. \xxi. 1875.
FLEMMING, W. Zellsubstanz, Kern und Zelltheilung. Leipzig. 1882.
Foi, H. La Quadrille des Centres. Un épisode nouveau dans Vhis-
toire de la fécondation. Arch. des Sct. phys. et nat. 15 Avril,
1891.
Foot, K. Yolk-Nucleus and Polar Rings. Journ. of. Morph. Vol.
xii, No. 1. 1896.
Foster, M. A Text-Book of Physiology. Fifth American edition.
Philadelphia. 1893.
GEGENBAUR, C. Ueber den Bau und die Entwickelung der Wirbel-
thiereier. Rezchert u. Du Bois-Reymond Arch. 1861.
GEGENBAUR, C. Anatomische Untersuchungen eines Limulus. 4éA.
naturf. Ges. Halle. Bd.iv. 1858.
Go6TTE, A. Entwickelungsgeschichte der Unke. 1875.
GUIGNARD, L. Nouvelles études sur la fécondation. Ann. d. Sct-
ences nat. Bot. Tome xiv. 1891.
GUIGNARD, L. L’origine des sphéres directrices. Journ. de Bot. 1894.
HACKER, VALENTINE. Die Vorstadien der Eireifung. Arch. f. mtkr.
Anat. und Entw. Bd. xlv, Heft 2. June, 1895.
HACKER, V. Ueber die Selbstaéndigkeit der vaterlichen und mitter-
lichen Kernbestandtheile wahrend der Embryonalentwicklung von
Cyclops. Arch. f. mikr. Anat. Bad. xlvi. 1895.
HENKING, H. Untersuchungen iiber die ersten Entwicklungsvorgange
in den Eiern der Insekten. Zezt. f. wiss. Zool. 1891.
HENKING, H. Untersuchungen iiber die Entwicklung der Phalangiden.
Zeit. f. wiss. Zool, Bd.xlv. 1887.
HENKING, H. Beitrage zur Anatomie, Entwicklungsgeschichte und
Biologie von Trombidium. Zezt. 7. wiss. Zool. Bd. xxxvii. 1882.
204 MUNSON. [VoL. XV.
'93
'93
‘77
"92
‘73
'38
'93
"90
'93
'93
'92
‘78
89
‘89
'96
‘70
HenneGuy, L. F. Le corps vitellin de Balbiani dans l’ceuf des
vertébrés. Journ. de l’ Anat. et de la Physiol, No.1. 1893.
Herta, V. Etudes des variations de la mitose chez l’Ascaride mega-
locephale. Arch. de Biol. Tome xiii. 1893.
HERTWIG, O. Weitere Beitrage zur Kenntniss der Bildung, Befruch-
tung und Theilung des thierischen Eies. Morph. Jahrb. Bad. iii.
1877.
HERTWIG, O. Die Zelle und die Gewebe. Jena. 1892.
His, W. Untersuchungen iiber das Ei und die Eientwicklung bei
Knochenfischen. 1873. .
HoEVEN, J. VAN DER. Recherches sur ’histoire naturelle et l’Anato-
mie des Limules (Lyde). 1838.
Hout, M. Sitzungsber. k. k. Akad. Wiss. Wien. Bd. iii-vii. 1893.
Hori, M. Ueber die Reifung der Eizelle des Huhns. Sztzungsder.
hk. k. Akad. Wiss. Wien. Bd. xcix, Heft 4. 1890.
JorDAN, E. O. Embryology of the Newt. Journ. of Morph. May,
1893.
Jutty, J. Le corps vitellin de Balbiani et les éléments des Metazoaires
qui correspondent au Macronucleus des infusoires cilies. Bud/. Soc.
de la France et de Belgique. Tome xxiv.
KINGSLEY, J. S.. The Embryology of Limulus. Journ. of Morph.
Vol. vii. 1892.
KLEIN, E. Observations on the Structure of Cells and Nuclei. Q./.
M.S. Vol. xviii, N.S. 1878.
KORSCHELT, EuGEN. Ueber die Entstehung und Bedeutung der ver-
schiedenen Zellenelemente des Insekten-Ovariums.
KoRSCHELT, EuGEN. Beitrage zur Morphologie und Physiologie des
Zellkernes. Jena. 1880.
KOSTANECKI AND WIERZEJSKI. Ueber das Verhalten der sogenannten
achromatischen Substanzen im befruchteten Ei. Arch. (2 mikr.
Anat. Bad. xlii. 1896.
La VALETTE St. GEORGE. Ueber den Kernfleck und die Deutung
der Eitheile. Arch. f. mikr. Anat. Bad. ii. 1866.
Ler, ARTHUR BoLLes. La Regression du Fuseau Caryocinetique.
La Cellule. Tome xi, Fasc. 1. Jan. 4, 1895.
LEuCKART, Rup. Artikel ‘Zeugung.’ Wagner's H. W. B. Ba. iv.
LreypiG, F. Zum feineren Bau der Arthropoden. Arch. f. Anat. u.
Physiol. 1855.
LrypiG, F. Beitrage zur Kenntniss des thierischen Eies im unbefruch-
teten Zustande. Spengel’s Zool. Jahrbicher, Abtheil. f. Anat. u.
Ontogente der Thiere. Bd.iii. 1888.
LeypiG, F. Untersuchungen zur Anatomie und Histologie der Thiere.
Bonn. 1883.
Lockwoop, S. The Horsefoot Crab. Amer. Naturalist. Vol. iv-
1870,
"92
90
"95
93
95
fo
'72
‘73
'70
ifs
‘71
'86
86
» 22] THE OVARIAN EGG OF LIMULUS. 205
Lupwic, H. Ueber Eibildung im Thierreich. A7d. d@. zool. Inst.
Wirsburg. Bd.i. 1874.
MACFARLANE, J. M. A Comparison of the Minute Structure of Plant
Hybrids with that of their Parents, etc. Zrans. Roy. Soc. Edin-
burgh. Vol. xxxvii, Part 1, No. 14. 1892.
Mark, E. L. Studies on Lepidosteus. Bull. Mus. Comp. Zobl. Vol.
xix. 1890.
Meap, A. D. Some Observations on Maturation and Fecundation in
Chaetopterus pergamentaceus. Journ. of Morph. Vol. x. 1895.
MERTENS, H. Recherches sur la signification du corps vitellin de
Balbiani dans l’ovule des mammiferes et des oiseaux. Arch. de
Biol. Tome xiii. 1893.
MeEvVEs, Fr. Ueber die Zellen des Sesambeins in der Achillessehne
des Frosches (Rana) und uber ihre Centralkérper. Arch. fi mikr.
Anat. Bd. xlv. 1895.
MeEvEs, F. Ueber eine Metamorphose der Attraktionssphare in den
Spermatogonien von Salamandra maculosa. <Arch. f. mikr. Anat.
Bd. xliv. 1894.
OELLACHER. Die Veranderungen des unbefruchteten Keimes des
Hiihnereies. Zez¢. f. wiss. Zool. Bd. xxii. 1872.
OweEN, R. On the Anatomy of the American King Crab. Tvans.
Linn. Soc. Zool. Vol. xxviii. 1873.
PackarD, A. S. Morphology and Ancestry of the King Crab.
Amer. Naturalist. Vol. iv. 1870.
PacKARD, A. S. On the Embryology of Limulus polyphemus. Pvac.
Am. Assoc. Adv. Sci. Vol. xix. 1871. Q. J. M.S. Vol. xi.
1871.
Packarp, A. S. On the Embryology of Limulus polyphemus. 2. /.
M.S. Vol. xi, N.S. 1871.
PackarpD, A. S. The Embryology of Limulus polyphemus. 4 mer.
Naturalist. Vol. iv. 1871.
PACKARD, A. S. The Development of Limulus polyphemus. JZem.
Bost. Soc. Nat. Hist. Vol. ii. 1872.
PackArD, A. S. The Anatomy, Histology, and Embryology of
Limulus polyphemus. Axnnzv. Mem. Bost. Soc. Nat. Hist. 1880.
PacKarD, A. S. On the Embryology of Limulus polyphemus. 4 mer.
Naturalist. Vol. xix. 1885.
PATTEN, W. M. Artificial Modification of the Segmentation and
Blastoderm of Limulus polyphemus. Zool. Anz. No. 441. 1894.
PFLUGER. Ueber die Eierstécke der Saugethiere und des Menschen.
1863.
PLATNER, G. Ueber die Entstehung des Nebenkerns und seine Be-
ziehung zur Kerntheilung. Arch. f. mtkr. Anat. Bd. xxvi. 1886.
PLATNER, G. Die Karyokinese bei Lepidopteren. J#ternat. Monat-
schr. f. Anat. u. Histol. Bad. iii. 1886.
206 MUNSON. [Vor. XV.
'93
"95
'88
"75
‘87
'82
'93
92
'86
'83
"75
'93
'87
"95
7k)
‘88
‘70
PLATNER, G. Ueber die Befruchtung bei Arion Empiricorum. Arch.
f. mikr. Anat. Bd. xxvii. 1886.
Ras, C. Ueber Zelltheilung. Axat. Anz. No.1. 1889.
Ransom, W. H. On the Structure and Growth of the Ovarian Ovum
in Gasterosteus Leiurus. Q./. M@.S. 1867.
RAwWITZ, BERNHARD. Centrosoma und Attraktionsphare in der ruhi-
gen Zelle der Salamanderlarve. Arch. f. mikr. Anat. u. Entwick-
lungsgeschichte. Bd. xliv, Heft 4. March, 1895.
RHUMBLER, L. Ueber Entstehung und Bedeutung der in den Kernen
vieler Protozoen und in Keimblaschen von Metazoen vorkommenden
Binnenkorper (Nucleolus). Eine Theorie zur Erklarung der ver-
schiedenartigen Gestalt dieses Gebildes. Zezt. f. wiss. Zool. Bd.
Ivi. 1893.
RUckeErT, J. Ueber das Selbstandigbleiben der vaterlichen und
miitterlichen Kernsubstanz wahrend der ersten Entwicklung des
befruchteten Cyclops-Eies. Arch. fi mikr. Anat. 1895.
SCHARFF, ROBERT. Q./. M.S. Vol. xxviii. 1888.
SCHNEIDER, AIME. Nouvelles observations sur les Sporrilation du
Klossia Octopiana. Arch. de Zool. expér. et génér. Sér. 2. Tome
LtSzib
SCHULTZE, O. Untersuchungen tiber die Reifung und Befruchtung
des Amphibieneies. Zezt. f. wiss. Zool. Bd. xlv. 1887.
ScutTz, J. ‘Ueber den Dotterkern.” /nxaug. Dissert. Bonn. 1882.
STAUFFACHER, H. Eibildung und Furchung bei Cyclas Cornea.
Jena. Zeit. f. Naturw. Bd. xxviii. Bd. xxi, N. F. 1893.
STRAHL. Die Rickbildung reifer Eierstockeier am Ovarium von
Lacerta agilis. Verh. d. Anat. Ges. auf der sechsten Versammlung
in Wien. 1892.
STUHLMANN, FR. Die Reifung des Arthropodeneies. Ber. d. naturf.
Ges. zu Freiburg. Bd.i. 1886.
VAN BAMBEKE, C. Arch. de Biol. Tomeiv. 1883.
VAN BAMBEKE, C. Recherches sur l’Embryologie des Poissons osseux.
Mem. Cour. Acad. Belg. Tome xi. 1875.
Van BAMBEKE, C. Elimination des éléments nucleaires dans l’ceuf
ovarien de Scorpagna scrofa. Arch. de Biol. Tome xiii. 1893.
VAN BENEDEN, Ep. and Neyt, A. Nouvelles recherches sur la fécon-
dation et la division mitosique chez l’Ascaride megalocephala. Bzxd/.
Acad. roy. Belgique. Sér.3. Tome xiv. Brussels. 1887.
Von LENHOSSEK, M. Centrosome und Sphare in den Spinalganglien
des Frosches. Arch. f. mikr. Anat. Bd. xlvi. 1895.
WaGENER, G. R. Bemerkungen iiber den Eierstock und den gelben
Korper. Arch. f. Anat. u. Phys. 1879.
WALDEYER, W. Ueber Karyokinese, etc. Arch. f. mikr. Anat.
Bd. xxxii. 1888.
WALDEYER, W. Eierstock und Ei. Leipzig. 1870.
No.
'93
vid)
"95
‘78
85
"84
86
96
'96
195
49
87
"85,
‘hl
2.] THE OVARIAN EGG OF LIMULUS. 207
Warast, S. Homology of the Centrosome. /ourn.of Morph. Vol.
vill. 1893.
WEISMANN, AuGuUST. Zur Naturgeschichte der Daphnoiden. Zezt.
f. wiss. Zool. Bd. xxviii. 1877.
WHEELER, W. M. ‘The Behavior of the Centrosomes in the Fertilized
Egg of Myostoma glabrum. /ourn. of Morph. Vol. x. 1895.
Wuirman, C. O. The Embryology of Clepsine. Q. /. M.S. Vol.
xviii. 1878. :
WIELOWIEJSKI, VON. Zur Kenntniss der Eibildung der Feuerwanze.
Zool, Anz. No. 198. 1885.
WiLL, L. Ueber die Entstehung des Dotters und der Epithel-Zellen
bei den Amphibien und Insekten. Zool. Anz. Bad. vii. 1884.
WiLL, L. Oogenetische Studien. I. Die Entstehung des Eies von
Colymbetes fuscus. Zezt. f. wiss. Zool. Bd. xiii. 1884.
Wirson, E. B. The Cell in Development and Inheritance. The
Macmillan Company. New York. 1896.
Witson, E. B. Archoplasm, Centrosome, and Chromatin in the Sea
Urchin Egg. Journ. of Morph. Vol. xi. 1896.
Witson, E. B. Atlas of Fertilization and Karyokinesis. The Mac-
millan Company. New York. 1895.
WirticH, von. Die Entstehung des Arachnideneies im Eierstock.
Miiller’s Arch. 1849.
ZACHARIAS, E, Beitrage zur Erkenntniss des Zellkerns und der Sexual-
Zellen. Bot. Zeit. No. 21. 1887.
'87 ZACHARIAS. Ueber den Nucleolus. Bot. Zeit. 1885, 1887.
ZIMMERMANN, K. W. Ueber den Kerntheilungsmodus bei der Sper-
matogenese von Helix pomatia. Verh. d. anatom. Ges. 1891.
208
[Vou. XV.
yolk-nucleus.
carapace.
tunica propria.
peripheral bodies.
alimentary canal.
peritoneal coat.
follicular pouch.
metaplasm.
centrosome.
aster.
nucleus.
chorion.
nucleolus.
archoplasm.
sphere.
blue granules.
polar mitosome.
inner zone.
MUNSON.
REFERENCE LETTERS.
muscle. yn.
ovarian tube. ca.
anus. Lp.
ova. pb.
operculum. al.c.
oviduct. pile
genital opening. p-
follicle. mpl.
muscle coat. ¢.
egg stalk. ast.
germinal vesicle. n.
“ Hauptnucleolus.” ch.
“Nebennucleolus.” nel.
polar protoplasm. arch
yolk zone. sph.
vitelline-body. b.g.
yolk secretion. pm
secretion granules. 4.8.
No. 2.] THE OVARIAN EGG OF LIMULUS. 209
EXPLANATION OF PLATE XIII.
Fic. 1. Germinal vesicle with a crescent-shaped nucleolus, containing within
it a reticulum resembling that of the germinal vesicle.
Fic. 2. A female Limulus with dorsal carapace removed, showing the netted
ovary, the ovarian tubes on the right, ov.t., being filled with eggs. The left side
represents the condition in younger animals before eggs have been discharged into
the ovarian tubes; ov., young growing eggs; aw., anus; of., operculum; g.o.,
genital openings ; ca., carapace ; ov.¢., ovarian tubes extending along the alimentary
canal, a/.c., to the anus ; ov.d., terminal oviducts.
Fic. 3. Germinal vesicle showing “ Nebennucleoli,” and a body resembling
these within the ‘“* Hauptnucleolus.”
Fic. 4. Germinal vesicle showing a nucleolus having numerous internal
bodies, one of which is about to be extruded.
Fic. 5. An amoeboid germinal vesicle containing a large nucleolus, in which
there is a large vacuole containing nothing stainable.
Fic. 6. Germinal vesicle, containing a large hollow nucleolus in the form of
a deeply staining shell. Within the nucleolus there is a granular network resem-
bling the network of the germinal vesicle.
Fic. 7. Germinal vesicle showing diverticula, and containing a ring-shaped
nucleolus containing within it a network resembling that of the germinal vesicle.
Fic. 8. Germinal vesicle with diverticula, and containing a nucleolus having
a radial striation, a central granular mass, in which is imbedded a deeply staining,
homogeneous spherical body — the endonucleolus.
Fic. 9. Germinal vesicle; a nucleolus in form of a deeply staining crescent
containing a finely granular substance surrounding a vacuolated endonucleolus.
Fic. 10. An egg with its germinal vesicle containing a nucleolus, and also a
radial arrangement of the chromatin network about a center resembling a centro-
some.
Fic. 11. The nuclear pole (“ Kernpol ”) of an egg about to be discharged from
the follicle, showing the position of the germinal vesicle, and the spongy polar
protoplasm spreading out under the egg membrane.
Fic. 12. Mature egg of the ovarian tube, showing an amoeboid remnant of
the germinal vesicle, and its connection with a peripheral mass of protoplasm.
Fic. 13. Portion of an egg showing the formation of the egg membrane after
the first layer has been formed; the orderly radial arrangement of the protoplas-
mic fibers previous to the hardening of the interfilar substance, showing that the
radial striae of the chorion are due originally to protoplasmic fibers.
Fic. 14. Section of an egg about to be discharged into the ovarian tube,
showing the movement of the germinal vesicle and its relation to the “ Kernpol ”
area.
Fic. 15. Section of an adult ovarian tube, showing the folding of the germinal
epithelium and with it the tunica propria, 4/.; the variable size and form of the
epithelial cells ; the formation of empty follicles, 4., by evagination through the
fenestrae of the muscle coat, m.c.; the relation of the number and size of the eggs
to the number of empty follicles ; the position of the immature eggs with refer-
ence to the empty follicles and the point of attachment of the tube; the relation
of this latter to the enclosing peritoneal coat. f.c.
210 MUNSON.
Fic. 16. Portion of an ovarian tube taken from a living animal thirteen
inches long, and examined in the normal fluids of the ovary, drawn with a camera;
a sharply defined granular zone surrounding the germinal vesicle, as in 4., or
extended on the proximal side to the point of attachment of the eggs, as in c.,
or collected into a dense body in the cytoplasm, as in a.
Fic. 17. A portion of a mature egg in the ovarian tube, showing an opening
in the chorion, and some distance below it the last remnant of the nucleus in the
form of a spindle, the laminated structure of the chorion, and also its radial
striations.
Fic. 18. Structure of the egg membranes; #.¢, primary tunic, corresponding
to the tunica propria — the original egg membrane; the first egg membrane fully
formed ; the second having the form of radial protoplasmic fibers ; the interfilar
substance not yet hardened into the definite chorion. The primary tunic shows
the bright dots — the original points of insertion of the radial fibers.
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212 MUNSON.
EXPLANATION OF PLATE XIV.
Fic. 19. An egg showing an inner granular zone of the cytoplasm, and its
connection with the stalk of the egg. The usual chromatin network of the ger-
minal vesicle is wanting. No nucleolus is present. The tunica propria forming
the basement membrane of the epithelium and the outer boundary of the stalk of
the egg is continuous with the investing membrane of the egg ; m.c., muscle coat;
z“.p., tunica propria; y.z., internal yolk zone.
Fic. 20. An egg showing a hyaline inner yolk zone which is extended towards
the point of attachment of the egg.
Fic. 21. An egg showing an inner yolk zone, y.z., surrounding the germinal
vesicle ; in the dividing line between the inner and outer zone, the vitelline-body,
v.6., surrounded by a modified polar mitosome.
Fic. 22. The proximal portion of an egg showing the accumulation of yolk
secretion, y.s., at the base of the stalk of the egg, and the formation of the first
layer of the egg membrane.
Fic. 23. An empty follicle serving as a yolk gland, showing the secretion
products obscuring nuclei and cell boundaries; s.g., secretion granules; m.c.,
muscle coat.
Fic. 24. An egg showing an internal, hyaline protoplasmic zone surrounding
the germinal vesicle as a uniform ring, 7.z.; the germinal vesicle with diverticula
and the nucleolus containing a vacuole.
Fic. 25. An egg showing the peripheral bodies, #.é., and their gradual fusion
into the first layer of the egg membrane or chorion, ch. Low magnifying power.
Fic. 26. Small egg with a germinal vesicle containing two ‘“ Hauptnucleoli,”
A.n., each extruding a “* Nebennucleolus,” 2.72.
Fic. 27. The proximal portion of an egg, showing deeply stainable bodies
resembling nuclei — the yolk-nucleus, y.2. The basement membrane of the stalk
of the egg is seen to be continuous with the primary egg-tunic, “/.
Fic. 28. Tangential section of an egg, showing the form and distribution of
the peripheral bodies, #.d.,and the round bright dots or pores with which they are
pierced.
Fic. 29. Small egg showing the internal granular zone, y.z., in the form of a
ring surrounding the germinal vesicle, the latter containing no true chromatin
reticulum and no nucleolus.
Fic. 30. An egg in the third stage, filled with nuclei, and partly surrounded
by nucleated granular cells; no germinal vesicle.
Fic. 31. An egg (similar to Fig. 30) undergoing regressive metamorphosis ;
no germinal vesicle ; proximal portion of the yolk filled with nuclei and a hyaline
protoplasm ; egg surrounded by a false follicle epithelium of granular cells, as in
Fig. 30 ; chorion folded, distorted, and pierced in various ways.
Fic. 32. A small egg of an adult ovary (magnified ;'; oil immersion), showing
a very distinct cytoreticulum, and a sphere in the form of a central dense network
with very distinct radial fibers, many of which appear to be continuous with the
general cytoreticulum ; the cytoreticulum apparently continuous with the nuclear
network ; metaplasm (archoplasm) scant or absent.
Fic. 33. Section of an ovarian tube of a young Limulus (Leitz, oc. 1, obj. 7),
showing a group of cells undergoing the preliminary phases of karyokinesis pre-
vious to the formation of a growing oocyte.
THE OVARIAN EGG OF LIMULUS. 213
Fic. 34. Young Limulus, six-inch ; transverse section of ovarian tube showing
odgonia ; the spireme of karyokinesis, and a growing odcyte. The young germi-
nal vesicle contains a nucleolus ; in the cytoplasm, close to the germinal vesicle,
the crescent-shaped archoplasm. Method: nitro-picro-sulph., lithium-carmine,
Lyon’s blue. Archoplasm alone a deep blue.
Fic. 35. Six-inch animal ; longitudinal section ovarian tube ; chromatin thread
—spireme of karyokinesis. Method: Merkel’s fluid, Heidenhain’s iron-haema-
toxylin, oc. 1, obj. 7; 4/., tunica propria ; /.c., peritoneal coat.
Fic. 36. Six-inch animal; transverse section ovarian tube, cut obliquely ;
metaphase and anaphase of karyokinesis ; centrosome at the pole of one spindle,
Fic. 37. Seven-inch animal; transverse section ovarian tube; odgonia in
karyokinesis ; spindles ; equatorial plate. Method: Merkel’s fluid, Heidenhain’s
iron-haematoxylin.
Fic. 38. Seven-inch animal; transverse section of ovarian tube ; odgonia ;
growing odcyte with blue archoplasm. Method (3) (see method), Lyon’s blue,
lithium-carmine.
Fic. 39. Seven-inch animal ; ovarian tube ; odgonia ; growing odcyte forming
a diverticulum, and surrounded by the tunica propria and by the nucleated peri-
toneal mantle. Blue archoplasm conspicuous.
Fic. 40. Seven-inch animal ; oblique section of ovarian tube, showing three
growing odcytes, one of which has formed a diverticulum, and remains attached
only by a narrow stalk ; enclosed first by the tunica propria, and second by the
peritoneal nucleated coat. Method: No. 3, Lyon’s blue and lithium-carmine.
Blue archoplasm very distinct.
Fic. 41. Thirteen-inch animal; transverse section of ovarian tube, showing
the relation of the ovarian tube, lined with an epithelium, which is bounded by
the tunica propria, 4.., to the investing mantle or peritoneal coat, f.c. The point
of attachment of the tube is seen to be also the point of origin of the eggs. From
this point the eggs are seen to increase in size, regularly, to the point opposite
where the largest egg is found.
Fics. 42-47. Growing odcytes from ovary of animal seveninches. All drawn
with Leitz camera, ;, oil immersion, and showing the centrosome and archoplasm,
and the relation of these to the cytoreticulum. Method (3).
Fic. 42. Eosin and nigrosin.
Fics. 43, 44. Lithium-carmine and Lyon’s blue; archoplasm and centrosome,
blue; everything else, red.
Fics. 45, 46. Delafield’s haematoxylin and picric acid ; centrosome and sphere,
yellow ; everything else, blue ; sphere, very distinct.
Fic. 47. Weigert’s picro-carmine ; archoplasmic sphere; cytoreticulum and
central radial structure very distinct.
Fic. 48. One of the smallest eggs from an ovarian tube, like that shown in
Fig. 41. A distinct centrosome in the center of a light area, in the widest portion
of the metaplasmic zone surrounding the germinal vesicle. In the latter, a promi-
nent nucleolus.
Fics. 49-104, 109, 114. The entire series of figures from Fig. 49 is drawn on
the same scale, Leitz camera, oc. 1, obj. 5.
Fic. 49. One of the smallest eggs observed in the adult animal, showing a
germinal vesicle, g.v., with a granular nucleolus; a very distinct blue sphere, sf4.,
with bright-red central granules. Method: Merkel’s fluid, safranin, and Lyon's
blue. Sphere, sf., alone bright blue ; everything else, red.
214 MUNSON.
Fic. 50. One of the smallest eggs of an old animal having many empty folli-
cles. Cytoreticulum and aster, as¢, very distinct. Metaplasmic granules or
archoplasm, very scarce or absent. Method: Merkel’s fluid, Biondi-Ehrlich stain.
Fic. 51. Similar to Fig. 50 ; egg a little larger; metaplasmic granules more
abundant. Method same as Fig. 50.
Fic. 52. A very distinct, sharply limited aster, ast; g.v., germinal vesicle ;
nci., nucleolus, containing a central vacuole.
Fic. 53. Egg from adult animal, showing a blue sphere, sf/., consisting of a
central body, centrosome, surrounded by radial astral rays that become lost in a
zone of archoplasmic granules, which again is surrounded by compacted fibers that
merge gradually into the cytoplasmic reticulum.
Fic. 54. Egg showing a distinct cytoreticulum, in which there is a conspicuous
aster, ast. This is farther removed from the germinal vesicle, g.v., than the similar
structures seen in Figs. 50, 51.
Fic. 55. Section of an egg showing position of aster ina plane at right angles
to the primary egg axis.
Fic. 56. Section of an egg preserved in Flemming’s fluid and stained in acid
fuchsin. A large, bright-red body, c¢., in the cytoplasm, whose reticulum is
arranged radially around the centrosome and sphere.
Fic. 57. Section of an egg showing a central body or centrosome, and a con-
spicuous aster, ast. Method: corrosive acetic, haematoxylin, and acid fuchsin.
Fic. 58. Section of an egg showing sphere, sf/., with two central structures
surrounded by archoplasm. Method: safranin and Lyon’s blue. The sphere
alone, deep blue ; the rest of the cytoplasm and the germinal vesicle, red.
Fic. 59. Section of egg showing germinal vesicle, g.v.; nucleolus, zc/.; periph-
eral bodies, #.6.; and a centrosome and sphere, c., connected with a fibrous polar
protoplasm, or polar mitosome, 7.7.
Fic. 60. Section of an egg showing a large granular central body, c., a zone
of fibrous protoplasm, and a zone of granular metaplasm (archoplasm), .f/., from
which radiate many cytoplasmic fibers or astral rays.
Fic. 61. Section of egg showing archoplasm, avch., containing many vacuoles
and a central granular body, the centrosphere; g.v., germinal vesicle; zc/.,
nucleolus.
Fic. 62. Section of egg showing a conspicuous sphere, sf/., containing a cen-
tral body, centrosome (c.), and surrounded by a zone of blue granules, 4.¢. The
sphere, sf., red. At the proximal pole, the modified polar mitosome.
Fic. 63. Section of egg showing a conspicuous vitelline-body, vd. The
peripheral protoplasmic layer is seen to extend into the central granular mass, and
to thus divide the latter into three portions.
Vol. XV
Journal of Morphology
20.
Lith. Werner &Winter, Frankfort “MW.
.
.
216 MUNSON.
EXPLANATION OF PLATE XV.
Fic. 64. Section of egg showing a conspicuous, deep-red, homogeneous vitel-
line-body or sphere, containing central granular bodies or centrioles.
Fic. 65. A deep-red vitelline-body, whose fibers are arranged with reference
to two poles, at one of which there is a sphere with archoplasm and radial fibers.
Method: Merkel’s fluid, eosin.
Fic. 66. Section of egg showing peripheral bodies, a germinal vesicle with
nucleolus ; in the cytoplasm, archoplasmic sphere containing two large spherical
central bodies.
Fic. 67. Section of egg showing peripheral bodies, a conspicuous red centro-
some, surrounded by a clear zone, which again is surrounded by a zone of blue
granules, and this again surrounded byarchoplasm. Radial fibers proceeding from
this can be traced throughout the entire cytoplasm. Method: Merkel’s fluid,
Ehrlich’s haematoxylin and eosin.
Fic. 68. Section of egg showing germinal vesicle, containing a nucleolus, in
which is seen a central spherical body, the endonucleolus. In the cytoplasm is a
conspicuous deep-red centrosome, surrounded by a zone of blue granules, and
this again by a system of red astral rays extending to the periphery of the egg.
Method : Merkel’s fluid, Ehrlich’s haematoxylin, and acid fuchsin.
Fic. 69. Section of egg preserved in Hermann’s fluid, showing a conspicuous
homogeneous vitelline-body with indications of astral rays proceeding from it.
Method: Hermann’s fluid, acid fuchsin.
Fic. 70. Section of egg showing germinal vesicle and nucleolus; and in the
cytoplasm a conspicuous vitelline-body containing granular vacuoles, a system
of astral rays, and numerous blue granules. Method: Merkel’s fluid, Ehrlich’s
haematoxylin, and acid fuchsin.
Fic. 71. Section of egg showing germinal vesicle containing a large “ Haupt-
nucleolus ” and small “ Nebennucleolus.” Close to the germinal vesicle, a
sharply defined sphere with indistinct granular rays, and a large granular central
body. Method: Merkel’s fluid, eosin.
Fic. 72. Section of egg with germinal vesicle containing nucleolus; in the
cytoplasm a deeply staining sphere containing numerous granular vacuoles.
Fic. 73. Section of egg with germinal vesicle, nucleolus, a prominent vitelline-
body or sphere, consisting of a sharply defined granular body, partly surrounded
by a zone of fibrous archoplasm. Method: Merkel’s fluid, haematoxylin, and
eosin.
Fic. 74. Section of egg with germinal vesicle, a “‘ Hauptnucleolus,” and a
“ Nebennucleolus.” In the cytoplasm a conspicuous sphere with a central lighter
granular body, surrounded by a broad zone of deeply staining archoplasm.
Method: Kleinenberg’s picro-sulphuric, haematoxylin, and acid fuchsin.
Fic. 75. Section showing the same as above. The sphere, deep blue, shows
central body, centrosome, and a somewhat regular arrangement of radial fibers to
a peripheral concentric protoplasmic zone. Method: Merkel’s fluid, safranin, and
Lyon’s blue.
Fic. 76. Section with germinal vesicle, nucleolus; in the cytoplasm a vitelline-
body, consisting of a central granular body which is partly surrounded by a horse-
shoe-shaped archoplasmic zone. In the outer portion of this red archoplasm are
THE OVARIAN EGG OF LIMULUS. AF
three vacuoles filled with blue granules. The entire body is surrounded by a zone
of blue granules, which are more numerous at the opening of the horseshoe-shaped
archoplasm. Method: Merkel’s fluid, Ehrlich’s haematoxylin, and eosin.
Fic. 77. Section showing peripheral bodies, a central sphere with astral rays.
The center of the sphere consists of closely packed blue granules, and this is
surrounded by a thick dense red limiting membrane. Method: Merkel’s fluid,
erythrosin, and cyanin.
Fic. 78. Section showing a proximal polar mitosome, a conspicuous vitelline-
body, staining red, and surrounded by a zone of blue granules. Similar granules
are seen also at the pole opposite the stalk of the egg.
Fic. 79. Section of egg showing germinal vesicle with a prominent “ Haupt-
nucleolus,” and an extruded “ Nebennucleolus”’; peripheral bodies at the periphery
of the egg; in the cytoplasm, an archoplasmic sphere, containing two central
structures. Method: Merkel’s fluid, safranin, and Lyon’s blue. The sphere
alone, blue or greenish ; everything else, red. A deep-red circle at the proximal
pole.
Fic. 80. Section showing a vitelline-body, consisting of a central, irregular,
granular mass, from which radiate straight fibers, which at a certain distance from
the central body are again limited by the granules of the cytoplasm. A peculiarly
modified fibrous protoplasm exists in the neighborhood of the body, and this is
eonnected with three small refractive bodies imbedded in a strand of protoplasm.
Method: Merkel’s fluid, Biondi-Ehrlich.
Fic. 81. Section showing germinal vesicle with vacuolated nucleolus; a vitel-
line-body or sphere, consisting of fibrous protoplasm, containing two central
granular bodies. Method: Merkel’s fluid, erythrosin, and cyanin.
Fic. 82. Section showing germinal vesicle, nucleolus; in the cytoplasm a
large, sharply defined, deep-red centrosome, surrounded by two zones of archo-
plasm and astral rays, the latter on one side being modified into a conspicuous
polar mitosome. Method: Merkel’s fluid, haematoxylin, and acid fuchsin.
Fic. 83. Section showing germinal vesicle with vacuolated nucleolus ; in the
cytoplasm a large, homogeneous sphere containing vacuoles with blue granules,
partly surrounded by groups of blue granules resembling nuclei. Method: Merkel’s
fluid, Ehrlich’s haematoxylin. The homogeneous part of sphere, unstained.
Fic. 84. Section showing germinal vesicle, a vacuolated nucleolus ; in the
cytoplasm a large oval fibrous vitelline-body, containing granules, and connected
with the periphery of the egg by a modified fibrous protoplasm resembling that of
the vitelline-body. Method: Merkel’s fluid, Biondi-Ehrlich stain.
Fic. 85. Section showing germinal vesicle with diverticula; a large vesicular
nucleolus, containing granules, and in the cytoplasm a large, finely granular sphere,
which is partly surrounded by a dense zone of granular archoplasm. Method:
corrosive-acetic, haematoxylin, and picric acid.
Fic. 86. Section showing a conspicuous, deep-red, fibrous vitelline-body
(sphere), containing two central vacuoles with blue granules, and numerous
peripheral vacuoles with similar blue granules, the whole being surrounded by a
zone of larger metaplasmic granules and astral radiations. Method: Merkel’s
fluid, Ehrlich’s haematoxylin, acid fuchsin.
Fic. 87. Section showing germinal vesicle, a vacuolated nucleolus, and in the
cytoplasm one or two centrosomes surrounded by conspicuous silken astral rays
that are not sharply limited. Method: corrosive-acetic, haematoxylin.
218 MUNSON.
Fic. 88. Section showing germinal vesicle, a pale ‘‘ Nebennucleolus ”; in the
cytoplasm a sharply defined, deep-red, fibrous sphere, containing two centrioles.
Method: Merkel’s fluid, erythrosin, and cyanin.
Fic. 89. Section showing sphere with a distinct centrosome in a lighter space,
which is surrounded by a broad zone of archoplasm and astral rays extending
throughout the egg. Method: Merkel’s fluid, Weigert’s picro-carmine.
Fic. 90. Section showing germinal vesicle, nucleolus; in the cytoplasm a
granular elongated body at the apex of a fibrous cone of protoplasm. Around
the periphery are numerous peripheral bodies. Method: Merkel’s fluid, Biondi-
Ehrlich.
*
*
7 e
Ve
7
Journal of Morphology. Vol XV
THE OVARIAN EGG OF LIMULUS. 219
EXPLANATION OF PLATE XVI.
Fic. gt. Section showing germinal vesicle, nucleolus; in the cytoplasm a
granular yolk-nucleus at the proximal pole ; connected with it, a proximal polar
mitosome. Method: Merkel’s fluid, Biondi-Ehrlich.
Fic. 92. Section showing germinal vesicle ; in the cytoplasm a conspicuous
aster with a large, deeply staining granular centrosome. Method: Merkel’s fluid,
erythrosin, and cyanin.
Fic. 93. Section showing germinal vesicle with nucleolus ; peripheral bodies
at the boundary of the egg ; a peculiar vitelline-body consisting of a spherical mass
of fibrous protoplasm, connected by a stalk with a finely striated protoplasm at
the pole opposite the stalk of the egg ; a few refractive granules, near the stalk of
the spherical body.
Fic. 94. Section showing germinal vesicle with a large vacuolated nucleolus ;
in the cytoplasm a large homogeneous fibrous vitelline-body, having a mass of
granules atone pole.
FIG. 95. Section showing germinal vesicle with diverticulum, and a conspicu-
ous nucleolus ; a large vitelline-body, having numerous vacuoles, containing the
blue granules and a central structure, probably the centrosome. Method: Merkel’s
fluid, erythrosin, and cyanin.
Fic. 96. Section showing vitelline-body with astral rays, and surrounded by
groups of blue granules arranged radially; within the body, also, a group of blue
granules. The fibrous portion unstained. Method: Merkel’s fluid, Ehrlich’s
haematoxylin.
Fic. 97. Section showing vitelline-body as a large granular central body, sur-
rounded by a zone of archoplasm and a conspicuous system of radial fibers,
which extend to the periphery of the egg, and is especially pronounced at the pole
opposite the point of attachment of the egg. Method as above.
Fic. 98. Section showing vitelline-body with several granular central areas.
Method as above.
Fic. 99. Section showing germinal vesicle with a large vacuolated nucleolus ;
in the cytoplasm a large, homogeneous sphere staining deeply, and surrounded by
radial fibers. Method: Merkel’s fluid, erythrosin, and cyanin.
Fic. too. Section showing germinal vesicle, containing vacuolated nucleolus ;
in the cytoplasm a distinct sphere, containing a central granule, centrosome, with
radial fibers and granules, which is again bounded by layers of fibrous protoplasm
concentrically arranged ; the body, surrounded by a zone of large, stainable gran-
ules ; on the proximal side a modified polar mitosome. Method: Merkel’s fluid,
Biondi-Ehrlich.
Fic. 101. Section showing central sphere, consisting of a large, spherical, cen-
tral body staining blue, and composed of blue granules, and another zone of
concentrically arranged fibrous protoplasm; indications of astral radiations.
Method: Merkel’s fluid, Ehrlich’s haematoxylin, and acid fuchsin.
Fic. 102. Section showing germinal vesicle with large central nucleolus ; in
the cytoplasm a conspicuous sphere, consisting of large granules, a central granu-
lar body being separated from an outer granular zone by a light ring nearly free
from granules. Method: Merkel’s fluid and Biondi-Ehrlich stain.
Fic. 103. Section showing germinal vesicle with a large nucleolus. At one
pole of the germinal vesicle in the cytoplasm there is an area which shows a faint
220 "MUNSON.
radial striation and also a faint concentric striation. It is less granular than the
rest of the cytoplasm, and is no doubt the sphere. It resembles the condition
seen in Fig. 104, but is less conspicuous.
Fic. 104. Section drawn with a camera with the same magnifying power as
the preceding figures, and showing the sphere in connection with the germinal
vesicle. The latter contains a large nucleolus. The hyaline protoplasm, consti-
tuting the “ Kernpol” area, is seen near the point of attachment of the egg.
Method ; platinum chloride, erythrosin, and cyanin.
Fic. 105. Germinal vesicle with diverticula containing the nuclear network
and “ Nebennucleoli”; also a large “ Hauptnucleolus,” showing an opening into
the central cavity.
Fics. 106, 108, 115-118. Nucleoli taken from the living egg of an animal
thirteen inches long. In Figs. 115, 116 two “‘ Hauptnucleoli,” one large and one
small, are present. In Fig. 115 the two are closely united.
Fic. 107. Germinal vesicle with a nucleolus containing within it a network
resembling that of the germinal vesicle.
Fic. 109. Section showing a condition of the sphere similar to Fig. 104, cut
at right angles to the principal egg axis. Method: same as above.
Fic. 110. Germinal vesicle containing a “ Hauptnucleolus,” from which a
“ Nebennucleolus ” has been extruded.
Fic. 111. Germinal vesicle containing a nucleolus with the “ Nebennucleolus ”
partly extruded.
Fic. 112. Germinal vesicle with a “ Nebennucleolus”’ partly extruded. In one
diverticulum of the germinal vesicle is seen a pale ‘‘ Nebennucleolus,”’ and in the
cytoplasm near by are two similar bodies.
Fic. 113. Germinal vesicle with a “ Hauptnucleolus,” from which a “ Neben-
nucleolus ” is partly extruded.
Fic. 114. Section showing the germinal vesicle containing a large nucleolus ;
and in the cytoplasm a large sphere, in which a radial striation can be made out.
The center contains a large clear area, in which a centrosome is difficult to find,
when the other portions of the sphere are made prominent. In the present figure
the central body with its centrosome and archoplasmic zone was taken from
another section of an egg of exactly the same size as the present one, as in that
case the central portion was better preserved than the peripheral portion.
Method: platinum chloride and Biondi-Ehrlich stain. Excellently preserved.
ey
“4
is
y Vol.Xt
Journal of Morpholog:
ay
THE LATERAL LINE SYSTEM OF BATRACHUS
DAU.
CORNELIA M. CLAPP.
CONTENTS.
PAGE PAGE
Un trod ution eereccrrcsesetceresnssereceecnrensns 223 uw LNT Vat OMersescrescsrectsececssevesesrseveessers= 232
General Considerations ................... 237
I. ADULT Form.
General Description ..................... 225 II. LarvaL Forms.
Topography of the Lateral Line Origin of the Lateral Line System 238
System ....... ee poorer 226 (1) Lateral line sense organs...... 238
(1) Infraorbital line ...................... 226 (2) Lateral line nerves ................ 242
(2) Supraorbital line -.........0...... 227 (3) Formation of canals.............. 244
(3) Operculo-mandibular line...... 22 (4) Connecting strand is
(4) Body lines.......................-....-.. 228 | Comparison with other teleosts...... 249
Carvalls oieoticcesceectecnccteeen secs accteonccoeonecs 228 Comparison with ganoids................ 250
(Ti) BR ONES ter rstesrecesetcecessstemenseeae se 229 Comparison with selachians .......... 250
(2) Relation of canals to cranial Comparison with dipnoids............... 250
DON ES rete estercnet ree eae tens .. 230 Comparison with cyclostomes........ 250
Number and position of organs...... 231 Comparison with Amphibia............ 251
\WidtiatlOns easectrsccessearereceseeteaneste= <i 29 2s GENEL AICO UMN ALY cceseesesnsvacscerersensress 251
INTRODUCTION.
In a recent contribution to the “Skin and Cutaneous Sense
Organs of Teleosts,’’ Leydig (1) says: ‘‘ Every one who has
worked in this field shares the conviction that there is need
of the codperating participation of many observers before any
conclusive presentation of the subject is possible.”
As the discoverer of the true nature of the so-called “ lateral
line”’ of fishes, Leydig’s words have weight, when, after nearly
fifty years of investigation, he is obliged to confess that “the
points still obscure outnumber by far those well ascertained.”
Kupffer maintains that we are still on the very threshold of
a history of the development of the peripheral nervous system.
It is therefore the task of the investigator to furnish data for
the future work of generalization.
224 CLAPP. [Vor. XV.
In the following pages an attempt has been made to describe
the lateral line system of the toadfish, both in the adult and
developmental stages, for, as Mr. Allis (2) has well said, the
“purely descriptive part of the subject” has been too much
neglected.
Frequent reference will be made to the conditions existing
in Amia, and the nomenclature employed by Allis will be
adopted.
Ryder (3) describes the appearance of the lateral line organs
in the young toadfish at the time of the formation of canals on
the head, and speaks of the lines of free organs on the body
as canals! It is evident that his observations, though in many
ways valuable, were incomplete. So far as I am aware, this
preliminary “notice’”’ contains all we have on the subject of
the lateral line system of the toadfish.
In Jordan’s ‘Synopsis of Fishes of North America’”’ (4) the
only mention of this system is the statement that in Batrachus
there is ‘‘7o lateral line, nor conspicuous pores.”
My study of the lateral line system of Batrachus was begun
in the summer of 1888, under the direction of Prof. C. O.
Whitman, at the Marine Biological Laboratory of Woods Holl,
Mass., and completed at the University of Chicago,
I wish to express my deep feeling of obligation to Pro-
fessor Whitman for the interest he has taken in the supervi-
sion of my work ; and for the many courtesies and suggestions
received from instructors and associates, I wish here to make
acknowledgment.
For assistance in obtaining material at Woods Holl I am
greatly indebted to Mr. G. M. Gray, the Collector of the
Marine Biological Laboratory ; for specimens of Acanthias I
wish to thank Dr. A. D. Mead and Prof. A. D. Morrill.
The drawings for this paper were made after my sketches
by the following draughtsmen at the Marine Biological
Laboratory :
Figs. 1-3 and 7-11 were made by Mr. Crosby.
“ 4-6 oe « « Mr. Tokano.
GS ii) “ “© « Mr. John Walton.
O22 5523524: a3 “© ~Mr. Hayashi.
No. 2.] LINE SYSTEM OF BATRACHUS TAU. 225
I. Aputt Form.
General Description.
There is something singularly grotesque in the appearance
of the toadfish; and, as its name would imply, there is a
superficial resemblance to the familiar batrachian. The slug-
gish disposition, the mottled brown and gray of the wrinkled,
scaleless skin, the depressed head and toadish eyes do not
suggest the typical teleost. The young fish also are tadpole-
like in their form and motions.
From Pl. XVII, Figs. 1-3, it will be seen that there are quite
conspicuous projections of the skin on the head. Besides the
paired flaps found in connection with the sense organs, there
are other single, often longer projections to be found, which
become laciniated in the older fish. These are especially
prominent about the mouth, fringing the margin of the lower
mandible and opercular regions, while over each eye rises a
broad conspicuous flap, giving an owl-like facial expression.
The goosefish (Lophius) and the sea raven (Hemitripterus)
also possess these somewhat ornamental appendages about the
mouth. The function of these skinny tentacles seems evidently
to be for protection, as they strikingly resemble both in color and
form the seaweed (fucus) that abounds near their favorite haunts.
The toadfish frequents the shallow water of bays and inlets
of the sea, ranging on the Atlantic coast from Cape Cod to
Florida.
It is abundant at Woods Holl, Mass., and is easily obtain-
able in the month of June, during the spawning season. At
this time the fish resort in pairs to large stones, usually near
low watermark, and scooping out a cavity beneath, remain for
days in their retreat. The toadfish of the Eel pond near the
laboratory seem to prefer the dérvis of civilization to the
excavation beneath the rock; for example, tin cans, old boots,
broken jugs, etc. After depositing the eggs, the female
departs, while the male remains to guard the nest.
The young fish do not “attach themselves by a ventral disc
which soon disappears,” as has been supposed, but at the time
226 CLAPP. (Vou. XV.
of oviposition each egg is securely glued to the rock by means
of a secretion on the egg membrane at the pole of the egg
opposite the micropyle.
After hatching, the embryo fishes still remain attached to
the rock by the adhesion of the yolk sac to the inside of the
egg membrane over the disc area, until the yolk material
has been entirely absorbed —a period of three or four
weeks.
The largest toadfish seldom reaches a length of more than
twelve inches.
Dr. Goode (5) gives the following facts about the toadfish:
“In general appearance it resembles a sculpin. It possesses
the power of changing its color to lighter or darker shades
when exposed to light in shallow vessels with dark or light
colored bottoms. It probably becomes torpid in winter in the
more northern regions, is very hardy, and utters a loud croaking
sound when handled.”
In Storer’s description of Batrachus tau one finds certain
statements which are hardly correct. For example, he speaks
of the eggs as being “not larger than very small shot,” as
“‘increasing in size’’ after deposition, also as adhering by a
“disc acting as a sucker,” and finally he says of the fish which
remains to guard the eggs, that “it is in all cases the mother
of the young ones.”
Topography of the Lateral Line System.
1. Infraorbital line. — The first six organs of this line are
found on a semicircular fold of the skin, anterior to the nasal
tube (Pl. XVII, Fig. 2). These organs constitute the antorbital
portion of the infraorbital line. They are free organs pro-
tected by a pair of flaps of the skin, representing in their posi-
tion the sides of a canal. Each organ occupies a depression in
the skin, and on opposite sides are developed the pointed flaps
which arch over this depression, the tips of the flaps almost
meeting over the center of the organ (Fig. 1).
There is no anterior commissure between the infraorbital
lines of the two sides of the head as seen in Amia.
No. 2.] LINE SYSTEM OF BATRACHUS TAU. 227
At a point midway between the anterior and posterior nares
the infraorbital line branches. One division extending along
the border of the maxillary may therefore be called the maxil-
lary branch, the other being the suborbital portion of the main
line (Fig. 1). There are seven organs in this maxillary branch,
five being free organs and two enclosed in a short canal
(Pl. XX, Fig. 22). The suborbital portion consists of eight
free organs, bordering the lower half of the orbit (Figs. 22
and 23).
At the outer angle of the eye there are two free organs
(9, 10) continuing the line of the infraorbital and correspond-
ing to the otic portion (15, 16) as seen in Amia. (Compare
Pl. XX, Figs. 21 and 22.) Inthe temporal portion of the line
there is a single organ (11) enclosed in a canal (Fig. 22). The
infraorbital line is continued on to the body as the dorsal line
of free organs (Figs. 22 and 24).
2. Supraorbital line. — There are seven organs in this line.
The first, a free organ, is situated near the median line, a little
anterior to the opening of the posterior nares (Pl. XX, Fig. 22).
Organs 2-6 are enclosed in a canal, while the seventh is a free
organ occupying a position apparently outside the line and on
the top of the head (Pl. XVII, Fig. 2 ; Pl. XX, Fig. 22). There
is evidence of the presence of the supra-temporal cross-commis-
sure, although the canal seen in Amia is wanting in Batrachus.
In one specimen, 12 cm. in length, the line was conspicuous,
as two extra organs were present in this region of the head.
In Pl. XX, Fig. 22, st.com., the position of the line is indicated.
The middle pit line of Amia may be represented in Batrachus
by the organ just dorsal to the temporal canal (Fig. 22, m./.).
Four organs on the top of the head, extending on to the
trunk each side of the first dorsal fin, constitute what is desig-
nated by Allis as the dorsal body line in Amia. (Compare
Figs. 21-23.)
3. Operculo-mandibular line. — The first organ of this line is
found on the lower side at the symphysis of the mandible.
There is no commissural connection here between the two
sides of the head. Four organs, which never become enclosed
in a canal (Fig. 3), occupy a depression which appears as an
228 CLAPP. [VoL. XV.
open groove in the bone (Fig. 5). The succeeding organs,
5-7, are within a canal in the articular bone (Fig. 5). At the
angle of the jaw the opercular division begins, and consists of
four enclosed organs (8-11) with one (12) free organ near the
temporal region (Fig. 22). Outside of these twelve organs of
the operculo-mandibular line there are accessory lines of free
organs. On the mandible there is a short line of three organs
(Pl. XVII, Fig. 3, ac.md./.) anterior and parallel to the canal.
Near the pore at the junction of the two portions of the
operculo-mandibular line there are two free organs (#d/.) (Figs.
I, 3, and 22), while on the operculum two lines of free organs
diverge at right angles to the canal in the preoperculum, the
more dorsal (d.o./.) having four, and the other (v.o./.) three
organs (Fig. 22).
4. Body lines. — There are three lines of free organs on the
side of the body (Fig. 24); the most dorsal, of twenty-seven
organs, being a continuation of the infraorbital, the middle line
appearing as a branch from this line, represented by only a few
scattered organs, usually eleven, and the ventral line, of twenty-
seven organs, extending from a point in front of the ventral
along the border of the anal fin. Continuations of these lines
are found on the caudal fin, but the organs are somewhat
diminished in size toward the posterior end of the body. The
usual number on the caudal fin is four.
Canals,
The canals enclosing lateral line organs are found only on
the head, and these present a rudimentary, perhaps vestigial
condition in Batrachus.
From Fig. 22 it appears that the infraorbital line through-
out its extent has only two short canals, one on the maxillary
branch containing two organs, and the other in the temporal
region enclosing only one. The supraorbital line, on the other
hand, exhibits the opposite condition, in that all the organs of
the line are enclosed in a canal with the exception of a free
organ at each end of the line. The operculo-mandibular canal
is well developed, only five of the twelve organs being super-
No. 2.] LINE SYSTEM OF BATRACHUS TAU. 229
ficial. There is a direct union of the canals of the two sides
of the head between the eyes, but no organ is developed in
this commissural portion (Fig. 23; also Fig. 4).
Pores.
For a complete understanding of the relation of the pores
to the canals, a knowledge of their mode of development is
necessary. Each organ becomes enclosed in a short canal
(Cut 1), the two openings of which are called by Allis terminal
or half pores. By the union of these half pores, the so-called
primary pores of the young Amia are formed.
In the case of Amia there is a subsequent process of division
of these primary pores, resulting in the dendritic systems of
the adult fish, The pores in Batrachus correspond to the
terminal and primary pores of Allis, as shown in the diagram
Cur 1.— Diagrammatic representation of the formation of a primary pore: a, 4, and c, two
terminal pores approaching each other and fusing; @, primary pore.
representing the post-larval stage of Amia. (Compare Figs.
21 and 22.) In the supraorbital line of Batrachus the przmary
pores have become fused, so that only the two terminal pores
are present, and no pore marks the union of the canals between
the eyes, as seen in Cottus gobio. These pores, in process of
fusion, may be observed during the development of the canals
in the young fish.
It seems all-important that the term fove be restricted in its
application to the openings into the canals. In consequence of
the indiscriminate use of this word, it is often difficult to under-
stand the statements of some writers. A puzzling case is pre-
sented in a description of the canals of Polyodon by a writer (6)
on the Sensory System of Ganoids, where the ‘cluster pores”
ave described as openings of canals, and figured as sense organs !
A recent writer (7) in alluding to this subject says: “The
word fore is inappropriate in Amphibia if used in the same
230 CLA PP. [Vou. XV.
sense as in fishes,” as may be easily understood when it is
known that xo canals exist in the Amphibia.
Relation of Canals to Cranial Bones.
From an examination of the skull (Fig. 4) it appears that
grooves or open channels in the bones serve as_ protection
for the organs. In Batrachus the only cranial bones which
become modified to give protection to the lateral line organs
are the frontal, dentary, and articular bones, the preoperculum,
and an accessory membrane bone in the maxillary branch of
the infraorbital. The curious T-shaped arrangement of the
upper surface of the frontal bones where the canals of the two
sides of the head unite, has given the specific name (tau) to
the species under consideration. These channels are spaces
between ridges of bone projecting from the surface and
partially surrounding the membranous tube containing the
sense organs. They vary in diameter in the different regions
of the head. In the opercular region this membranous tube
occupies the space (Fig. 4) between the outer edges of the
two lamellae of bone forming the preopercle. In the canal
of the maxillary branch the accessory membrane bone appears
as though folded together to enclose the canal (Fig. 4, ac..).
In the mandible there is the nearest possible approach toa
closed bony canal (Fig. 5), while in the case of the temporal
canal there is no cranial bone involved. This short canal lies
outside the muscles which cover the squamosal and occipital
bones, and consists of a tough fibrous or semi-cartilaginous
covering within which is the lining epithelial layer (Fig. 22,
T.C.). Leydig (8) figures a similar formation in Chimaera.
The supporting substance is described as consisting of incom-
plete rings, one behind the other, comparable to the rings of
the trachea, and the free ends of these rings are represented
as branching. In cross-sections of the temporal canal in
Batrachus a very similar structure is seen.
At the anterior end of the supraorbital canal there is a
scale-like cartilaginous formation, by means of which the canal
is extended across between the two openings (Fig. 22) of the
No. 2.] LINE SYSTEM OF BATRACHUS TAU. 231
nasal tube. This scale bears some resemblance to the carti-
laginous tube of the temporal canal, yet is unlike it, and seems
to be a peculiar structure found in no other part of the canal
system of Batrachus. Something very similar is found in the
canal of the trunk in Cottus gobio (Cut 2),
as described and figured by Bodenstein (9).
The nasal tube itself is a canal belong-
ing to this system which never becomes
surrounded by any bony formation. In Cur 2.—Scale from trunk
this connection it may be stated that there 9 “™™ °F “™™ EP
is good reason for regarding the semicircular canals of the
ear as belonging to the lateral line system, although shut off
entirely from the surface of the body. This view has been
advocated by Ayers (10) and other writers.
Number and Position of Organs.
In Batrachus the organs in canals are identical with the
so-called free organs, the only difference being the fact that
the free organs, being situated on dermal papillae, have a
slightly different form.
The number of organs on the head is 128, and on the body
140, making a total of 268 organs on the entire surface of the
head and body. The number enclosed in canals is only 30,
making the number of free organs 238. There is no indication
that the number of organs increases by multiplication during
the life of the fish, and the “nerve ridges”’ described by Allis
(2) have never been found in Batrachus. The “pit organs’’ of
Amia, assigned to the same general class of nerve hillocks,
are yet said to differ greatly from the canal organs in “shape,
arrangement, and methods of multiplication.” From the
description, however, there seems little evidence of greater dif-
ference than between the enclosed and free organs of Batra-
chus, except, possibly, in the size. It seems quite impossible to
arrange them in two separate groups in the case of Batrachus,
as they replace so constantly the regular canal organs. The
enclosure of organs within a canal seems quite incidental and
secondary. The absence of accessory lines of pit organs is
232 CLAPP. [Vou. XV.
quite noticeable in Batrachus, as also the numerous ‘surface
sense organs” (terminal buds) described by Allis (2) on the
head of Amia.
Variations.
Frequent variations in the number and position of the organs
have been noted. There may be five, six, or seven organs in
the antorbital portion of the infraorbital line. The number
in the suborbital may be eight or nine. In the mandibular
line at the place of union of the opercular and mandibular
divisions one organ is often wanting.
Two extra organs—one on each side of the head —
occurred in the case of one specimen, confirming the opinion
that the free organs of this region are homologous with those
of the commissural canal in the occipital region of Amia.
(Compare Figs. 21 and 22.)
On the body the variation is still more marked, the two sides
seldom having exactly the same number or arrangement of
organs.
On one large specimen there was the following arrangement :
In the dorsal body line of the right side, 25, left 29.
“6 “ec middle “ce “ck “6 “c ““ it, “c oe
“ “ ventral “ GGG “ “ «“ 26, “ Dif
At the anterior end of the ventral line in another specimen
one organ was lacking on each side. The number may be four,
five, or six on the caudal fin.
Innervation.
The method most successfully employed for determining the
course of the nerves was maceration of the adult fish in nitric
acid. After being kept for some time in a weak solution, not
only the large nerve trunks could be easily followed, but the
bundles composing these trunks could be separated, the con-
nective tissue sheath having been dissolved. It thus became
possible to demonstrate the course of the different components
of nerves enclosed in the same sheath,
No. 2.] LINE SYSTEM OF BATRACHUS TAU. 233
By reference to the diagrams (Figs. 22 and 23) which represent
the side and dorsal view of the head and anterior part of the
body of an adult toadfish, the course of the nerves may be
traced after their exit from the skull. Fig. 21 is reproduced
from Allis’s plate for purposes of comparison, as it is of interest
to note the general resemblances and slight differences which
appear in comparing the teleost Batrachus with the ganoid
Amia. As may be observed, the number of organs in the
different lines and their mode of innervation correspond in a
surprising manner.
The lateral line system in the head of Batrachus is innervated
by dorsal branches of the VII and X cranial nerves.
The VII Nerve.
The supraorbital line is innervated by the R. ophthalmicus
superfacialis facialis (Fig. 22). This branch arises from the
ganglion lying above the Gasserian ganglion (Fig. 13), and
runs along the inner margin of the orbit in close association
with the ophthalmic branch of the trigeminus. There is
an evident interchange of fibres in one place, and the two
nerves are included in the same sheath for a short distance
near their peripheral termination. Each organ is supplied by
a short branch, which enters the bony canal immediately below
the organ. Organ No. 7 being a free organ, and on the top of
the head, yet belongs to this supraorbital line of organs, as
may be seen by tracing its development and its innervation.
As Allis has shown in Amia, the supraorbital line is widely
separated, at an early period of development, from the infra-
orbital, at the point where later there is a union of the two
canals.
Infraorbital line. —The organs in the pre-auditory part of
the infraorbital line are innervated by the R. buccalis facialis
(Figs. 22 and 23, due.f.). This branch arises from the facial
(Fig. 13), lying above the Gasserian ganglion, and immediately
divides, sending a comparatively small number of fibres (Fig.
13, duc f.*), to the outer angle of the orbit to supply the eight
organs of the suborbital portion of the infraorbital. The
234 CLAPP. [Vor. XV.
main portion of the buccalis passes directly downward to the
floor of the orbit (Fig. 13, duc.f.1) enclosed for some distance
in the same sheath with the maxillaris of the fifth. It then
again divides, one branch being directed toward the median
line supplying the six antorbital organs, while the other sends
branches to the seven organs which constitute the maxillary
portion of the infraorbital line (Fig. 23). The two organs
(9, 10) of the infraorbital line, corresponding to those inner-
vated by the otic branch in Amia (Fig. 21), are in Batrachus
supplied by a branch from the R. buccalis facialis (Figs.
22 and 23).
Operculo-mandibular line. —The organs of this line are
innervated by the R. mandibularis externus facialis. Organ 12,
—a free organ,—together with the most dorsal of the two
branch lines of free organs on the operculum (Fig. 22, @.0./.),
are innervated by a branch of the hyoideo-mandibularis facialis,
before the externus has separated from it. It leaves the main
trunk through the foramen at the base of the opercular
spine.
Organs 8-11 are supplied by branches which pass from the
externus between the bony lamellae of the preoperculum to
the canal occupying the space between the outer edges of these
lamellae. Between organs 9g and 10 a branch is given off to
the three free organs forming the ventral line on the opercu-
lum (Fig. 22, v.0.2.).
There are two free organs situated near the large pore which
marks the union of the opercular and mandibular portions
of the line, and which seem to correspond to the mandibular
pit line of Amia (Fig. 21, .d@./.), which are innervated by
branches from the externus. In the same way the two free
organs at the angle of the mouth in Batrachus may easily be
identified with the vertical cheek line in Amia (Fig. 21, c./.),
also supplied by a branch from the externus.
The three canal organs of the mandible are innervated by
the externus, as also the four organs in the groove at the ante-
rior part of the mandible (Fig. 3), while the three superficial
organs in a line parallel with them are also supplied by a
branch of this same nerve (Fig. 22, ac.md./.).
No. 2.] LINE SYSTEM OF BATRACHUVUS TAU. 235
There seems to be an interesting peculiarity in the innerva-
tion of the body lines of Batrachus. The N. lineae lateralis
does not supply the line of sense organs continuous with the
infraorbital of the head, but it does send branches to some of
the scattered organs of the middle line. The dorsal and ven-
tral lines of the body are innervated, in part at least, by the
R. recurrens facialis. This nerve emerging from the veztral
branch of the dorsal VII (Fig. 13) turns directly backward
within the cranial cavity; it passes obliquely through the cranial
wall and through a loop in the glosso-pharyngeal, beyond
which it forms an anastomosis with an ascending branch
from the posterior root of the vagus, at a point just behind the
auditory capsule. The position of the R. recurrens facialis is
superficial to the N. lineae lateralis, and it extends on to the
body just underneath the skin. It divides immediately (Fig.
22), sending one branch toward the dorsal region supplying
the organs of the anterior portion of the dorsal line, while the
ventral branch curves around behind the base of the pectoral
fin innervating the anterior organs of the ventral line.
The X or vagus nerve.— The anterior root of the vagus
nerve arising from the dorsal region of the medulla (Fig. 12,
X an.yr.), does not possess any distinct ganglion. It runs
backward and outward, crossing the main root of the vagus,
with which it is connected by a few fibres, and after leaving
the cranium by the foramen in the occipital is continued on
the body as the N. lineae lateralis. It courses deeply under-
neath the muscles for some distance, becoming superficial at
the posterior portion of the body.
Although this is the main lateral line nerve, zt seems to
innervate only a few of the organs on the body of Batrachus.
The supratemporal branch of the vagus is composed mainly
of anterior root fibres (Fig. 13, s¢.v.1), It arises intracranially,
passing upward and leaving the skull through a foramen in the
supraoccipital (Fig. 4, o.cf.). It then turns forward, supplying
the canal organ of the temporal region (Figs. 22 and 23) and
three other organs on the top of the head. The most anterior
of the three organs may be considered as representing the
middle dorsal line of pit organs, which in Amia are innervated
236 CLAPP. [Vor. XV.
by branches of the IX, while the two others are probably
homologous with the organs forming the cross-commissure of
Amia. (Compare Figs. 21 and 23.)
Another branch (v.?), arising from the anterior root of the
vagus just outside the cranium, and taking a course upward and
backward, innervates the four organs of the dorsal line (Fig.
22, Np.
Fig. 13 shows an intracranial commissure between the VII
and X. The branch from X which anastomoses with the R.
recurrens facialis (Fig. 13) arises from the posterior root of the
vagus. It is also evident that the fibres of the R. recurrens
facialis emerge with the ventral root fibres of the VII. It is
probable that the components of this nerve have a different
central termination in the medulla from the dorsal branches
of the VII and the anterior root of the X. The innerva-
tion of the body lines in Batrachus presents a somewhat
puzzling problem, which can only be solved by a careful study
of the terminations or origin of the fibres in the medulla. The
apparent course of the nerves is often deceptive, as fibres havy-
ing different central connections are enclosed in the same sheath
outside of the medulla, or, the central connections being the
same, the fibres follow different pathways to their destination.
A case in point is that of the glosso-pharyngeal, which seems
to take no part in the innervation of sense organs in Batrachus,
although in Amia one canal organ and a line of pit organs are
supplied by that nerve.! It is probable that the fibres inner-
vating the organs are enclosed sometimes with the IX and
sometimes with the X nerve.
The attempt to homologize the body lines of Batrachus
seems useless until a better knowledge of the components of
the so-called R. recurrens facialis is obtained. It seems prob-
able that this nerve is identical with the R. dorsalis recurrens
trigemini (Stannius), which is said to innervate a line of end
buds at the base of the dorsal fin in Siluroids, but the dorsal
body line of organs in Batrachus would hardly seem homologous
with this line of end buds.
1In a recent paper (Journal of Morphology, vol. xii, p. 747) Allis has shown
that the ‘so-called dorsal root” of the IX is composed of fibres from the lateral
line root of X.
No. 2.] LINE SYSTEM OF BATRACHUS TAU. 237
General Considerations,
If one may judge from the contributions to this subject by
various investigators, it is becoming evident that the lateral
line system may take rank among the organs of special sense.
The connection of the olfactory, optic, and auditory organs
with the central nervous system is effected by means of special
pairs of cranial nerves originating in definite centers within the
brain. On the other hand, the sensations of touch are medi-
ated by cutaneous nerves which seem to be so universally dis-
tributed as to suggest the idea that the skin itself may be
regarded as an immense sense organ and its innervation corre-
spondingly general,
The system of the lateral line has usually been regarded as
composed of organs of the more generalized type. Their wide
distribution over the entire head and body would favor this
conclusion, but the study of the cranial nerves of Amphibia
brings into view several significant facts. In his recent paper,
Strong (11) calls attention to a “most beautiful extirpation
experiment in nature.’ The tadpole has the sense organs
found in fishes and the Urodeles, and these organs are inner-
vated by certain dorsal branches of the cranial nerves. When
the tadpole becomes a frog, and these organs disappear from
the skin, the dorsal branches supplying them become atrophied.
As regards the innervation of the lateral line organs, there
seems to be a remarkable agreement between the Urodela, larval
forms of Anura, and the fishes. In general, the arrangement
seems to be the same, inasmuch as dorsal branches of the VII
and X cranial nerves supply these organs. This has been
shown in the case of amphibians (11), selachians (12), two
ganoids and one dipnoid (13), but among teleosts the mat-
ter has been in doubt. Batrachus is certainly one teleost in
which the dorsal branches are present and innervate the lateral
line organs.
In his analysis of the cranial nerves of Amphibia, Strong
gives a description of the different nerve components distin-
guishable by the nature of their fibres, peripheral distribution,
and internal origin:
238 CLAPP. [Vou. XV.
He describes a general cutaneous component and a sfeczal
cutaneous or lateral line component, thd dorsal branches of
which innervate the organs of the lateral line. These branches
are coarse fibered and therefore distinguishable in sections,
while their internal origin or termination is the tuberculum
acusticum, a portion of the medulla which is greatly developed
in fishes. If the lateral line component has its origin in the
tuberculum acusticum, we have good reason to conclude that
the localization of function in the medulla is as definite for
these widely distributed organs as it is for the more circum-
scribed patches of sensory epithelium seen in the case of the
ear, eye, or olfactory organ.
The ear furnishes a fine illustration of this subject, and seems
like a connecting link between the system of lateral line
organs from which it has probably originated, and the most
highly modified sensory structure in Vertebrates —the eye.
Ayers (10) has shown that the auditory organ is in reality a
series of canal organs innervated by two distinct cranial
nerves which he regards as possibly dorsal roots of VII and IX.
II. Larvat Forms.
Origin of the Lateral Line System.
1. Lateral line sense organs.—Sections of early stages in
the development of Batrachus show thickenings of the ectoderm
in the region behind the eyes. In sections of later stages these
areas of thickened ectoderm have become invaginated to form
the auditory vesicles. Immediately after the closure of the
auditory pits, thickenings of the lower layer of the ectoderm
are observed on each side of the head in the pre-auditory region.
From this thickened area two cords extend, one above and
another below the eye. These cords are the rudiments of the
supraorbital and infraorbital lines of sense organs.
Simultaneously in the post-auditory region there appear
similar thickenings of the ectoderm which extend rapidly
backward on to the trunk. In very young embryos this line
advances along the side of the body with an enlargement at the
growing end.
No. 2.] LINE SYSTEM OF BATRACHUS TAU. 239
These thickenings of the ectoderm are described by H. V.
Wilson (14) as “‘sensory tracts,’ and he maintains that in the
bass, unlike what has been observed in selachians, in Amia
and in the trout, the lateral line originates in the form of
“sensory sacs,” which later on become flattened out into the
“patches’’ described by other authors. There is no dissent
from the view that the auditory region is the place where the
lateral line system originates, but the occurrence of these
“sensory sacs’’ appears to be peculiar to the bass. Wilson
states that the ear, branchial organ, and the first of a series of
organs extending on to the body, are derived from this ‘‘ common
sensory furrow.’’ In Batrachus there is no definite furrow
present, and the “branchial sense organ” described as “ func-
tional in the later stages of embryonic and during larval life”
is certainly not “histologically differentiated”’ as in the bass.
There is no sign of an organ composed of ‘sense cells with
short stiff hairs,’ as described by Wilson in an embryo of
fifty-nine hours.
Fig. 17 shows the growing end of this line as seen in a
preparation of the skin of an embryo 5 mm. in length. After
fixing in picro-sulphuric acid and slight maceration in water,
if the skin is removed, stained in alum cochineal, and mounted
in glycerine, the proliferating cells in the lower layer of the
ectoderm may be clearly seen. A horizontal section of this is
shown in Fig. 18, at the time when the growing point has
reached only a short distance behind the pectoral fin. A
more highly magnified view of a portion of the same is seen in
Fig. 19. A comparison of the lateral line of Batrachus at
this stage with the same structure in an Acanthias embryo is
of interest (Fig. 14). In Acanthias the lateral line is quite
conspicuous. In an embryo of 22 mm. (Cut 3) it is easily seen
with a hand lens, as a prominent, somewhat flattened ridge,
extending backward above the branchial region and along the
sides of the body. There is a curious fold of the epidermis,
the so-called “pocket,” which covers the growing end of the
line. PI. XIX, Fig. 14, shows a horizontal section of an embryo
17 mm. in length, from which it is evident that the “ pocket”
consists of a reduplicature of the skin accompanying the
enlarged growing point.
240 CLAPP. [Vor. XV.
The branches of this system on the head also show this
peculiar fold (Cut 3), the significance of which it is hard to
discover. In connection with this fold, Mitrophanow (15)
describes the formation of canals, but gives no figures that
illustrate the manner of their formation. Behind the dorsal
fin, as seen in older embryos, the ‘pocket’’ becomes greatly
elongated, and suggests the existence of a canal in that region,
but furnishes no clew to the condition that is supposed to exist
in the anterior part of the body of the adult Acanthias. Cut
4 is a cross-section of the anterior part of the line at this stage.
as
See
Cut 3.—Acanthias embryo, 22 mm. in length.
This subject has not been sufficiently investigated to afford a
satisfactory basis for comparisons. Beard (16) mentions this
growing end of the line as “ plowing its way backward through
the indifferent ectoderm.’’ The appearance of the structure
in Acanthias would suggest this idea.
Balfour (17) describes the lateral line of Syllium as appearing
‘cin the form of a linear thickening of the inner row of cells of
the external epiblast on each side, at the level of the notochord.”
He says that at this time it shows no segmental character, and
he also notes the interesting fact of the “broadening at the
growing point.” He probably has reference to this remarkable
fold of the epiblast when he speaks of the “ perfectly abrupt ”’
termination of the line. He also mentions the contrast between
the narrow anterior and the broad terminal portion of the line.
This thinning out of the anterior portion of the lateral line is
noticeable in Batrachus. Allis describes and gives figures of
surface preparations showing the same appearance of the line in
Amia. ‘The ends of these lines are enlarged, that of the
lateral line sometimes forming a large and prominent swelling.”
Hoffmann (18) regards the sense organs as arising seg-
mentally, and gives no account of the growth of the rudiment
No. 2.] LINE SYSTEM OF BATRACHUS TAU. 241
of sense organs on the side of the body. H. V. Wilson (15)
has evidently found the line only in the form of a slender cord
on the posterior part of the body, and makes no mention of
any enlargement at the growing end. From figures in a
recent paper on Necturus, by Miss Platt (19), this enlarged
growing point is shown as quite conspicuous. In selachians,
ganoids, and amphibia we have evidence of this mode of
growth of the sense organ rudiment, but no figures or descrip-
tions of the enlarged growing point of the lateral line of any
teleost have been published, so far as I am aware.
Fig. 6 represents the condition of a Batrachus embryo
about the time of hatching and when the embryo is still
attached to the yolk sac. The principal organs of the different
lines can now be distinguished in surface preparations, but a
more satisfactory showing of the exact number and position of
the organs, as well as of the connecting strand, can be obtained
from preparations of the skin, as previously described.
Regarding the canal and free organs as identical, the devel-
opment may be briefly outlined as follows: In Batrachus, as
in Amia, the growing line of sensory epithelium begins to
present the appearance indicated in Fig. 15, which is a camera
drawing from a preparation of the skin at a stage somewhat
earlier than that shown in Fig. 6. The cells destined to
form the sensory portion of the organ begin to elongate and
arrange themselves in a definite manner, suggesting the name
“hillock” given by Merkel (20) to this class of organs. At
the summit there soon appears a clear vacuolar space toward
which the upper portion of the cells is directed. The
“hillock” formed in the lower layer of the skin soon pushes
its way to the surface and gradually takes on the characteristics
of an adult organ. This process has been fully described by
Allis (2).
The sensory and supporting cells are very much alike in the
organs of Batrachus, although the cells in the center of the
“hillock”? are pear-shaped and somewhat shorter than those
of the peripheral part of the organ. From Fig. 16, which is a
section of a side organ of a fish of one year old, the shape of
the adult organ is evidently that of a cone hollowed at the base.
242 CEAPE. [VoL. XV.
Soon after the organ has reached the surface, there appears
on the summit a structure known as the “hyaline tube” or
“cupola.” This was seen on specimens 2 cm. in length, being
easily observed with low magnifying power on the living fish,
some chloroform being added to the water to quiet the fish
during the observation. This tube measured .10 mm. in length
and .or in breadth. From sections through the canal organs of
older fish, it is apparent that this “hyaline tube” is present
after the enclosure of the organs in canals. There is little
doubt in regard to the nature of this hyaline structure. The
cells of the organ, in common with other epidermal cells, may
secrete a cuticular substance on the free ends of the cells. In
the case of the hair cells this secretion is in the form of hairs
or bristles. These hairs may coalesce, forming a continuous
membrane, surrounding the central portion of the hillock,
thus forming the so-called tube, which is frequently present.
The hairs of the most central cells may remain separate within
this tube, as Leydig (1) observes in his most recent paper on
this subject.
The ‘terminal buds” of Merkel or ‘end buds” of other
writers are not found on the surface of the head or trunk of
Batrachus, but they occur in the mouth and branchial cavities.
These organs are much smaller than the hillocks on the surface
of the body, and very little is known in regard to their
innervation.
2. Lateral line nerves. — In his “ Elasmobranch Fishes” Bal-
four (17) says that, in considering the subject of the lateral line
system, we are dealing with two distinct structures, (1) the
modified epidermis seen in certain lines along the sides on
the head and body, and (2) the accompanying nerves.
The origin of the organs from the modified ectodermal cells
has been demonstrated, but the mode of origin of the accom-
panying nerves is not so well understood. Balfour (17), follow-
ing what he believed to be the analogy of cranial nerves in
general, held that the dorsal branches which supply the sense
organs grow out from the brain to these organs. On the other
hand, Gotte (21), Semper (22), van Wijhe (23), and Beard (16)
consider it certain that the cells from the ectoderm contribute
No. 2.] LINE SYSTEM OF BATRACHUS TAU. 243
to the formation of these branches. According to Hoffmann
(18), the lateral nerve in Salmo arises from a string of cells in
the nervous layer of the ectoderm some time previous to the
development of the organs. This string gradually moves out
of the ectoderm, coming to lie at some distance internal to it,
but connected at intervals
by short side branchlets with
the locality where the future
segmental sense organs are
to arise. Hoffmann’s (18)
observations were made on
embryos of a teleost, which
he regards as a less favor-
able form than the selachian,
in which, according to Sem-
per (22), it is uncommonly
easy to show the origin of
the lateral nerve.
In the section of the
Acanthias embryo (Fig. 14),
there is an evident exten-
sion of nerve fibres from
the vagus ganglion accom-
panying the lateral line
rudiment. In cross-section
these fibres are seen to con- Cur 4. — Cross-section of lateral line of
: ‘ Acanthias embryo.
stitute at this stage a part
of the lateral line (Cut 4). It will be necessary to study the
changes taking place in the later stages before a final conclusion
can be drawn, but it would appear that the thickened ectoderm
forming the lateral line and the extension of the outgrowths of
the ganglion cells were associated during the early history
of the structure.
Hoffmann’s (18) description of the origin of the nerve
becomes more intelligible after the study of the selachian
embryo, although in both the case of Salmo and Acanthias the
exact mode of origin of the sense organs remains uncertain. In
Batrachus, on the other hand, the origin of the sense organs
OLY 90 mmm nm mn
244 CLAPP. (VoL. XV.
is easily demonstrated, while the origin of the nerves and thetr
connection with the organs becomes the great problem, as in the
case of other teleosts.
In stages represented in Fig. 18 it is impossible to detect
any nerve fibres accompanying the growing line on the side
of the body. The whole line has the appearance of being an
extension of the mass of ganglion cells. This seems the more
striking as the entire string of cells constituting the rudiment
of the lateral line in Batrachus seems to occupy the same relative
position as the extension of ganglionic fibres in Acanthias.
Wilson (14) states that he has been unable to trace the
origin of nerves in the Bass. He says in regard to the lateral
‘branch of the vagus, that he could not distinguish it “during
embryonic life’’ nor “in the larvae of two or three days.”
It is difficult to reconcile Hoffmann’s (18) observations on
Salmo with the facts brought out by Wilson (14) or with the
conditions existing in Batrachus. That the origin of the sense
organ rudiment precedes the appearance of the nerve in both
the Bass and Batrachus can hardly be doubted, while from the
description of the ‘growing sensory tissue” in the skin of
Amia, Allis (2) surely conveys the idea of the early appear-
ance of the sense organ rudiment.
3. Formation of canals. — Figs. 7-11 represent the appear-
ance of the head of Batrachus during the period of the
enclosure of the organs in canals. The plates of Allis show in
detail the different steps of this process of canal formation,
and avery full account of the same is to be found in the admi-
rable paper on the development of the lateral line organs of
Amia. Previous to this paper, we have the accounts of canal
formation in Cottus gobio by Bodenstein (9), and in Plateria
by Schulze (24) and Solger (25), but the illustrations of the
subject, as well as the accounts, are not so full and clear as in
the case of Amia.
The appearance of the head during these stages is almost
identical in Amia and Batrachus. At the time of hatching,
the organs are not apparent on the surface, but after treatment
with picro-sulphuric acid they may be seen below the surface
as whitish spots or irregular lines (Fig. 6).
No. 2.] LINE SYSTEM OF BATRACHUS TAU. 245
The canals are formed in sections, as described by Allis (2):
«After a developing canal organ has reached the surface, it
begins to sink, carrying with it the surrounding tissues, thus
forming a small pit at the bottom of which the organ lies.
Lips grow upward and inward from the edges of the pit, and
meeting above the organ, form a short canal, the openings of
which are inclined to the general surface and give to the canal
a tunnel-like appearance.” In Figs. 7 and 8 the organs have
begun to sink below the level of the surface and form linear
areas of depression.
In Figs. 9, 10, and 11 the process has been continued and
the organs are partially enclosed by the approaching lips of the
canal, but complete fusion has not taken place. This condition
is permanent in some forms, as Chimaera and Polyodon,
open grooves taking the place of canals in the adult. The
process of enclosure goes on unequally; the most anterior organs
are the first to become enclosed. In Fig. 10 the line of fusion
of the nasal tube is distinctly seen, and the two half pores
which are formed constitute the anterior and posterior nares.
In the supraorbital line the process of fusion is carried out
most completely, the short canals coalescing and therefore no
primary pores formed, the terminal or half pores only being
present (Figs. 2 and 23). A comparison of the commissural
canal between the eyes, so prominent at this stage (Fig. 10),
with the bony channels on the frontal bones (Fig. 4) is instruc-
tive, as showing the effect of the flattening of the head and
the closer approximation relatively of the eyes in the adult.
In the case of the operculo-mandibular line (Fig. 9) the oper-
cular portion is seen to form independently of the mandibular
division, and the double or primary pore which marks their
union remains larger than the others of the line (Fig. 22).
In the mandibular portion of the line the four anterior organs
are never enclosed in a canal, but retain the open groove
condition in the adult (Fig. 3).
4. Connecting strand, — While examining adult specimens of
Batrachus which were partially macerated in nitric acid my
attention was attracted by a very well-defined strand connecting
the organs on the side of the body. This structure had the
246 CLAPP. [VoL. XV.
appearance of the commissures connecting the ganglia of the
sympathetic system, and from the fact that it resisted the action
of nitric acid I inferred that it was nerve tissue. In direct sun-
light, by aid of an ordinary lens, this cord was plainly to be
seen, and its connection at either end with the cells near the
summit of the sense organs was evident.
The appearance of this structure in connection with a free
organ is shown in a section from the side of the body of a
specimen 10 cm. in length (Fig. 16, cow.s¢.). As may be seen,
the strand near the organ has a diameter greater than that
of the nerve supplying the organ, and it extends in a sort of
festoon from the summit of one organ to that of the next in
the line, becoming narrower midway between the organs. It
extends below the skin into the thick felt-like layers of connect-
ive tissue occupying the space between the skin and the mus-
cles. This cord is also found connecting the organs of the
supra- and infraorbital lines in the head, as well as those of
the operculo-mandibular line, and even where free organs seem
quite disconnected, as in case of organs on the top of the head,
there is at least a short extension of this cord on each side
of the organ. In Figs. 22 and 23, the strand is represented in
blue. A cord of cells is found on the floor in the epithelial
lining of the canals
(Figs. 22 and 23). It
therefore becomes
evident that in the
case of Batrachus tau
the connecting strand
constitutes a promi-
nent feature through-
out the lateral line
system in the adult
Cur 5.—A cross-section of temporal canal of Batrachus, fish (Cut 5). Boden-
showing the strand in floor of canal. Higa (9) Heccubes
this structure in the adult Cottus gobio, and says that the
strand extends from the cezter of one organ to the next. This
is hardly the case in Batrachus (Fig. 16), as the cord evidently
terminates at the swmmt of the organ, among the supporting
No. 2.] LINE SYSTEM OF BATRACHUS TAU. 247
cells, having no connection with the nerve, as suggested by
Bodenstein, when he speaks of the possibility of its forming
anastomoses with the nerves in the series of sensory hillocks.
Emery (26) describes what he calls “epithelial canals” in the
adult Fierasfer, and
his figures leave no
doubt as to their
homology with the
connecting strand of
Batrachus (Cuts 6
and 7). No mention
is made of any con-
necting canals be- Cur 6.—Copied from Fig. 58, Emery (26).
tween the canal
organs, but they are evidently well developed between the
“nerve buttons” (pit organs) of the accessory lines.
The fact that these “canals” sometimes branch and end
blindly (Cut 7) is a peculiar characteristic if these canals are
functional. Exactly similar peculiarities are noticed in the
case of the strand in Batrachus. The free organs situated in
a line parallel with the canal on the mandible have the strand
—__,.___o—__»_, © _e—. con. St.
Cut 7. — Copied from Fig. 6, Emery (26).
directed at right angles to the canal, and in one case the end
of the cord was branched in a similar way to that figured
by Emery. In a series of cross-sections the irregular out-
line of this strand in Batrachus becomes evident (Cut 8).
There is some indication of its being fibrous in structure, and
248 CLAPP. [Vor. XV.
often near the organ a suggestion of a lumen is noticed,
especially in longitudinal sections.
According to Leydig (1), Feé (27) seems to have figured this
connecting strand, but makes no allusion to it. Solger (25)
refers to the “side organ chains,” in the case of Acerina cer-
nua and Lota vulgaris, and speaks of the chain as consisting
of “marrowless nerve fibres enclosed in a nucleated sheath.”
Merkel (20) speaks of ‘ modi-
fied (cutis) epithelium” and
suggests that the connecting
strand may be the vestige
of a canal! The presence,
however, of both the canal
and the connecting strand,
one found within the other,
as in Batrachus, would over- Cur 8.—Cross-section of strand between organs
eae any sich supposition. g and 10 of infraorbital line of Batrachus.
Carriére (28) thinks there is no possibility that this “chain” is
composed of nerve fibres. Ryder (29) speaks of “faint fila-
mentous prolongations” from the organs. In a figure of
Savi’s (40) vesicles there is a connecting cord shown and
described as “filament anastomotique,’ which suggests the
same structure.
Leydig (1) has examined this peculiar structure in Gobio,
Rhodeus, Salmo, and Anguilla, and although reaching no con-
clusion as to its significance, says that the strand does not
consist of nerve elements, but principally of epithelial cells
which enclose a space that may be considered a lymph passage,
or, in some cases, no lumen being present, the strand presents a
fibrous or ligamentous appearance. He regards the “ epithelial
canals” of Fierasfer as lymph channels. Leydig (1) further
observes that although he has not seen the epithelial thick-
enings out of which the sense hillocks arise, it is probable that
the strand is derived from these thickenings. From this point
of view the strand would be a remnant of an epithelial growth
which separates from the epidermis and forms the foundation of
the sense hillocks. Leydig (1) utterly discards the idea that this
structure is in any way connected with the later forming canal.
No. 2.] LINE SYSTEM OF BATRACHVS TAU. 249
In regard to the origin of the strand, my observations on
the embryos and larval forms of Batrachus would tend to con-
firm the opinion expressed by Leydig. Whatever the function
may be, z¢s origin from the sense organ rudiment is not to be
doubted. In very young embryos the growth of the sensory
tissue is easily demonstrated, as shown elsewhere. In the
larval fish 15 mm. long, just after the yolk has become
absorbed, the strand is distinctly seen in preparation of the
skin, the cells of the strand between the organs still retaining
much the same appearance as in earlier stages (Fig. 15, coz.st.).
Later, however, the cells undergo a change so that the tissue
appears as seen in Fig. 16, cov.st.
Comparison with Other Teleosts.
1. Lophius piscatorius.— The goosefish resembles the toad-
fish in being destitute of scales and in having similar tentacu-
lar appendages in various parts of the body (Guitel, 30). The
sense organs are not enclosed in canals, but are protected
by projections of the skin, as in the case of the free organs
of Batrachus. The maxillary portion of the infraorbital line of
organs is greatly developed and the suborbital is wanting.
The innervation is quite similar in the two forms, the dorsal
branches of the VII being quite distinct from the V.
2. Cottus gobio. — Bodenstein (9g) has described the ‘“con-
necting strand”’ in the adult Cottus and represents it in his
figures as on the floor of the canals. From his description of
the skin and the appearance of the canal organs, there is a
striking similarity between the two forms,
3. Amturus. — Batrachus and this common fresh-water form
have several characteristics in common. The naked skin,
closely studded with gigantic gland cells, the depressed head,
and general shape of the body is the same, but the sense organs
of the trunk in Amiurus are, for the most part, in canals. The
interesting comparison is in respect to the course of the
R. dorsalis recurrens facialis, which has been wrongly called
“trigemint.’ In Amiurus, according to Wright (31) and Pol-
lard (32), this arises from a “posterior dorsally placed gangli-
250 GIEATZES [VoL. XV.
onic extension and passes upwards intracranially to the parietal
bone,” and from thence on to the body innervating a dorsal line
of sense organs. This nerve is undoubtedly homologous with
the R. recurrens of Batrachus, although taking a somewhat
different course. It receives a branch from the vagus, and in
this respect resembles the R. recurrens facialis of Batrachus.
4. In Fierasfer the “connecting strand” is well developed, and
although Emery (26) describes this structure as an ‘epithelial
canal,” still the evidence is hardly conclusive from his figures.
5. Ganotds. — As already shown, Batrachus and Amia have
many of the same characteristics, but in Batrachus the canals
are never entirely enclosed within the bones of the skull, nor
is the elaborate system of branching canals with their numer-
ous groups of pores to be found. Allis (2) has shown that the
trigeminus takes no part in the innervation of the canal organs
of Amia. The terminal buds found in such abundance on the
surface of the head of Amia are not present in Batrachus.
6. Selachtans. — The comparison between the lateral line of
Acanthias and Batrachus, which has already been made, shows
the differences that will probably be found to exist in the mode
of origin of this system in the two groups of fishes. So far as
the innervation is concerned, there is great similarity between
Batrachus and selachians. Ewart (12) has shown that the
lateral line organs are supplied by the dorsal branches of
the VII and X cranial nerves.
7. Dipnoids. — Pinkus (13) has shown that the commissure
connecting the VII and X is quite prominent in Protopterus.
He does not describe this nerve as connecting with the branches
extending on to the body, but shows its union with the vagus
ganglion. This commissure is undoubtedly homologous with
the R. recurrens facialis of Batrachus. A few of the sense
organs of Protopterus are enclosed in canals, but they are, for
the most part, on the surface of the body, as in Batrachus.
8. Cyclostomes. —The commissure between the VII and X
has been found in Petromyzon and figured by Ahlborn (33).
Stannius (34) speaks of the N. lateralis as “formed partly by
a vecurrent branch from the facialis passing around outside the
auditory capsule, a thing which does not occur in the N. lateralis
No. 2.] LINE SYSTEM OF BATRACHUS TAU. 251
in higher forms.’ This is a complete description of the course
of the R. recurrens facialis in Batrachus.
9. Amphibia.—While Batrachus is a true teleost, there are
certain superficial resemblances to the Urodeles, the sense
organs of both having much the same appearance and arrange-
ment on the body.
As regards the course of the cranial nerves, Strong (11)
has pointed out the remarkable homologies that are presented
in the tadpole and the teleost ; the dorsal branches corre-
sponding to those of teleosts being present in the tadpole but
becoming aborted in the aduit frog.
General Summary.
Development of organs and canals.— The sense organs of the
lateral system in Batrachus arise from special cords of cells
formed in the lower layer of the epidermis. These cords origi-
nate from certain thickenings which make their appearance in
the auditory region of very young embryos, and proliferate
along definite lines on the head and trunk. The enlarged
growing end of one of these cords pushes its way from the
auditory region to the extreme posterior part of the body,
the swollen appearance remaining conspicuous for some time
in the region of the caudal fin.
These thickenings of the ectoderm give rise to the sense
organs; each organ arising as a “local proliferation” of cells
along the cord (Fig. 15). These cells push through the over-
lying epidermal cells and gradually take on the form and char-
acter of the adult organ, having the hair cells well developed,
and the so-called “cupola” structure surmounting the organ.
In a later stage each organ sinks slightly below the surface,
and a pointed fold of the skin projects on either side of it.
This is the permanent condition of the majority of the sense
organs of Batrachus. On each side of the head, however, four
short canals are formed. They enclose organs identical with
those remaining on the surface, and the canals may be regarded
as a fusion and extension of the paired flaps which serve to
protect the free organs. In the adult the canals lie in open
252 CLAPP. [Vor. XV.
channels of the dermal bones and only primary pores are
present.
Tnnervation. — The dorsal branches of the VII and X cranial
nerves supply the lateral line system. The supraorbital line
of organs are innervated by the R. ophthalmicus superfacialis ;
the infraorbital by the R. buccalis facialis ; the operculo-man-
dibular by the R. mandibularis externus; while the vagus sends
branches to the single canal organ in the temporal region, as
well as to the organs on the top of the head.
The anterior organs of the trunk are supplied by the R. re-
currens of the VII, which forms an anastomosis with a branch of
the vagus just outside of, and posterior to, the auditory capsule,
and extends on to the body, occupying a position directly super-
ficial to the N. lineae lateralis. The R. recurrens in Batrachus
is probably the same as the R. dorsalis recurrens facialis (tri-
gemini) of the Siluroids, or of the cutaneous quinti in Gadus,
although following a different course on the side of the body.
It remains for future investigation to determine the exact
innervation of the organs on the body of Batrachus.
The complexity of the peripheral nervous system grows more
apparent with every step of advance in methods of investiga-
tion. In Kupffer’s words, ‘‘ The researches of the last decade
in comparative embryology have shown that the development
of the peripheral nervous system is a far more complicated
process than it was formerly supposed to be” (36). This
is especially true in the case of the vertebrate head, as the
recent work on Amphioxus by Hatschek (37), and the important
investigations by Kupffer (38) on Ammocetes clearly show.
In the views of Hatschek (37) we encounter a slightly modi-
fied form of Balfour's hypothesis in regard to the origin of
both cranial and spinal nerves from a type of segmental nerves
which had only dorsal, yet mzved dorsal roots. According to
Hatschek the spinal nerves have lost certain elements, while the
cranial nerves have retained more of the primitive characteris-
tics. In Petromyzon, Kupffer (38) has shown that the “dorsal
spinal nerve root” and the “mixed head nerve root” exist
together side by side as codrdinate components of a typical
head nerve.
No. 2.] LINE SYSTEM OF BATRACHUS TAU. 253
In the researches of Kupffer (36), we gain important addi-
tions to our knowledge of the development of the cranial
ganglia in connection with the thickenings of the ectoderm
which have long been recognized, but little understood. Since
Beard’s (16) and Froriep’s (39) simultaneous discovery of “ bran-
chial sense organs” in the embryos of sharks, and the corre-
sponding transient structures in embryos of higher forms, there
has been much controversy in regard to the question of the
ectoblast elements entering secondarily into the formation of
the cranial ganglia and nerves. There has been much hesita-
tion on the part of investigators in accepting this fact, for, as
Froriep (39) has said, “It would certainly bring about a
fundamental change in our views, were we to be convinced that
during a long period of embryonic development, the whole
ectoblast possessed the capacity to act as ‘ Nervenkeim.’”
It is now settled beyond dispute that these ‘“placodes”’ in
Ammocetes do furnish material to the processes growing down
from the neural ridge, and subsequently forming the cranial
ganglia and nerves. The peripheral portion of the “ placodes”’
may become the “foundations of the primary sense organs.”
The sense organs of the lateral line, although distributed over
the entire length of the trunk, are connected with ganglia
formed in the head region, and are therefore innervated by cranial
nerves. There seems every reason for considering the system
as belonging with the more highly specialized sense organs.
In his admirable paper on “The Cranial Nerves of Am-
phibia,’ Strong (11) has shown the extensive modification
which takes place in the nervous system of Rana, due to the
disappearance of the lateral line organs in the adult, and sug-
gests “the importance of taking into full consideration, as a
factor, the cutaneous sense organs in the attempt to obtain a
philosophical understanding of the changes undergone by the
peripheral and central nervous systems. The development
and specialization of these structures have probably played an
important part in the changes leading to the organization of
the vertebrate peripheral and central nervous systems.”
UNIVERSITY OF CHICAGO,
May, 1896.
254
33:
10.
16.
26.
12.
37:
18.
CLAPP. [Vou. XV.
BIBLIOGRAPHY.
AHLBORN, F. Ueber den Ursprung und Austritt der Hirnnerven von
Petromyzon. Zezt. f. wiss. Zool. Bd. xl. 1884.
ALLIS, EDWARD PHELPS, JR. The Anatomy and Development of
the Lateral Line System in Amiacalva. Journ. of Morph. Vol. ii,
No. 3. 1889.
AYERS, HowaArpb. Vertebrate Cephalogenesis. II. A Contribution
to the Morphology of the Vertebrate Ear, with a Reconsideration of
its Functions. Journ. of Morph. Vol. vi, No.1. 1892.
BatFour, F. M. Elasmobranch Fishes. 1877.
BEARD, JOHN. The System of Branchial Sense-Organs and their
Associated Ganglia in Ichthyopsida. Q./. M@. S. Vol. xxvi, N.S.,
No. tor. 1885.
BopENSTEIN, Emit. Der Seitenkanal von Cottus gobio. Zezt. f.
wiss. Zool. Bd. xxxvii, Heft 1. 1882.
CARRIERE, G. Postembryonale Entwicklung der Epidermis des
Siredon pisciformis. Arch. mikr. Anat. 1884.
COLLINGE, W. E. The Sensory Canal System of Fishes. Part I.
Ganoidei. Q./. M.S. Vol. xxxvi,N. Ss. 1894.
Emery, C. Le Specie del Genere Fieras nei Golfo di Napoli e
Regimi limitrofe. Fauna und Flora des Golfes von Neapel. Il.
Monographie. 1880.
Ewart, J. C. The Lateral Sense-Organs of Elasmobranchs. I.
The Sensory Canals of Laemargus. II. The Sensory Canals of the
Common Skate (Raia batis). Trans. Roy. Soc. Edinb. Vol. xxxvi,
Parti. 1892.
Fef£. Recherches sur le nerf pneumo-gastrique chez les poissons. 1869.
Froriep, AuGust. Entwicklungsgeschichte des Kopfes. Jerkel
und Bonnet’s Anat. Hefte. Bd.i. 1891.
Goope, G. Brown. Natural History of Useful and Aquatic Animals.
1884.
GOTTE, ALEXANDER. Die Entwicklungsgeschichte der Unke. 1875.
GuITEL, F. Recherches sur la ligne laterale de la Bandroie (Lophius
piscatorius). Arch. de Zool. ex. et Gen. Sér.2. Tome ix. 1881.
HaTSCHEK. Die Metamerie des Amphioxus und des Ammocoetes.
Verh. ad. anat. Gesell. Sechste Versammlung. 1892.
HorrMANN, C. K. Zur Ontogenie der Knochenfische. Arch. f.
mikr. Anat. Bd. xxviii, Heft 1. 1883.
JORDAN AND GILBERT. Synopsis of the Fishes of North America.
Bull. U.S. Nat. Mus. No. 16. 1883.
Kinespury, B. F. The Lateral Line System of Sense-Organs in
Some American Amphibia and Comparison with the Dipnoans.
Proc. Am. Micr, Soc. Vol. xvii. 1895.
No. 2.] LINE SYSTEM OF BATRACHUS TAU. 255
36.
38.
KUPFFER, C. von. Entwicklungsgeschichte des Kopfes. JMJerkel
und Bonnet’s Anat. Hefte. Bad. ii. 1892.
KUPFFER, C. voON. Studien zur vergleichenden Entwicklungsge-
schichte des Kopfes der Kranioten. Heft 2. Die Entwicklung
des Kopfes von Ammocoetes Planeri. Miinchen und Leipzig. 18094.
LeypiG, F. Integument und Hautsinnesorgane der Knochenfische.
Weitere Beitrage. Zool. Jahrb. Bad. viii, Heft 1. 1894.
LeypiG, F. Zur Anatomie und Histologie der Chimaera monstrosa.
Miiller’s Archiv f. Anat. u. Phys. 1851.
MERKEL, Fr. Ueber die Endigungen der Sensiblen Nerven in der
Haut der Wirbelthiere. Rostock. 1880.
MITROPHANOW, PavL. Etude embryogenique sur les Selaciens.
Arch. de Zool. ex. et Gen. Sér.3. Tomei. 1893.
Pinkus, F. Die Hirnnerven des Protopterus annecteus. Schwadlbz’s
Morph. Arbeiten. Bd. iv, Heft 2. 18094.
Piatt, J. B. Differentiations of Ectoderm in Necturus. Q. /. M.S.
Vol. xxxviii, Part 4. 1896.
POLLARD, H. B. The Lateral Line System in Siluroids. Zool. Jahrd.
Bd. v. 1892.
RYDER, JOHN A. A Preliminary Notice of the Development of the
Toadfish (Batrachus tau). Bull. of U.S. Fish Comm. 1886.
RypDER, JOHN A. Embryography of Osseous Fishes. Report of
U.S. Fish Comm. for 1882.
SAvI, PAuL. D’études anatomiques sur le systtme nerveux et sur
Vorgane electrique de la Torpillo.
SCHULZE, FRANZ EILHARD. Ueber die Sinnesorgane der Seitenlinie
bei Fischen und Amphibien. Arch. f. mikr. Anat. Bd.vi. 1870.
SEMPER. Merkel und Bonnet’s Anat. Hefte. Bd. i, p. 592. 1891.
SoLGER, B. Neue Untersuchungen zur Anatomie der Seitenorgane
der Fische. Arch. fi mik. Anat. Bad. xvii, Heft 1, iv, and Bd. xvii,
Heft 3.
StTannius, H. Das peripherische Nervensystem der Fische, anato-
misch und physiologisch untersucht. Rostock. 1849.
STRONG, OLIVER S. The Cranial Nerves of Amphibia. A Contri-
bution to the Morphology of the Vertebrate Nervous System. Journ.
of Morph. Vol. x. 1895.
VAN WHE, J. W. Ueber die Mesodermsegmente und die Entwick-
lung der Nerven des Selachierkopfes. 1882.
Witson, H. V. The Embryology of the Sea Bass (Serranus atra-
rius). Bull. of U.S. Fish Comm. Vol. ix. 1891.
WRIGHT, R. Ramsay. On the Nervous System and Sense-Organs
of Amiurus. Proc. Can. Just. Vol. ii, Fasc. No. 3. Toronto. 1884.
256 CLAPP. [Vou. XV.
REFERENCE LETTERS.
A, =anal fin.
ac.b. = accessory membrane bone.
ac.md.l. = accessory mandibular line.
a./. = anterior pit line of head (Amia).
an.r. = anterior root.
an.na. = anterior nasal aperture.
ar. = articular.
buc.f. = ramus buccalis facialis.
cb. = cerebellum.
C.H. = cerebral hemispheres.
c./. = cheek line.
com.VIZ-X = commissure between V// and X.
con.st. = connecting strand.
. = dentary.
d.b.V II = dorsal branch of V// nerve.
@.6.di.n.= dorsal branch of nervus lineae lateralis.
d.b.rec f. = dorsal branch of recurrens facialis.
d.l. = dorsal line.
d.o.J. = dorsal opercular line.
ep. = epiphysis.
F.R. = frontal.
fg. = facial ganglion.
gl. = glossopharyngeal nerve (Amia).
gg. = Gasserian ganglion.
h.l. = horizontal pit line of cheek (Amia).
7.0.C. = infraorbital canal (Amia).
71.y. = lateral line rudiment.
m. == muscles.
MX.C.= maxillary canal.
md.l. = mandibular line.
m.e.f.= ramus mandibularis externus facialis.
m.i. = middle dorsal pit line of head.
nll. = nervus lineae lateralis.
2.c. = noto chord.
oc.f. = supraoccipital foramen.
oll. = olfactory lobes.
OM.C. = operculo-mandibular canal.
OP. = operculum.
op.f- = ramus ophthalmicus superfacialis.
op.l. = optic lobes.
O.S. = opercular spine.
ot.n. = otic nerve (Amia).
p- = pore of canal.
P.= pectoral fin.
pigm. = pigment.
pl. = posterior pit line of head (Ainia).
No. 2.] LINE SYSTEM OF BATRACHUS TAU.
p-7.a. = posterior nasal aperture.
P.OP. = preoperculum.
p-r. = posterior root.
vec.f. = ramus recurrens facialis.
5.07". = sense organ.
SO.C. = supraorbital canal.
sp.c. = spinal cord.
st.com. = supratemporal cross-commissure.
stv’, = supratemporal branch of vagus.
7.A.=tuberculum acusticum.
7.C. = temporal canal.
V. = ventral fin.
v.b.rec.f. = ventral branch of recurrens facialis.
v.o./. = ventral opercular line.
v.g. = vagus ganglion.
v2 = second branch of vagus.
wad. = wolffian duct.
257
258 CLAPP.
EXPLANATION OF PLATE XVII.
Fic. 1. Side view of head of Batrachus tau, one year old. X 6. Showing
the appearance of the lines of sense organs in the adult, also the position of the
paired flaps and other projections of the skin on the head.
Fic. 2. Dorsal view of same. X 6.
Fic. 3. Ventral view of same. X 6.
Fic. 4. Dorsal view of skull. Natural size.
Fic. 5. Ventral view of mandible. Natural size. The grooves show the
position of the lateral line organs.
In Fig. 4, for /X read FR.
Journal of’, Vorphology. Vol. xv.
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EXPLANATION OF PLATE XVIII.
Fic. 6. Embryo of Batrachus at time of hatching, showing the different lines
of organs well defined. X 15.
Fic. 7. Side view of head of larva, showing sense organs on the surface. X 6.
Fic. 8. Front view of same.
Fic. 9. Side view of head, a few days later, showing canals in process of
formation.
FIG. Io.
FIG. 11.
FIG. 12.
FIG. 13.
X 6.
Front view of same.
Dorsal view of same.
Dorsal view of brain, showing roots of cranial nerves. X 6.
Diagram showing connections between the V/7 and X. The intra-
cranial commissure, and the anastomosis of the recurrens facialis with a branch
from the X. The VZ// has been omitted, as also the portion of the X innervating
the branchial region.
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262 CLAPP.
EXPLANATION OF PLATE XIX.
Fic. 14. A horizontal section of an Acanthias embryo 17 mm. long, showing
the growing point of the lateral line with its “pocket” and the nerve. Cam.
Z. 16, Oc. 3.
Fic. 15. Drawn from a preparation of the skin, showing the appearance of
the cells of the organs at an early stage of their development. Cam. Z. 4, oc. 3.
Fic. 16. A vertical section through a sense organ, showing the relation of the
connecting strand to the organ. Cam. Leitz 7, oc. 3.
Fic. 17. Preparation of skin showing the appearance of the rudiment of the
lateral line organs with its enlarged growing end. Cam. Z. 4, oc. 3.
Fic. 18. A horizontal section of an embryo of Batrachus 7 mm. long, show-
ing the position of the growing point of the line in its relation to the outer layer
of ectoderm and to the muscles. Cam. Z. 16, oc. 3.
Fic. 19. View of same magnified. Cam. Z. 4, oc. 3.
Fic. 20. Cross-section through the enlarged portion of the line. Cam. Leitz
FOCHS:
In Fig. 16, for m read x.
oo»
Journal of Morphology Vol. XV.
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204 GEAPR:
EXPLANATION OF PLATE XX.
Fic. 21. Diagram showing the innervation of the lateral line organs of Amia.
Journ. of Morph., Vol. ii, No. 3, Pl. XLII (reduced).
Fic. 22. Diagram showing innervation of the lateral line organs of Batrachus.
The lines in blue indicate the sense organ with the connecting strand.
Fic. 23. Diagram showing dorsal view of the same. The course of the nerves
is shown on the right, and the position of the connecting strand, in blue, on
the left. The short lines on each side of the organ represent the position of the
paired flaps.
Fic. 24. View of left side of body of Batrachus one year old. Showing posi-
tion of sense organs in adult. Natural size.
In Fig. 21, for st.chm. read st.com.
In Fig. 22, in supraorbital line, supply 1, 2, 3, 4, 5, 6, as indicating the organs
of that line.
For d.b.nec.f. read d.b.recf.
For 6.%.//. read d 0.2.11.
Sournal of Morphology Vol. xv: . ; PN
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COMPARATIVE CYTOLOGICAL STUDIES, WITH
ESPECIAL REGARD TO THE MORPHOLOGY
OF THE NUCLEOLUS.
THOS. H. MONTGOMERY, Jr., PH.D.
(Lecturer 1N ZooLoGy, UNIVERSITY OF PENNSYLVANIA.)
FROM THE LABORATORY OF THE WISTAR INSTITUTE OF ANATOMY AND BIOLoGy,
PHILADELPHIA.
CONTENTS.
TD pe INTRODUCTION Scccecesececsass sos terec cones soc to tercc sone eacascatver oes aubuvacarasparsssvesscogewvesmssesseees: ZOO
II. REVIEW OF THE LITERATURE UPON NUCLEOLI.......0...::::ecccesssecsseeeeeeesse 267
PAR OOO RAG AMM Lal be Kel CG seeeeeerncteceern een meeeneie ders semsenescohessssesecenssreresvecen smears 268
Pam Ocal Calm leit d At Le sense ecreenetsservareenenaserseestaceesestreuseveecastramsctereasnaees 375
Ga Synonym srorithe NermlNuCleolus!2eercrs:ccecesseresseccsnensnssarenenatoresnaseres 399
DDT Tet OBSERVATIONS soescee ces essteceeeer cae cen sore ceerar a coee tons satercea snes cosicectecactncernneervesereecesone 400
PANU EEL OCS OfmIS CLG Y reece ne ce rereree te opceerethe circ meectcsrerent revert yecrtissecnccsuvesssstet=s 400
US PPYOCOZOA es cnccecersacestonean cave svc cacss wer otveccesecoeedscucpsuescouscccbvestceteests Sonituseven 402
1. Gregarine from Lineus gesseremsis..........2....:.:0+00-e:eeceseee 402
2. Gregarine from Carinella annulata................cscececeeseseeeeees 400
Ga Metazoae rece cescsrerese es pact lens steamer masters
a. Egg Cells
1. Montagua pilata (Verr.)... FA
DTV OLO Sere a eae a os oe ane ee ar oea rete cece cena ue peneedls des San cceceacovetions 418
3. Amphiporus EinGnoses (Wen) Se soccdesastuscasiecsecnsec cos sissssetcnse 418
4. Tetrastemma catenulatum (Verr.) Mont... 423
HemletkastemImavelepansi(\VELi) saceecssssscssssieracsacscovsrsecreenneraeees 4.3L
6. Zygonemertes virescens (Wen) Money 433
7. Stichostemma eilhardi (Montg.) ..........2.-..-----s:-ccsseeeeeceeeeneee 437
8. Lineus gesserensis (O: FF. M.)..........2..-:ecsessscesssorsesesseeneveeee’ 446
Qh Siphonophore: (ROGalia?)) -cc.ccc sccqccseccsasessacnanassesdenenewecterunnte 451
TOME OLY. O Yet pearerer eee revere aes creeneeresseneveccsmnzaccnssasssecencessursuvenseneemenecerad
11. Piscicola rapax (Verr.)
DS OWA tC Gell seeeee reer ene aera y etna as esencscranrenccas a snansucavenaes atecestarezar’
12. Ganglion Cells of Doto ..
13. Ganglion Cells of } Vionaccn mie Cia. stecawerewAgS
14. Ganglion Cells of Piscicola rapax (Verr.) .....2.....00-2.. 475
15. Muscle Cells of Lineus gesserensis (O. F. M.)..........026 475
16. Muscle Cells of Piscicola rapax (Verr.).....-...--2:00--:ee0- 477
17. Blood Corpuscles of Doto
TOs Grlanits GENS OF WL) OO ier ccrecrces serene cececeeserecoreeecteceneenses
266 MONTGOMERY. [VoL. XV.
PAGE
19. Gland cells of Piscicola rapax (Verr.) ............ --- 483
20. Mesenchym Cells of Cerebratulus tars (SAR - 494
21. Ganglion Cells of Nemerteans 490
IV. GENERAL COMPARISONS AND CONCLUSIONS ... 497
APPENDIX) TO) DHE LITERATURE REVIEWS peccccccerestscsescerctecesesdete seteondeeveesen=aten - 539
LITERATURE LIST . saya tascaans Wereuaieeenca tous nate coe sncee aateste od asatoreoncanteetotaenee aoreeee eA
EXPLANATION OF PLATES . fees seas Seto St eectctas tants seseee aaterevntcesernce eee terpenes SOL
I. INTRODUCTION.
Tue following studies are based upon animal cells, both egg
cells and somatic cells having been investigated. They were
made, primarily, with a regard to the morphology of the true
nucleoli (plasmosomes), though numerous other points in onto-
genetic cellular development have been considered. In con-
nection with these observations the zodlogical literature upon
the subject of nucleoli has been reviewed as thoroughly as
possible, and, less completely, the literature from the botan-
ical standpoint as well; reviews are given of these observa-
tions of previous writers. No attempt has been made to
review the literature from the pathological standpoint. Under
the caption “ General Comparisons and Conclusions”’ are com-
pared together the more important deductions from my own
observations, and these are compared with those of previous
investigators.
The nucleoli are cellular structures which have been studied
to much less extent than other constituents of the cell, and
though there are numerous observations upon them, these are
so scattered through works of more general import that it is
well-nigh impossible to collect together all the previous inves-
tigations upon the subject. I hope that this explanation may
be taken as an apology by any authors whose papers I have
chanced to overlook.
At the laboratory of the Fish Commission at Woods Holl,
the following species were collected by me : Moxtagua, Ampht-
porus glutinosus, Tetrastemma catenulatum, Zygonemertes,
Lineus, Polydora,and Piscicola, At Sea Isle City, at the labo-
ratory of the University of Pennsylvania: Tetrastemma elegans,
Doto, and certain of the species found at the former locality.
Stichostemma was collected in the aquaria of the University
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 267
of Berlin; and the preparations of the siphonophore Rodalia
were kindly placed at my disposal by Dr. E. G. Conklin.
Doto and Montagua belong to the family of the Acolzdizdae ;
Amphiporus, Tetrastemma, Zygonemertes, and Stichostemma are
Metanemertini; and Lineus and Cerebratulus are Heteronemer-
tint; Polydora is a Polychaete; and Piscicola a rhynchobdellid
leech.
The present paper was sent to Dr. Whitman, editor of
the Journal of Morphology, on Feb. 3, 1897; on receiving the
MSS. again in March, 1898, I was able to incorporate in the
text reviews of the literature of the whole year 1897. No
other changes of importance, however, were then made in the
original text, except brief mention of observations which I had
made in the past year. It is my intention to follow this paper
by others on nucleolar structures, particularly on structures
which have received but little consideration in the present
paper, namely, ‘double”’ nucleoli and chromatin nucleoli.
II. REVIEW OF THE LITERATURE UPON NUCLEOLI.
In this review shall be considered separately, first, those
papers from the zodlogical, and, second, those from the
botanical standpoint. The references from zodlogical papers
I have endeavored to make as complete as possible, while my
citations from the observations of botanical observers are
much less numerous, though even in this I have consulted the
more important papers from 1880 to the present time. In
referring to the zodlogical papers, I have taken them up in
chronological order; and in doing so, shal] treat separately
the periods 1781-1860, 1861-69, 1870-79, and from the year
1880 to the present time I shall treat the literature for each
year separately, in order that the reader may more conven-
iently be able to turn to the citations from a given paper.
Under each year papers are reviewed according to the alpha-
betical sequence of the authors’ names. The _ botanical
literature, on the other hand, shall simply be treated in
chronological order, without regard to any division into periods.
The full titles of the papers referred to are to be found on
268 MONTGOMERY. [Vou. XV.
page 542, where their arrangement is according to the alpha-
betical order of the authors’ names, both the zodlogical and
botanical papers being in this one list. A certain number
of contributions dealing with nucleoli are entered into the
literature list, which I was unable to find in the libraries
at my disposal; all such papers have been distinguished by
an asterisk (*); the contents of some of the latter I have
reviewed from the citations of other writers.
Literature reviews are here given of all papers, with the
object of furnishing a reference library on the subject; in
Chapter IV, consequently, brief allusions only are made to the
views of particular authors, and readers can compare their
views by referring to the present section. This arrangement
of the literature appears the most practical.
A. ZooLoGIcAL LITERATURE.
1781-1860.
Fontana (1781, cited by Carnoy, ’84) was the first to figure
the nucleolus in the nucleus, which he describes as “un corps
oviforme, pourvu d’une tache en son milieu.”
The discoverer of the nucleolus in germinal vesicles is
R. Wagner (’35), and he termed it “ Keimfleck” or “macula
germinativa.’”’ He notes that the germinal vesicle of Uzzo and
Anodonta “zeigt constant zwei Flecke in Form von Kreisen,
welche sich schneiden, selten finden sich Abweichungen; der
grossere derselben méchte eine gewisse Aehnlichkeit mit dem
Keimfleck haben.” In his ‘‘ Nachtrag”’ to the same paper, he
states: “‘Der Keim ist bei seinem ersten Auftreten eben das,
was ich Keimfleck genannt habe. Es ist eine Schicht korniger
Masse, welche bald einfach (Saugethiere, Schnecken, Insekten
etc.) als Fleck erscheint, bald mehrere zerstreute Kiigelchen
bildet (Flusskrebs, Fische, Batrachier), . . . die an der inneren
Wand des Keimblaschens angeheftet sind.” In two subsequent
communications (’36, '37) he notes the occurrence of nucleoli
in the germinal vesicles of Coryna, Lucernaria, Cyanea, Chry-
saora, Asterias, and Insecta, and finds in Melolontha vulgaris
one large and one small nucleolus. Finally he remarks : “ Viel-
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 269
leicht bildet das Material des Keimblaschens und der Keim-
flecke die Grundlage zum serésen Blatt und zum Fruchthof
der Keimhaut.’”’ (Jones, '35, '37, does not mention the nucle-
olus; accordingly, he is not the discoverer, as is claimed by
Bischoff.)
Valentin ('36, cited by Carnoy, '84) describes the nucleolus
as a “rundes Korperchen, welches eine Art von zweitem Nucleus
bildet.”” (On this historical ground Carnoy considers the term
“nucleolus ”’ should be limited to his “ nucléole-noyau.”’)
Valentin (39, mentioned by Carnoy, ’84) introduces the
terms “nucleolus” and “Kernk6érperchen’’; the latter term
was proposed also by Schwann ('39) in the same year.
Bischoff ('42) found in the egg of the rabbit one nucleolus,
“ein schwach granulirtes Kornchen,” which he considers to be
a “ Zellenkern.”
Vogt ('42) found several nucleoli (six to twelve) in the ova of
Coregonus ; these subsequently migrate into the yolk to form
the first cells of the blastoderm.
Leydig ('49) describes in the germinal vesicle of Wephelis
one nucleolus, in C/epszve one or numerous ones, in Piscicola
two to four, while in Haemopis ‘der Keimfleck war einfach,
8-formig oder doppelt.”’
Kolliker ('49) studied numerous Gregarines, and concludes
that the nucleoli (‘‘Kornchen’’) “bei manchen Gregarinen
gewisse bestimmte Entwickelungen durchlaufen, namlich bei
jungen Individuen einfach vorhanden sind, bei alteren allmalig
in zwei, drei oder mehr Korner zerfallen.”’ In G. ¢terebellae,
clavata, saenuridis, and enchytraet there is a single nucleolus ;
G. sipunculi has from one to six; G. heeri, six to eighteen ;
G. sieboldiz, one to seven, which are either homogeneous or
vacuolar, or else only one or two are present, and each of these
is composed of a mass of smaller ones ; G. dvevirostra has from
six to nine nucleoli.
Lovén (49) studied the eggs of Modiolaria, Cardium,
Patella, and Solen, and found that during the process of
fecundation the nuclear membrane ruptures, and the nucleolus
passes out through the vitelline membrane. (It is very prob-
able that he confused the nucleolus with a pole body.)
270 MONTGOMERY. [VoL. XV.
Quatrefages (49) found that preceding the first maturation
division of Zeredo the nucleolus dissolves in the nucleus.
v. Wittich (49) found that in the germinal vesicles of
Lycosa, Theridium, Epeiva the “Keimfleck” first appears
“als ein matt gelblicher, nicht immer scharf begrenzter, aber
durchaus homogener Fleck, wird immer entschiedener rund,
verliert seine Homogenetat, indem er hie und da den Schein
von unregelmassig rundlichen Aushohlungen bietet, und neben
ihm treten zuletzt zerstreut ungleich geformte Korperchen
auf, die dem ersteren sehr ahnlich, an Zahl immer mehr
zunehmen, je mehr sich das Blaschen [Kern] seinem ganz-
lichen Schwinden niahert.” In Gasterosteus aculeatus the
number of the ‘ Keimflecke”’ increases with the size of the
egg. In the youngest germinal vesicles of Fringz//a there is
at first no nucleolus, later a single large, excentric one.
Leydig (50) finds that in the ovarial egg of Paludina vivt-
para there are two widely separated nucleoli, while in the ripe
egg they are in contact with each other : “so muss wohl ange-
nommen werden, dass der achterformige Keimfleck des reifen
Eies durch Aneinanderriicken und theilweises Verschmelzen
der friiher getrennten Korperchen entstanden sei.”’
Leydig ('52), ovum of Synapta digitata: there is a single
nucleolus with a vacuole ; ‘‘ was aber als eigenthiimlich hervor-
tritt, ist, dass er constant an einem Pol des Keimblaschens
liegt und zwar in einer tellerformigen Grube desselben.”
Leuckart ('53) states: ‘Der Keimfleck bildet eine zusam-
menhangende Masse von feinkérniger Beschaffenheit und
opakem Aussehen, die unter dem Deckglaschen mancherlei
Formen annimmt und ohne Umhiillungshaut ist. Nicht sel-
ten lassen sich im Innern auch einzelne gréssere Molekiile
ganz deutlich unterscheiden. In manchen Fallen nehmen
diese Molekiile an Zahl und Selbstandigkeit in einem solchen
Grade zu, dass der ganze Keimfleck eine haufenformige
Aggregation von Kornern darstellt.”
Hessling (54) finds in the youngest eggs of Uzzo a single
large nucleolus ; in larger ova there is a larger and a smaller
nucleolus, the latter having divided off from the former, and
showing a different reaction to acetic acid.
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 271
Lacaze-Duthiers ('54) finds in the eggs of Lamellibranchs
either one nucleolus, or when two are present they are of
unequal size.
Leydig (55a) says that in the egg of Cyclas “der Keimfleck
hat constant die Bisquitform.’’ In a second paper of the same
year ('55b) he makes the following notes on the ova of Rotato-
ria: in Notommata myrmeleo there are about 100 finely granu-
lated nucleoli; in WV. szeboldiz “Die Keimflecke erscheinen
als Haufen von kleinen, hellen Kiigelchen,” and disappear in
the ripe egg ; in WV. centrura and Brachionus bakeri there isa
single large nucleolus.
Agassiz (57) in studying the egg of the turtle introduces
the following terms: “ectoblast”’ for cell membrane, “ meso-
blast’? for nucleus, “entoblast,” or “ Wagnerian vesicle,” for
the nucleolus, and “ entosthoblast,” or ‘“ Valentinian vesicle,”
for the body sometimes enclosed in the latter. In the youngest
ova the nucleoli are absent, later they become numerous and
large, though they disappear in the ripe egg. The excentric
vacuole (‘“ Valentinian vesicle”’) of the nucleolus “increases in
size at a greater proportionate rate than its parent, the
“ Wagnerian vesicle,” till at its final stage it oftentimes occupies
three-fifths of the diameter of the generating medium.”
Lacaze-Duthiers ('57), ovarian egg of Dentalium: at first
there is but a single nucleolus, later a second one appears and
apposes itself to the former; the volumes of the two are
different. (Cf. Fol, ’89.)
Remak ('58), blood cells of Gallus: “Es kann kaum einem
Zweifel unterliegen, dass die Theilung der Blutzellen mit der
Theilung des Kernkérperchens beginnt. .. . Die Regel ist,
dass das Kernkorperchen sich in zwei Theile abschniirt, und
ebenso der Kern in zwei Kerne. Wie es aber zuweilen vier
Kernkorperchen giebt, so finden sich auch zuweilen vier Kerne
in einer Zelle.”
1861-60.
Pfliiger ('63) found one nucleolus in the egg of the calf.
While in the “Urei” of the cat he makes the interesting
observation that after a division of the nucleus, whereby one
272 MONTGOMERY, [VoL. XV.
of the daughter-nuclei retained the original nucleolus, in the
other a new nucleolus soon appeared, first in the form of a
granular mass.
In the paper by Balbiani ('64) movements of nucleoli are
described for the first time, and these observations were made
upon the living eggs. The first kinds of movements which he
distinguishes are exhibited by the eggs of spiders: “ces
mouvements de la tache germinative sont caractérisés par la
production de prolongements transparents ayant presque tou-
jours la forme de lobes arrondis qui s’allongent et se rétractent
alternativement.”” The second kind of movements is shown
in the egg of Phalangium, where there is a single large,
spherical nucleolus, which appears spongy, owing to the pres-
ence of a number of vacuoles, some of which ‘s’élévent plus
ou moins au dessus de la surface en soulevant sous forme
d’une ampoule la couche la plus externe de la substance du
corpuscule.... Lorsqu’un porte son attention sur une de ces
vésicules superficielles, on ne tarde généralement pas a la voir
grossir insensiblement, en méme temps que la couche de sub-
stance qui forme sa paroi extérieure se souléve en s’amincis-
sant de plus et plus; puis, assez brusquement, cette paroi se
rompt comme sous la pression d’un liquide intérieur, et ses
bords se rétractent vers la base adhérente de l’ampoule qui se
trouve ainsi transformée en une petite cupule ou excavation
superficielle, . . . et bientdét il ne reste plus aucune trace de
Yampoule ni de l’excavation qui lui a succédé.” All the periph-
eral vacuoles discharge themselves thus in succession, while
at the same time the smaller central vacuoles increase in size
and wander towards the periphery to take the place of the
preceding. Balbiani compares these movements to those of
the contractile vacuoles of the Rizzopoda, but notes this dif-
ference: in the latter forms the vacuole always forms itself
at the same place again. In the eggs of Geophilus and of
Helix pomatia he finds that the vacuole discharges through a
small orifice.
Balbiani (’65b) describes some remarkable structures in ger-
minal vesicles, all studied in life. In Geophilus longicornis
there is an external infundibular canal extending from the sur-
No.2.] COMPARATIVE CYTOLOGICAL STUDIES. 273
face of the nucleus to the surface of the vitellus, its larger
opening being apposed to the nucleus. A smaller inner infun-
dibular canal extends from the nucleolus into the outer canal.
The numerous vacuoles of the nucleolus are contractile, and
empty into the inner canal. He believes that these canals
disappear at the time that the nucleolus does. ‘Dans les
ovules de la Chienne, aprés la séparation des follicules primor-
diaux, la vésicule et la tache germinative offrent chacune un
prolongement canaliculé dont l’un est intérieur a l'autre,
comme chez le Géophile. ... Chez la Raie, ot les ovules
renferment généralement d’un a quatre petits corpuscules
germinatifs creusés d’une vacuole centrale, chacun de ceux-ci
émet un nombre variable de petits canaux, ordinairement de
deux a quatre, lesquels traversent dans différentes directions
la cavité de la vésicule, percent sa paroi et vont se perdre
dans le vitellus ambiant. ... Chez les poissons osseux
et les Batraciens, dont les ceufs renferment ... un grand
nombre de taches germinatives adhérentes a la paroi interne
de la vésicule, celle-ci est entourée d’un systéme de canaux
rayonnants vers la surface de l’ceuf, légérement flexueux et
de longuer inégale suivant le trajet quils ont a parcourir
pour atteindre cette surface. Chaque canal est en rapport
avec un des corpuscules précédents, et présente un calibre
correspondant au diametre de ce dernier. . . . Quelquefois,
ainsi que je l’ai observé chez quelques Crustacés (Ecrevisse,
Cancer moenas), ces taches multiples s’ont paru en outre
réunies, dans l’intérieur de la vésicule, par des canaux qui
s’etendaient de l’une a l’autre.... Chez plusieurs Annélides,
Turbellariés, Mollusques et Acaléphes, dont j’ai examiné les
ceufs, ceux-ci ne renfermaient pour la plupart qu’une tache
germinative simple, souvent assez volumineuse, en rapport
avec un canal unique renfermé dans l’intérieur d’un deuxiéme
canal émanant de la vésicule germinative.”’ In the germinal
spots of Helix, Vorter, and Prostomum he noticed one or
several contractile vacuoles.
Schrén (65) finds in the eggs of the cat and rabbit one or
two “Korner” in the nucleoli of the larger eggs, though not
in those of the smaller eggs. He considers the ‘ Korn” dif-
’
274 MONTGOMERY. [VoL. XV.
ferent in structure and substance from the rest of the nucleo-
lus, and that it is characteristic for a certain stage of the cell.
Stepanoff (65) describes for the youngest germinal vesicles
of Cyclas two nucleoli which are unequal in size, while in more
mature ova there are usually two (seldom one) large ones. He
figures, further, in one nucleus a smaller nucleolus in contact
with a larger one.
La Valette St. George ('66) studied in iodized serum the
germinal vesicles of various animals. In the egg of the kitten
there is one large nucleolus, either homogeneous or finely
granular, containing sometimes a large vacuole. In that of
the embryo of a sheep he noticed one or several nucleoli, with
slight differences in size, finely granular in structure, and con-
taining each a clear vacuole. In the egg of a larva of Lzdella
there was a small and a large nucleolus, the latter being darker
and more refractive, and spherical or irregular in form ; “seine
Substanz war entweder homogen oder zeigte je nach der Ein-
stellung des Mikroskopes hellere oder dunklere Flecken von sehr
verschiedener Zahl und Grosse, von unmessbarer Kleinheit bis
zu zwei Drittel des Keimfleckes.... Anfangs war der grosse
Keimfleck unregelmassig geformt fast viereckig und zeigte in
der Mitte eine hellere Stelle, etwa ein Drittel so gross wie der
ganze Keimfleck und daneben ein zweites kleineres Fleckchen.
Nach einer Viertelstunde hatte er seine Form geandert,
der kleinere Fleck war verschwunden, der gréssere nach der
Spitze zu geriickt. Nach Verlauf einer halben Stunde war er
kuglig geworden und jene helle Stelle verschwunden.” (In
this last stage the nucleolus touches the nuclear membrane,
according to his Fig. 2c.) In the egg of Porcellio scaber the
nucleolus is an irregular granular mass, and later becomes a
massive body; ‘“zuweilen stellt er einen nach einer Seite
geoffneten Ring dar, oft auch eine ausgehdhlte Kugel.” By
these observations he believes he has proved what Schroén
termed a solid granule (“Korn”) to be a vacuole.
Ransom ('67), egg of Gasterosteus: young eggs with numer-
ous peripheral germinal spots, which are spherical and homo-
geneous. He supposes these ‘are soluble in some of the
constituents of the yolk, and we may thus explain their disap-
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 275
pearance in ripe ova.” A 1.5% solution of NaCl gives rise to
vacuoles in the nucleoli (this antedates the observation of
Morgan, '96).
Van Beneden (69) studied Gregarina gigantea: ‘Le nombre
de nucléoles varie 4 chaque instant ; quelques-uns disparais-
sent, tandis que d’autres se forment ; ils apparaissent sous
forme d’un petit point presque imperceptible ; ce point grandit
jusqu’a certaines limites; il devient un véritable corpuscule
formé d’une substance homogéne trés-réfringente, puis le
corpuscule diminue de volume ; il réfracte de moins en moins
la lumiére, enfin il disparait.”
Claparéde ('69) found in the egg of Lumbricus terrestris
that the nucleolus ‘ist doppelt, indem er aus zwei einander
beritthrenden ungleich grossen Kiigelchen besteht.”
1870-79.
Eimer ('71), epithelial cells of the snout of Za/pa: each
nucleolus is surrounded by a clear space (‘‘Hof”’), and the
outer boundary of this space “war bezeichnet durch zahl-
reiche kleine Piinktchen.... Im _ optischen Querschnitt
stellten diese Kornchen einen Kreis um den hellen Hof des
Kernes dar.”
Eimer ('72) finds in the earlier stages of the egg of Lacerta
that all the nucleoli are grouped near the center of the
nucleus, while in more advanced ova there are numerous larger
peripheral nucleoli, and smaller ones in the other portions of
the nucleus ; around each of the large peripheral nucleoli are
situated concentric rows of smaller ones. Here, as well as in
Cistudo, Testudo, and Tropidonotus, the smallest nucleoli are
homogeneous, while the larger contain vacuoles. He con-
cludes that “die complicirt gebauten Keimflecke aus einfachen
Kornchen”’ are built up.
Kleinenberg (72) : in the egg of Hydra the single spheri-
cal nucleolus contains “ein auffallend stark lichtbrechendes
Korperchen. .. . Nach kurzer Zeit schwindet es wieder.”
The nucleolus then becomes irregular in form, breaks into
small granules, and he supposes that these latter become
dissolved.
276 MONTGOMERY. (VoL. XV.
Eimer (’73), nervous system of Beroé: each nucleus contains
one large nucleolus. ‘Aufmerksamer Beobachtung kann es
nicht entgehen, dass jede Epithelzelle von einer Primitiv-
fibrille versorgt wird... . Ich kann nur so viel sagen, dass
ich dieselbe [Primitivfibrille] stets auf das Centrum des Kerns
zugerichtet sah, so dass ich zu der Ansicht hinneige, es werde
sich spaterhin ihre Endigung im Kernkorperchen feststellen
lassen.”
Fol ('73) noticed in the egg of Geryonia fungiformis one
large nucleolus, containing one large, or several smaller
vacuoles. :
Auerbach (74). This important paper I have been unable
to consult in the original, and quote from citations by
R. Hertwig ('76) and Flemming ('82). According to Auerbach
the nucleus is originally a vacuole in the protoplasm, around
which a layer of the latter becomes differentiated to form a
nuclear membrane. In this vacuole a nucleolus appears
later, being derived from the protoplasm, either by a separa-
tion of particles from the nuclear membrane or is produced
out of those protoplasmic particles which had penetrated
from the protoplasm into the original vacuole. He distin-
guishes “enucleolar,” “uninucleolar,” and ‘“ multinucleolar”’
nuclei, the first being the more primitive state. The nucleo-
lus has the value of an elementary organism: as long as it
is homogeneous, it is comparable to a cytode ; when a vacu-
ole appears in it, the latter stands in the same relation to
the nucleolus as this does to the nucleus, so that that vacuole
may be considered the nucleus (“Kern”) of the nucleolus.
The original single nucleolus can divide into numerous nucle-
oli, and the latter, by the disappearance of the nuclear
membrane, become free, and each develops into a separate
cell. Auerbach considers this theory as “eine vorlaufige,
noch mit Vorbehalt aufzustellende und weiter zu priifende.”
A. Brandt (74) observed in life (in the blood fluid) slow
amoeboid motions of the single nucleolus of the egg of Blatta.
Flemming ('74) investigated the egg of Anodonta. In young
eggs the nucleolus consists of two apposed spheres of equal
diameter; in larger eggs one of these spheres is much larger
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. Tif
than the other. “Der kleinere Theil ist starker lichtbrechend,
auch etwas starker tingirbar, und beim Zerdriicken resistenter
als der grosse: beide zeigen sich hierbei als eine homogene,
zahe Masse.” The smaller has usually one large vacuole; the
larger has several smaller vacuoles. ‘Bei Anodonta scheinen
mir ausserhalb der Fortpflanzungszeit die beiden Theile normal
zusammenzuhangen. ... Kurz vor Eintritt der Befruchtungs-
zeit gewahrt man viele (aber nur reife, grosse) Eier, an deren
Kernkorpern eine wirkliche Trennung vorgegangen ist ; aber in
der Art, dass der kleinere Biickel stiickweise abgesprengt wird.”
Haeckel ('74) notes in the nucleolus of some egg cells “ein
innerstes Piinktchen, einen Nucleolinus, welchen man Keim-
punkt (Punctum germinativum) nennen kann. Indessen haben
diese letzteren beiden Theile (Keimfleck und Keimpunkt), wie
es scheint, nur eine untergeordnete Bedeutung,” only the
yolk and the nucleus being of fundamental importance.
Ludwig ('74) gives notes on the number of nucleoli in
various germinal vesicles. In the Coelenterata “Das Keim-
blaschen umschliesst durchgangig einen einzigen Keimfleck,
welcher haufig nochmals ein Kérnchen beherbergt.” There
is one germinal spot in Lchinus, Amphidetus, Solaster,
Branchiobdella, and in Trematodes and Rhabdocoeles.
Van Beneden ('75) remarks in regard to the egg of the
rabbit, that there is one nucleolus, and ‘deux ou trois petits
corps arrondis qui j'ai appelés pseudonucléoles.” When the
nucleus, during the maturation of the egg, has reached the
“zone pellucide”’ of the yolk, ‘le nucléole s’accole 4 la mem-
brane de la vésicule du céte de la surface de l’ceuf, la ot la
vésicule est appliquée contre la membrane. II s’aplatit contre
la membrane et se soude avec elle; sa substance plastique
s’étale en une plaque qui présente d’abord un épaississement
médian. Cette lame je lai appelée plaque nucléolaire.”
Shortly afterwards the latter body ‘grace probablement a la
contractilité inhérente a sa substance, . . . se ramasse en un
corps de forme variable, souvent ellipsoidal, quelquefois lenti-
culaire ou en forme de calotte, que j’ai appelé le corps nucléo-
laire.” The latter is the first pole body (‘corps directeur”’), the
nucleoplasm plus the pseudonucleolus constituting the second.
278 MONTGOMERY. [VoL. XV.
Eimer ('75) studied the egg cells of Sz/urus in eye fluid,
and found the nucleolus to present amoeboid movements.
Kidd ('75) found slow amoeboid movements of the nucleoli
of the epithelial cells from the mouth of the frog. These
cells were placed in humor aqueus, and studied on a stage
heated to 39° C.
A. Schneider ('75) says: “Les nucléoles ne sont pas un
elément constant de la structure des Grégarines ; beaucoup
d’espéces en sont normalement privées. Dans les genres
Clepsidrina, Euspora, Gamocystis, il n’y a jamais qu'un nucleé-
ole, permanent, trés-volumineux et sphérique. . . . Dans tout
ces genres, jamais deux individus ne sont semblables a eux-
mémes au point du nombre, de la grandeur, de la configu-
ration, de l’opacité ou de la transparence de leurs nucléoles.”’
F. E. Schulze ('75) noticed in life that an equal division
of the nucleolus precedes that of the nucleus, in Amoeba
polypodia.
Auerbach ('76) repeats some of his previous observations
(74) and adds that the nucleoli show a further similarity to
the cytoplasm, in that they have a tendency to produce
vacuoles.
Balbiani ('76) describes certain structures in the egg of Steno-
bothrus, which may be chromatic filaments, though I may give
a brief citation in regard to them in this place. The con-
tents of the nucleus in the fresh state appear “rempli de
petites hachures pales, tantét paralleles les unes aux autres,
tant6ot distribuées plus ou moins irrégulierement dans la cavité
nucléaire. . . . A l'aide de l’acide acétique, on s’assure que
ces hachures sont déterminées par les corpuscules en forme de
batonnets étroits . . . chaque batonnet parait formé de petits
globules réunis en série.’’ At the time of nuclear division,
these ‘batonnets’’ become less numerous but larger.
Van Beneden in the same year ('76) gives the results of obser-
vations on the egg of Asteracanthion. There is one large nucle-
olus, and eight to fifteen small “pseudonucléoles.” He did not
notice amoeboid motions in these, but found change of form
and successive re- and disappearance of the nucleoli in Raza,
Polystomum, Gregarina, and Monocystis. ‘Mais je ne doute
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 279
pas que les différences constatées dans la forme de la tache
germinative ne doivent étre attribués a la contractilité de la
substance des nucléoles.’”’ The vacuoles in the nucleoli are
probably “le résultat de l’union momentanée de certaines
parties de la substance nucléolaire avec le suc nucléaire.’’
Before its disappearance the nucleolus breaks into fragments,
which then dissolve in the “substance nucléaire.” In this
fragmentation one fragment is always larger than the others,
and contains the vacuole of the primitive nucleolus; it persists
until all the smaller fragments have disappeared.
Biitschli ('76) found that the nucleolus disappears before the
formation of the first pole spindle in Zylenchus, Anguillula,
Notommata, Brachionus, Triarthra, Aphis. He mentions that
von Siebold, in 1848, first introduced the name “nucleolus” for
the micronucleus of the /zfusorza, and compared it with the
nucleoli of metazoan cells. He also cites some of the earlier
writers who compared the pole bodies with nucleoli.
O. Hertwig ('76) calls the nucleolus “das wichtigste Form-
element des Kerns,’”’ and terms its substance “ Kernsubstanz”’
in opposition to the “ Kernsaft’’ (compare his brother’s paper
of the same year). In the process of maturation he holds that
“der Eikern der aus dem Keimblaschen frei gewordene oder
ausgewanderte Keimfleck ist.’’ He noticed vacuoles in, but not
amoeboid movements of, the germinal spot of Toxopneustes
lividus; he observed such motions, however, in the germinal
spots of Rana and Pterotrachea.
R. Hertwig (’76) terms the dense substance of the nucleolus
“ Kernsubstanz.” ‘Entweder leiten sich die vielen Kern-
korper direkt aus dem homogenen Zustand des Kernes ab,
indem die Aussonderung der Kernsubstanz an verschiedenen
Punkten gleichzeitig begonnen hat; oder die zahlreichen
Nucleoli sind, . . . durch Theilung aus einem urspriinglich
einfachen Nucleolus entstanden.” He believes that the
“Nucleoli die Trager der Kernfunction sind. ... Somit
miissen wir in allen den Fallen, in denen sich ein oder mehrere
Nucleoli im Kerne differenziren, in diesen die Thatigkeitscen-
tren des Kernes erblicken.”
Schwalbe ('76) studied the nuclei of retinal ganglion cells of
280 MONTGOMERY. [VoL. XV.
the ox, rabbit, and sheep: in the smallest nuclei there is no
nucleolus within the nucleus, but there are small peripheral
prominences on the inner surface of the nuclear membrane ;
when a nucleolus is present within the nucleus it is jagged in
outline, with fine, thread-like processes. The substance of the
nuclear membrane ‘stimmt in allen Eigenschaften mit der des
Kernk6rperchens vollstandig iiberein, und ist mit ihr continu-
irlich.”” Further, the substance of the peripheral prominences
is quite identical with that of the nucleolus, and “ Man konnte
in dem Falle, wo ein innerer Nucleolus fehlt, geradezu davon
reden, dass als Ersatz dafiir wandstandige Kernkorperchen vor-
handen seien.”’ In similar cells of the calf, there are no nucleoli
in the smallest nuclei; in larger ones there are from two to
four, one or two lying within the nucleus, the others being mere
thickenings of its membrane; ‘‘beim Wachsen des Kernes
(12.5) nimmt die Hohe und Zahl dieser Wandverdickungen
immer mehr ab, wahrend im Innern ein gut ausgebildeter
zackiger oder eckiger Nucleolus von 2.7 bis 3.6” das gewohn-
liche ist.” He considers the substance of the nucleoli and of
the nuclear membrane to be at first identical, and to be diffused
in the “ Kernsaft.” In the sympathetic ganglion cells of the
frog, he noticed, on the heated stage, that the nucleoli exhibited
slow changes of form; and in these nuclei he distinguishes
«‘Nucleolarsubstanz, den Kernsaft und die reticulare Sub-
stanz.””
O. Hertwig ('77a) found in the egg of Haemopis one large
nucleolus, with usually one large vacuole; and also a number of
small nucleoli,;.some of which contain each a small central
vacuole. In the production of the pole bodies: ‘Aus den
Theilstiicken des Nucleolus und einem Rest des Kernsaftes
entsteht ein fasriger, spindelformiger Kern . . . es muss dahin-
gestellt bleiben, ob der ganze Nucleolus oder nur ein Theil des-
selben und ob die Nebenkiigelchen [Nebennucleolen ?] in die
Zusammensetzung der Spindel mit eingehen.”
v. Kennel ('77) remarks of the ripe egg of Malacobdella:
“der Kern . . . enthalt eine mehr oder minder grosse Anzahl
stark lichtbrechender runder Troépfchen, die sich meist an
seiner Peripherie befinden.”
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 281
Mark ('77): the salivary gland cells of Chzonaspis contain
each forty to fifty nucleoli; corresponding cells of Aspidiotus
have a single large one which may contain from two to seven
“nucleoluli.” In cells of the oval gland of Chionaspis the
nucleus contains a true nucleolus, usually without nucleoluli,
and also a ‘‘ Fetttropfchen,” which differs from the former in
color and refraction. (The Fig. 32 of the salivary gland cells
of Aspidzotus shows each nucleus to contain a double nucleolus,
containing a larger sphere apposed to, in one case separate
from, a smaller colorless sphere.)
A. Brandt ('78) gives observations on the germinal vesicles
of different forms. In Aeschna grandis: “Der vom Keimblis-
chen umschlossene Keimfleck ist wie dieses, urspriinglich
rund, aber in noch viel hoéherem Grade, und zwar unstreitig
bei allen von mir beobachteten Insekten, amédboid beweglich,
so dass seine Form meist sehr verschieden erscheint. Nicht
selten ist er in einige Theile zerfallen. ... In einzelnen
Keimblaschen lagen ausser dem Keimflecke noch ein oder
mehrere Kornchen von verschiedener Grésse ;—nur ein Paar
Keimflecke wurden aufgefunden, welche anscheinend aus zwei
aneinandergedrangten und theils iibereinander geschobenen
Kugeln bestanden.” In Periplaneta vacuoles as well as solid
“secundare Keimflecke”’ occur in the nucleolus. In the egg
of Nemura, after the action of acetic acid, the vacuoles in the
nucleolus increase in size and in each a small granule is to be
seen. In Gryllus, Lepisma, and Holostomzs the germinal spot
is amoeboid : ‘Die améboide Beweglichkeit veranlasst nicht
selten das Loslésen einzelner Partikel, welche, wie der Keim-
fleck selbst, améboid-contractil sind. Die Zahl und Grésse
dieser gelegentlich wieder zusammenfliessenden Partikel ist
eine dusserst. verschiedene”’; thus the nucleolus may break
into a number of equal-sized pieces, or into a mass of very
fine granules. In the egg of Zegenaria there is usually a
single vacuolated nucleolus, though sometimes there may
be present also two ‘“ Nebenkeimflecke.” In Déstomum the
“Keimfleck . . . ist in sehr hohem Grade mit amébenartiger
Beweglichkeit begabt,” and there is a central body in the
nucleolus which changes its form periodically. Brandt observes
282 MONTGOMERY. [Vou. XV.
in regard to the frog’s egg : ‘Der Keimfleck des Froscheies,
in den allerjiingsten Eianlagen meist ein zusammenhangendes
Gebilde, erscheint bekanntlich spater, in eine gréssere Anzahl
von rundlichen Kliimpchen zerfallen—und diese fand ich
(bei Rana esculenta) améboid gestaltet”; and adds, against
Biitschli ('76), ‘ist einzuwenden, dass dieser Zerfall des Keim-
flecks als amoboide Erscheinung keineswegs auf ein Absterben,
sondern im Gegentheil auf eine erhohte Lebensthatigskeit
hinweist.”’
Brock (78) : the immature ovum of Axguz//a has one or two
large nucleoli; the number of the latter increases with the
size of the egg.
Eimer (78) notes the great relative and absolute size of the
nucleus and nucleolus in ganglion cells, and finds it to be
paralleled only in egg cells.
O. Hertwig ('77b, '78a) noticed in the nucleolus of the
maturing egg of Asteracanthion certain changes, “die darin
bestehen, dass die in seinem Innern bisher zahlreich vorhan-
denen kleinen Vacuolen verschwinden und in seiner Mitte oder
mehr der Peripherie genahert eine gréssere Vacuole erscheint,
die fast ganz von einem kugligen aus Kernsubstanz bestehenden
Korper erfillt wird. . . . Plotzlich verschwinden die in ihm
gelegenen Vacuole mit ihrem kugligen Kérper unter dem Auge
des Beobachters,” and in consequence the nucleolus begins to
gradually shrink in size, and 1% hours afterwards has com-
pletely disappeared. The body within the large vacuole of the -
nucleolus corresponds to the smaller, more deeply staining
portion of the original nucleolus, and during the nuclear division
reaches out of an opening in the vacuole beyond the surface of
the nucleolus, takes on the form of a long, thin rod, and occupies
the middle point of the first pole spindle; while at the same
time the remaining portion of the nucleolus gradually breaks
into a granular mass, which then disappears. Also in Sphae-
vechinus, Ascidia, some Coelenterata, and various JJollusca, he
noticed a similar differentiation of the nucleolus into two sub-
stances, namely, a smaller, deeply staining portion apposed to,
or enclosed by, a lighter, larger portion.
O. Hertwig, in still another paper ('78b), investigated the
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 283
germinal vesicles of various animals. In Excope polystyla there
is one nucleolus in small eggs, several in riper ones: “Es
liess sich hier feststellen, dass die zahlreichen Nucleoli durch
Ablosung vom urspriinglichen einfachen Keimfleck entstehen.”’
Klein (78) studied the stomach cells of the newt, and con-
cludes “that in most cells the so-called nucleoli are local
accumulations of the intranuclear network, that they are incon-
stant in size and number, and that they are only transitory
appearances.”
Schindler (78), Malpighian tubules of insects: after a cell
has become obliterated by the outflow of its secretion, its
nucleus becomes a new cell, and its nucleolus a new nucleus.
Whitman (78) found in the egg of Clepsime one to three
nucleoli, each ‘composed of several highly refractive pieces.”
Bergh ('79) found in the egg of Gonothyraea (Campanularia)
a single large nucleolus, which is usually round, but sometimes
with irregular outlines caused by slow amoeboid movements
(observed in life), these motions being most vigorous later,
when the nucleolus begins to divide. It increases in size, and
acquires one or two vacuoles. In a later stage, but before the
production of the pole bodies, there are a number of irregular
nuclear bodies (staining as the original nucleolus), which had
been produced by division of the nucleolus; in one case he
actually observed the division of the nucleolus, which lasted
half an hour, and at the same time the vacuole of the primitive
nucleolus seemed to divide into two, so that each daughter-
nucleolus received a daughter-vacuole. ‘Oft macht es den
Eindruck, als ob das Volum der secundaren Keimflecke zusam-
mengenommen grésser ware, als das der primaren fiir sich
. eine active Wanderung der Nucleoli durch den Kernsaft,
wie dies Auerbach ['74] bei gewissen Nematoden in den Vor-
kernen gesehen hat, kommt wahrscheinlich hier nicht vor.”
The nucleolus also divides in the egg of C/ava. In the eggs
of Psammechinus and Echinocardium, the single nucleolus
begins to fragment before the chromatic network has disap-
peared. The Phallusta egg contains one large germinal
spot, which probably disappears without fragmenting: ‘ich
habe namlich unter Eiern, die im Keimblaschen einen scharf
284 MONTGOMERY. [VOL. XV.
begrenzten, durch die Osmium-Carminbehandlung rubinroth
gefarbten Keimfleck zeigten, auch solche gefunden, welche
statt dessen eine sehr feinkornige, bisweilen rubinroth, bisweilen
weniger intensiv rothgefarbte Masse enthielten, die nicht scharf
contourirt war, aber von derselben Grosse wie der Keimfleck.
Falls diese Deutung, es schwinde der Keimfleck ohne sich
vorher zu theilen, richtig ist, beginnt die Aufldsung desselben
mit dem Schwinden der Vacuolen in seinem Innern.”
Klein maintains his previous views in regard to the nature
of the nucleoli in two papers published in the following year
('79a, '79b).
I880.
Van Beneden (80) studied the egg of the bat, and found one
nucleolus (rarely two): “on trouve en outre quelques granules
trés petits, tous d’égales dimensions, répandus dans le corps
de la vésicule (pseudonucléoles)”’; the latter have no resem-
blance to any part of the chromatic filament.
Biitschli ('80) incorporates in his great ‘‘ Protozoenwerk”’ the
observations of preceding authors. In Ayalosphenia there
may be as many as six spherical nucleoli; in certain other
Rhizopoda the “Binnenkorper kann den von der Kernhiille
umschlossenen Raum nahezu vollig ausfiillen.” In the He/z-
ozoa the nucleoli are much as in the preceding group. In the
Radtolaria (for which Biitschli follows some of the observa-
tions of R. Hertwig, '79) there is usually a number of rather
large nucleoli, frequently containing vacuoles. The nucleus
of Thalassicola ‘“enthalt einen ansehnlichen, strangférmigen
und unregelmassig verastelten Nucleolus, dessen Masse nicht
ganz homogen, sondern ausserlich feinkornig ist”; it later
breaks into a number of segments. In Acanthometra the nucleo-
lus is at first spherical, while later “Aus dem Nucleolus-Pol,
welcher der Einstiilpungsstelle der Kernmembran zugewendet
ist, bildet sich eine helle homogene Masse aus, welche den
dunkleren Haupttheil des Nucleolus wie eine Kappe bedeckt
oder auch wie eine Vertiefung desselben eingesenkt erscheint.
Der Nucleolus erscheint demnach jetzt von zwei verschiedenen
Substanzen zusammengesetzt.” In many Flagellata a nucleo-
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 285
lus is absent, in others there is a single one, sometimes with
a vacuole; in the Choanoflagellata there is always one large,
spherical nucleolus, in the Cys¢oflagellata several of various
sizes; and in the Denoflagellata there may be several small
nucleoli, which are sharply localized from the chromatin, but
show the fine reticulation of the latter element. In the (7/ata
and Swctorta there are nucleoli of varying size and number in
the macronucleus, but none in the micronucleus.
Chun (go) finds in the egg of all Crenophora a single large
nucleolus, very rarely two.
Engelmann ('g80) figures the nucleoli of certain ciliated cells
of various invertebrates as each surrounded by a clear space,
the outer boundary of which is marked on optical cross-section
by a circle of granules.
Flemming (80) concludes in regard to the nature of the
nucleolus: ‘Dass die Nucleolen iiberhaupt keinerlei mor-
phologischen Antheil an der Kernvermehrung nehmen”’; and
«Dass die Dinge, die wir Nucleolen nennen, vielleicht gar keine
morphologisch wichtige Theile des Kerns sein mégen, sondern
nur Ablagerungen von Substanzen, welche fiir den Stoffwechsel
im Kern verbraucht und wieder neugebildet werden; sie wiirden
damit gewiss physiologisch wichtige Theile des Kerns bleiben,
—was ohnehin durch ihr fast allgemeines Vorkommen bewahrt
wird, — aber doch keine eigentlich organischen, d. h. morpholo-
gisch-wesentlichen Kernbestandtheile.”’
O. Hertwig (80) found in the eggs of Chaetognatha numerous
small nucleoli.
Shafer (80), ovum of Gallus: there is a single nucleolus,
which in young germinal vesicles consists of a homogeneous
matrix which stains slightly with haematoxylin, and a number
of coarse granules which stain deeply; in larger ova the
nucleolus is homogeneous throughout and stains deeply. The
threads radiating from the periphery of the nucleolus may be
either artefacts or may be regarded as extrusions of the homo-
geneous substance of the nucleolus. Ovum of Lepus: in
younger nuclei the nucleolus has the same general structure
as in the fowl, though it is more irregular in form. In some
larger ova the nucleolus “is represented by a number (a dozen
286 MONTGOMERY. [Vou. XV.
or so) of globules of varying size which appear to lie loose
within the germinal vesicle. An intravesicular network is
sometimes present, and serves to unite the granules of the
macula. ... It is possible that the homogeneous matrix
above described may represent the remains of such a network,
the filaments of which have shrunk up into a mass on contact
with the hardening reagent ” (picric acid and alcohol).
Trinchese (’g0, according to Platner, '6) found in the germinal
vesicle of Amphorina coerulea a “macchia germinativa laterale
o accessoria,’ and a ‘“macchia germinativa principale,’ the
latter being about seven times the size of the former.
I88T.
Balbiani ('81) investigated the salivary gland cells of the
Chironomus larva. There are here ‘‘ Deux gros nucléoles irré-
guliers, larges de 0.03 a2 0.04 mm., bosselés a leur surface, et
formés d’une substance réfringente granuleuse, creusée d’un
plus ou moins grand nombre de vacuoles isolées ou confluentes.
I] arrive assez souvent que les deux nucléoles se confondent par
une partie plus étroite qui les réunit comme une sorte de pont;
d'autres fois enfin, ils se fusionnent plus ou moins intimement
en un seul nucléole, dont le diamétre est le double de celui des
nucléoles isolés.” The ends of the chromatin filament are
apposed against the nucleolus; and the latter differs both
chemically and morphologically from this ‘cordon nucléaire.”
Giard ('81) observed in the egg of a Sfzonzd during life a
single central nucleolus. A certain time before completed
maturation an ‘élément cellulaire” appears in the nucleus,
which is a little smaller than the latter, and encloses in its
center a small “noyau”’: “ D’abord fort éloigne du nucleéole, il
s’en approche progressivement et vient s’appliquer a sa surface,
ou il s’aplatit et prend la forme d’une double calotte. En
s’appliquant de plus en plus contre le nucléole, il perd son
noyau et finit par se réduire a une double membrane qui entoure
le nucléole,” . . . and finally its substance fuses with that of
the nucleolus.
Hubrecht (81), egg of Proneomenia: “in all the different
stages of development of the ovum the germinal spot is double:
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 287
a larger and a smaller sphere may be distinguished, which,
however, are not connected in any way whatever... but
perfectly free and independent of each other.”
Mark (81) finds that during the maturation of the egg of
Limax campestris the male as well as the female pronucleus
may contain as many as fifty or sixty “ pronucleoli,”’ which dis-
appear before the copulation of the two pronuclei. In an
undetermined species of Lzmax he “observed in both female
and male pronuclei a single nucleolus of much greater size and
more deeply stained than the other nucleoli.”
Pfitzner (81) finds that the “‘Kernsubstanz” is contained in
the reticulum and the nucleoli; the latter lie within the meshes
of the former, and their réle is problematical. ‘ Wahrend des
weiteren Verlaufes der Karyokinese verschwinden sie, werden
anscheinend allmalig aufgezehrt, ohne direkt mit dem Geriist
in Verbindung getreten zu sein.”
Retzius ('81, cited by Van Bambeke, ’85): the nucleoli are
simple local accumulations of the chromatin, derived from the
nuclear reticulum.
7882.
Blochmann ('82) observed in the egg of (Veritina one large
nucleolus containing a vacuole. Preceding the pole body pro-
duction, the nuclear membrane vanishes, and the nucleolus at
first retains its original size, then breaks up into several equal-
sized fragments. ‘Dass die Elemente der Kernplatte aus
Theilstiicken des Nucleolus entstehen, kann bei unserem
Objekt keinem Zweifel unterliegen, da ich alle Uebergangszu-
stande vom unversehrten Nucleolus bis zur ausgebildeten
Kernplatte beobachtet habe.’ After the two pole bodies have
divided off, the remaining chromosomes in the female pro-
nucleus fuse together to form a deeply staining, spherical
body, which resembles the original nucleolus.
Flemming in his classical work ('82) gives the following defini-
tion of nucleoli: ‘Substanzportionen im Kern von besonderer
Beschaffenheit gegeniiber dem Geriist und dem Kernsaft, fast
immer vom starkeren Lichtbrechungsvermogen als beide, mit
288 MONTGOMERY. [Vou. XV.
glatter Flache in ihrem Umfang abgesetzt, stets von abgerun-
deter Oberflachenform, meist in den Geriistbalken suspendirt,
in manchen Fallen ausserhalb desselben gelagert.’”’ A mem-
brane is absent around all nucleoli. He (erroneously) attrib-
utes the discovery of the nucleolus to the botanist Schleiden.
Flemming holds it probable that with the possible exception of
spermatozoa one or more nucleoli occur in every nucleus, of
which it is therefore an important organ (in this conclusion
he departs from the views expressed in his previous contribu-
tion, 80). “Die Zahl ist bei Thierzellen selten iiber 8 (mit
Ausnahme der Kerne meroblastischer Eier), bei den meisten
Arten von Thierzellen durchschnittlich 3-5. ... Es ist der
haufigste Fall, dass einer der Nucleolen an Groésse besonders
vorwiegt,”’ this being then the ‘“ Hauptnucleolus,” the others
‘“Nebennucleoli.” In the “ Hauptnucleolus”’ of the egg of
Lepus two parts are distinguishable, but he leaves it undecided
whether “die Unterscheidung von Haupt- und Nebennucleolen
eine durchgehende Geltung beanspruchen kann.”’ This inves-
tigator notes further: ‘Die absolute Grosse der Nucleolen
steht bei den meisten Zellenarten in, annahernder Proportion
zur Grosse der Kerne selbst.” The nucleolar vacuoles are
filled with fluid. In regard to the apparent clear spaces around
nucleoli, we read “dass dieses Phanomen nichts anderes ist als
ein Reflex, bedingt durch die rundliche Flache und starkere
Lichtbrechung des Nucleolus.” He did not find amoeboid
changes of form, but concedes that they may occur. The true
nucleolar substance differs from the chromatin. The nucleoli
are ‘“specifische Produkte des Kernstoffwechsels und zugleich
auch specifische Formtheile des Kerns . . . so kann man die
Nucleolen ganz wohl Organe des Kerns oder der Zelle nennen.”
They appear to be “ besondere Reproductions- und Ansamm-
lungsstellen des Chromatins. . . . Entweder ist also in den
Nucleolen noch ein anderweitiges Substrat vorhanden, in
welchem das Chromatin verarbeitet wird und mit dem es in
ihren durchlagert liegt, oder . . . die Substanz der Nucleolen
mag zwar in sich homogen sein, ist aber dann nicht identisch
mit Chromatin resp. Nuclein, sondern eine chemische Modifi-
cation, Vorstufe oder Doppelverbindung derselben.”’
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 289
Graff ('82) figures in the eggs of Proporus, Plagiostoma, and
Vorticeros a single nucleolus containing vacuoles.
Nussbaum ('82) studied the nuclei of gland cells (stomach
nucosa of various Vertebrata, epidermis glands of Argulus).
“Es liess sich im Allgemeinen feststellen, dass wahrend des
ungestorten Ablaufs der Secretion die mononucleolaren Kerne
vorherrschten, dass nach langerem Hunger die multinucleo-
laren Kerne an Zahl vermehrt waren. . .. Ein Driisenzellenpaar
der Saugscheibe von Argulus foliaceus hatte am 12. Oktober
mononucleolare Kerne; am 18. Oktober zeigten sich viele
Kernkoérperchen im Kern; nach und nach ging die Granulierung
der Zellen, die Strahlung verloren und die Kerne waren platte
Ovoide mit mehreren glanzlosen Korperchen darin.” From
these observations Nussbaum concludes: “So wird man den
Kern mit vielen Kernkorperchen als den Ausdruck einer Ruhe-
pause der Kernfunctionen auffassen kénnen, die entweder zum
kraftigen Leben oder zum Tode iiberleitet.”
Rauber (’82) figures the nucleoli in the ova of various verte-
brates, and distinguishes the following kinds of nuclei, with
regard to the mode of distribution of the “‘chromophile Sub-
stanz”’ (chromatin together with pyrenin): “globulare,” “ tra-
bekulare, “ filoide,” and “ gemischte.”
Seeliger ('82) finds that in C/avelina the nucleus of the loose
mesoderm cell (from which the ovum is derived) becomes the
nucleolus of the ovum, and its cytoplasm becomes its nucleus.
In the germinal vesicle there is then one large nucleolus, in
which nucleolini lie, and also (to judge from his figures)
vacuoles.
Vejdovsky ('82), egg cells of Sternaspis scutata: the young
nucleus contains at first one small nucleolus, bounded by a
membrane (though the latter structure would appear from his
figures to be a clear space enveloping the nucleolus). “ Beim
fortschreitenden Wachstum des Keimblaschens vergrossert
sich auch der Keimfleck, und zwar in der Weise, dass die ihn
umgebende Membrane einseitig sich verdickt und schliesslich
auf dem runden sich in Pikrokarmin stark farbenden Keimfleck
als ein glanzendes, gelbliches Biickelchen erscheint.” The
nucleolus disappears in the ripe egg.
290 MONTGOMERY, [VoL. XV.
1883.
Balbiani ('83) renewed his observations on the egg of Geophilus
longicornis, making several emendations. In very young eggs
there are two or numerous nucleoli, in larger eggs only one large
one, containing one or several vacuoles. In his previous paper
referred to, he assumed that the double tubular structure in
these eggs served for the purpose of an intraovular circulation ;
but in the present paper he offers another explanation: that
the double tubular structure later develops into a knotted cord,
the distal portion of which then divides into irregular frag-
ments, which become scattered through the yolk ; and then each
of these fragments, with the exception of one which becomes
the “noyau vitellin,” differentiates into cytoplasm, nuclear and
nucleolar substance, and then represents a cell of the follicular
epithelium.
Van Bemmelin (83) states of the eggs of Brachiopoda: “Sie
haben meist zwei Kernkorperchen, die enganliegend und stark
lichtbrechend sind. Ausser diesen nimmt man oft noch mehrere
lichtbrechende Kiigelchen in dem gefarbten Inhalte der Eikerne
wahr. Von Boraxkarmin werden sowohl diese Korperchen als
die Nucleoli stark gefarbt.” (Certain of his figures show one
of the nucleoli imbedded in another.)
Van Beneden (83), ovum of Ascaris megalocephala: there is
a single ‘corpuscle germinatif,”” which contains all the chro-
matin of the nucleus, and is contained within a special portion
of the nucleus termed the “ prothyalosome’”’; from one to three
“pseudonucléoles” also occur in the nucleus, but they play no
important part in the maturation of the egg.
Fol (83a), egg of Ciona intestinalis: there is here one large,
very refractive nucleolus containing a number of vacuoles
which he believes are artefacts, since they cannot be found in
the living egg, though their appearance after the action of
reagents would show that the substance of the nucleolus is
chemically not homogeneous. The nucleolus consists of a more
refractive cortical substance, and of a less refractive, clearer
medullary portion ; in the latter, the vacuoles are produced.
Fol maintains that the follicle cells arise by budding from the
No.2.) COMPARATIVE CYTOLOGICAL STUDIES. 29gI
egg nucleus : ‘Ce nucléole a une tendance bien évidente a se
placer dans le voisinage immédiat des noyaux folliculaires en
voie de formation. Le fait n’est pas constant, mais il est trop
fréquent’’; he did not actually observe that the nucleolus gives
off a part of its substance to the follicle cell, but supposes this
to be the case.
Fol, in a second paper ('83b) of the same year, finds that in
Ciona during the “production endogéne”’ of the follicular cells
a segment (diverticulum) of the egg nucleus breaks off, while
the (then peripherally situated) nucleolus gives a part of its
substance into this diverticulum, and the nucleolus then wanders
back to another portion of the nucleus. “Chez Asczdia mam-
millata, le bourgeonnement de l’enveloppe a lieu simultanément
en une foule de points, et il est tout ou moins admissible que la
substance de la tache germinative dispersée a la formation de
ces bourgeons.”’
Gruber (83) describes in Actinosphaerium the growth of a
supposed nucleolus and its division during mitosis into two
equatorial plates; though his figures would show that he
mistook true chromatin masses for a nucleolus.
Jensen (83) studied the ovum of Cucwmaria; there are from
fifteen to thirty nucleoli flattened against the nuclear membrane,
and containing vacuoles. As shown by treatment with acetic
or picrosulphuric acid, the outer layer of the nucleolus seems to
be a continuation of the nuclear membrane, so that the inner,
less refractive portion of the nucleolus appears to be situated in
a depression of the outer surface of the nuclear membrane.
La Valette St. George ('83, quoted after Platner, '6) found in
the egg of an /sofod one nucleolus which is at first homogene-
ous, later granular, and which may enclose a vacuole and show
amoeboid movements. In other cases there are either several
smaller vacuoles or one or two larger ones.
Leydig (83), from comparative studies, concludes that the
nucleoli ‘“‘sind Theile des Kernnetzes,”’ and that each of them
is enclosed ina small, clear cavity of the nucleus. “ Die Nucle-
oli kénnen als eine Vielzahl von Kornchen erscheinen, die
unter sich gleichwerthig sind.... Nicht selten lasst sich bei
genauem Zusehen in der Menge kleiner und unter sich gleicher
292 MONTGOMERY. [VoL. XV.
Kernkorper ein grosserer Nucleolus . . . auffinden (Epithel des
Eierstocks von Ag/ia tau).... Wahrhaft riesige Kernkorper
kommen zu Stande, wenn viele Nucleoli zu einem einzigen
Korper zusammenfliessen.... Priifen wir Herkommen und
Beschaffenheit der Kernkorper naher, so ist beziiglich der
kleineren Nucleoli leicht festzustellen, dass sie aus Verdich-
tungen oder Knotenpunkten des Kernfadennetzes den Ursprung
nehmen. Daher schon im frischen Zustande solche Kernkér-
perchen einen zackigen Saum haben, auch durch Spitzen und
Striche sich verbinden, die bis zum Rande des Kernes gehen.
Aber selbst die groésseren Nucleoli... erweisen sich als Um-
bildungen von Partien der Kernfaden.” In the ganglion cells
of the brain of Lzmax and Avion the nucleoli are jagged in out-
line, with long fibers. In the cells of the salivary gland of Vepa
they are three or four in number, bent and elongated in form.
Those of the corresponding gland in Vaucorts have often the
shape of a half ring, or may be lobular or band shaped, with
cross striation. In the salivary gland of Chironomus plumosus
there is usually a single nucleolus, spherical, lobular or tubular,
its radiating cavity filled with a homogeneous, refractive sub-
stance ; its wall contains vacuoles, ‘‘und starke Linsen lassen
deutlich werden, dass der ganze Kernkorper eben wieder
die Struktur eines Schwammgebildes besitzt.” Besides the
nucleoli there are in these cells several looped or contorted
bodies, one of which is always in connection with the nucleolus,
and all of which evince a cross striation, the nature of which is
as follows: ‘Mit Tauchlinsen unterscheiden wir abwechselnd
je eine dunkle und helle Querlinie und sehen die erstere, welche
leicht gekerbt ist, zusammengesetzt aus einzelnen kleinen
Stiickchen, vergleichbar den Elementen einer Muskelscheibe.
Die feinen Abtheilungslinien der den Querstrich bildenden
Stiickchen erstrecken sich ferner durch die helle Zwischenzone,
so dass dadurch auch eine Art von zartesten Langslinien zum
Ausdruck kommen kann.” He believes that these cross-stri-
ated structures “durch Umbildung des den Kern durchziehen-
den Maschenwerkes entstanden sind.” In young larvae these
structures are not seen immediately in life, but “nach und
nach, wahrend das Thier noch lebt, tauchen die querstreifigen
No.2.] COMPARATIVE CYTOLOGICAL STUDIES. 293
Bildungen auf.... Man darf wohl annehmen, dass die frag-
lichen Gebilde, bevor sie dem Auge sichtbar werden, schon
dagewesen sind und nur erst jetzt sich abheben, weil die Licht-
brechungsverhialtnisse sich gedndert haben.” Similar cross-
striated bodies were noticed in the cells of the Malpighian ves-
sels of Chironomus. In the ovarial egg of Lzbella Leydig
found one nucleolus, which consisted of a mass of granules
grouped around a central cavity, these granules being connected
together by fine threads ; ‘der lebende Nucleolus zeigt ferner
langsam ablaufende Gestaltsveranderungen, wobei sich nach
und nach einzelne Kliimpchen mehr oder weniger absondern.”’
Ogata (83) investigated the pancreas cells of man, which had
been treated with various poisons and with the induction current,
then fixed in aqueous solution of corrosive sublimate, and with
osmic acid. One to more than eight nucleoli may be present:
“Die einen farben sich wie die Kernmembrane tief mit Haema-
toxylin.... Die anderen oder vielmehr das andere, denn es ist
in der Regel nur eins, farbt sich nicht mit Haematoxylin, son-
dern mit Eosin. ... Manchmal hat es einen ganz feinen blauen
Saum, als habe es selbst wieder eine Membran. Es ist viel grés-
ser als die anderen Kernkorperchen, und das Feld, in dem es
liegt, ist durch eine starkere Linie von dem iibrigen Kern
getrennt.... Man wird es am unbefangensten wegen seiner
Farbung als Plasmosoma von den iibrigen die Kernfarbung
annehmenden Karyosomen des Kerns unterscheiden.” Some-
times several smaller plasmosomata are also present. Close to
the nucleus is a body he terms “ Nebenkern,” which stains as
the plasmosoma, but is much larger, and is apposed to the sur-
face of the nucleus like a hat ; its substance is homogeneous,
refractive, enclosing small cavities in which minute spherules
occur, the latter having a resemblance to zymogen granules.
The “ Nebenkern” is produced by a plasmosoma which has
wandered out of the nucleus, and there becomes the nucleus of
a new cell. (This process is called “ Zellneuerung.’’)
Pfitzner ('83) found in the resting nuclei of the ectodermal
cells of Hydra usually one central, spherical nucleolus. Its sub-
stance is not identical with the chromatin in the resting stage
of the nucleus, but becomes metamorphosed into the latter
294 MONTGOMERY. (VoL. XV.
substance during the following mitosis ; wherefore he suggests
the term ‘“ Prochromatin” for nucleolar substance. In the pro-
phase of the mitosis only one nucleolus is present in the
nucleus, while in the “ Riickkehr der Tochterkerne zum Ruhe-
stadium waren dagegen stets mehrere vorhanden. In einem
gewissen Stadium, wo die Nucleolenbildung beginnt, ist eine
ganze Anzahl vorhanden ; jemehr sich der Tochterkern dem
Ruhestadium nahert, desto mehr vermindert sich die Zahl
unter gleichzeitiger bedeutender Grossenzunahme der ubrigge-
bliebenen, bis fiir das ausgesprochenste Ruhestadium das Vor-
handensein eines einzigen grossen central gelegenen Nucleolus
geradezu typisch wird,” and this he concludes to be a process
of fusion. The nucleolus plays only a passive réle in mitosis,
“‘namlich die eines aufgespeicherten Nahrungsmaterials zur
Neubildung von Chromatin.”
Rein (83) studied the eggs of Lepus and Cavia. In each
there is one large nucleolus which disappears during matura-
tion and is succeeded by several smaller ones, which have the
same consistency as the first, and at the time of their first
appearance occupy a central position inthe nucleus. ‘So weit
ich den Vorgang am Saugethiere verfolgen konnte, machte mir
derselbe eher den Eindruck eines successiven Zerfalles des
urspriinglichen Keimflecks in immer kleinere Stiickchen, welche
schliesslich in der Substanz des Keimblaschens verschwinden.”
Roule’s ('83) conclusions are, in the main, confirmatory of
Fol’s ('83) observations in regard to endogenous cell formation.
In the egg of Czona there is one large and two or three smaller
nucleoli, the latter being ‘formés pendant |l’évolution des
cellules endotheliales en cellules ovulaires.” In eggs a little
larger these “ nucléoles adventifs ’’ become more numerous (five
to six), and certain of them show a limiting membrane. Later
still some of these adventive nucleoles are found in the yolk,
where each becomes surrounded by a clear zone ; these he con-
siders at this stage to be the nuclei of endogenetically formed
cells (follicular cells), the clear zone around each representing
its cytoplasm.
Schauinsland (83) noticed in the egg of Distomum a single
large nucleolus.
No.2.) COMPARATIVE CYTOLOGICAL STUDIES. 295
A. Schneider (83) studied A7Zossza, one of the Cocczdia. One
or several nucleoli are present, ‘‘ formant un ensemble souvent
trés complexe que j’appelerai le corps nucléolaire.’ Sometimes
the largest nucleolus is enveloped on one side by a number of
secondary, much smaller ones (‘‘nucléolites”’), which are portions
loosened from the inner substance of the large nucleolus, from
which they break out through a “canal micropylaire””’ (such a
canal was not observed in life, and on only a single fixed prepa-
ration; cf. his Fig. 7). ‘ Corrélativement a la multiplication du
corps nucléolaire, le nucléole principal diminue de volume... .
Touts les petits nucléoles qu’on observe dans le corps nucléo-
laire me paraissent descendre aussi sirement du nucléole
primitif ou ancétre que les jeunes d’une espéce de leurs parents.
Les nucléolites, une fois produits, grossissent et, d’_homogénes
qu ils étaient d’abord, peuvent offrir a leur tour la différencia-
tion d’une couche corticale et d’une zone centrale et faire office
de producteurs nouveaux . . . j'ai de bonnes raisons de penser
qu’a ce moment tous les nucléolites produits sont de taille
sensiblement égale et qu’ils paraissent tous homogénes. . . .
Je n’ai pas vu ce que deviennent ces fragments du nucléole,
quelque soin que j’aie mis a scruter leur destinées. Je suppose
que l’enveloppe du noyau se rompt, que les nucléolites mis en
liberté gagnent par des mouvements propres la zone super-
ficielle de la masse granuleuse pour s’y diviser activement. . . .
Si ma hypothése était fondée, le corps nucléolaire mis en
liberté dans le plasma du kyste représenterait en réalité les
débris de la fortune d’un noyau; ce serait le noyau lui-méme,
segmenté, morcelé, et le nom employé, celui de nucléoles, serait
complétement impropre.”’
Weismann (83), ova of Hydromedusae: in all the genera
studied there is always a single large nucleolus, which some-
times contains one or several vacuoles.
7884.
Ayers ('84) germinal vesicle of Oecanthus niveus: in smaller
eggs a single nucleolus, in larger ones several; these nucleoli
he considers as “nodules of nuclear filaments.”
296 MONTGOMERY. [VoL. XV.
Carnoy (84) distinguishes three kinds of nucleoli: (1) “nuclé-
oles nucléiniens,” which are parts of the chromatin network ;
(2) “nucléoles-noyaux,’ which contain all the elements of a
normal nucleus (namely, a membrane, chromatic filament, and
nucleolar substance), while the substance in the remainder of
the nucleus is allied to cytoplasm; such nucleoli occur in
Gregarines, large Radiolaria and Rhizopoda, Spirogyra, the asci
of lichens, testicle cells of Lz¢tobius, and eggs of Pleurobrachia,
Ascidia, and Nephthys,; (3) ‘“nucléoles plasmatiques,” which
contain no chromatin, but consist of a plastin network in
which an albuminous enchylema is imbedded.
Frommann ('84) studied fresh ganglion cells from the anterior
horn of the medulla of the ox; their nucleolus shows “eine
Zusammensetzung aus feinen und derberen Kornchen und aus
sehr kurzen Faden, mitunter auch einen netzformigen Bau mit
theils ganz engen, theils etwas weiteren Maschen.’”’ In the
ganglion cells (of the ganglion Gasseri) of the rat, the nucleolus
is usually homogeneous, as are those of the sympathetic ganglion
cells of Bufo.
R. Hertwig (84), Actinosphacrium: in the resting nucleus
there is one central nucleolus which consists of deeply staining
nuclein and faintly staining paranuclein. The nucleolus is
rarely spherical; when so, it consists mainly of nuclein, except
for a small portion of paranuclein superimposed on the margin.
In other cases the larger nuclein portion is of a curved dumb-
bell shape, and “ gleichzeitig bildet das Paranuclein ein schwach
gekriimmtes Stabchen, dessen Kriimmung zur Kriimmung der
Nucleinmasse senkrecht gestellt ist.”” The connecting portion
of the dumb-bell may disappear, “so dass sich zwei Nucleoli
bilden, welche von einander durch ein queres Stabchen Para-
nuclein getrennt werden. . . . Hiermit beginnen die plurinu-
cleolaren Kerne, wie sie fiir gewohnlich bei Actinosphaerium
beobachtet werden.”” In most nuclei there lies a mass of from
six to twenty nucleoli, which are smaller as they become more
numerous: “ Hier ist es sehr schwer festzustellen, was aus dem
Paranuclein geworden ist, und . . . bin ich zu dem Resultat
gekommen, dass es als ein Korn im Centrum des Haufens von
Kernkoérperchen ist, dass es mit einem Fortsatz an jedes
No.2.] COMPARATIVE CYTOLOGICAL STUDIES. 297
derselben herantritt und alle somit unter einander zu einer
Rosette vereinigt. . . . Die staubformigen Nucleoli sind
urspriinglich vorhanden, erst allmahlich vereinigen sie sich
zu grosseren Stiicken, bis endlich nur ein einziger Nucleolus
und Paranucleolus gegeben ist; dann tritt die Theilung ein.” In
the resting nucleus all the chromatin is contained in the larger
nucleolus.
Jijima (84) found that there are one or several nucleoli in
the ripe eggs of Triclad TZurbellaria, but none in younger
germinal vesicles.
Korschelt (84), following Balbiani ('81) and Leydig ('83),
investigated the interesting structures in the cells of the
salivary gland of Chzvonomus. The form and number of the
nucleoli is mainly such as was described by Balbiani, ‘“ meist
aber sind sie ausgehohlt und von der Form einer mit sehr
dickem Boden versehenen Schale.... Die Convexitat der
Schale richtet sich immer nach der zunachst gelegenen Aussen-
flache des Kernes. ... Der Kernkorper besteht aus einer
feinkornigen Masse, in welcher Vacuolen auftreten. ... Von
den Vacuolen fliessen oft einander benachbarte zu einer gros-
seren zusammen.” The cross-striated structures described by
Balbiani are not to be seen in the fresh nucleus, but, as noted
by Leydig, first appear after the nucleus has remained under
the microscope for some time; thus they may be possibly prod-
ucts of coagulation. ‘ Dass sie sich, wie dies Balbiani zeichnet,
mit ihren fransenartig gebildeten Enden an die sog. Kern-
membranen anheften oder dass (nach Leydig) Anheftungsfaden
von ihrer Oberflache zur Umgrenzung des Kernes hingingen,
habe ich allerdings nie bemerken konnen. ... Ich muss nach
meinen Befunden ... sagen, dass die ‘‘ Querstreifung ” der
Bander auf einer Faltung ihrer Oberflache beruht und dass eine
Zusammensetzung aus verschiedenartigen Schichten nicht vor-
handen ist.” Further, Korschelt did not observe the envelop-
ing membrane of these structures, described by Balbiani, though
he corroborates the observation of this author that the end of
the band gradually fuses into the mass of the nucleolus. From
experiments on starving larvae, he concludes: ‘Es scheint
demnach das eigentliche Chromatin nicht die ganze Masse der
298 MONTGOMERY. [VoL. XV.
Bander auszumachen, sondern nur einen Bestandtheil derselben
zu bilden, der bei mangelhafter Ernahrung der Gewebe zuerst
schwindet.”’
Lang (84) remarks of the egg cells of Polyclad 7urbellaria:
“Das Kernkorperchen oder der Keimfleck ist stets als ein
kugliger, relativ sehr grosser, intensiv gefarbter Korper zu
unterscheiden.”’
Vejdovsky (84) noticed a single nucleolus in the eggs of
Oligochaeta.
Wielowiejski ('84) studied the egg cells of various Arthropoda.
In the Avaneina and Acarina the larger nucleolus contains a
single large or several smaller vacuoles, though no pulsating
or amoeboid movements were noticed (in opposition to the
observations of Balbiani). In Drassws and Lycosa there is a
small mass of granules in place of a germinal spot ; in Oxzscus,
a single large nucleolus; in Astacws, numerous peripheral ones;
and in Musca, a large, irregularly spherical one. (He notes
that the germinal vesicle differs from all other nuclei in that
its contents do not stain at all, or only faintly, with acetic acid
methylen-green solution.)
Will ('84) studied in life the eggs of Bufo and Rana. Larger
and smaller nucleoli may be distinguished; the latter increase
somewhat in size, but never attain the dimensions of the
preceding. Those nucleoli, then, which lie close to the nuclear
membrane cause small protuberances (‘ Knospen”’) of this
membrane, each such bud next breaks off from the nucleus, and,
still enclosing a nucleolus within itself, wanders towards the
periphery of the cell, and there becomes a “ Dotterkern,” the
disintegration of which furnishes the yolk granules.
7885.
Van Bambeke (85) reviews the opinions of the following
writers in regard to the nature of nucleoli: Flemming (82),
Strasburger, Pfitzner ('81), Retzius ('81), Leydig ('83), Balbiani
(81), Korschelt (84), R. Hertwig ('84), Van Beneden (83),
Frommann (84), Carnoy (84), Brass, Wielowiejski (84), and
Rabl (84). Nucleoli are rarely absent, and hence they must
be regarded as an essential element of the nucleus. “Le
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 299
mode d’origine des nucléoles généralement admis explique le
rapport de ces éléments avec la charpente nucléaire. . . .
Flemming est dans le vrai en disant que si les nucléoles sont
généralement suspendus au reticulum, ils ne sont pas en con-
tinuité de substance avec ce dernier, mais constituent des
éléments spéciaux. . . . Nous croyons devoir rapprocher du
nucléole principal la formation récemment désignée par Ed.
Van Beneden, sous le nom de corpuscule germinatif, et plusieurs
de celles appelées par Carnoy nucléoles-noyaux.’ The nucleoli
are probably reservoirs for masses of chromatin.
Van Beneden and Julin (85) found, in contradiction to
Roule, that in the ovum there is only a single large “corpuscule
germinatif” in C/avelzna, and neither smaller nucleoli nor any
migration of nucleoli into the cytoplasm.
Biitschli (85), Cevatium tripos: most of the nucleoli of the
individuals examined contained no nucleoli; only occasionally
are one or two present, and then these evince a honey-combed
(““wabige’’) structure. In many Flaged/ata there is no trace of
a nucleolus.
Carnoy (’85) amplifies his observations of the preceding
year, in which he had distinguished the following four types
of nucleoli: (1) “nucléoles nucléiniens”; (2) ‘nucléoles plas-
matiques”; (3) “nucléoles mixtes”’ (‘‘qui sont constitués
par la réunion des deux espéces précédentes en un corps
unique”); (4) ‘“nucléoles-noyaux.’” Types 1, 3, and 4 are
closely related, and all are sharply demarcated from type 2.
The “nucléoles plasmatiques”’ are plasmatic, albuminoid accu-
mulations, and not chromatin material in reserve (in opposition
to the views of Heuser, Guignard, and Pfitzner) : ‘‘ Nous pré-
férons dire qu’ils concourent avec les autres éléments plas-
matiques du noyau a l’élaboration du fuseau, dont les filaments
constituants sont formés d’une substance, ou de diverses
substances, présentant beaucoup d’analogie avec la plastine.”’
The “nucléole nucléinien’”’ may be composed of amorphous
masses or of a skein of chromatin (the latter is the case in
the testicle cells of Chzlopoda, ova of Pleurobrachia and
Cymbulia): “Le nucléole central de beaucoup de cellules
ganglionnaires est de nature nucléinienne et présente souvent
300 MONTGOMERY. [VoL. XV.
la méme constitution filoide’’; and similar nucleoli occur in the
Protista and in various cells of the Arthropoda. The “nucléole-
noyau”’ of the eggs of Cymbulia and Lithobius has a fine
external membrane and a convoluted chromatin filament. In
the amitotic division of the capsular ovarial cells of Gryllotalpa
the nucleolus (formed of a central portion of chromatin and a
peripheral layer of plastin) divides first so that each daughter-
nucleus receives one nucleolus. But in the amitosis of the
intestinal cells of Aphrophora the “nucléoles plasmatiques ” do
not divide; and in the testicle cells (‘«métrocytes”’) of Sco/o-
pendra there is also a “nucléole plasmatique,” and at the
commencement of the mitosis the ‘“nucléole se liquéfie pour
enrichir le caryoplasma,” and is not to be found later. The
amitotic division of the fat cells of Geotrupes is introduced by
a division of the ‘ nucléole-noyau.”’
Frenzel ('85) studied the cells of the mid-gut in insects at
various stages of development. Bombyx dispar, larva: one
large nucleolus containing a vacuole, in which lies a small
spherical ‘“Nucleollolus.” Zachina, larva: here is one large
nucleolus, “mit kurzen zackigen Auslaufern. In seinem
Innern umschliesst er fast stets wenigstens einen, in der Regel
aber mehrere, etwa 6 bis 12, kugelige oder matt aussehende
Gebilde, welche nicht gerade den Eindruck von festeren
Korpern, sondern vielmehr von Vakuolen machen.” In cylin-
der and gland cells of various insect larvae the nucleus is
filled with a homogeneous fluid, ‘in welche sowohl echte
Nukleolen, wie auch nucleolenartige Korper (‘ Keimflecken’
oder ‘Nukleolide’) einerseits und andererseits zahlreiche ver-
schieden angeordnete sehr klein aber stets gleich grosse
Kornchen eingelagert sind, die hier ‘Kerngranula’ oder
‘-granulationen’ heissen mégen.”
Leydig ('85) noticed in ganglion cells of Astacus a large,
spherical, granular nucleolus, in which is a large cavity ; this
nucleolar cavity stands in communication with that of the
nucleus itself. We read further: ‘Die Koérper im Kern, die
man Nukleoli nennt, sind Bildungen verschiedener Art”’ ; some
arise out of the nodal points of the nuclear network, others out
of the “ Kernplasma.”
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 301
Rabl (85) studied mitoses in cells of the larva of Sa/amandra,
and found that in the prophases of mitosis the nucleoli gradu-
ally vanish and take part in the production of the chromatin
threads. In the unripe germinal vesicle of Proteus, on the
inner surface of its membrane, “sieht man in unregelmassigen
Abstanden’ von einander kugelige, stark glanzende, wie Oel-
tropfen aussehende Korperchen,”’ which he assumes are neither
nucleoli nor masses of true chromatin.
Will ('85) studied the ovogenesis of (Vo¢onecta and WNepa.
The young “Ooblast”’ contains one nucleolus bounded by a
membrane and surrounded by smaller ‘“Chromatinballen”’ ;
subsequently the latter bodies fuse together and form a closed
ring around the nucleolus. The nuclear division of the odblast
is an amitotic one, and is preceded by a division of its nucleolus ;
in each daughter-nucleus, then, the divided half of the primitive
nucleolus breaks up into fragments, which become distributed
through the nuclear sap, and the daughter-nucleus produces a
new nucleolus without the aid of these particles. When the
ovum proper is ripe, the nucleolus finally disappears.
7886.
Van Bambeke ('s6) found that in the germinal vesicles of
Arachnida, Tsopoda, Hymenoptera, and Meconema, the nucleoli
and the chromatin do not stain with methylen green (corrobo-
rating Wielowiejski) though they stain with carmine and
haematoxylin; ‘ Rien ne s’oppose, me semble-t-il, A ce que l’on
considére le corpuscule germinatif comme étant équivalent a
l’ensemble de la charpente chromatique des noyaux ordinaires
[somatiques].’”’ He concludes that there is no proof of the
identity of the true nucleoli of the somatic cells with the ger-
minal spots of egg cells. Two stages in the formation of the
nucleolus may be distinguished in the ova of various Arachnids
(Lycosa, Amaurobius, Argyronecta, Tegenaria, Attus, Theridium,
Epetra, Zilla, Phalangium): (1) there is a single large nucleolus
(sometimes accompanied by smaller accessory ones), in which
at first a few vacuoles arise, which later fuse to produce a single
voluminous vacuole; and (2) the nucleolus becomes replaced
by a mass of fine granules. In the ovarial egg of Amaurvobius
302 MONTGOMERY. [VoL. XV.
ferox the nucleolus consists of (1) a peripheral, less deeply
staining portion ; and (2) of a more deeply staining and more
highly refractive central portion, in which one large and several
smaller vacuoles lie: ‘Chose remarquable dans la vacuole
centrale se voyait, a l’état frais, un granule foncé, doué d’un
mouvement trés vif’’; in this germinal vesicle a small, finely
granular nucleolus is also present. Amoeboid movements of
the germinal spot of Periplaneta were noticed. In the egg
of Zzlla there are from one to three homogeneous, spherical
nucleoli, as also a large “tache principale”; the latter is com-
posed of two or three different substances, somewhat as in
Amaurobius.
Carnoy (86), egg of Spzvoptera strumosa: there is one large,
central “nucléole nucléinien,’” sometimes also one or two small
“nucléoles plasmatiques”’; the former nucleolus is the only
part of the nucleus which stains deeply with methyl green;
it is bounded by a fine membrane, and contains eight ‘ baton-
” (chromosomes), so that it is comparable to a “ nucléole-
noyau.” Vematode from the stomach of Scy/lium canicula: in
the “ceufs trés jeunes. . . le filament nucléinien y est assez
puissant, il parait continu. . . . Nous n’avons pu voir s’il se
scindait d’abord en trongons; nous croyons plutét qu'il se
localise par le retrait de ses anses, pour constituer un nucléole
nucléinien pelotonné. Ainsi nait la tache de Wagner. Elle
est toujours simple; elle se colore peu par le vert de méthyle”’;
no “nucléoles plasmatiques”’ are present in this nucleus. In
the egg of Frlavoides mustelarum one or two “nucléoles plas-
matiques”’ occur; but in that of Ascaris lumbricotdes such
nucleoli are usually absent, and the chromatic filament extends
through the whole nucleus. In Ascaris sp. (from the dog) there
is one “nucléole plasmatique” in young eggs.
Heathcote ('86) noticed in the egg of /#/us one nucleolus with
vacuoles ; it disappears before the production of the pole bodies.
Knappe ('86), ovarian ova of ufo: The nucleoli show amoe-
boid movements in life, and these movements probably lead to
the dissolution of the nucleoli, by causing the latter to first
break into fragments, these fragments afterwards dissolving in
the nuclear sap.
nets
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 303
Pfitzner ('86a) distinguishes in the nucleus: “ Das Achroma-
tin, eine geformte farbbare Substanz, das Chromatin (mit der
Unterart der Nucleolensubstanz, des Prochromatins) und eine
geformte nicht farbbare Substanz, das Parachromatin.” In a
second paper ('86b) he studied Ofatina: here are several
nucleoli flattened against the nuclear membrane; “bei der
Kinese verschwinden sie allmahlich, aber spater als bei anderen
Objekten bisweilen sind sie noch bis zur Metakinese vorhan-
den.” Though they are occasionally found at the poles of the
spindle they take no part in the formation of the chromatin
elements, and in the daughter-nuclei reappear at a distance
from the latter elements. For denoting the substance of the
nucleoli he substitutes for his earlier term ‘“ Prochromatin”
the term “ Pseudochromatin,” since “das Chromatin und die
Nucleolensubstanz wohl nichts Anderes mit einander gemein-
sam haben, als die untergeordnete Eigenschaft, sich bei den
meisten Farbemethoden gleicherweise stark zu farben.”
Platner (’86) investigated the ovogenesis of Avzon and Helix.
In Avion there appears first in the “primitives Ei” a small,
completely spherical nucleolus, to which he limits the name
“Nucleolus”; ‘weiterhin enthalt das Keimblaschen den
eigentlichen Keimfleck. Dieses ist zu Beginn seines Auftre-
tens meist rundlich mit hervorspringenden Erhabenheiten, als
sei er durch Contraktion eines Knauels entstanden. Zuweilen
erscheint er auch mehr ringf6rmig oder ganz unregelmissig.
Immer aber verdichtet er sich bald zu einem vdllig runden
homogenen Element, welches Kernfarbstoffe begierig auf-
nimmt und den Nucleolus bedeutend an Ausdehnung iiber-
trifft.” (His figures show the two to be in close contact.) A
number of clear vacuoles begin to appear in the “ Keimfleck”’:
«Sie sind rund und von verschiedener Grosse . . . und schei-
nen nur dazu zu dienen, weitere Verdnderungen einzuleiten.
Sie verschwinden namlich alsbald wieder, und in dem stetig an
Grosse zunehmenden Keimfleck scheidet sich mit wachsender
Deutlichkeit eine heller gefarbte und eine dunklere Partie.
Letztere, dem ‘“corpuscle germinative’’ van Benedens ent-
sprechend, ist von geringer Ausdehnung, rundlich oder lang-
lich oval und liegt excentrisch in der von runden Contouren
304 MONTGOMERY. [Vou. XV.
begrenzten hellen Substanz, die demnach auf dem Querschnitte
halbmondformig erscheint. Sie diirfte dem von van Beneden
als ‘prothyalosome” bezeichneten Gebilde entsprechen. Es
sei mir daher gestattet, sie Hyalosoma zu benennen. In vollig
entwickelten Eiern ist dieses Element nahezu vollig farblos
und erscheint aus feinen K6rnchen zusammengesetzt. Die
gefarbte Partie des Keimflecks tritt dadurch um so scharfer
hervor, man kann sie im Anschluss an van Beneden Keim-
korperchen nennen.” The nucleolus of the ripe egg “liegt
excentrisch und besteht wieder aus dem runden zart granulirten
Hyalosoma, sowie in dem peripher in demselben gelagerten
Keimkorperchen, welches sich stark farbt und keine weitere
Differenzierung erkennen lasst. Dem hellen Hyalosoma meist
dicht anliegend findet sich der intensiv sich farbende Nucleolus
oder der kleinere Keimfleck.’’ Platner considers that by the
last division of the ovocyte the ‘‘ Nebenkern”’ disappears and
becomes a constituent of the nucleus. ‘ Bei Ausbildung der
Furchungsspindel konnte ich mit Sicherheit constatiren, dass
die Spindelfasern aus der unfarbbaren Substanz des Eikerns
hervorgingen. Diese ist bei sich entwickelnden Eiern im
Keimfleck enthalten, in welchem sie sich bald als Hyalosoma
differenzirt.” In He/zx the “primitive Eier . . . entbehren
des schénen grossen Nucleolus. . . . Daher enthalt ihre defi-
nitive Form auch nur einen Keimfleck, welcher weiterhin
dieselben Veranderungen zeigte wie bei Avzox.” It may be
noted in conclusion that in the spermatogonium of Avzox the
nucleolus appears in the nucleus at the same time that the
“Nebenkern”’ appears in the cytoplasm.
Schauinsland ('86) found one or two large nucleoli in the egg
of Bothriocephalus rugosus.
Stuhlmann ('86) investigated the early stages of the ovum in
a large number of species, more particularly of the Arthropoda.
Carabus memoralis: there are numerous “chromatin” granules
in the young eggs, which increase in number and size; later a
granular nucleolus appears : “Es ist schwer zu entscheiden, ob
der Haufe von chromatischen Koérnern zu einem grossen Ballen
zusammenschmilzt, oder ob sich einer, wohl das urspriinglich
central gelegene, zum Nucleolus ausbildet oder endlich ob
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 305
3
letzterer eine ganz neue Bildung ist... . Wenn aber schon
Dotter ausgeschieden ist, hat der Nucleolus fast stets eine
Form, die aufs Tauschendste einer Eichel gleicht.... Wir
sehen an dem Nucleolus einen helleren, vollig homogenen
Theil und einen dunkler gefarbten, welcher fein granulirt ist
und wie mit einer Menge von winzigen Vacuolen durchsetzt
erscheint. Dieser dunklere Theil umgreift wie die Cupola
einer Eichel den helleren Theil. Um die Formahnlichkeit
ganz zu vollenden, sitzen haufig auf der Kuppe der homogenen
Halfte noch einige dunkle Kornchen. ... Auf einem Aequa-
torialschnitt sieht man nun, dass der dunklere Theil eine Zone
um den helleren Theil bildet.”” This enormous nucleolus
measures 674; it disappears when the nucleus wanders to
the periphery of the egg. Cavabus auratus and Pterostichus
elatus: one spherical nucleolus, containing a few small vacu-
oles, and its size increases with that of the nucleus; later it
assumes a peripheral position, “und in seiner Nahe treten mehr
oder weniger kleine Chromatinkugeln auf, wahrend der Nucle-
olus selbst kleiner zu werden scheint’’; the nucleolus dis-
appears, then the small “Kugeln” unite to form a larger
spherule, and finally the latter also vanishes. In the egg of
Dytiscus marginalis there are no true nucleoli, only irregular
masses of chromatin. Egg of Sz/éza: one granular nucleolus,
which increases in size up to a certain point, and later, when
vacuoles arise in it, a number of small spherules become appar-
ent outside of the nucleolus: “Ob dieselben aus dem Nucleolus
stammen oder ob sie als Paranucleolen des Kerngeriistes auf-
zufassen sind, weiss ich nicht,” though he does not think that
they are products of the nucleolus; the nucleolus, as well as a
portion of the nucleus, disappears later. MVecrophorus vespillo:
several non-homogeneous germinal spots, later a single nucleolus,
which finally vanishes. Eggsof Geotrupes and Cetonia: several
small, spherical or elongated nucleoli, which occupy a central
position in the nucleus, and increase in number and size;
“Dieselben liegen in concentrischer Anordnung um einen
homogenen Kern, der etwas dunkler als die Kerngrundsubstanz
gefarbt ist.” Lina populi: at first there is one large and one
small nucleolus; in this stage “sind im ganzen Keimblaschen
306 MONTGOMERY. [VoL. XV.
mit Ausnahme der Randzone ganz feine klare Blaschen ver-
theilt, welche ich jedoch als Kunstprodukte ansehen méchte”’ ;
later there lies in one part of the nucleus a group of minute
nucleoli; then a portion of the nucleus breaks off and wanders
into the cytoplasm, while the remaining portion of the nucleus
retains one small nucleolus; and lastly, when the nucleus
becomes amoeboid in shape, it contains one large vacuolated
nucleolus, “sowie mehrere kleinere chromatische Korper.”
Lycus aurora: at first there is no nucleolus, later a large and
a small one (both granular); when the nucleus wanders to the
periphery of the egg it retains one of the nucleoli, which
subsequently disappears at the same time as the nucleus
does. Periplaneta orientalis: at first there is no nucleolus,
“derselbe bildet sich erst allmahlich heraus. . . . Wir sehen
ausser dem etwas kérnigen Nucleolus eine Anzahl kleinerer
stark farbbarer Kiigelchen, die wohl als Bestandtheile des
Kerngeriistes, als Paranucleolen aufzufassen sind.” Gvryllotalpa
vulgaris: in the immature egg “ein eigentlicher Keimfleck ist
nicht vorhanden; vielmehr liegen in der Kerngrundsubstanz
zerstreut Chromatinpartikel von 4 Durchmesser bis zu
unmessbarer Feinheit’’?; when the nucleus has assumed a
peripheral position a large nucleolus is produced in it, “ wohl
durch Verschmelzung mehrerer kleinerer.” Locusta viridissima:
in maturer ova a large but lightly staining nucleolus, “ von dem
aus ein Kernnetz seinen Ursprung nimmt.” Pzeris brassicae:
one large, homogeneous germinal spot, which later acquires
vacuoles and divides into three parts. Sphinx ligustris: in the
immature germinal vesicle lies a large, excentric nucleolus, con-
taining vacuoles; “ausser letzterem finden sich noch einige
wenige Paranucleolen”’; at the time when the nuclear frag-
ments break off, the nucleolus becomes paler and then vanishes.
Zygaena filipendulae: at first no nucleolus is present, later
there is a larger one with vacuoles, as well as a smaller one,
“«der sich wohl von dem grossen abgelést zu haben scheint” ;
subsequently both disappear. Mzusca vomitoria: there is at
first in the germinal vesicle a single, large, excentric nucleolus,
but later appear in it “eine Anzahl von Paranucleolen und ein
Nucleolus, . . . von welchen letzterer aus einem Haufchen von
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 307
kleinen, gefarbten Kiigelchen besteht.” In the egg of Axabolia
there is one large nucleolus, but in those of Vespa germanica
and V. media apparently no true nucleoli are present. There
is a large granular nucleolus in the larger germinal vesicles
of Bombus terristris. Trogus lutorius: there is one large,
irregularly shaped nucleolus and two smaller ones; all these
finally disappear, and their place is taken by smaller granules.
Banchus fulvipes: at first no nucleolus is present, later one or
three large nucleoli appear, but all of them vanish subsequently.
In the egg nucleus of Pzmpla sp. only a number of small
granules are to be found, and at a later period still smaller
granules. Axomalon circumflexum: in the youngest germinal
vesicles no nucleolus is to be found, in older ones there is
a single large one; this has nothing to do in the formation of
the ‘“ Dotterkerne,’’ and disappears when the nucleus does.
There is one spherical germinal spot in Ophion ventricosum,
but not in O. luteum. Ephialtes liturater: in the smaller nuclei
a considerable number of “ chromatic’’ bodies occur, while in
the older ones there is a single large nucleolus. Asdyteles
castigator: one large nucleolus, in older ova also several smaller
ones. Lfetva diademata: here is one large spherical nucleolus,
which later becomes jagged in outline and evinces vacuoles,
which may unite to produce a single larger vacuole: ‘In sel-
tenen Fallen kann man einen Zerfall des Nucleolus in mehrere
kleinere sehen, was jedoch wohl eine pathologische Erscheinung
sein diirfte.” Glomeris marginata: one large, spherical or
angular nucleolus, and later also a smaller one: ‘ Héchst
wahrscheinlich stammt dieser von dem grossen Nucleolus ab” ;
the smaller nucleolus disappears subsequently. In the egg of
Peripatus edwardsii one nucleolus forms itself gradually, and
vacuoles begin to appear in it. In Amaroectum rubicundum a
single large nucleolus is present; while in Clavelina lepadi-
formis the nucleolus is probably formed out of the central
chromatin masses. From these numerous observations Stuhl-
mann draws the conclusion: ‘ Aus Allem schien mir hervorzu-
gehen, dass das Schwinden des Nucleolus nicht zum Wesen
der Eireifung gehort, besonders weil ich ihn bisweilen (so bei
Silpha) so lange verfolgen konnte, als noch ein Rest des
Keimblaschens im Ei sichtbar war.”
308 MONTGOMERY. [VoL. XV.
Vigelius ('g6) finds in the egg of Bugula one large nucleolus,
containing vacuoles.
Will ('86) studied the maturation of the egg of Colymbetes.
“Dem Kernkorperchen oder Nucleolus . . . kann nach meinen
Untersuchungen keinerlei morphologische Bedeutung zukom-
men. Was wir Kernk6rperchen nennen, ist nach meiner
Auffassung nichts als ein besonders grosses Stiick Chromatin-
substanz. So konnen wir es verstehen, dass bald eines, bald
mehreres, bald gar keine vorhanden sind.”
1387.
Boveri (87): in the ovum of Ascaris megalocephala bivalens
there are no true nucleoli when the tetrads are formed. In the
variety wnzvalens there is usually one “ achromatisches kugeliges
Ko6rperchen. Von dem “ Prothyalosoma,”’ das an den van
Beneden’schen Eiern den Keimfleck [Vierergruppe] umgiebt
und welches im weiteren Verlauf bei ihm eine so grosse Rolle
spielt, habe ich weder auf diesem Stadium, noch spater die
geringste Spur wahrgenommen.”
Eisig (87) remarks in regard to the egg of Capztellids: “Der
urspriinglich rundliche, jederzeit durch Dichtigkeit und hohes
Tinctionsvermégen auffallende Keimfleck erleidet im Laufe
seines Wachsthums offenbar Theilungen; denn man findet ihn
in spateren Stadien mit ein oder zwei verschiedengradig abge-
schniirten Kuppen besetzt; ausserdem trifft man schon friihe
mehrere Pseudonucleoli, welche offenbar Produkte des Haupt-
nucleolus darstellen, in dem Keimblaschen zerstreut.’”’ He
notes, further, that in the maturing ovum the nucleolus does
not increase in size in equal proportion to the size of the
nucleus. (To judge from his figures, the nucleoli are not
homogeneous.)
Fraipont (87) found in the germinal vesicle of Polygordius
several nucleoli of unequal size.
Henking ('87) studied the eggs of Pkalangids. In the ovarial
egg a sickle-shaped body lies at one pole of the nucleolus: “es
scheint, als wenn in ihm und dem Keimfleck die Chromatin-
substanz des Keimblaschens sich koncentrirt hatte.’ In the
nearly ripe egg there is one large nucleolus, which is not homo-
r}
No.2.) COMPARATIVE CYTOLOGICAL STUDIES. 309
geneous, and a number of smaller globules, these latter stain-
ing as the former, and some of them containing vacuoles: “ sie
stellen einerseits eine Zusammenballung der bisher ganz unre-
gelmassigen, im Keimblaschen vertheilten Chromatinsubstanz
dar, rihren andererseits aber wohl vom Keimfleck her.’’ These
bodies have all disappeared in the ripe egg.
Hubrecht ('87) noticed only a single nucleolus in the egg of
Cerebratulus sp.; as to the egg of Pelagonemertes, he figures
one nucleus containing one large and several smaller nucleoli,
and another nucleus with only numerous small nucleoli.
Kosinski ('87, '93, mentioned by Lavdowsky, '94): within the
nucleolus of cancerous cells there is sometimes a vacuole, and
within the latter a small body which Kosinski considers may
correspond to Carnoy’s “nucléoles-noyaux’’; such nucleoli have
the faculty of division, and of wandering through the nuclear
membrane into the cytoplasm.
Lukjanow ('87a), stomach epithelium of Amphibzans : in the
cytoplasm of the cylinder epithelium are structures of various
form (‘“* Nebenkerne’’), which stain in general like the nucleoli.
In some of the nuclei of the deep layer of gland cells each
nucleolus is joined with a karyosome.
Lukjanow ('87b) distinguishes three kinds of nucleoli in
muscle cells of Vertebrates: (1) ‘“ Plasmosomen’”’; (2) “ Karyo-
somen’’; (3) ‘“‘Kernkorperchen von gemischtem Charakter.’’
The first stains deeply red (eosin), the second blue-violet (haema-
toxylin), while the third stains a mixed color with these two
stains (when used together). He remarks also: “dass in
manchen Kernen die Kernkérperchen gianzlich fehlen, in
anderen entweder nur eine Kategorie derselben, oder mehrere
zugleich vertreten sind... . Zuweilen liegt das Kernkorper-
chen sogar ganz ausserhalb des Kernes.”
Nussbaum (87) found in smaller eggs of Hydra a single large
nucleolus, while in larger ova several are present. ‘In frischem
Zustande sieht man in den allerersten Stadien neben den Keim-
flecken noch eine blasse Kugel, die im Gegensatz zu den Nucle-
olen des Keimblaschens keine Farbstoffe in sich aufnimmt.”’
O. Schultze ('g7) studied the maturation of the egg in Rana
and Zyviton. In the unripe germinal vesicle there are larger
310 MONTGOMERY. [VoL. XV.
nucleoli near the nuclear membrane, and smaller ones at the
center of the nucleus: ‘Dass sie sich durch Theilung vermehren,
kann keinem Zweifel unterliegen, denn nicht nur sind dieselben
in ganz jungen Ejiern grosser und weniger zahlreich, .
sondern die grésseren Keimkorperchen weisen durch Ein-
schniirung und Zerkliiftung auf eine Vermehrung durch Thei-
lung hin.” He does not consider that such daughter-nucleoli
are again capable of division, but that the process is rather a
«“Losungsphanomen.” All the nucleoli are homogeneous, but
vacuoles are produced in them by % per cent normal salt solu-
tion. In larger ova a considerable number of nucleoli lie periph-
erally, and there is also a central group of them; and, still later,
the peripheral nucleoli commence to stain less intensely, and
the greater number are centrally situated. The nucleus of the
maturing egg consists of ““Membran, Kernsaft und Keimkor-
perchen,’’a chromatin network being absent; andthe microsomes
of the chromosomes are formed from the smallest, most centrally
placed nucleoli.
T&88.
Bohm (as) found in the egg cell of a 5 cm. long Ammocoetes
of Petromyzon a homogeneous nucleolus, “an dem sich sehr oft
eine kleine Vacuole zeigt, welche mit einer feinen Strasse bis
an die Oberflache des Fleckes [nucleolus] reicht.” At the
animal pole of the nucleus lies a disc-shaped mass (‘‘ Deckel’’):
“rathselhaft ist die Bedeutung des Deckels.’’ (Compare the
extranuclear structure found by Lukjanow, 'gs.)
Boveri (88): in the female pronucleus is neither a prothyalo-
soma nor a hyalosoma, such as were described by Van Beneden
(83); the hyalosoma is probably “ein durch Schrumpfung
entstandenes Artefakt.” Just before copulation “zeigen sich
die ersten Spuren achromatischer KernkGrperchen als ganz
kleine Kornchen, die... stets...in nachster Nachbarschaft
der chromatischen Elemente sich finden, . . . so dass die Ver-
mutung nahe gelegt wird, dass sie sich aus diesen absondern.”’
Fiedler (gs) studied the egg development of Sfongilla: one
large homogeneous nucleolus is present in the germinal vesicle.
In the nuclear division (which is intermediate between the
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 311
mitotic and the amitotic) ‘der gesammte sonstige — iibrigens
sparliche — Chromatininhalt des Kernes vereinigt sich . . . mit
dem Kernkorperchen zu einem kugeligen Gebilde, und erst
dieses zerfallt dann durch allmahliche Zerschniirung in zwei
kleinere, unter sich gleich grosse Kernkorperchen, welche an
die beiden Pole des Kernblaschens riicken.”’
Graff (88) found in the egg of Spznther either a mass of
granules or a single nucleolus; the nucleolus may be either
granular or contain a large vacuole.
R. Hertwig (gs): in nuclei occur “chromatische ”’ nucleoli,
and “das unter gewohnlichen Verhialtnissen nicht farbende
Paranuclein, welches zumeist rundliche K6rper, die Paranucleoli,
bildet. Die Paranucleoli kénnen entweder die einzigen Kern-
korperchen im Kern sein (gewohnliche Gewebszellen, reifes Ei
und Furchungszellen) oder sie finden sich neben den chromati-
schen Nucleoli, unter Umstanden auch als Einschliisse derselben
(Keimblaschen der unreifen Eier, Kerne von Actinosphaerien
und anderen Protozoen) vor.... Zweifelhaft wird es dagegen
gelassen, ob auch der Substanz des achromatischen Geriistes
. nicht . . . vielleicht auch Paranuclein [ist], welches sich
durch seine Anordnung von den Paranucleoli unterscheidet.”
The centrosomes are probably derived from the paranucleoli,
and the paranuclein is ‘die befruchtende Substanz” (these
views have subsequently ('96) been retracted).
Kultschitzky (ss) found in the youngest eggs of Ascaris
marginata one ‘ Kernkérperchen,” which afterwards “in zwei
Stiickchen zerfallt, deren eines sich intensiv mit Karmin farbt
und alle Eigenschaften des Chromatins bewahrt, das andere
sich in die blasser gefarbte gewohnliche Kernkorperchen ver-
wandelt” ; the latter he terms the true ‘“ Kernkorperchen,”
which from this stage on gradually decreases in size, and finally
disappears.
Leydig (88) gives the results of numerous comparative inves-
tigations on germinal vesicles; most of these observations
were made on the living egg, fixing reagents having been little
employed. Nephelis vulgaris: here there is one nucleolus,
which sometimes has a long process, ‘in dessen Nahe kleine
rundliche Ballen von gleicher Art, wie er selber ist, liegen, so
312 MONTGOMERY. [VoL. XV-
dass man die Entstehung der letzteren durch Abschniirung
von dem Fortsatz sich denken darf.” Argulus foliaceus: in
young eggs there is one large nucleolus with clear spaces in it,
showing that the nucleolus “aus Theilen besteht, die allmah-
lich von einander weichen, so dass man alsdann in anderen
Thieren anstatt eines Keimflecks eine ganze Anzahl kleinerer
vor sich hat’’; these nucleoli are often jagged in contour ; by
treatment with chromo-acetic acid “ bekommen die Keimflecke
eine Querzeichnung, so dass sie wie aus Querstiicken zusam-
mengesetzt erscheinen.” Tetragnatha: one large nucleolus
with dark contours, and several smaller pale, granular ones,
which gradually disappear during the maturation of the egg.
Lycosa: “Ein einziger, grosserer Keimfleck zeigt sich . .. und
dieser bietet das Bild eines Knauels dar.” Theridium : the large
“Hauptkeimfleck hat die Beschaffenheit eines stattlichen,
aus scharf geranderten kleinen Korpern zusammengesetzten
Ballens. Von ihm nun weg zieht sich ein Strang solcher Kor-
perchen oder Theilstiicke tiber die Grenze des Keimblaschens
hinaus in den Dotter hinein. In einzelnen Eiern, deren gros-
ser Keimfleck das Bild gewundener und geknauelter Faden
giebt, konnen die kleinen Theilstiicke zusammenhangend oder
in bereits abgelosten Gruppen abermals in den Dotter sich
erstrecken. Jaich glaube an dem lebenden Ei verfolgt zu haben,
wie Theile der geknauelten Faden sich zu einzelnen Ballen
zusammenschoben und in den Dotter vordrangen’’; there are
present also one or several pale ‘“‘ Nebenkeimflecke.” Phalan-
gium: the young ovum has one large nucleolus containing
vacuoles ; ‘‘ Wiederholt habe ich beobachtet, dass ein solcher
Keimfleck — das lebende Ei mit Mundspeichel befeuchtet —
unter dem Mikroskop allmahlich verblasste und zuletzt fiir das
Auge vollig verschwand.” Lzthobius: there may be one granu-
lar nucleolus, or numerous nucleoli, each with a granular core:
“Wieder eine andere Form ist die, dass die amdbenartigen
Gebilde in ihrem Innern einen hellen, kernartigen Fleck mit
centralem Piinktchen zeigen und am Rande feinstrahlig sind”’;
in other germinal vesicles there may be present numerous
small nucleoli, either irregularly grouped or arranged in “kurze,
goldrollenahnliche Saulchen , . .; ein andermal stésst man auf
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 313
langere fadige Aufreihungen, deren Strange zu Schlingen gebo-
gen oder geknickt sind.” In these ova two kinds of nucleoli
may occur, namely, numbers of the small ones just described,
and a large one with dark contours, which has a central
vacuolar, granular portion, and is peripherally homoge-
neous; but nucleoli also occur which are intermediate between
these two kinds. Geophilus electricus: here are numerous
small, pale nucleoli and a large one, which has a finely granu-
lated core, and an outer homogeneous layer, the latter portion
consisting of concentric layers; further, he noticed the infundib-
ular structure first found by Balbiani on the outer surface of
the nucleus, though he remarks that it is especially apparent in
eggs in which post-mortem changes have commenced (!), and
concludes : ‘‘ Wir haben es sonach beziiglich des Trichters mit
einer Ausbuchtung jenes Hohlraumes oder Lichtung zu thun,
welche von der Hohlung um das Keimblaschen herum in den
Dotter dringt.” The basis of this infundibulum empties into
a space around the nucleus, and not into the nucleus itself (as
opposed to Balbiani’s observations); Leydig also thinks that
particles of finely divided nucleoli penetrate separately out of
pores which are present in the nuclear membrane, and that
these particles, arrived in the cytoplasm, fuse together to form
a large “Ballen.” Stenobothrus: in the ova of the proximal
portion of the egg tube there are either numerous small nucleoli
or a dense mass of very fine granules ; in riper germinal vesicles
they are much larger and resemble somewhat the nucleoli
in the salivary glands of Chzvonomus,; masses of nucleolar
substance wander out of the nucleus into the cytoplasm. In
Pemphigus bursarius there is one compound nucleolus, with fine
radiating processes ; and in JZeloé violaceus there are numer-
ous nucleoli, each of which has the structure of the single one
of the preceding species. Gasterosteus aculeatus: in the month
of May there are numerous germinal spots, sometimes densely
grouped, sometimes arranged in rows ; the gradual thickening
of the nuclear membrane takes place at the cost of nucleolar
substance. Triton taentatus: the germinal vesicle at the end
of October contains numerous nucleoli of unequal size, many of
which are arranged in columns ; the peripheral ones probably
314 MONTGOMERY. [VoL. XV.
wander into the cytoplasm. Salamandra maculosa, \arvae :
the “ Urei” has a single large nucleolus. Bzfo cinereus, larvae
of several months : concludes “ dass die Keimflecke, wenn noch
winzig klein, aus den Knotenpunkten des Spongioplasmas
entstanden sind, und nachdem sie eine gewisse Grosse erreicht,
die Form und Sonderung einer Amobe besitzen. Dieselben
stellen sich jetzt dar wie hiillenlose, kleine Zellen, ar denen wir
einen homogenen kérnigen Korper, der feinzackig oder selbst
in feine Strahlen ausgezogen ist, unterscheiden und im Innern
einen lichten, kernahnlichen Fleck, in dem sich noch ein Kor-
perchen abzeichnet”’; numbers of such nucleoli may later fuse
together, ‘unter Vermittelung ihrer Zackenspitzen.” Rana
esculenta: in the smallest ova there is only a single large
nucleolus, with a vacuolar central portion and peripheral
radiating strands ; in larger eggs there are a number of smaller
nucleoli, each of which has the same structure as the primitive
one ; Leydig believes that nucleoli wander out of the nucleus,
since he found a granular mass on the outer surface of the
latter. The ova of Sus scrofa, Myoxus nitela, and Talpa euro-
pea contain each a single nucleolus.
Lukjanow (gg) investigated the stomach mucosa of Sa/aman-
dra. There are several, usually club-shaped nucleoli (“ Nucleoli
claviformes’’), the smaller, often funnel-shaped, end of which
is in contact with the nuclear membrane. He concludes
“dass die kolbenahnliche Form des Nucleolus . . . auf eine
Vorbereitung zur Inhaltsentleerung hinweist. Der Kolben
entleert seinen Inhalt etwa ebenso, wie die Becherzelle ihren
Schleim entleert” ; and he supports this conclusion with the
observation that a mass is often found on the outer surface of
the nuclear membrane which stains like the nucleolus.
Nagel ('88) studied the human egg. The “ Primordial-Ei”
has a single nucleolus ; those which contain no nucleoli he
believes do not develop further. In the ripe egg amoeboid
motions were noticed in life (studied in liquor folliculi).
Sanfelice (88) terms the nucleolus of the spermatoblast
“nucleus,” and the nucleus, “cell.’’ What he calls the nucleus
then divides karyokinetically (but that this process is a division
of the nucleolus may be deduced from his figures 60 and 62).
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 315
Scharff ('88) studied the maturation of the eggs of various
Teleosts. In the smallest ova examined (.o1! mm.) there are
numerous peripheral nucleoli, and a few which are central in
position. In larger eggs (.03 mm.) ‘the nucleoli show an
inclination to gather still more towards the periphery of the
nucleus . . . one or more of the nucleoli become larger than
the others, and in their interior refractive specks are visible
which have sometimes been described as endonucleoli.” In
still larger ova (.08 mm.) ‘‘in some cases the big nucleoli dis-
appear almost completely, leaving an unstained part around
them.” In Cozger he “noticed a small nucleolus being con-
stricted off from a larger one.” He figures outside of the
germinal vesicle of Gadus certain granules, and these he
considers are emigrated nucleoli which are destined to become
dissolved there, though he holds it possible that “some find
their way to the surface of the egg to form the nuclei of the
follicular epithelium” ; in eggs which have attained the
dimensions of .132 mm. the nucleoli become very irregular
in shape. In the 77ig/la egg of .13 mm. the surface of the
nucleolus is raised into small protuberances, most of which
contain a nucleolus ; these protuberances later break off and
become the yolk spherules (in corroboration of Will, '84),
Schewiakoff (88), Euglypha: the nucleolus gradually dis-
appears in the prophasis of mitosis.
Steinhaus (88), intestinal cells of Sa/amandra: karyosomes
and plasmosomes are distinguished within the nucleoli, and are
usually combined in pairs with one another. Plurinucleolar
nucleoli are formed by continued divisions of a single nucleolus,
“et les nouveaux nucléoles s’éloignent l’un de l'autre, probable-
ment a l’aide de mouvements amoeboides ou d’autres qui leur
sont propres.” Plasmosomes when extruded into the cytoplasm
increase greatly in size, though this increase is due to mere
imbibition of some substance; each such extruded nucleolus,
combining with a karyosome, develops into a new nucleus.
Vejdovsky ('88) studied the maturation of the egg of Rhyn-
chelmts. The embryonal genital cells contain no true nucleoli.
The nucleolus does not stain when it first appears (in very
young stages). Subsequently it is always excentric in position,
316 MONTGOMERY. [VoL. XV.
perfectly spherical, and consists of a central, homogeneous,
deeply staining portion, and an outer unstaining envelope
(judging from his Fig. 5, Tab. 3, I would consider this sup-
posed envelope to be a vacuole in which the nucleolus lies).
In the more advanced ovum this envelope has disappeared, and
the nucleolus has increased in size, but is no longer homogene-
ous, since it contains a number of deeply staining granules.
When “das Kernkorperchen die oben angedeutete Grosse
[013 mm.] erlangt hat, beginnt es sich einzuschniiren, was
gewiss auf dessen Theilung hinweist’”’; he believes that this
division is rapid, ‘‘dass es aber thatsachlich so geschieht,
beweist die Thatsache, dass in den reiferen Ejiern in der Regel
zwei Kernkorperchen vorhanden sind. Das neu entstandene
Kernkorperchen liegt anfanglich in der Nahe des alteren und
ist etwas kleiner als dieses ; spater entfernt es sich mehr oder
weniger und wachst zu der Grosse des ersteren heran.’’ In the
ripe egg two nucleoli are present, or there may be three or four,
the latter two having been divided off from the former ; each
of these consists of an inner chromatic portion and an achro-
matic envelope ; the latter is porous, and “man kann voraus-
setzen, dass durch die Poren die fliissige Nahrung in das Innere
des Kernkorperchens eindringt.”” When this envelope has
vanished, each nucleolus is formed of (1) a hyaline, homogene-
ous fluid, in which (2) a delicate network arises, the nodal
points of which are represented by the previous granules of the
nucleolus ; “kurz und gut, die Kernkérperchen unserer Eier
sind chromatische Kernfaden. ... Die intensive Farbung
sowohl der Knotchen als des Fadenwerkes erleichtert die Ver-
folgung des metamorphosirten Kernk6rperchens, welches jetzt
ganz und gar den Kernen des spateren Blastomeren gleich-
kommt.” (The descriptions do not enable one to determine
whether all the nucleoli become thus metamorphosed.)
Waldeyer in his “ Referat’’ (88) agrees with Klein “dass
die Nucleolen nur stark verdickte Knotenpunkte des Netz-
werkes der Geriistfaden [chromatin], also mit den letzteren
identisch seien. . . . Die Bedeutung aller dieser Dinge fiir das
Zellenleben ist noch fast vollkommen dunkel,”
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. a7,
T8809.
Bergh ('89), Uvostyla; the fragments of the macronucleus
contain true nucleoli, while the micronuclei do not.
Brass ('89) states: “ Fiir gewohnlich erscheint jedes Kernkor-
perchen rund, sehr haufig kugelrund; es besteht entweder aus
einer gleichartigen Masse oder es sind in derselben einige
hellglanzende Kérnchen ausgeschieden, oder aber es finden sich
in ihm dichtere, weniger glanzende Kornchen. ... Im Umkreis
der Kernkorperchen ist vielfach ein heller Hof, der von feinen
Kornchen kugelschalenartig umgeben wird. Der Hof wird als
Kernkérperchenhof beschrieben; er ist in sehr vielen Fallen
sichtbar zu machen.”
Davidoff ('89) observed in the egg of Dzstaplia a single
large, spherical nucleolus, consisting of a homogeneous mass in
which a few granules are imbedded. These nucleoli increase
in size as follows: “Sie werden gréssere Partien des
Reticulums in sich aufnehmen, sich mehr und mehr verdichten
und demgemass sich immer deutlicher und deutlicher farben.”
Subsequently, but antecedent to the production of the pole
spindle, the nucleolus contracts, and its contour becomes
irregular, often with regular branched processes: “ Vielleicht,
ja sogar wahrscheinlich, werden sie dadurch hervorgerufen,
dass der Nucleolus Fliissigkeit ausscheidet”; and the central
portion of the nucleolus becomes lighter in color. Next, first
the lighter portion, then the whole nucleolus, becomes filled
with fine granules (‘‘Chromatosomen’’). Then these chroma-
tosomes collect and form in the center of the nucleolus
a compact, granular body, in the middle of which is one
especially large chromatosome, and the whole is surrounded by
a membrane. And finally, other chromatosomes, not con-
cerned in the formation of the central granular body, form a
reticulum around it. Davidoff concludes “dass aus dem
Nucleolus ein Kern mit Kernnetz, mit einem Nucleolus und
Nucleolinus hervorgegangen ist. Wir konnen diesen Kern
weder als Keimblaschen, noch als Nucleolus bezeichnen. Es
ist eben ein neues Gebilde, dass wir einstweilen mit dem Namen
Polkern belegen wollen”; out of this ‘“ Polkern”’ the first pole
spindle is formed.
318 MONTGOMERY. [VOL. XV.
Fol (89), ovarian egg of Dentalium ; the nucleolus is at first
absent, and single. In larger nuclei there are two apposed
nucleoli (which disappear when the nuclear membrane has
vanished), ‘Le nucléole présente d’abord deux parties dis-
tinctes, dont l’une, plus volumineuse et moins foncée, entoure
l'autre un peu comme un bonnet posé sur la téte. La partie
foncée est sphérique ; elle retient ]’hématoxyline ou le carmin
alunique avec une nuance rougedtre ou vineuse. Sa texture
est compacte. L’autre partie est formée des corpuscules plus
clairs [vacuoles] et d’un réseau plus foncé; elle prend les colo-
rants que nous venons de nommer avec une teinte violacée tirant
sur le bleu... . Lorsque l’ovule approche de 1’époque ou la
vésicule germinative va se dissoudre, les deux nucléoles, au lieu
de s’emboiter, sont simplement accolés, et le nucléole clair s’est
accru beaucoup plus que l'autre.”
Hermann ('89a) investigated the spermatogenesis of the
mouse. The ‘“ Spermatoblastkern”’ (nucleus of a v. Ebner’s
cell) possesses one nucleolus, which is made up of two parts,
‘einen von Safranin sehr intensiv gefarbten, und einen unge-
farbt bleibenden Bestandtheil. Letzterer tritt stets in Form
einer einfachen Kugel auf, die chromatische Substanz aber
besteht entweder aus zwei kleinen, leuchtend roth tingirten, an
zwei gegeniiberstehenden Polen der farblosen Kugel liegenden
Kiigelchen, oder das chromatische Element stellt eine einzige,
in diesem Falle gréssere Kugel dar, die dem ungefarbten
Bestandtheile des Nucleolus sich innig anschmiegt. Im
ersteren Falle erscheint dann das ganze Kernkorperchen als
ein annahernd spindelformiges Element, im anderen als eine
Doppelkugel, und ist in beiden Fallen die Langsaxe des Nucleo-
lus stets in dem gréssten Durchmesser des Zellkerns einge-
stellt.” The nucleoli of the spermatogonia are sometimes
biscuit-shaped. Those of the spermatids are at first multiple
in number, but later they unite to form a biscuit-shaped one.
Still later, by the formation of the spermatozoén out of the
spermatid, the two parts of this nucleolus wander apart,
“dabei aber noch durch eine chromatische Briicke mit einander
in Verbindung stehen.’’ He observed in the follicle cells of
the testicle of Sa/amandra “neben kleinen Nucleolen einen
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 319
grosseren, . . . der vollkommen die gleichen Strukturverhalt-
nisse zeigt, wie sie oben von dem Kernkorperchen der Sperma-
toblastkerne der Maus beschrieben wurden und wie dies fiir
den Frosch von Sanfelice angegeben wird.”’
Hermann ('89b), testicles of immature white mice : the nuclei
of the follicle cells contain compound nucleoli, similar to those
of the cells of v. Benda of Salamandra.
Korschelt (9) made observations on the germinal spots of
Epetva, Dolomedes, Phalangium, Spinther, and Ciona. In Epeira
the nucleolus is at first a compact mass of granules “von stark
lichtbrechenden Kornchen umlagert.... Ich will damit nicht
sagen, dass eine direkte Aufnahme von [nutritiven] Kornchen
stattfande, welche letzteren sich dann unmittelbar zum Keim-
fleck formirten, sondern méchte vielmehr glauben, dass die
Substanz in fliissiger Gestalt aufgenommen und erst im Kern
wieder geformt wird’’; in later stages small vacuoles are fre-
quently present in the nucleolus. In Dolomedes the nucleolus
is at first homogeneous, it later contains vacuoles, and finally
becomes simply a membrane surrounding a cavity. In Sp7n-
ther there is a single large nucleolus with a vacuole. Korschelt
draws the general conclusion: ‘Ich muss es nach meinen
Erfahrungen, . . . als zweifelos hinstellen, dass eine Auflésung
der Nucleolarsubstanz stattfindet. Die Erklaérung dieser
Erscheinung fand ich darin, dass die Nucleolarsubstanz in
und vielleicht ausserhalb des Kerns zur Verwendung gebracht
werden sollte.”
Lukjanow ('g9) describes the nucleoli ( “ plasmosomata’’) of
the germinal vesicle and cleavage nuclei ; they disappear during
mitosis.
Platner (89a), Malpighian tubule cells of Dytzscus, fixation in
Kleinenberg’s fluid : there are one or several nucleoli, of irregu-
lar form, and around each one usually “ein hellerer Hof,
welcher aussen von einer Anzahl grésserer unregelmassiger
Chromatinbrocken eingefasst wird.” The division of the nucle-
oli introduces the amitosis of the nucleus: “Der anfangs
mehr runde Nucleolus zeigt eine Abplattung zur Scheibe,
welche der umgebende Hof mitmacht. Zugleich tritt in der
Richtung seiner kiirzern Durchmesser eine Streifung an dem-
320 MONTGOMERY. [VoL. XV.
selben auf, als wenn er aus einer Anzahl nebeneinander liegen-
der schmaler Elemente zusammengesetzt ware. Weiterhin
tritt eine Spaltung in der Richtung des langsten Durchmessers
auf.... Die auf diese Weise entstehenden Tochterplatten
zeigen an den einander zugewandten Seiten spitze Hervorrag-
ungen, an den abgekehrten Flachen dagegen mehr abgerundete
Erhabenheiten. Beide besitzen wieder eine langsgestreifte
Struktur, als seien sie aus parallelen Stabchen zusammengefiigt.
Den auseinanderweichenden Tochterplatten passt sich der
helle, umgebende Hof an, der also in der Richtung dieser
Bewegung sich verlangert.”
Platner (89) contends, in opposition to the views of Ogata
(83) and others, that in the pancreas cells the nucleoli do not
wander out of the nucleus.
Platner’s ('89c) observations on the egg of Awlastomum shall
be mentioned in the course of our observations on the egg
of Piscicola. In accord with O. Schultze (87) he finds in
amphibian ova that the contents of the nucleus are composed
only of “ Kernsaft und Keimkorperchen,” a portion of the latter
forming the nuclear filament, the rest being extruded from the
nucleus ; the true chromatin loops were not seen by him.
Weismann and Ischikawa (’s9) find in the ovarial winter ova
of Leptodorva one large nucleolus (rarely is a smaller one
apposed to it), containing a large vacuole ; it wanders out of
the nucleus and becomes the ‘Nebenkern, Paranucleus,”
which ultimately disappears, and corresponds to the nucleus
alone of the paracopulation cell of the other Daphnids. In
nearly ripe ova of Bythotrephes “findet man ... innerhalb des
Keimblaschens und dem Nucleolus desselben ganz nahe einen
scheibenformigen Korper, der sich wie der Nucleolus farbt.
Etwas spater, wenn das Keimblaschen bereits an die Ober-
flache des Eies gestiegen ist, liegt dieser Korper ausserhalb des
Keimblaschens und ist in einen Protoplasmahof eingebettet ”;
then it rapidly disappears.
Wheeler (89), ovarial follicle cells of B/atta: there is a
“nucleolus of unusual structure. The latter consists of an
irregular mass, not stainable in carmine or methyl green, and is
regarded as plastin by Carnoy.... The mass of plastin encloses
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 321
a smaller mass of chromatin, or at least of a substance which
does not differ in its reactions from the chromatin of the coiled
filaments in the same nuclei.’’ This nucleolus divides first
in mitosis.
1890.
Auerbach (90) distinguished two kinds of chromatin sub-
stance: ‘“erythrophile,” ze, substances staining with eosin,
fuchsine, aurantia, carmine, picrocarmine; and “kyanophile,”
substances staining with methyl] green, aniline blue, haematoxy-
lin. The nuclear reticulum is not the fundamental portion of
the nucleus, but the nucleoli are its important elements. He
finds “dass in einer Grundsubstanz, die im frischen Zustande
homophan, im geharteten ... hdchstens feinkornig erscheint,
grossere, scharf begrenzte, isolirte, starker lichtbrechende und
starker farbbare Kérperchen, Nucleoli, von wechselnder, aber fiir
die verschiedenen Zellarten und Thierspecies typischer Anzahl
eingebettet sind’; thus in the Batrachia most of the nuclei
contain numerous nucleoli, and when they are particularly
abundant the greater number are peripheral in_ position.
There are two kinds of “ Kernk6érperchen,” those which stain
blue (or green) and those which stain red (or yellow) ; both
kinds occur in most nuclei. In the giant nuclei of the gland
cells from the skin of Vrode/ea are found (1) numerous small cyan-
ophilic nucleoli, and (2) from one to fifteen (usually two to five)
much larger, erythrophilic nucleoli, which sometimes contain vac-
uoles. Embryonal nuclei contain only cyanophilic nucleoli, while
in maturer nuclei erythrophilic nuclei become differentiated
from the former. Thus in the blood corpuscles of frog larvae
there is at first only one large nucleolus, which later differenti-
ates into. an inner erythrophilic and an outer cyanophilic por-
tion. The peripheral layer next breaks up and divides into
small cyanophilic nucleoli, while the central portion remains
as a large erythrophilic nucleolus. Subsequently the smaller
cyanophilic nucleoli (“ Nebenkiigelchen”’) may fuse together
so as to produce six or eight larger cyanophilic nucleoli, each
of which attains the size of the original “ Stamm-Nucleolus”’;
at the conclusion of the larval period of the frog, the latter
322 MONTGOMERY. [VoL. XV.
nucleolus entirely disappears, becoming dissolved in the nuclear
sap. ‘Die erythrophile Kernsubstanz ist iibrigens dem Pro-
toplasma des Zellleibes offenbar ahnlicher als die kyanophile.”
Birger (90) made observations on the maturing ovum of
various Memerteans. Carinella: there is one large, spherical
nucleolus. In the ripe egg of Cerebrvatulus marginatus “in der
Regel kann man zwei umfangreiche Keimflecke konstatiren,
welche aus einer schwarzlich-griinen kornigen Substanz zusam-
mengesetzt sind, aber einen membranartig scharfen Kontour
besitzen. Die beiden Keimflecke sind nicht von gleicher
Grosse.” In the immature germinal vesicle of Drepanophorus :
“Dem wenig tingirten Binnenraum des Kernes durchflicht ein
zartes Netzwerk feiner Faserchen ; peripher sind grobere dunk-
lere Kornchen angeordnet”’; the ripe ovum of this Nemertean
contains one finely granular, central nucleolus, in which are found
“‘kuglige, noch intensiver gefarbte Koérperchen.” Pvrosadeno-
porus janthinus: constituting the inner portion of the wall of
the genital ducts are seen numerous cells, ‘welche ganz wie
in der Entwicklung im friihen Stadium stehen gebliebene
Geschlechtsprodukte aussehen,” and each of these cells has
one large nucleolus ; while in the ripe egg the “ Keimblaschen
ist ausgezeichnet durch eine Menge kugliger Blaschen von iiber
5“ Durchmesser mit scharf kontourirter und stark gefarbter
Peripherie.”
Eimer (90, cited by Mann, 92), recalls his previous observa-
tions (73, 78) in regard to the termination of nerve fibrils in
the nucleolus ; he mentions further that such radiating fibers
are also to be found in the nucleolus of the egg cell, such
fibers serving at first as paths for nourishment, and later
becoming nerve fibrils.
Henking (90), spermatogenesis of Pyrrhocoris: the single
peripheral nucleolus of the first spermatocyte gradually
becomes smaller in the prophase of division, and it is considered
probable “dass er spaterhin eine Einschniirung erfahrt.”
O. Hertwig (90), Ascaris megalocephala: in the spermato-
cytes of the growth zone the nucleolus is usually flattened
against the periphery of the nucleus, or it may be irregularly
elongated, or in addition to it a “« Nebennucleolus”” may be also
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 323
present ; from these differences in form he concludes that the
nucleolus may be capable of amoeboid movements. Subse-
quently it wanders towards the center of the nucleus, becomes
larger and more spherical. When the chromatin has assumed
the characteristic radial distribution, before the first maturation
division, the nucleolus passes again towards the periphery, and
there becomes gradually smaller, partly by fragmentation, and
so gradually disappears.
Holl (90) found one spherical nucleolus in ova of the newly
hatched chick: “Da das Kernkorperchen so auffallend verschie-
den vom Kernnetze und Kernsafte hinsichtlich des Verhaltens
zur Farbe sich zeigt, so muss es wohl aus einem anderen Stoffe
bestehen als jene. Auch bei Sa/amandra, Rana, und Lacerta
fand ich das Kernkorperchen immer sich verschieden halten
von den anderen Theilen des Kernes.” The nucleolus is always
situated excentrically at the upper pole of the nucleus. Towards
the end of the spirem stage the nucleolus lies on the periphery
of the chromatin, with which it stands in no close connection ;
it is no longer present in ova of 491 diameter.
Kastschenko ('90) investigated the maturation of the ova of
Pristiurus, Scyllium, and Torpedo: there are numerous nucleoli,
which attain a diameter of 16m, and all disappear at the
spirem stage (in the prophase of the first pole spindle). Each
nucleolus contains a large unstaining globule (but in his Fig. 1,
in several of the nucleoli, all of which had been stained with
borax carmine, this globule is colored blue, while the peripheral
portion of the nucleoli is red).
Masius (90): in the ovum of Asp/anchna the nucleolus forms
the greater part of the nucleus. In Lacznu/aria it is at first as
in the preceding genus, but at a later stage several much smaller
nucleoli are found.
Mellissinos and Nicolaides (90), pancreas cells of Canzs: The
“ Nebenkern” is a plasmosome which has wandered out of the
nucleus; this migration is caused by an injection of pilocarpin
into the living gland. ;
Sheldon (90) found one germinal spot in Peripatus capensis,
which disappears when the nucleus reaches the periphery of
the egg.
324 MONTGOMERY. [Vou. XV.
Smirnow ('90), sympathetic ganglion cells of Rana and Bufo:
a ‘“Kernkorperchenkreis”’ is figured around the nucleoli of
some of the cells.
I8or.
Brauer ('91) studied the maturation of the ovum of Aydra.
As a rule in the smaller eggs there is a single large nucleolus
which occupies an excentric position within the nucleus ; in
larger ova numerous small nucleoli arise, which gradually
become grouped near the large one. ‘“ Die Anzahl [der kleinen]
wechselt, was zum Theil darin seinen Grund zu haben scheint,
dass der grosse — selten sind zwei grosse vorhanden — wahr-
scheinlich durch Aufnahme kleinerer wachst . . . zum Theil
aber auch darin, dass in verschiedenen Keimblaschen die
Masse der Nucleolen eine verschieden grosse ist, was mit der
Ernahrung zusammenhangen mochte. . . . Sehr oft lag in der
Nahe des grossen Nucleolus eine etwa halb so grosse blasse
Kugel . . . moglich ware es, dass diese sich vom grossen Nucle-
olus abgespalten hat, und den achromatischen Theil derselben
vorstellt.” Just before the formation of the first pole spindle the
large nucleolus breaks into fragments, which, together with the
smaller nucleoli, wander towards the periphery of the nucleolus :
“Ein Theil scheint im Keimblaschen selbst aufgelost zu werden,
ein Theil tritt unverandert nach dem Schwinden der Membran in
das Eiprotoplasma iber.’’ Brauer contends that the nucleoli
have no morphological significance in the maturation of the egg.
Cuénot (91), ovarial egg of Synapta inhaerens: ‘la tache
germinative primitive bourgeonne une quantité de petits nuclé-
oles secondaires, qui errent dans le protoplasma clair de la
vésicule germinative; presque toujours la tache a un aspect
mamelonné par suite de la formation de ces nucléoles.”
Davenport ('91) figures in the germinal vesicle of Plamatella
a double nucleolus.
Macallum (91), following Ogata (83), distinguishes two kinds
of nucleoli, namely, plasmosomata and karysomata. He finds
the “ Nebenkerne”’ of Nussbaum to be abnormal structures.
In the pancreas cells of Amphibia an extrusion of plasmosomata
occurs, but it is not a normal process, and the extruded portion
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 325
does not become a “ Nebenkern”’ (in opposition to the views
of Ogata). In the eggs of Rava and Necturus the chromatin is
“ principally collected in the form of nucleoli at the periphery,”
but it is also contained in certain threads in the nucleus. He
concludes from the study of the reactions of the substances
to the indigo-carmine stain: “the peripheral nucleoli generate
a substance, therefore, which diffuses gradually through the
nucleus, then into the cell protoplasm, the point in time of the
latter occurrence corresponding with the formation of the yolk
spherules. The mode of origin is through a process of deposi-
tion from the nucleus of a substance allied to chromatin in the
cytoplasm. ... I regard the yolk spherules as formed by the
union of a derivative of the nuclear chromatin with a constituent
of the cell protoplasm.”
C. Schneider ('91) concludes that the true spherical nucleoli
“ebenso wie die Klumpen [of the chromatic network] aus
[achromatischen] Geriist und Chromatin bestehen und die
Unterschiede beider nur morphologischer Natur sind.’ In the
testicle cells of Astacus the nucleoli are spherical, with ‘eine
deutliche Membran an und durch welche genau wie bei der
Kernmembran [achromatische] Geriistfaden treten.... Der
ganze Unterschied zwischen Nucleolus und Klumpen besteht
also hier darin, dass um ersteren die Fasern zu einer Membran
sich zusammenlegen . . . was man ringformig am Rande des
Nucleolus wahrnimmt, ist sicher nicht die optische Wiedergabe
einer Membran, sondern durch das Brechungsvermégen der
Wandung des Nucleolus veranlasst.”” The nucleolus in eggs of
Echinodermata is homogeneous only in the final stages of its
formation. Nucleoli are only metamorphosed portions of the
true chromatin, and represent reserve masses of this substance :
“die Zusammenballung kann nur eine Befreiung der chroma-
tischen Substanz von ihrer Arbeitsleistung bedeuten.”
Wolters ('91) studied the sporulation of MJoxocystis: in the
youngest individuals there is one nucleolus, which “in seinem
Innern sich starker tingirende chromatische Kugeln fiihrt.”” In
larger individuals the nucleolus consists of eight spheres, ‘ Diese
Kugeln fiihren in ihrem Innern wieder Stabchen und Korner.”
Just before the conjugation of two individuals this compound
326 MONTGOMERY. [VoL. XV.
nucleolus breaks into a number of nucleoli of various sizes.
After the copulation and encysting the nucleoli fuse together
and gradually disappear (but I am unable to determine from
his description whether the substance of the chromosomes is
derived from the nucleoli). Shortly after the nuclei themselves
copulate, the nucleoli reappear in them. In Clepsidrina blat-
tarum there is a single primitive nucleolus, formed as in
the preceding species; later there are numerous smaller
nucleoli, which have probably arisen by division from the
primitive nucleolus.
1892.
Bannwarth ('92) figures a division of the nucleolus in leuco-
cytes from the spleen of the cat.
Born (92) finds that in the Amphibian egg, in opposition
to the observations of O. Schultze (’87), the chromatic “ Faden-
knauel” has no origin in the nucleoli, but is directly derived
from the chromatin network of the “ Urei.”
Brauer ('92) made observations on the maturation and fecun-
dation of the egg of Branchipus. Each germinal vesicle from
the ‘“ Wachsthumszone” of the ovary has one large, slightly
staining nucleolus, and near it a much smaller, deeply staining
one. Each ‘ Nahrzelle,” however, contains numerous nucleoli,
and its nuclear sapalso stains deeply. When the chromosomes
are being produced, the larger nucleolus of the egg cell gradu-
ally ceases to stain, and it finally disappears. In the male
pronucleus small nucleoli are present.
Frenzel ('92) noticed in Carcznus moenas and in a species of
Amphipod, in the ferment cells and “ Fettzellen” of the hepa-
topancreas, amitotic division of the nucleus, but no division of
the nucleolus ; “‘sondern dass vielmehr an geeigneter Stelle
des Tochterkernes noch vor der Abschniirung desselben ein
ganz neuer Nucleolus entstehe, der alle Charaktere des ersten
besitzt”’ ; in this nuclear division one of the daughter-nuclei
retains the whole original nucleolus. In similar cells of /dotea
tricuspidata he found the nuclear division to be as in the pre-
ceding species (but his Figures 8b, 10, and especially 11, would
seem to represent stages of division of the nucleolus).
No.2.) COMPARATIVE CYTOLOGICAL STUDIES. 327
Hacker (92a) studied the early development of Aeguorea
forskalea: in the ripe egg there is one spherical or kidney-
shaped nucleolus, containing vacuoles. At the time of the
first pole body mitosis the nucleolus does not accompany the
nucleus, but remains behind in the cell, at the place previously
occupied by the nucleus ; and from this time on he applies to
it the name “ Metanucleolus.” It is to be observed in one of
the cleavage cells until about the 32-cell stage. ‘Zur Zeit
wenn sich dann in der schwarmenden Blastula die Zellen des
hinteren Poles . . . zu differenziren beginnen, kann man in
einzelnen von ihnen neben dem chromatischen Fadenknauel
kleine nucleolenahnliche Korper beobachten, welche den nicht
differenzirten Blastula-Elementen fehlen. Es ware denkbar,
dass man es hier mit den Abkémmlingen des Metanucleolus zu
thun hat, ich vermag aber weder hierriiber, noch tber das
weitere Schicksal dieser Gebilde etwas bestimmtes zu sagen.”
Hacker assumes that what Metschnikoff ('86) supposed to be
the “ Sperm-nucleus ” in A/ztrocoma was in reality a Metanucle-
olus ; and also that the “ Paracopulationszelle,’ described by
Weismann and Ishikawa (89) in the winter egg of Daphnia,
to have been also a nucleolus.
Hacker in a second paper ('92b) studied the maturation of
the ovum of Canthocamptus. In the smallest eggs the nucleolus
is large and contains vacuoles. Later it becomes differentiated
into a lighter central portion and a denser peripheral part con-
taining small vacuoles. At this stage the nucleolus presents a
concavity facing the chromatic spirem. Then ‘aus dem Kern-
k6rper tritt unter plotzlicher Verkleinerung desselben eine Masse
aus, welche vermuthlich dem grossen, bis dahin in den meisten
Kernkoérpern kugligen Einschluss entspricht.” The nucleolus
apparently disappears when the first pole spindle is perfected.
Heidenhain ('92), cells of Salamandra: the nucleoli lie
enclosed within the chromatin and linin network ; he was unable
to decide whether each nucleolus has a particular chromatin
envelope. The nucleolus has no processes, and ‘nur die ihm
auflagernde, von ihm selber stofflich differente Schicht ist mit
dem Chromatin- und Lininfadengeriist kontinuierlich verbun-
den... . Mirist es wenig wahrscheinlich, dass die Substanz
328 MONTGOMERY. [VoL. XV.
der Nukleolen etwas dem Chromatin ahnliches sei. Zwar sind
sie durch einige Chromatinfarbstoffe stark farbbar, wie z. B.
durch Safranan, allein auf eine andere Gruppe derartiger Farb-
stoffe reagieren sie nicht, hierher gehort das Methylgriin.”
O. Hertwig ('92) in his recent text-book materially changes
some of the views expressed in his previous papers. The
true nucleoli consist of ‘ Paranuclein” (Pyrenin), and he
uses the term “ Nuclein” for chromatin. ‘ Nuclein und Para-
nuclein betrachte ich als die wesentlichen Substanzen des
Kerns. . . . Beide scheinen mir in irgend welchen Bezie-
hungen zu einander zu stehen.” Further, he distinguishes
“ Keimflecke” from “echte Nucleolen.” ‘Je nach dem Alter
oder der Entwicklungsstufe einer Zelle kann der ruhende Kern
. in der Zahl, Grosse und Beschaffenheit seiner ‘ Nucle-
olen’ erhebliche Veranderungen erleiden.”
Kostanecki ('92a) is preliminary to his ’92b.
Kostanecki ('92b) studied mitoses “ in samtlichen embryonalen
Zellen”’ of Lepus, Cavia, and Eguus, with especial regard to the
central spindle; I quote this paper here, since the “ Central-
spindelkorperchen’”’ may have some relation to nucleoli. ‘Im
Bereich dieser Centralspindel sieht man in diesem Stadium
[Dyaster] in der Nahe der beiderseitigen Tochterfiguren der
Chromosomen kleine Ké6rperchen auftreten, die ich als ‘Cen-
tralspindelkérperchen”’ bezeichnet habe. Grédsse und Zahl
dieser K6rperchen zeigen ganz betrachtliche Schwankungen.
Meist fand ich nun jederseits vier, fiinf oder sechs grés-
sere Korperchen, . . . daneben aber immer noch eine gréssere
Anzahl kleinerer Kornchen. Diese Kérnchen sowohl als auch
die grésseren Koérnchen standen in inniger Beziehung zu den
Faden der Centralspindel.” These granules then wander from
both sides towards the equator of the spindle, ‘so dass sie. . .
eine aquatoriale Kornchenplatte bilden. . . . Sobald die Ein-
schniirung des Zellleibes bis zur Centralspindel vorgeschritten
ist, werden die mehr peripher gelegenen Centralspindelfasern
gerade im Aequator da, wo die Centralspindelkérperchen liegen,
durchschnitten, und man sieht die Korperchen zugleich mit den
verkiirzten und undeutlich werdenden Fasern sich wiederum
polwarts begeben.’”’ At each pole, then, these granules become
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 329
so densely grouped that often only one or two large granules
appear to be present. ‘Mit der volligen Durchschniirung
der Zellen wird schliesslich . . . der Zwischenkorper in zwei
Theile getrennt, von denen jeder einer Tochterzelle angehért.”’
Similar in the main points is also this process in the Chick,
Frog, Axolotl, Triton,and Salamandra: ‘Wenn wir uns nun
fragen, ob diese Vorgange bei tierischen Zellen mit Recht mit
den Vorgangen der Zellplattenbildung bei den pflanzlichen
Zellen homologisiert wurden, so kann ich diese Frage nur zum
Teil bejahen”’; for two processes take place together, ‘‘ namlich
eine aquatoriale Differenzierung der Centralspindelfasern zum
Zweck ihrer Halbierung und eine eigentliche Zellplattenbildung,
aus der die Zellscheidewand hervorgeht. Von diesen beiden
Prozessen ist der eine, namlich eine eigentliche Zellplatten-
bildung zum Zweck der Scheidewandbildung, bei tierischen
Zellen gar nicht vertreten, wodurch der zweite desto deutlicher
und unverhiillter zu Tage tritt.” (Kostanecki mentions the
following observations of previous authors on the occurrence
of such a granular aequatorial plate in animals: Van Beneden,
germs of Dicyemida ; Balbiani, epithelial cells of an Orthopterous
larva; Fol, eggs of echinoderms and Cymdula; Flemming,
eggs of echinoderms ; Biitschli, egg of Vephelis ; Mark, egg of
Limax ; van Gehuchten, egg of Ascaris megalocephala,; Pre-
nant, testicle cells of Scolopendra and Lithobius; Henking,
similar cells of Pyrrhocoris ; numerous observations of Carnoy ;
Van Beneden, ectoderm of vertebrate embryos ; Strasburger,
cartilage cells of vertebrates; Mayzel, corneal epithelium of
Fringilla ; Schleicher, cartilage cells of Batrachia ; Carnoy,
Triton ; Biitschli, embryonal blood corpuscles of chick ; Schott-
lander, inflamed epithelium of the cornea of the frog.)
Kraepelin (92, cited by Braem, '97) noticed in the Bryozoan
egg a division of the nucleolus.
Loénnberg ('92) studied the nucleoli of various ova and somatic
cells. In the liver cells of Mytilus there are two “ Nebennu-
cleoli” and one “ Hauptnucleolus.” In the cells of the intestinal
epithelium of Ze//imva a granule is sometimes found on the
outer surface of the nucleus, which resembles a small nucleolus,
and stains in the same manner. Doris, egg: “eine starker
330 MONTGOMERY. [VoL. XV.
sich farbende Kugel (meist auch eine oder mehrere kleine
Vacuolen) in einer grésseren hineingesenkt war und so den
Nucleolus darstellte.” /ytzlus; “In den Einucleolen von
Mytilus liegt oft eine (oder bisweilen zwei) grosse, blasse
Kugeln in der Mitte oder ein wenig excentrisch, aber von der
starker sich tingirenden Substanz vollstandig umschlossen ; es
ist schwer zu unterscheiden, ob es sich hier nur um Vacuolen
handelt. . . . Bei Aeolzdia papillosa [Ei] . . . zwei, ein wenig
abgeplattete Kugeln die in einanden teilweise eingesenkt sind.
Diese Kugeln sind aber hier beinahe gleich gross und die
blasse ist in der gefarbten eingesenkt, bei Unzo [nach Flem-
ming] umgekehrt. . .. In den jungen Keimzellen fand ich
nur einen einfachen Nucleolus, und dieser farbte sich stark.”
In the liver cells of Doris proxima there are two nucleoli:
“Der eine von diesen ist ganz kugelrund und stark licht-
brechend, glanzend; dieser, der sich auch intensiv tingiert, muss
als eigentlicher Nucleolus aufgefasst werden. Der andere ist
blasser und grosser, seine Gestalt ist bald rundlich, bald lang-
lich, bohnenformig also mehr unregelmiassig ; diesen méchte ich
als Nebennucleolus bezeichnen”; the two stain differently ;
“Die Lage der beiden Kernkérperchen ist auch wechselnd,
indem sie bald ganz neben einander liegen oder sogar der Nucle-
olus im Nebennucleolus hineingesenkt, bald vdéllig getrennt
sind... . Der Nebennucleolus, der immer scharf begrenzt
ist, enthalt oft eine kleine Vacuole. Ein Paar Mal traf ich in
demselben Kern zwei Nebennucleoli.” The latter are homoge-
neous, with an outer clearer layer, while the “ Hauptnucleolus”’
is granular. L6nnberg found similar nucleoli also in the liver
cells of Polycera and Acolidia. Liver cells of the “ Krebs”
(Astacus ?): “Meist sieht man... einen blassen Korper,
der sich schwach wie der Nebennucleolus bei den Nudibranchi-
aten farbt, und daneben einen oder mehrere kleine K6rper-
chen, die sich intensiv tingiren und sich wie Nucleolen verhal-
ten; .. . bald liegt ein stark gefarbtes Kiigelchen an einem
Pole des Nebennucleolus, bald eins an jedem Pole desselben und
in wieder anderen Fallen schmiegen sich drei Nucleolkorper-
chen dem Nebennucleolus an. Bisweilen treten Nebennucle-
olen in zwei- oder dreifacher Zahl auf.” Lonnberg concludes
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 331
that the “ Nebennucleoli”’ may play a part in the acquisition
of nourishment or may hold reserve nourishment.
Marshall ('92) studied the sporulation of Gregarina blattarum
v. Sieb. In the youngest individuals there is one large nucle-
olus. In larger ones there are one large and two or three
smaller nucleoli, or four or five smaller ones of equal size ;
these now increase in size, accompanying the growth of the
nucleus. He believes that the smaller nucleoli which are sub-
sequently produced, arise in only one (as a rule) of the four or
five original nucleoli: “Im Innern dieses Formationsnucleolus
erscheinen dann klare, runde Ballen von verschiedener Grosse,
welche keine bestimmte Grosse haben. Sie sind in wechselnder
Zahl vorhanden und etwas heller als die itibrige Masse des
Nucleolus. Bei vielen Formationsnucleoli . . . waren alle
Stadien der Entwickelung zu finden; kleine und grdéssere
Ballen im Innern, und einige, die schon halb nach aussen
getreten waren.’ After leaving the ‘“ Formationsnucleolus ”
they stain like the latter, and become either irregularly or
spirally grouped together. ‘Die Vermehrung dauert bis zum
Beginn der Cystenbildung fort. . . . Am Anfang der Encys-
tierung enthalt jeder Kern etwa 25-40 deutlich erkennbare
Nucleoli, welche bald in dieser, bald in jener Art angeordnet
sind. In beiden Fallen liegt der jetzt unregelmassig gestaltete
Formationsnucleolus der von ihm ausgegangenen Gruppe ge-
geniiber” ; the latter is smaller than heretofore, ‘doch zeigt
er noch Ballen im Innern.” The smaller nucleoli increase
in number, but now by repeated divisions of their own; the
small granules resulting from these divisions are termed
«‘Chromatink6rner” : “‘Jedes Chromatinkorn bildet nun eine
Hille um sich, nachdem es sich vorher mit einer Schicht Plasma
umgeben hat. Auf diese Weise vollzieht sich die Bildung der
jungen Sporen.... Kurze Zeit, nachdem die Spore gebildet ist,
nimmt dieses Chromatin-Korn die Gestalt einer 8 an und teilt
sich in zwei Halften, die beide an die entgegengesetzten Seiten
der Spore treten.’”’ Later each of these divides into two, and
each of the resulting four then divides into two, so that eight
is the result; then one such “Chromatin-Korn”’ is allotted to
each “Keim” (young Gregarine) and represents its nucleus.
B82 MONTGOMERY. [VoL. XV.
Riickert (92) studied the maturation of the eggs of Scy//ium,
Pristiurus, and Torpedo. In young germinal vesicles there
are a few small nucleoli, most of them peripheral in
position. In larger ova they have increased in number and
size, and become grouped in a cluster at that part of the nucleus
which is nearest the animal pole of the egg; this cluster may
occupy one-fourth of the whole space of the nucleus. Later,
but still antecedent to the formation of the pole spindles, the
nucleoli decrease in size and commence to stain very faintly.
Riickert considers the nucleolus of an egg cell as strictly com-
parable to that of any somatic cell. From the fact that the
nucleoli are largest, and color most intensely, at the same time
that the chromosomes do, and simultaneously with the latter
become gradually invisible later, he concludes : “dass es die
Stoffwechselvorgange der Chromosomen sind, zu welchen die
Nucleolen in direkter Beziehung stehen, sei es nun, dass sie
notwendige Stoffe an die letzteren abgeben (vielleicht das Chro-
matin, wie schon Flemming vermutete), oder dass sie Stoffe
von ihnen aufnehmen, oder endlich dass beides zugleich der
Fall ist.... Spater freilich, wenn die Chromosomen merklich
an Substanz verlieren, wird man eher geneigt sein, die betref-
fenden Nucleolen als Trager von Zerfallsprodukten der Chromo-
somen anzusehen.” He also observed that the number of the
nucleoli varies in different germinal vesicles of the same age,
that a number may coalesce to form a larger one, and that a
few wander out into the cytoplasm, where they become paler
and finally vanish.
Wireén (92) found that the smallest germinal vesicles of Chae-
toderma contain no nucleoli ; in nuclei of about 15 diameter a
nucleolus appears for the first time, and consists of a dense
mass of granules, which stain differently from the other nuclear
granules. More than one nucleolus is never to be found.
189}.
Van Bambeke ('93) found one to five homogeneous nucleoli
in the germinal vesicles of Scorpaena scrofa, and notes that in
older eggs they do not stain as deeply with carmine as in
younger ones.
No.2.] COMPARATIVE CYTOLOGICAL STUDIES. 333
Bohmig (93), Riodope veranii: the single nucleolus in older
eggs contains one or several vacuoles.
Brauer ('93) investigated the spermatogenesis of Ascarzs
megalocephala: there is one homogeneous nucleolus in the
spermatogonium, which becomes smaller in the spermatocyte
and often evinces a large vacuole. The nucleolus is smaller
than the centrosome (which is at this stage enclosed in the
nucleus), and stains differently from the latter.
Brooks ('93), Sa/pa. the single large nucleolus of the ovarian
ovum ‘is suspended near the center of the nucleus by a net-
work of fine threads.’’
Fick (93) studied the maturation of the egg of the Avo/ot/.
In the germinal vesicle lies a group of nucleoli, which vary in
size from 3u to 16; some contain a single vacuole, and some
stain more deeply than others. The greater number of them
disappear at the time of the longitudinal division of the chro-
mosomes, though a few may remain in the yolk for a certain
time. ‘Bei den Nucleolen des Keimblaschens liegt es sehr
nahe mit Strasburger und Pfitzner daran zu denken, dass sie
vielleicht eine Art Reservestoffbehalter darstellen”’ ; further,
he holds that the nucleoli “in einer allerdings noch nicht auf-
geklarten Beziehung zu den Veranderungen des Chromatins
stehen, da sie bei der Ausbildung der Chromosomen fiir die
erste Spindel vollstandig verschwinden.”’
Frenzel ('93a) studied the nucleoli of various Gvregarines.
In Gregarina statirae the single nucleolus, which he terms
“Morulit,” appears “eigenthiimlich glanzend mit einem
schwach gelblichen Schimmer und dabei an der Oberflache
rauh und warzig-runzelig. . .. In seinen Reaktionen verhalt
er sich an allen Orten ahnlich wie Nuklein.” In G. dergza
single Morulit is present. Inthe embryo of Pyxinia crystalligera
there is a single Morulit; in older individuals the nucleus
“enthalt mehrere helle, klare, glattrandige und lebhaft glan-
zende Nucleoli . . . die oft noch einen Fliissigkeitsraum im
Innern bergen.” In Gregarina portunt, Callyntrochlamys, and
Aggregata portunidarum there are several nucleoli inthe nucleus.
Frenzel ('93b) hepatopancreas cells of Astacus: in the fat
and ferment cells a single nucleolus is present ; in the amitosis
334 MONTGOMERY. [Vou. XV.
of the nuclei he concludes that the nucleolus divides (‘«nukleo-
lare Kernhalbierung’’), since the nucleoli of the daughter-cells
are of equal size.
Hacker ('93a) divides the maturation stages of the ovarian
eggs of Moina, Cyclops, and Szda, into two periods, “von denen
der erste gekennzeichnet ist durch die Anwesenheit eines
einzigen ‘Nucleolus’ und durch die leichte Farbbarkeit des
Fadenspirems (chromatische Stufe), der zweite durch die
Anwesenheit mehrerer ‘Nucleolen’ und die Abneigung der
chromatischen Substanz, die Mehrzahl der Farbungsmittel
anzunehmen (achromatische Stufe).’” In the first period
(‘‘Wachstumsphase”’) there is one excentric, deeply staining
nucleolus (‘‘ Hauptnucleolus”’), which possesses a “ Hiillmem-
bran’’; in the second period, in addition to the “ Hauptnucle-
olus’’ there are also one or two “ Nebennucleoli” of greater
size than the former, but staining less deeply, and somewhat
irregular in form. Both kinds of nucleoli contain vacuoles.
The “Nebennucleolus ... stellt sich vielfach als hohles
Gebilde von ellipsoidischer Gestalt dar, dessen einem Pole der
Hauptnucleolus kappenformig aufsitzt.’’ Only the outer shell
of this nucleolus stains deeply. Subsequently the “ Haupt-
nucleolus’? grows gradually smaller and finally disappears ;
and at the same time the “ Nebennucleolus”’ increases in size
and becomes irregularly lobular in shape, and finally breaks
into pieces. The nucleolar relations in Mozna are as in Cyclops
(just described). In Szda only a “ Hauptnucleolus”’ is present,
and this contains a large central and several smaller peripheral
vacuoles. Hacker distinguishes the following types of ova
with regard to their nucleolar structure : (1) Lamellibranchiate
type, with one “ Hauptnucleolus’’ and one or two “ Neben-
nucleoli,” the latter larger and less chromatic than the former,
but both frequently in close connection (Naja, Anodonta,
Cyclops brevicornis); (2) Echinoderm type, with one large
‘«‘Hauptnucleolus,” which increases in size, and only towards
the close of the “Keimblaschenstadium” do a few smaller
nucleoli appear (Zoxopneustes, Sida crystallina, primiparous
Cyclops strenuus and C. signatus); (3) Vertebrate type, with
several nucleoli varying in size, number, and form (Raza and
No.2.) COMPARATIVE CYTOLOGICAL STUDIES. 335
numerous other Vertebrates, Sagitta, Motna, Cyclops brevicornis,
multiparous C. stvenuus). ‘Aus der obigen Zusammenstel-
lung ... geht ... hervor, dass das unter der Bezeichnung
‘Nucleolus’ oder ‘Keimfleck’ im Eikern auftretende Gebilde
hauptsdchlich in zweierlei Gestalt auftritt : entweder stellt
dasselbe einen in der Einzahl vorhandenen, stetig seine Grosse
verandernden, formbestandigen K6rper dar, oder aber finden
sich als ‘ Nebennucleolen’ Blaschen oder Trépfchen von wech-
selnder Zahl, Grodsse und Gestalt vor.” The ‘ Hauptnucle-
olus”’ remains in the nucleus until just before the formation of
the first pole spindle ; after that it either diminishes rapidly in
size, or it passes out of the nucleus into the cytoplasm, where
it remains for a time as a “ Metanucleolus”’; the ‘“ Hauptnucle-
olus” is phylogenetically derived from a “ Nebennucleolus,”
and has developed into “einen membranumhiillten, formbe-
standigen und stetig durch Diomose wachsenden Organ.”
“Es diirfte vielleicht zunachst die Thatsache heranzuziehen
sein, dass ein Auftreten von ‘ Nebennucleolen’ von wechselnder
Zahl, Form und Grésse und von analoger chemischer Reak-
tion auch in den ruhenden Furchungskernen der betreffenden
Thierformen festzustellen ist, und dass diese ‘ Nebennucleolen ’
hier nicht mit einem als Hauptnucleolus anzusprechenden
Korper vergesellschaftet sind. Nebennucleolen treten folglich
auch da im ruhenden Kerne auf, wo kein Zellenwachsthum
stattfindet.” Hacker considers that the ‘“‘ Nebennucleoli” are
not drops of a nutritive fluid, but ‘““Abspaltungsprodukte oder
Sekretstoffe der chromatischen Substanz. Diese Auffassung
findet vor allem in der Thatsache eine Stiitze, dass die Neben-
nucleolen, z. B. bei Mozva und Cyclops strenuus (mehrgebarend),
im Lauf der Wachsthumsphase stetig an Grdésse und Massigkeit
zunehmen und dass sie das Maximum ihrer Entwickelung erst
in dem Moment erreichen, wenn bereits die Vierergruppen zur
Ausbildung gelangt sind, wenn also von einem Wachsthum der
chromatischen Substanz kaum mehr die Rede sein kann.”
Hacker ('93b) : a preliminary contribution to the following
paper.
Hacker ('93c) found in the germinal vesicle of Echinus micro-
tuberculatus, in addition to the “ Hauptnucleolus,” a few small
336 MONTGOMERY. [Vor. XV.
globules which stain in the same manner as, and are probably
homologous to, the “Nebennucleoli” of other animals ; the
‘«‘Hauptnucleolus”’ increases in size by the absorption of these
latter. “Der Hauptnucleolus des Echiniden-Keimblaschens
ist ... ein pulsirendes Organulum, in welchem periodisch eine
grosse Hauptvacuole sich durch Zusammenfluss_ kleinerer
Vacuolen bildet, um dann wieder langsam abzunehmen....
Was die Dauer der Perioden anbelangt, so wurden solche von
vier bis zu solchen von acht Stunden beobachtet ’’; the central
vacuole at the time of its maximum size passes from the cen-
ter to the periphery of the nucleolus: ‘ Die Centralvacuole
tritt also in Beziehung zur aussersten Wandschicht des Haupt-
nucleolus, anscheinend um ihren Inhalt mit ... den Kernsaft
in Kommunikation zu bringen.” Accordingly, the “ Haupt-
nucleolus”? may be “als ein osmotisches System betrachtet
werden, in welchem die feste Substanz (Rindensubstanz) nach
zwei Seiten hin, einerseits mit dem Kernsaft, anderseits mit
den Vacuolen, in diosmirender Verbindung steht. Sobald
jedoch ein Korper nach zwei Seiten diosmirt, so ist eine An-
haufung in demselben nur durch das Eingehen einer neuen
Verbindung méglich [Pfeffer (91)]. Es folgt schon hieraus,
dass die aus dem Kernsaft aufgenommene Fliissigkeit in der
Nucleolarsubstanz nicht nur eine Verdichtung, sondern auch
eine weitere chemische Umsetzung erfahren muss.’”’ The fluid
vacuoles of the ‘ Hauptnucleolus” represent an excretion
which in Echinus is periodic, while in the Copepoda “im Laufe
der Eireife wachst unter Mitwirkung der Rindenvacuole die
Centralvacuole Jangsam heran, nimmt allmahlich eine excen-
trische Lage an und entleert sodann kurz vor der Bildung der
Richtungskorper ihren Inhalt nach aussen.’’ He compares the
vacuole of the “ Hauptnucleolus”’ to the pulsating vacuole of
the /zfusoria : “so wiirden die Centralvacuole des Hauptnucleo-
lus mit der eigentlichen pulsirenden Vacuole des Protozoen-
korpers, die Rindenvacuolen des Hauptnucleolus mit den
Bildungsvacuolen zu vergleichen sein.” From a study of the
pole-body mitoses he concludes: ‘dass der Hauptnucleolus
wahrend der Auflésung der Keimblaschenwandung zunachst
noch in seiner urspriinglichen Grosse erhalten bleibt und sich
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. Qa
von dem Kernplasma langsam zu trennen beginnt.” He could
not exactly determine how long the nucleolus remains in the egg
after that, but considers the fact important, ‘dass der Haupt-
nucleolus zur Zeit der Umbildung der Keimblaschensubstanz
ohne bemerkbare Volumverminderung fort besteht’’; it is at
this time (after the disappearance of the nuclear membrane),
to use his terminology, a ‘“ Metanucleolus.”
Henneguy ('93) studied principally the genesis and occur-
rence of the yolk nuclei, “corps vitellin de Balbiani,” in the
ovarian egg of various vertebrates. This body is absent in the
ova of the Rabbit, Bitch, Mole, Rhinolophus, Cow, Antelope,
Baboon; and in Lizards, Galeus, Raja, and Scyllium. In the
rat it consists of a peripheral clearer portion and a central
denser core ; it stains with eosin, safranin, and haematoxylin,
but not with methyl green or gentian violet. In the bat it
encloses a spherical corpuscle. It is also present in the chicken.
Though absent in Bufo and Triton, it is found in Rana tempo-
varia, where it is much the same as in birds, enclosing a more
deeply staining portion. In the trout the corpuscle of Balbiani is
as in the rat, but larger (twenty). Sygnathus: the very young
egg contains a nucleus “ renfermant un réseau chromatique bien
développé”’ (his Fig. 20 would show three small nucleoli enclosed
in this ‘réseau ’”’); in older eggs the nuclear membrane is lined
by a large number of nucleoli, and “le centre du noyau est
occupé par une petite masse finement granuleuse et teintée en
rose par la safranine, tandisque le reste du contenu demeure
incolore. Le protoplasma ovulaire est également faiblement
coloré et renferme un corpuscule arrondi, réfringent comme les
taches germinatives et retenant la safranine avec la méme
intensité que ces derniéres”’ ; this corpuscle at the time of its
first appearance is flattened against the outer surface of
the nuclear membrane. Subsequently this intravitelline body
becomes elliptical in form, with its long axis parallel to the surface
of the egg : “il est de plus au contact immédiat par son bord
externe avec une amas arrondi, constitué par une substance
fondamentale d’apparence homogéne, mais remplie de granula-
tions trés colorées. A un stade plus avancé tout le corps
réfringent s’est transformé en un amas let qu’on l’observe dans
338 MONTGOMERY. [VoL. XV.
la plupart des ovules des Poissons’’; in this manner it develops
into a Balbianian corpuscle, and later breaks into small granules.
In no eggs of any of the species studied are more than one
of these corpuscles present; and it always arises during the
maturation period of the ovum, before fecundation. “C'est
trés probablement une partie de la tache germinative, ou une
tache germinative enti¢re, qui sort la vésicule [germinative]
pour penétrer dans le vitellus. . . . C’est un organe ancestral
qui, avec les éléments nucléolaires de la vésicule germinative,
correspond au macronucleus des Infusoires, le micronucleus
étant représenté par le réseau chromatique, prenant seul part
aux phénomeénes de fécondation.”
Heuscher (93) noticed in the ovum of Proneomenia either
one nucleolus, or two of different size which were usually
separated from each other.
Holl (93) studied the maturation of the ovum of the mouse.
“Die Faden [Chromatin] zeigen eine innige Verbindung mit
dem Kernkorperchen derart, als ware dasselbe ein Centrum,
von welchem die Faden des Netzwerkes auslaufen.”’ The
nucleolus is not homogeneous, but contains granules (“ Schroen’-
sche Korner’) to the number of twenty ; these gradually become
stained during the growth of the nucleus, until the whole nu-
cleus becomes evenly stained. ‘Im weiteren Verlaufe der
Entwickelung treten die Schroen’schen Korner aus dem Kern-
korperchen heraus und gelangen als chromatische Ballen in
das Kernnetz, wo sie sich mit den Faden desselben verbinden.
Endlich wird das Kernkorperchen von seinem Inhalte ganz
frei; es bleibt nur die Kernkorperchenmembrane iibrig, und im
Kernraume liegen zerstreut eine gréssere Anzahl der chroma-
tischen Ballen. Dieselben sind anfangs klein und schwach
gefarbt, wachsen auf 2m heran und farben sich immer besser.
Die chromatischen Ballen wandern aus dem Kerne aus,
und das ibrige [Fadennetzwerk] riickt als ‘Kernrest’ ganz an
die Oberflache der Eizelle. Die chromatischen Ballen liegen
in 6 Gruppen von je 4 neben einander, und jeder Ballen wandelt
sich in eine dicke, kurze Schleife um,” z.¢., a chromosome of the
“« Richtungsspindel.”
Jordan (93) studied the development of the ovum of the
No.2.] COMPARATIVE CYTOLOGICAL STUDIES. 339
newt. He thinks ‘that certain deutogenic substances are
formed in the [germinal] vesicle, perhaps through the agency
of the nucleoli, and are then sent forth to share in the building
up of the cell,” 2.2. of the yolk particles. ‘The nucleoli in
the young egg appear arranged along the chromatin threads,
and possibly originate from the thread substance.” Later they
lose this connection, grow larger, and assume a_ peripheral
position within the nucleus. There is apparently no division
of the nucleoli; they ‘attain their maximum size shortly
before their centripetal movement.” Having arrived at the
periphery of the nucleus, the nucleoli commence to stain less
deeply, their contours become uneven, and they then wander
back to the center of the nucleus, where they disintegrate.
He does not agree with O. Schultze (87) that the nucleolar
particles build up the chromosomes.
Kaiser ('93) found in the egg of Echinorhynchus bipennis one
large, spherical, peripherally situated nucleolus. It disappears
before the pole spindle is produced.
Lustig and Galeotti ('93), mentioned by Lardowsky ('94), con-
sider that the centrosome does not proceed from the nucleolus.
Mertens ('93), ovum of Yomo: two or three nucleoli are present,
consisting of a central clearer and a peripheral darker portion;
it is probable that several smaller ones may fuse together to
form a larger one ; they are at first in intimate connection with
the chromatin filaments, but later lose this connection and gradu-
ally cease to stain with safranin. The Balbianian corpuscle
is an extruded nucleolus: “c’est alors aussi que nous nous
étondrons quelque peu sur l’expulsion des parties chromatiques
du noyaux, expulsion qui parait affecter les mémes charactéres
chez les oiseaux et les mammiféres’’; eliminated nucleoli
(‘grains chromatiques ’’) as well as attraction spheres have been
described as Balbianian corpuscles. Ovum of Pica: in young
ovules there is one nucleolus which arises as follows: at one
point in the nucleus the reticulum concentrates itself, and
here a certain number of the filaments fuse together, thus
producing the nucleolus. The chromatin is at first irregularly
arranged in the nucleolus, but “finit par étre également dense
dans toutes les parties de la tache germinative,’ and subse-
340 MONTGOMERY. [VoL. XV.
quently accumulates on its surface. ‘Le nucléole devenu
indépendant [from the chromatin reticulum] est expulsé : les
chromosomes s’écartent pour lui livrer passage. II n’est pas
rare d’en rencontrer qui, arrivés a la périphérie, sont coiffés par
un filament nucléinien. . . . Le filament se rompt bientot et le
nucléole est libre.’”” The presence of a vacuole in the nucle-
olus is explained by the assumption that the chromatin wanders
to the periphery of the nucleolus, thereby leaving a clear
space at the center of the latter. (Safranin the only stain
employed.)
Minchin ('93) states that the single nucleolus of Gregarina
irregularis “consists of a darkly stained ground substance con-
taining an immense number of clear vacuoles of all sizes. One
of the vacuoles is much larger than the others, and being
excentrically placed, constitutes the clear spot seen in the
thick sections.” The nucleolus of G. holothuriae has a similar
structure.
Pizon (93), ova of Botryllida: a single large nucleolus con-
taining several vacuoles.
Repiachoff (93) figures a large vacuole in the single nucleolus
of the ovarial cells of a pelagic, acoelic Rhabdocoele (species
undetermined).
From Rhumbler’s contribution (93) to the morphology of the
nucleoli, or ‘“ Binnenkorper,”’ the following extracts are impor-
tant: “Mir scheint es. ..noch keineswegs sicher, ob die
Nucleolen der Gewebszellen und die Nucleolen der Keimzellen
bezw. vieler Protozoen (vielleicht ausgenommen die Ciliaten
und Suctorien) analoge Gebilde sind; obgleich auch das Gegen-
theil wegen des ahnlichen Verhaltens der beiderlei Nucleolen-
arten wahrend der Mitose sehr zweifelhaft bleiben muss.”’ In
Saccamina sphaerica there are from 1 to 300 nucleoli: “ ahnlich
wechselnd wie ihre Zahl ist ihre Grosse, ihr Lichtbrechungs-
vermogen und ihre Gestalt.’”’ The largest of them “zeigen
meist eine, durch starkeres Lichtbrechungsvermégen ausge-
zeichnete Innenmasse, in welche kleinere, noch starker bre-
chende und oft von der Kugelgestalt abweichende unregelmas-
sige Kérperchen eingelagert sind, und eine dunklere, weniger
lichtbrechende Aussenmasse, die in gleichmassiger Dicke wie
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 341
eine feste Membran um die Innenmasse herum gelagert ist ”’;
this latter portion also stains more intensely with eosin.
Rhumbler concludes that the “ Binnenkorper. . . durch Zu-
sammenfliessen anfanglich leicht fliissiger, dann zahflissiger
und schliesslich erstarrender Massen entstanden sind. Ich
nehme an, dass die Binnenk6rpersubstanz an allen oder auch
nur an bestimmten Stellen (das Letztere da, wo eine fixirte
Nucleolenzahl Regel ist) des Kernplasmas zuerst in Gestalt
kleinster, erstarrender Trépfchen abgeschieden wird, die auf
verschiedenen Stadien ihrer Erstarrung an einander treffen,”
this deduction being based in part on an observation of A.
Schneider ('75). He explains why the nucleoli are not evenly
distributed in the nucleus, on the ground ‘dass die einzelnen
Tropfchen jedenfalls nicht an allen Stellen des Kernraumes zu
genau derselben Zeit entstehen.” The nucleoli probably repre-
sent “ Reservestoffe,’”’ which are consumed in the later growth
of the nucleus, and since in Saccamina they decrease in size
as the amount of the chromatin increases, it is probable ‘dass
die Nucleolensubstanz [die sehr verschieden sein kann] in irgend
welcher Beziehung zum Chromatin steht.” Further, he holds
that the nucleolar substance is produced in the nucleus, ‘und
dann erst erzeugt wird, wenn sie in kleinen Tropfchen auf-
tritt.” But it is not yet possible to decide whether the nucle-
oli of the J/etazoa also arise in this manner, and hence the
use of the general term “ Binnenkérper”’ instead of the more
specific one ‘“‘ Nucleolus.”” That amoeboid movements of nucle-
oli have been noticed is not contradictory to his theory, since
changes of form would be caused by the processes of fusion,
or these motions might denote ‘“‘ Aufldsungsvorgange”’: ‘“ Die
Auflésung der Binnenkérper muss nach unserer Annahme von
zwei, im Kernsaft enthaltenen, sich gegen die Binnenkorper
kontrar verhaltenden Substanzen, auf eine Ueberschreitung
des angestrebten Mischungsoptimums von Seiten der lésenden
Substanz zuriickgefiihrt werden. ... Der Verschmelzungsvor-
gang ist schon von mehreren Forschern erschlossen oder ver-
muthet worden —neu diirfte nur die Annahme einer allmahli-
chen oder auch rascheren Erstarrung der urspriinglich fliissigen
Binnenkorpersubstanz sein.” Rhumbler concludes that the
342 MONTGOMERY. [Vou. XV.
“Binnenkorper’’ are not organs, since they show no fixed
organic structure, but represent accumulations of various sub-
stances. There is more nucleolar substance, ‘ Reservestoff,”’
accumulated in the nucleus before mitosis than is necessary
for it, so that after a mitosis some always remains to serve for
the production of daughter-nucleoli (this being an explanation
for the reappearance of nucleoli after mitosis).
Stauffacher (93), maturation of the egg of Cyclas: the
“Urei”’ contains a single large nucleolus ; later one or two
“ Nebennucleoli”’ also appear in the nucleus. When the ovum
has so increased in size that it adheres to the wall of the ovary
only by a narrow thread of cytoplasm, two nucleoli are present,
which are of unequal size but are in close contact with each
other; in one case the nucleolus was trilobular. After borax-
carmine staining, the smaller one appeared more refractive and
deeply stained than the larger. Subsequently the two became
separated, and both vanished before the formation of the first
pole spindle.
Strasburger’s paper (93) presents a general discussion of
certain problems of mitosis in animals and plants; his
remarks on the aequatorial plate are apropos here. He believes
that the ‘Kornchen” found by Kostanecki (92) in the equator
of the central spindle are similar to, and comparable with,
structures found by himself in the mitoses of plants, and are
masses of nucleolar substance (these bodies being termed
“Centralspindelkorperchen’”’ by Kostanecki, ‘ Zwischenkor-
per” by Flemming, and “ Zwischenkiigelchen” by O. Hertwig,
92), « Vergegenwartige ich mir nun das, was ich seinerzeit bei
der Bildung pflanzlicher Zellplatten beobachtet habe [/7/zs‘o/.
Beitr., vol. i, p. 161], namlich das Fortschreiten jener tingir-
baren Substanz, die ihrem Auftreten und ihren Tinctionen
nach nur als Nucleolarsubstanz gelten konnte, zwischen den
Verbindungsfaden bis zum Aequator, so muss in mir die Vor-
stellung erwachen, dass es sich in der von v. Kostanecki
geschilderten Erscheinung um einen entsprechenden Vorgang
handle. . . . Mit den durchschnittenen [achromatischen] Ver-
bindungsfaden . . . wanderte dann auch die Substanz der
halbirten Zwischenkorper nach den Zellkernen zuriick, ahnlich,
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 343
wie wir das fiir die unverbrauchte Nucleolarsubstanz bei Pflan-
zen angeben konnten. .. . Bei Pflanzen treten die Elemente
der Zellplatten als Anschwellungen der Verbindungsfaden im
Aequator der Zelle auf. Diese Anschwellungen bilden sich
dort erst, wenn jene tingirbare Substanz.. .den Aequator
erreicht. Diese Substanz wird in geloster Form zwischen den
Verbindungsfaden dorthin befordert. Aus den verschmolzenen
Elementen der Zellplatte geht die Scheidewand hervor. . . .
Man konnte denken, dass in tierischen Zellen ein mittlerer
Teil der ‘Zwischenkorper’ in eine ldsliche Substanz sich
verwandle und so die Halbierung der Zwischenkorper und
damit auch der Verbindungsfaden bewerkstelligte.”
Ver Eecke (93), pancreas cells of Rana and Canis: he
distinguishes one “nucléole nucléinien”’ and several “ nucléoles
éosinophiles,” or plasmosomes, the latter being the larger.
When the cell enters on its functional activity “le plasmosome
unique devient plus volumineux; il n’est pas rare d’en trouver
plusieurs dans un seul noyau; ils se rapprochent de la mem-
brane nucléaire, la soulévent, la perforent et se placent en
définitive a céte du noyau pour former un noyau accessoire.
D’ordinaire le plasmosome dans sa migration est accompagné
de petits karyosomes qui lui forment parfois une véritable
couronne’’; the mother-nucleus subsequently degenerates.
Against the opinion of Platner (that the supposed migration
of the nucleoli is artificially produced) “il suffit de faire remar-
quer que la migration ne s’observe pas ou trés rarement a
l’état de repos pour ne se manifester dans tout son éclat
qu’au début de l’activité sécrétoire.” In the cytoplasm the
nucleolus and its attendant karyosomes gradually change into
a nucleus.
Wasielevsky ('93) found the “‘ Urgeschlechtszelle”’ of Ascaras
with one or two nucleoli. While in the resting state of the
nucleus only one nucleolus is present, two are regularly seen
in the spirem stage, and these he believes have originated by
division of the primitive one. He noticed no difference in size
or stain between these nucleoli and the centrosomes, and hence
concludes that the latter are identical with, or have some
genetic relation to, the former.
344 MONTGOMERY. [Vou. XV.
1894.
Blochmann (94) gives a preliminary account of the results
of the observations of Keuten (95).
Born (94) investigated the maturation of the ovum of T7yz¢on.
In the “ Urei”’ are one or several large, spherical nucleoli. In
the second stadium of the maturation (production and degenera-
tion of a ‘ Chromatinfadenknauel ”’) there are at first ten nucle-
oli, then they become more numerous, increase in size, and
lie close to the nuclear membrane. In the third stadium (eggs
of from 200m to 350m in diameter) the nucleoli increase still
more in size. In the fourth stadium (eggs measuring from 350"
to 800, first appearance of yolk in the cytoplasm) most of the
nucleoli lie in the peripheral “‘ Karyohyaloplasma,” only a few
pale ones being in the center of the nucleus (this part of the
nucleus he terms ‘‘Centralkérper”’). At the commencement
of this stage the nucleoli increase, at its conclusion decrease,
in number, and “wahrend der ganzen Periode steigt die Zahl
der verkleinerten und abgeblassten Nucleolen im Centralkér-
per,” only a few of these pale ones being situated at the
periphery of the nucleus. Thus while at the beginning of this
period the nucleoli attain their maximum size, at its end most
of them wander towards the center of the germinal vesicle,
become smaller, and lose their staining power. Fifth stadium
(the nucleus passes to the periphery of the egg): the nucleoli
decrease still further in size, and continue to wander to the
center of the nucleus; some of the larger ones contain vacuoles,
and for the first time appear granular; the smaller, lightly
staining nucleoli are division products of the larger ones. At
the commencement of the sixth stadium (formation of the first
pole spindle) all the nucleoli lie in irregular rows around
the “Centralkérper,” stain quite intensely, and are regularly
vacuolated ; the few in the midst of the “ Centralkorper”’ are
smaller and stain more faintly ; when the nucleus has decreased
still further in size, all the nucleoli vanish at once. Born con-
cludes as follows: “Eine sichere Herleitung der peripheren
Nucleolen von den Nucleolen des Ureies, bin ich freilich nicht
im Stande zu geben.... Die Nucleolen stehen in Beziehung
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 345
zum individuellen Zellleben, nicht zur Fortpflanzung; denn beim
Beginn der Mitose verschwinden sie, um nach Beendigung der-
selben — im Ruhestadium des Kerns — wieder aufzutreten.”
He notes that their peripheral position is “ Eine Lage, die fiir
eine Wirkung auf den Zellleib die denkbar giinstige ist.’
Brauer ('94), Actznvusphaerium .; in the cyst of the second order
(“Ruhecyste”’) there is a nuclear reticulum consisting of
chromatin granules imbedded in a linin network, and usually
numerous nucleoli of irregular form, arranged either in rows or
circles. Probably the nucleoli take no part in the formation
of the chromosomes, and are equivalent to those of metazoan
cells ; they disappear in the prophase of mitosis.
Bunting (94) found in the eggs of Hydractinia and Podoco-
ryne a single large nucleolus, containing one central vacuole of
large size.
Flemming’s (94) ‘“Referat”? includes some of the more
recent papers on nucleoli.
Foot (94), egg of Al/olobophora: during the first maturation
division the nucleoli are distributed in the cytoplasm. Each
pronucleus contains from one to seven nucleoli: “the nucle-
oli persist during the cleavage spindle, but how much later I
am unable at present to state.”’
Hodge ('94), nerve cells of Raza stimulated by the electric
current : amoeboid movements of the nucleolus were noticed ;
“it was possible to make out granules in the nucleolus which
moved slowly about and in several instances were seen to be
extruded into the nucleus’’; and in cells which had not been
stimulated, but simply fixed in osmic acid and stained with
safranin, “the granules were stained brighter red _ than
the body of the nucleolus, and several were found partially
extruded.”
Lavdowsky (94) studied nuclei from the epidermis of the
fins of Amphibian larvae, as well as various tissues of plants.
The nucleolus consists of : (1) an outer, thick “ Pyrenin-Chro-
matinschale’’; (2)an enclosed vacuole; and in the latter (3)
the “Nucleololus” (“das noch in Entwickelung begriffene
Centrosoma”’), The animal nucleolus varies from a spherical
to an angular or star shape. In the resting nucleus the chro-
346 MONTGOMERY. [Vou. XV.
matin and pyrenin shells are the largest, since ‘die Bestand-
theile noch nicht fiir die Karyokinese verbraucht sind.” The
centrosomes “sind wahrscheinlich Teile von Kernkorperchen
und wandern zur Zeit der Karyokinese von den Kernelementen
aus” (these centrosomes are spherical or oval, homogeneous
and compact, and stain very slightly). He concludes “dass die
Kernkorper nicht zu jeder Zeit des Zellenlebens persistieren,
dass ihr Verschwinden wahrend der Karyokinese keinem Zweifel
unterliegt und dass dies in innigem Zusammenhang mit dem
Erscheinen des Centrosoma steht.” The nucleoli divide amito-
tically (not seen in life, however) into very small pieces, which
“scheinen in das Geriistnetz eingeschaltet und verwandeln sich
in den Vorbereitungsstadien der Karyokinese in Chromatin-
faden’’; other ‘“ Kernkérper”’ pass out of the nucleus, at the
points where its membrane is broken. The nucleoli are not
sufficient for supplying the whole mass of chromatin necessary
for the mitosis ; ‘es muss also eine andere Quelle der Chromatin-
entwicklung da sein und hauptsachlich im Eidotter und in den
pflanzlichen Samen muss man die Quelle aufsuchen... .
Durch nichts unterscheiden sich die Chromosomen von den
zerteilten Dotterkornchen und den geteilten Nucleolen. Alle
diese Gebilde . . . konnen somit als ‘ Kariosomen’ betrachtet
werden,”
Metzner (94), cells in the testicle of Sa/amandra: he con-
cludes “dass die Nucleolen in keinem Stadium der Mitose
fehlen, obwohl sie von sehr verschiedener Grosse sind.’ In
resting nuclei the smaller nucleoli stain entirely with gentian
violet (after Flemming’s triple stain), the larger ones with
safranin except for a blue-stained peripheral zone. Smaller
nucleoli are budded off from the surface of the larger ones, and
the “ Leitkorper”’ (granules which serve to attach the chromo-
somes to the spindle fibers) resemble such buds in stain and
size ; ‘es ist mir vorerst nicht moglich zu entscheiden, ob diese
Leitkorper von dem Nucleolus stammen, doch ist es wahr-
scheinlich, dass gerade an ihm sich die ersten, den Kern- und
Zelltheilungsprocess einleitenden Vorgange abspielen. Denn
an den Zellen mit ziemlich gleichmassiger Vertheilung der
Chromatingranula und geringer Anzahl der Nucleolen kann
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 347
man immer schon den Vorgang der Ausstossung kleinster
Kiigelchen beobachten.” In mitosis the nucleoli wander into
the cytoplasm, where the larger of them disappear, while the
smaller persist ; ‘dass aber diese Nucleolen in den Tochterkern
einwandern, ist nicht sehr wahrscheinlich, denn es liegen in den
Tochterzellkernen nur die gelésten Leitkorper.... Vielleicht
persistiren nur einige von ihnen in der jungen Zelle und zwar
als Nucleolen.... Eine ‘Vermischung von Nucleinsubstanz’
kann ich... an meinen Praparaten .. . fiir Chromatingranula-
strange nicht annehmen, denn die augenscheinlich von den
Nucleolen stammenden Leitkorperchen adhariren nur den
Segmenten und erfiillen ihre. . . Function als Anheftungs-
punkte der Spindelfibrillen; sie lésen sich intakt in den
Tochterknaueln wieder ab. Dass sie noch andere Functionen
ausiiben (als Nucleolen) ist wahrscheinlich, doch nicht ganz
sicher.... Anden Nucleolen treten die ersten Erscheinungen
der Zelltheilung auf. Sie lassen aus sich eine Menge kleiner
Kiigelchen hervorgehen, die z. Th. aus dem Kerne in das Pro-
toplasma wandern, z. Th. aber als Leitkorperchen iiber den
Kern sich vertheilen und so wohl den Anstoss geben zur
Strangbildung der Chromatingranula. Dem Nucleolus fiele also
fiir die Fortpflanzung der Zelle eine wichtige Function zu.”
Murbach ('94) considers it probable that the “ Kapselkeim”’
of the nettle capsules of hydroids is derived from one of the
two nucleoli of the parent cell, in accordance with his view that
the capsule is of nuclear origin.
Purcell ('94) describes the nucleolus of the retinula cells of
Acantholophus as structurally “ wabig.”
Reinke (94) found in the cells of the spleen of the mouse
one oval or elongate nucleolus ; during the prophase of mitosis
this divides into three or four smaller ones, while at the end of
mitosis each daughter-nucleus has a single nucleolus.
Riickert ('94) studied the maturation of the ovum in three
species of Copepoda. Cyclops strenuus (his species he assumes
is not identical with the “C. strenuus”’ of Hacker) : in the
«“Wachsthumszone”’ of the ovary there is one large, sometimes
also two smaller nucleoli, which stain with haematoxylin as does
the chromatin, and together represent the ‘‘ Hauptnucleolus ”
348 MONTGOMERY. [Vou. XV-
of Hacker. The single “ Nebennucleolus ” appears a little later,
and is regular in its occurrence, both in females with egg
sacks (“mehrgebarend,” after Hacker) and in those without
egg sacks (‘‘erstgebarend,” according to Hacker); Hacker found
the “ Nebennucleolus ” only in the ova of the former category
of females. It is paler and much larger than the several
“ Hauptnucleoli,’ and has a more central position within the
germinal vesicle, while the latter are usually peripheral. When
the “ Hauptnucleoli”’ have disappeared the “ Nebennucleolus”’
increases in size and thereby at first assumes a mulberry shape,
or is produced into long processes (though at the start it was
spherical). ‘Wahrend er anfanglich ein kompaktes Gefiige
besitzt, lockert er sich spater auf. Schon friihzeitig sieht man
in seinem Innern einen lichteren Raum, und spater entrollt er
sich zu knauelartig gewundenen Ziigen, die ein sehr wechselndes
Ansehen bieten, sehr haufig bilden sie eine einzige, ziemlich
einfach verschlungene Figur, ein Achtertour, ein S u. a., neben
der jedoch noch ein oder ein paar kleinere kugelige Stiicke im
Keimblaschen liegen kénnen.... Er ist . . . nicht einheitlich
gebaut und homogen, wie ihn Hicker abbildet, sondern zusam-
mengesetzt aus rundlichen Anschwellungen, die in einer Reihe
hinter einander liegen, stellenweise getrennt durch schwach
gefarbte, schmalere Zwischenstiicke. Man konnte daher das
Ganze als eine Kette von Kugeln bezeichnen. ... In etwas
spateren Stadien verlieren diese Bildungen an Farbbarkeit,
erscheinen aber zunachst immer in sehr wechselnder Form.
Man trifft entweder einen mehr kompakten Substanzhaufen
oder meistens eine Anzahl durch das Keimblaschen zerstreuter
Stiicke. . . . Haufig sieht man ein vielfach verschlungenes,
sehr unregelmassig angeordnetes Fadensystem. ... Es ist
schwierig zu entscheiden, ob die beschriebenen, sehr wechsel-
vollen Bilder der Ausdruck nur fiir verschiedene Functions-
zustande des Nucleolus sind, oder fiir einzelne, zum _schliess-
lichen Zerfall fiihrende Entwicklungsstufen”’; they disappear
before the true maturation processes commence. In Heterocope
robusta and Diaptomus gracilis there is a single large, vacuolated
nucleolus; it disappears when the chromatin has arranged
itself into “ Vierergruppen.”’
No.2.] COMPARATIVE CYTOLOGICAL STUDIES. 349
Schaudinn ('94) finds in Amoeba crystalligera a large nucle-
olus, with “wabiger Struktur’’; in the mitosis it divides into
two equal parts.
Watasé ('94), in the course of his theoretical deductions as to
the structure of the cell, concludes in regard to the nucleolus:
“The nucleolus is not a permanent body in the nucleus. It
may exist at one stage of the cell, and may disappear at the
next. The micro-chemical reaction of the nucleolus is entirely
different from that of the chromosome. It appears probable
that three or more different bodies are included under the name
of nucleolus. Indeed, one sees no reason why the inside of the
nuclear membrane may not be used as a depository for some
solid products of cell metabolism. . . . And thus some of the
bodies included under the generic name of zzcleolus may
belong to the group of metaplasm.”
H. V. Wilson ('94), Zedanione foetida: the youngest germinal
vesicle contains a single, centrally placed nucleolus. Later
there are two nucleoli, “which are invariably placed on opposite
sides of the nucleus and adhere to the inner surface of the
nuclear membrane. In eggs which have reached the adult size
it is the rule to find either one nucleolus peripherally placed,
. . or the nucleus contains no nucleolus at all. It sometimes
happens that an egg of full size is found with two nucleoli, but
this is rare. From this evidence it would seem that the two
nucleoli present in the developing egg are lost, one after the
other, at the time when the egg reaches its full size. As to
how the first of the two is lost, I have no evidence, but the
second nucleolus may often be seen lying just outside of the
nucleus in the yolk, . . . showing that it has been extruded
from the nucleus.’”’ What Fiedler (88) described as polar
bodies in Spongilla are probably extruded nucleoli. In the egg
of Hircinia acuta the nucleolar changes are as in Tedanione.
895.
Balbiani ('95), reviewed by v. Erlanger in Zool. Centralol.,
1895, macronucleus of Spzvochona ; the nucleolus of the authors
arises in a vacuole of the chromatin, and is formed by the
separation of microsomes which fuse together to form one
350 MONTGOMERY. [Vor. XV.
or two nucleoli. The nucleolus then wanders through the
chromatin to take position in the center of the achromatic
substance ; it combines the qualities of a true nucleolus with
those of a centrosome. There is thus no fundamental differ-
ence between a nucleolus and a centrosome ; when it remains
in the nucleus it has the value of the former, when in the
cytoplasm it has the significance of a centrosome.
Bohmig (95) noticed in the ovarial eggs of Haplodiscus that
the nucleolus is at first small and homogeneous, while later it
becomes larger, and one or more vacuoles appear in it.
Bremer (95a), blood cells of Zestudo and Chelydra: there is
normally one paranuclear corpuscle to a cell ; “seiner Natur
nach ist das Paranuclearkorperchen ein vom Innern des Kernes
in das Diskoplasma [Cytoplasma] ausgewanderter Nucleolus
oder vielleicht ein Nucleolusfragment, umgeben von einer dem
Kerne entnommenen Hiillsubstanz. ... Seine Grosse, die
Schwierigkeit der Farbung und seine Lage sprechen fiir den
nucleolaren Charakter.”” In a second paper ('95b) he identifies
this corpuscle with a centrosome, and states: ‘“ Hertwigs
Vermuthung, dass ein Zusammenhang des Centrosoms mit dem
Nucleolus existire, wird durch meine Beobachtungen wahr-
scheinlicher gemacht.”
In Biirger’s monograph (’95) of the Memerteans the following
statement in regard to the structure of the germinal vesicle is
of interest: “Im Keimblaschen findet man ausser den intensiv
farbbaren Korperchen, den Nucleolen, von denen meist zwei,
ein grésseres und ein kleineres, vorhanden sind, ein Netzwerk
feiner Faden, in welche sehr feine Kiigelchen aufgehangt sind.”
Coe (95), ova of Cerebratulus lacteus: “as the ovum
increases in size its nucleus develops into the germinal vesicle
which has many germinal spots, of which one or two are much
larger than the others.” In the mature ovum the nucleus
‘often contains a highly refractive germinal spot one-third as
large as the vesicle itself.”
Cunningham (95), ovarial eggs of fishes: in the youngest
ova there is a single large nucleolus, in older ova a number of
peripheral ones ; the latter are produced in part by a division
of the primitive nucleolus, in part by an increase in size of
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 351
“ minute nucleolar granules”’ which were present in early stages.
In contradiction to the view of Scharff ('88) he finds that no
nucleoli wander out of the nucleus to form yolk globules.
Delage (95) opposes the view that the nucleoli and the
centrosomes are genetically related (as against the theory of
Julin (93b) and Wasielevsky).
Galeotti (95), embryonal cells of 7rzton and Spelerpes (fixa-
tion in Hermann’s fluid with chloride of palladium substituted
for chloride of platinum ; stained for five minutes in sat. sol.
acid fuchsine in aniline water at 60° C., then stained in 1% %
sol. methylen green in equal parts of water and alcohol for three
or four minutes): “Auf diese Weise erhalt man roth gefarbt
die Kornchen des Cytoplasma und alle Elemente des Kerns mit
Ausnahme des Nucleolus. ..; gelblichgriin erscheint der
protoplasmatische Grund der Zelle und lebhaft griin die baso-
philen Granulationen.”’ In the pancreas cells of Spelerpes the
green-stained nucleolus passes out of the nucleus and persists
as “ Nebenkern,” which in the cytoplasm seems to increase by
continued division; and from this he concludes ‘dass der
Nucleolus ein endonucleares Arbeitsprodukt des Kernes ist,
bestimmt aus der Kernmembran auszutreten und im Cytoplasma
so umgeadndert zu werden, dass er in Secretionsprodukte umge-
wandelt wird.”
Hacker ('95) first describes the nucleolar relations in the eggs
of Canthocamptus, and then gives expression to general views,
based on his numerous previous observations, in regard to the
nature of nucleoli. Canthocamptus staphylinus : in the smallest
eggs there is one large nucleolus, which increases in size, but not
to same relative extent as does the nucleus itself ; subsequently
vacuoles arise in it, one of which becomes a “ Hauptvacuole’”’;
smaller ‘‘ Kernkorper”’ appear first when the chromatin elements
commence to thicken ; ‘wenn endlich die Kernsubstanz aut
das Minimum ihres Volumens zusammengedrangt ist, so fehlt
in der Regel jede Spur von nucleolarer Substanz.” Then
follows his general conclusions in regard to the physiology and
structure of the nucleoli: ‘Die Nucleolen sind nach meiner
Ansicht im allgemeinen als nicht strukturirte Gebilde aufzu-
fassen.... Sie stellen als solche. . . ein Abspaltungsprodukt,
352 MONTGOMERY. [VoL. XV.
welches wahrend der vegetativen Thatigkeit der Zelle und des
Kerns in oder an den chromatischen Elementen zur Abschei-
dung gelangt und zu Beginn der Mitose aus dem Kernraum
entfernt wird. Wie bei allen organischen Wachsthums- und
Umbildungsprocessen, so wirden . . . Sekret-Substanzen zur
Abspaltung kommen, welche in Form eines Hauptnucleolus
oder mehrerer Nebennucleolen auftreten. . .. Die Griinde,
welche theils fiir die Auffassung der Nucleolen als nicht
organisirter Stoffwechselprodukte sprechen, theils speciell
darauf hinweisen, dass es im Kern entstandene und dem Kern
verlassende secretartige Stoffe sind,” are the following : (1)
“Die bedeutende Entfaltung der nucleolaren Substanz in den
Kernen solcher Zellen, fiir welche eine intensive vegetative
Thatigkeit angenommen werden muss (Keim-Mutterzellen,
Driisenzellen, Ganglienzellen, Wimperzellen), wiirde zum min-
desten dafiir sprechen, dass die Nucleolarsubstanz ein Stoff-
wechselprodukt darstellt, dessen Erzeugung in einem gewissen
Abhangigkeitsverhaltniss zur Intensitat der vegetativen Leis-
tungen von Kern und Zelle steht.” He cites numerous cases
to show that all germ cells with little yolk and with usually
adequal cleavage have a large ‘“ Hauptnucleolus’’ (sponges,
Hydromedusae, Siphonophora, Acalephae, Ctenophora, Echino-
dermata, Copepoda, Tomopteris) ; while all large ova with a
considerable amount of yolk and with discoidal or superficial
cleavage have numerous ‘“‘ Nebennucleoli’’ (most /zsecta, many
Crustacea, lower Vertebrata). He explains the time of the
appearance of the ‘ Nebennucleoli” in the egg of Canthocamptus
in this way: “ Zur Erklarung dieser Erscheinung ist anzuneh-
men, dass irgend welche die ganze Eizelle betreffenden Ver-
anderungen physiologischer Natur, die um diese Zeit eintreten,
die weitere Apposition der neu sich bildenden Nucleolarsub-
stanz an den Hauptnucleolus verhindern und das Auftreten
mehrerer Verdichtungscentren hervorrufen, welche haufig nicht
mehr das Farbungsvermégen des urspriinglichen Hauptnucleo-
lus erlangen. . . vom rein morphologischen Standpunkt aus
darf man aber wohl mit diesen in den Endstadien der Eibildung
auftretenden Bildern jeden intermediaren Keimblaschentypus
vergleichen, welcher sich im Lamellibranchiaten-Ei vorfindet.”
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 353
He notes that “die Bildung nucleolarer Substanz auch unab-
hangig vom Zellwachsthum in erheblichem Masse stattfinden
kann. Bekanntlich treten namlich auch in den zur Copulation
sich anschickenden Geschlechtskernen Nucleolen auf, welche
nicht selten betrachtliche Dimensionen annehmen, und dasselbe
gilt fiir die Kerne der friiheren Furchungsstadien. Hier ist
von einem Zellwachsthum nicht die Rede.” Accordingly, he
concludes: (1) “dass die Menge der nucleolaren Substanz in
einem direkten Verhaltniss steht zur Intensitat der Wechselbe-
ziehungen zwischen Kern und Zelle’’; (2) “hier méchte ich
nur wiederholen, dass ich aus den verschiedenen Bildern eine
Entstehung der Substanz der Nucleolen an oder in den Chro-
matinschleifen und die Méglichkeit einer Verschmelzung der-
selben ableiten und mich so entschieden gegen die Auffassung
aussprechen mochte, dass die Kernkorper aus dem Zellplasma
in den Kern hereingelangen und hier in die Bildung des Chro-
matins eingehen, sowie im allgemeinen dagegen, dass die
kleinen durch Theilung der grosseren entstehen”; (3) he
brings a few observations to show “dass der Kern die nucleo-
lare Substanz an das Zellplasma abgiebt, dass es sich also hier
wohl kaum um Stoffe handelt, welche als Nahrmaterial dem
Chromatin zugefiihrt werden, sondern um solche, die wahrend
der Veranderungen des letzteren zur Abspaltung und dann zur
Ausscheidung aus dem Kerne kommen.... Ichdenke...,
dass sie [die vorhergehenden Erorterungen] in ihrer Gesamt-
heit sehr wohl eine Stiitze fiir die Kernsekret-Theorie bilden
konnen.” Finally, Hacker gives his own explanation of the
maturation stages of Zyzton, based on the description of Born
(94), and, comparing the changes here with those observed by
himself in the maturation of Canthocamptus, generalizes the two
as follows: 1. Stadium (growth of the germinal vesicle), “« Ab-
scheidung einer dunkel tingirbaren Nucleolarsubstanz”’; 2.
Stadium, “ Verdichtung der chromatischen Substanz und Con-
centrierung in die Kernmitte. Beginn der Aufldsungsvorgange.
Der neu sich bildende Nachschub an nucleolaérer Substanz
erlangt .. . nicht mehr das urspriingliche Farbungsvermogen ”’;
3. Stadium, ‘“ Groéssenreduktion des Keimblaschens : Die chro-
matische Figur liegt unmittelbar im Zellplasma.”’
354 MONTGOMERY. [Vou. XV.
Held (95) finds that in the ganglion cells of vertebrates,
when stained with erythrosin followed by methylen blue, the
nucleolus stains blue and the “ Nebennucleoli”’ violet.
Herrick ('95) found that the nucleolus of Homarus contains
one large and several smaller vacuoles ; the gravitation of the
nucleolus in the caryolymph, 2.¢., its movement to the lower
side of the nucleus, may be post-mortem phenomena (at least I
learned as much from Dr. Herrick during a brief conversation).
Keuten (95) investigated the nuclear division of Eug/ena
viridis. In the nucleus there is an elongate body, the “ Nucle-
olo-Centrosoma,” which stains more intensely than any other
portion of the nucleus. At the commencement of mitosis it
elongates, “und wahrend die Segmente [Chromosomen] bisher
eine annahernd senkrechte Richtung zur Oberflache des Nucle-
olo-Centrosomas eingenommen hatten, bilden sie jetzt einen
spitzen Winkel mit demselben,” and gradually come to lie
parallel to it. At this time the middle piece of the ‘ Nucleolo-
Centrosoma”’ stains more lightly than its ends, so that these
latter parts are sharply demarcated from it (with the stain of
Heidenhain, namely, Bordeaux R. followed by haematoxylin).
“In der folgenden Phase riicken die parallel zum Nucleolo-
Centrosoma gelagerten Chromosomen von beiden Polenden her
nach dem Aquator zu, so dass die Enden des Nucleolo-Cen-
trosomes nunmehr frei in die Kernhohle hineinragen, wahrend
die Chromosomen als breite aquatoriale Zone das Mittelstiick des
Nucleolo-Centrosomes umgeben.” Next, the nucleus assumes
the form of a rotation ellipse, in the short axis of which the
“Nucleolo-Centrosoma” lies. After the longitudinal splitting
of the chromosomes, from three to five vacuoles appear in each
end of the “ Nucleolo-Centrosoma’’; then the latter structure
elongates and breaks into two parts, while at the same time the
long axis of the nucleus gradually changes so as to coincide
with the long axis of the ‘ Nucleolo-Centrosoma,” and part of
the chromosomes become grouped around the one end, the
remainder around the other end, of the ‘‘ Nucleolo-Centrosoma.”
Keuten believes his ‘‘ Nucleolo-Centrosoma”’ to be comparable
to the nucleolus of Amoeba crystalligera (Schaudinn), to the
“Centralspindel”’ in Dzatomea (Lauterborn), and to the centro-
No.2] COMPARATIVE CYTOLOGICAL STUDIES. 355
some plus central spindle of Ascaris megalocephala; it is
probably an important mechanical factor in the mitosis.
Korschelt ('95) finds that in the amitosis of the intestinal
cells of Ophryotrocha puertlis the “ Kernkorper”’ divides into
two. Ovarial and cleavage stages of the same annelid: the
« Kernkorper ” in the cleavage cells arises as “eine Anhaufung
von Chromatin, die sich zu einer Kugel abrundet. In ihr taucht
bald eine polygonale Felderung als Ausdruck einer schon ganz
friih beginnenden wabigen Struktur des Kernkorpers auf.” The
« Kernkorper’”’ increases in size rapidly, attaining its maximum
size and staining intensity when the chromatin filament for
the next mitosis becomes well marked. From this time on
“‘beginnt sein allmahlicher Verfall”’; it stains less intensely,
owing to the walls of its meshes becoming thinner ; the regu-
larity of the latter becomes lost, and granules appear within
and between them, while at the same time the “ Kernplasma”’
[‘‘Kernsaft”] stains more deeply: ‘Wahrend vorher das
Kernplasma hell und der Nucleolus dunkel gefarbt erschien,
hebt sich jetzt umgekehrt der helle Kernkorper von dem
dunklen Kernplasma ab. ... Immerhin halte ich es fiir
moéglich und sogar fiir wahrscheinlich, dass zu dieser Zeit ein
Austausch zwischen dem Kernsaft und der geformten Substanz
des Kernes stattfindet, bei welchem vielleicht ein Theil des
vorher im Kernkorper niedergelegten Chromatins dem Kern-
faden beigefiigt wird.” Similar nucleolar changes take place
in the male and female pronuclei, antecedent to the stage of
the first cleavage spindle ; in the male pronucleus “ man sieht
... bei dem aus dem Kopf des Samenfadens sich herausbil-
denden Spermakern im Geriistwerk den Nucleolus auftauchen.”’
The younger germinal vesicles contain one deeply staining,
homogeneous ‘“ Kernkérper”’; later vacuoles arise in it, so that
it eventually evinces an alveolar structure ; the time when the
nucleolus disappears is quite variable, thus it may sometimes
remain when the chromatin filament is perfected: “ Dieser kann
ibrigens auch noch vorhanden sein, wenn die vier Kernschleifen
bereits gebildet sind. Das letztere Verhalten méchte man
entschieden so denken, dass die Substanz des Kernkorpers von
keinerlei Bedeutung fiir die Ausbildung der chromatischen
356 MONTGOMERY. [Von XV.
Substanz ist. Das oben eingehend besprochene Verhalten der
Embryonalkerne liess dagegen eine ganz andere Auffassung zu,
obwohl es auch bei diesen allerdings abnormer Weise vorkommt,
dass neben den bereits gebildeten Chromosomen (sogar in der
angelegten Spindel) der Kernkorper noch vorhanden ist. .
Was die erwahnten Verschiedenheiten des Verhaltens der
Nucleolen in dem Ei- und Embryonalzellen betrifft, so liessen
sich diese vielleicht durch die recht verschiedenartige Ausbil-
dung und Funktion der Kerne in den beiderlei Zellen erklaren.”
Lauterborn (95a), nuclear division of Ceratzum hirundinella:
from one to four oval nucleoli are present and are frequently
apposed to the nuclear membrane. One nucleolus is still
present in the spirem stage (the mitosis advances no further
than this); but he was unable to decide whether this nucleolus
divides into two.
Lauterborn ('95b), M/u/tzcz/ia : each nucleus contains a rela-
tively large nucleolus, which frequently shows a “netzig-
wabige”’ structure.
Macallum (95) concludes that less iron is contained in the
nucleolus than in the chromatin, as is shown by its lighter stain
with haematoxylin. Nucleoli ‘are always attached to the
chromatin network, and sometimes there appears about them a
membrane derived from, and continuous with, the fibrils with
which they are connected.” Ina nucleus of a gland cell from
the kidney or liver of Vecturus “which is passing into the
mitotic phase, the nucleolar body disappears, apparently by
solution into the chromatin threads, for in the nucleus of a renal
cell, in which the meridional disposition of the chromatin
filaments obtained preparatory to the formation of the loops, I
saw, attached to one of the filaments and partly embraced by
its substance, what appeared to be the remains of such a body.”
The nucleoli of the amphibian ovum are derived from the
chromatin of the nuclear reticulum. In support of his previous
observations ('91) he adds, “that the iron in the cytoplasm of
the ovum makes its appearance only after the solution of the
peripheral nucleoli commences.” In plant cells (Evythronium)
there are at least three kinds of nucleoli: the first stain
intensely with eosin ; the second are composed of chromatin ;
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 357
and the third kind, which occur in the embryo sac, “are not
present in the mitotic nucleus, but in the retrogressive stage
[metaphase] they appear on the course of the filaments as
spherical elements enclosing one or more refracting corpuscles
and containing but a small amount of iron, which, however, in
later stages ... is more abundant. These nucleoli are eventu-
ally formed chiefly of chromatin, and in stained preparations
appear to contain nearly all the chromatin of the nucleus.
When mitosis again commences the filament forms at their
expense, the increase in size of the filament keeping pace,
apparently, with the decrease in the quantity of chromatin
which the nucleoli contain. Finally, before their disappearance,
when they contain but a minimal quantity of iron, they take
the eosin stain deeply. All these forms of nucleoli take up
safranin from solutions as readily as do the chromatin elements
in the same nuclei, and they hold the stain as tenaciously when
they are washed with alcohol. They are in this respect differ-
ent from the eosinophilous nucleoli in the animal cell, which
appear to be unrepresented in the vegetable cell.”” In Spzvogyra
and Corallorhiza “the greater portion of the chromatin in each
nucleus forms a single large spherical element unconnected with
the chromatin network.’’ He corroborates Leydig’s view of
the structure of the chromatin loops in the nuclei of the salivary
glands of Chzronomus,; the nucleolus is often vacuolar and
amoeboid, and may be transversed several times by the chro-
matin loop; “the presence of granules and vacuoles .. .
appears to indicate that it is physically active, which cannot be
postulated of the vast majority of the nucleoli of Vertebrate
cells.” In Zuglena the nucleolus stains deeply with eosin
(except after fixation in picric acid), but does not stain with
safranin ; it is “intermediate in composition between the nucle-
olus of higher animal cells and the chromatin of the nuclear
reticulum.”
Mead ('95), egg of Chaetopterus ; ‘in the second cleavage, as
in the first, the nucleoli are dropped out into the cytoplasm in
the equatorial plane.”
Montgomery ('95) described the various arrangements of the
nucleoli (“Chromatinmassen”’) in the ova of Stichostemma
358 MONTGOMERY. [Vo. XV.
etlhardt. ‘*Was diese Chromatinmassen chemisch darstellen,
ist mir vollig unklar: vielleicht sind sie als von dem Dotter
aufgenommene Nahrsubstanzen zu betrachten, oder vielleicht
stellen sie Konglomerate mehrerer Kernsubstanzen dar.’”’ (In
my present paper I have no corrections to make to these
previous observations, but add only fuller descriptions of the
genesis of these nucleoli.)
Moore (95), spermatogenesis of Se/achzz: the resting nuclei
of the first spermatogenetic period contain each a single large
nucleolus, which disappears in the following mitosis. In the
subsequent resting stage the nucleolus reappears, and also there
appears a smaller “secondary nucleolus” surrounded by a
vacuole. The larger one then “takes a position, generally, but
not always, in line with the long axis of the archoplasm. . . .
These two peculiar forms of nucleoli are always to be found
after the transition from the first into the second sperma-
togenetic period, and throughout all the generations of the
latter.”
Pfliicke (95), ganglion cells of Zxvertebvata:; “Ob... die
zum Nucleolus tretenden Lininfaserchen mit der Substanz
desselben verschmelzen oder jener dem Vereinigungspunkt der
Geriistbalkchen nur aufgelagert ist, muss ich unentschieden
lassen. Die Nucleolen erhalten sich hierin komplicirter als
die Chromatinkoérnchen, und die Méglichkeit, dass der intensiv
farbbaren Substanz des Kernkérperchens ein eigenes stiitzendes
Liningeriist zu Grunde liegt, ist nicht ausgeschlossen.” Nucleo-
lar vacuoles are normal structures, and are especially abundant
in the cells of gasteropods ; he followed in life the process of
the detachment of smaller vacuoles from a larger one, as well
as the process of fusion of two vacuoles. In Helix “kommen
neben drei bis fiinf grésseren Hauptnucleolen mit einem oder
mehreren Hohlraumen im Inneren, sehr zahlreiche ganz zer-
streut liegende kleinere Nebennucleolen bis zur Grdsse eines
Chromatinkornes herab vor, denen Vacuolen ganz fehlen und
die sich von Chromatinkérnchen nur durch die Farbung unter-
scheiden.” He also observed (cells of gasteropods) the
“‘Kernkorperchenkreis” first described by Eimer, and found
that the circle of granules around the nucleolus was connected
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 359
with it by linin fibers ; but he was unable to decide whether
these granules are thickenings of linin fibers, or whether they
correspond to “ Nucleolen bezw. Nebennucleolen . . . , welche
sich vielleicht vom Mutterkérper getrennt haben und durch
Wirkung centraler Lebensherde in jener typischen, regel-
massigen Stellung verharren.”
vom Rath (95a) studied the maturation of the ovum of
Euchaeta marina: on Pl. VII he figures a number of various
sized, all rather large nucleoli, in the germinal vesicle, at the
stage when the chromosomes are longitudinally cleft.
vom Rath (95b) finds that the secretion of the gland cells
of the head in Awz/ocra stains exactly like the nucleoli, and
concludes that both substances are probably chemically related.
He briefly mentions (footnote, p. §) having seen double nucleoli
in liver cells of molluscs and Amphzbia,; these dumbbell-
shaped nucleoli may be either regarded as states of fusion or
of division. In liver cells of Astacus the nucleolus consists of
“zwei verschieden tingirten einander dicht anliegenden Kugeln
einer dunklen und einer blassen.”’ There is no relation between
centrosomes and nucleolar substance.
Rhumbler (95) studied the nucleolar relations of Cyphoderia.
From three to nine “ Binnenkorper ” lie within the nucleus, the
largest nuclei having the smallest number; so that accompanying
the increase in size of the nucleus, a gradual fusion of the
“ Binnenkorper” takes place, though without an appreciable
increase in the total volume of their substance.
Sacharoff (95) concludes that since the eosinophilic granules
of the blood have the same appearance as the nucleoli, “und
weil diese Kernkérperchen bei dem Herausfallen der Kerne auch
herausfallen miissen, um dann unweigerlich von Leukocyten
verschlungen zu werden, so ist mit grésster Wahrscheinlichkeit
anzunehmen, dass bei Saugern die eosinophilen Granulationen
auf dem Wege der Phagocytose von aus Hamatoblasten heraus-
gefallenen Kernkérperchen entstehen.”’ In birds the nuclei
do not fall out of the erythrocytes, but the eosinophilic cor-
puscles are nucleoli which have wandered out of the nucleus;
these nucleoli are rod shaped. (Only medical literature is cited
in this paper.)
360 MONTGOMERY. [VoL. XV,
Sala (95), ovum of Ascaris; in the first maturation mitosis
the single nucleolus breaks into small pieces of various size,
which gradually become scattered throughout the nucleus ;
then they become smaller and spherical, and come to lie directly
under the nuclear membrane. These fragments may possibly
stand in a genetic connection with the corpuscles which are
subsequently found at each pole of the spindle. And since the
latter corpuscles may stand in some connection to a centrosome,
“‘es ist . . . nicht unmdglich, dass eine enge Beziehung besteht
zwischen der Auflosung des Nucleolus und dem Auftreten des
Centrosoma.”
Schloter (95), gland and liver cells of Salamandra: in
the nuclei may be distinguished, besides the chromatin and
paralinin, red-staining spherical corpuscles, the larger of which
are regarded as plasmosomes.
Sobotta (95), ovum of J/us: in contradiction to the view of
Holl, the chromosomes are not derived from the nucleoli only,
but from the whole chromatic substance of the nucleus.
van der Stricht ('95) observed in the larger ovarial eggs of
Amphioxus that each contains a large nucleolus with an excen-
tric vacuole ; it disappears at the time of formation of the pole
spindle.
Vejdovsky ('95a) found large, homogeneous nucleoli in the
yolk cells of Prorhynchus hygrophilus, die nicht die gewohn-
liche kugelige Gestalt bewahren, sondern immer in Theilung
begriffen sind. Man findet meist doppelte Kernkorperchen,
deren Halften durch eine ziemlich tiefe Furche von einander
getrennt sind und die eine centrale Hohlung erkennen lassen.
Nebstdem findet man in Drei- selbst Viertheilung begriffene
Kernkorperchen. . .. Ich glaube. . . , dass man es hier mit
einer Hypertrophie der normalen Kernkorperchen zu thun hat,
welche schliesslich zur Degeneration der Kerne fiihrt’’; these
nucleoli occupy more than two-thirds of the space within the
nucleus. In the ovum the nucleolus is much smaller, and
shows a division into two parts (Fig. 89), but here these two
parts are not of equal size.
Vejdovsky ('95b) found in the egg of Bothrioplana a spherical
nucleolus, ‘‘mit einem Nucleolinus.”
No.2.) COMPARATIVE CYTOLOGICAL STUDIES. 361
Waldeyer ('95), cited by Flemming (96), regards the nucleoli
as morphologically distinct from the chromatin reticulum.
Wheeler (95) observed in Myzostoma glabrum that the
nucleolus is large and vacuolated, and after the reduction
mitosis, “remains in the cytoplasm as an inert mass, gradually
melting away, but not disappearing until about the eight-cell
stage, when it may often be found in the largest blasto-
mere.”
Wilcox ('95) holds that ii the spermatocytes of Czcada the
nucleoli stand in genetic connection with the centrosomes,
and adds, “It is probable that different structures have been
called nucleoli by different authors.”
1896.
Auerbach (96) studied the spermatogenesis of Paludina :
the nucleus of the spermatogonium contains a number of
large, more or less spherical bodies (‘‘Karyosomen”’); each
nucleolus (of the resting spermatogonium), after simultaneous
staining with acid fuchsine and methylen greén, shows a
central red portion and a blue peripheral shell. ‘Es besteht
also eine Zeit lang der Nucleolus aus einer erythrophilen Cen-
tralmasse und einer kyanophilen Rinde.” In the subsequent
nuclear division of these cells the nucleoli disappear. ‘“ Fest
steht nur, dass in dem Netzstadium die Nucleoli als solche
verschwinden, und dass ihre Rindensubstanz auf die angegebene
Art zu einem Teile des intranuklearen Netzwerkes wird, der
anfangs noch unterscheidbar ist, dann aber durch Auseinander-
riicken der Knotenpunkte sich in dem iibrigen Fadennetze ver-
liert.””. In the spirem stage there are one or two small, spherical,
red-staining bodies in the nucleus; he was unable to determine
whether these stand in any genetic relation to the nucleoli,
which had previously vanished. In the spermioblast (which
changes directly into the hair-shaped spermatozo6n) a small,
red-staining body lies within the nucleus, but subsequently
disappears ; Auerbach supposes that it wanders out of the
nucleus and fuses with the “ Nebenkern.”
Doflein ('96), maturation of the egg of Tubularia larynx:
the single large nucleolus is suspended by achromatic fibers in
362 MONTGOMERY. [Vou. XV.
a clear, structureless space within the nucleus ; at first homo-
geneous, it later contains from one to five unstaining “ Korper-
chen,” which he thinks are not vacuoles, on account of their
refractibility. In the amitotic division of those nuclei which
degenerate and eventually become absorbed by a definitive egg
cell, division of the nucleolus precedes that of the nucleus.
Floderus ('96) studied the maturation and embryonal develop-
ment of various 7wnzcata. A ‘“‘ Hauptnucleolus” and “ Neben-
nucleoli” are present. The former is homogeneous in only
very young cells, and later differentiates into two different sub-
stances : (1) a refractive, larger portion, which encloses (2) a
less-refractive, paler portion. He considers the small vacuoles
of the nucleoli to be ‘ Kunstprodukte,” though the large one is
normal. ‘Nicht selten findet man in dieser grossen, allem
Anscheine nach mit Fliissigkeit erfiillten Hohlung eine Anzahl
fester, lichtbrechender Kornchen, vielleicht Coagulationspro-
dukte, die wahrscheinlich bei der Fixierung entstanden sind.”
As a rule there is one, but sometimes two ‘ Nebennucleoli”
in most though not all eggs; these rarely attain half the
diameter of the ‘“‘ Hauptnucleolus,” and appear in the germinal
vesicle shortly before the yolk granules arise in the cytoplasm ;
they are similar to, but paler than, the refractive portion
of the large nucleolus. The ‘“ Nebennucleoli” are absent in
Clavelina ; they probably arise by gemmation from the “ Haupt-
nucleolus,” and he figures to this effect a lobular “ Hauptnu-
cleolus.”” In the cytoplasm of the ova of Styelopszs and Ciona
(but not Clavelina and Corella) certain spherical “ intravitelline
Korper” occur, usually one to a cell, and frequently close to
the nuclear membrane; in size and staining reactions these are
similar to the “‘ Nebennucleoli,” and, following Roule, “sehe ich
mich genothigt, anzunehmen, dass sie von Nebennucleolen
herriihren, die aus dem Kern des Eies in den Dotter hinausge-
wandert sind,”’ thereby supposing that they press out through
a preliminarily produced pore in the nuclear membrane, and
that the larger intravitelline bodies are probably fused masses
of smaller ones. In accord with Henneguy (93) and Roule he
considers the intravitelline bodies not as ‘ Dotterkerne”’ nor
astrospheres, but as atavistic or rudimentary organs, which
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 363
together with the nucleoli correspond to the macronucleus of
the /ufusoria.
Gerould (96), ovarial eggs of Caudina; in the youngest ova
there are numerous peripheral nucleoli ; these increase in size
as the nucleus grows, and subsequently each contains a vacuole,
but they are always ciose to the nuclear membrane.
Greenwood ('96), macronucleus of Carchesium polypinum :
the nucleoli (‘‘protomacrosomes,” in distinction to the “ pro-
tomicrosomes,”’ or chromatin granules) are numerous and
vacuolated, and stain like true metazoan nucleoli. They vary
in size and form, and are probably amoeboid, though this point
could not be determined in the living nucleus, which is first
rendered visible by reagents. The vacuoles are fluid accumula-
tions, and arise first in the center of the nucleolus. ‘No
vacuoles surround the macrosomes of Carcheszum at any time,
nor do they ever show general increase of fluidity or swelling
such as might accompany the penetration through them of some
secretion from without ; . . . the deposition of vacuolar fluid is
centrifugal ; ...thus the macrosome may become a bladder-
like or honey-combed structure, its residual solid (?) forming a
well-defined membrane-like investment for fluid contents.”
Henneguy (96) distinguishes true and false nucleoli (the
latter being ‘‘nceuds du réseau,” in the sense of O. Hertwig,
'92). He reviews the observations upon nucleoli made by
several previous authors.
R. Hertwig (96), unfecundated ova of echinoderms poisoned
with strychnine : the nucleoli vanish within the nucleus as the
chromosomes appear. ‘Meine eigenen Untersuchungen lassen
es mir ausgeschlossen erscheinen, dass im Ei der Seeigel Nu-
cleolen und Centrosomen irgend etwas mit einander zu thun
haben. ... Dagegen ergeben sich unzweifelhafte Beziehungen
der Nucleoli zur Entwicklung der Chromosomen.... Dieses
Wechselverhaltniss ist nun nicht so zu verstehen, als ware das
gesammte Material der Chromosomen in den Nucleoli enthalten.
Dagegen spricht die geringe Masse der Nucleolar-Substanz und
ihr verschiedenes Verhalten den ublichen Chromatin-Farbungs-
mitteln gegeniiber. .. . Die Nucleolen k6nnen somit den
Chromosomen ein zur endgiiltigen Fertigstellung nothwendiges
304 MONTGOMERY. [VoL. XV.
Erganzungsmaterial liefern.” ‘Chromatin-Nucleoli”’ are such
as contain the whole chromatin of the nucleus (Actcénosphae-
rium, Spirogyra, salivary glands of Culex); ‘solche Kerne
zeigen dann ein achromatisches Geriist und in demselben einen
grossen chromatischen Korper, im iibrigen Nichts, was man den
Nucleoli oder den Chromosomen der Gewebszellen vergleichen
konnte. . . . Derartige Nucleoli waren dann nicht, wie mein
Bruder annimmt, und auch ich friiher geglaubt habe, von
den echten Nucleoli als etwas wesentlich Verschiedenes zu
unterscheiden ; sie wiirden Nucleoli sein, die ausser der specifi-
schen Nucleolensubstanz noch das Chromatin des Kernes
enthalten. .. . Bei der Umwandlung zur. Spindel losen sich
Chromatinkérnchen vom Nucleolus ab und treten auf das
Kernnetz iiber, ein Substrat hinterlassend, das man wohl den
echten Nucleolen vergleichen muss. Spater wird auch dieses
aufgelost.”
Korschelt (96), employing a modification of the Ehrlich-
Biondi stain, finds in the spinning glands of caterpillars that the
macrosomes stain green and hence consist of chromatin, while
the microsomes stain red and so must be regarded as nucleoli
(cf. '97).
List (96) made comparative studies on various nucleoli,
principally with a view to their chemical constituents, by apply-
ing a new staining method, whereby Berlin blue is produced in
the fixed tissues. ‘Wir sind zu dem Resultate gekommen,
dass die Nucleolarsubstanzen nach ihrem chemischen Verhalten
3 verschiedene Gebilde darstellen, von denen jedes wahrschein-
lich wieder eine eigene complicirte chemische Zusammensetzung
besitzt. Nach der bisherigen Bezeichnungsweise sind zu unter-
scheiden : Hauptnucleolus, Nebennucleolus und Nucleolus
schlechtweg”’; the substance of all the nucleoli differs from
that of the nuclein (chromatin) proper. ‘ Wir haben gesehen,
dass (bei Mytilus und Pristiurus) die Umsetzung des Ferro-
cyankaliums durch Salzsaure, wodurch Ferrocyanwasserstoff-
sdure und hieraus durch den Sauerstoff der Luft Berlinerblau
entstand, allein geniigte, um die Nebennucleolen zu farben. .. .
Wenn wir die Reagentien concentrirter anwenden, . . . so tritt
in jeder Zelle die Substanz des Nucleolus in Gestalt eines oder
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 305
mehrerer blauer Kiigelchen hervor.... Nach ihrem chemischen
Verhalten stehen also Nebennucleolus und Nucleolus einander
naher als Haupt- und Nebennucleolen”’; he concludes that the
“Nucleolus” and the ‘ Nebennucleolus . . . mindestens ver-
schiedene Modificationsstufen des Paranucleins . . . darstellen.”
Mytilus egg: what Lonnberg supposed to be vacuoles within
the nucleoli, List holds are « Nebennucleoli,” and these alone
evince the characteristic Berlin-blue reaction ; by afterwards
staining the preparation with carmine, “die Masse des Haupt-
nucleolus, das Nuclein, hatte sich scharf roth gefarbt, die
Nebennucleolarsubstanz, das Paranuclein, rein blau.” Even in
eggs where no yolk was as yet present, both these substances
could be demonstrated. The “ Hauptnucleolus” represents
the greater part of the nucleolus, and is usually single ; in it
may lie one spherical “ Nebennucleolus,” or the latter may
cover, cap-like, one pole of the former; sometimes “‘ Nebennucle-
oli” occur in the nuclear cavity, separated from the “ Haupt-
nucleolus’’; occasionally there are true vacuoles within the
latter. Pholas egg: (treatment with iron chloride, nitric
acid, then “ Ferrocyankaliumlésung’’) the “ Nebennucleolus”’
is much larger than the “ Hauptnucleolus,’ except in very
small ova, where they may be equal in size. The last-named
nucleolus may enclose a large, excentric vacuole, or in place
of this, a “ Nebennucleolus”’; in the chromatin network of
the nucleus there are small nodules of paranuclein, and some-
times a free “ Nebennucleolus.” “In alteren Eiern tiberwiegt
bei Weitem der Nebennucleolus den Hauptnucleolus an Masse”’;
the latter is either apposed to one end of the former, or there
may be a large “‘ Nebennucleolus”’ with a small ‘“ Hauptnucle-
olus” at each end of it. Pyistiurus egg: in the youngest
germinal vesicles the minute nucleoli all lie at the nuclear
periphery, the larger ones being central; in larger ova all the
nucleoli are placed at the periphery of the nucleus. Sphaere-
chinus egg: the supposed (Hacker, '93b) vacuole of the “‘ Neben-
nucleolus” is in reality the “‘Hauptnucleolus”’: ‘“ Jedoch weichen
meine Resultate von denen Hiacker’s darin principiell ab, dass
eben festgestellt werden konnte, dass das, was H. Hauptnucle-
olus nennt, wie ein Nebennucleolus reagirt, und die Vacuole
366 MONTGOMERY. [VoL. XV.
wie ein Hauptnucleolus.”’ With the three staining methods
employed (all used on material fixed with corrosive sublimate),
only the “ Nebennucleolus”’ is plainly stained, “nicht aber der
Nucleolus schlechthin, wie er in jeder Zelle vorkommt.” By
treatment for half an hour with a drop of .5 % iron chloride
solution, then stained by the Berlin-blue reaction, in each
somatic cell the nucleolar substance appears in the form of
bluish-green spherules. ‘Im Mollusken- wie im Vertebraten-
gewebe hatte jede Zelle einen rundlichen Nucleolus; in secer-
nirenden Zellen, z. B. Darmzellen, traten 2 oder 3 auf, oder
Grossenunterschiede, wie z. B. der Nucleolus in der Leberzelle
von Mytilus durch seine Grosse auffallt.”
Michel (96), ova of Wephthys and Spiophanes: each double
nucleolus consists of (1) a darker, more granular, portion, which
in Spzophanes contains either a small granule or a vacuole (he
is undecided which it is), and in Wephthys is vacuolated; and
(2) of a clearer, refractive, unstaining portion. In Wephthys
there are usually two double nucleoli, “la substance colorable
recouvrant plus ou moins compléetement la masse claire comme
d’une calotte’’; but other states were also found: “trois nuclé-
oles doubles, une sphére claire entre deux parties sombres
presque a l’opposé; inversement, une partie sombre et deux
sphéres claires presque opposées, nucléoles plus composés avec
plusieurs spheres claires et méme comme spumeux, sphérules
claires libres en plus de celles des nucléoles doubles jusqu’a
une douzaine. . . . Les masses claires, avec leur aspect, jeur
forme sphérique et leur déformation temporaire par la pression,
leur variation de taille suivant les conditions osmotiques,
l’épaississement de leur paroi par réduction de volume, apparais-
sent comme des vésicules 4 contenu liquide spécial,” while the
colorable portions are composed of pyrenin, and hence are true
nucleoli (the pyrenin proved “par l’absence de gonflement par
l'eau et par le gonflement par les acides, par l’insolubilité dans
le sulfate de cuivre ou le ferrocyanure de potassium. . .~
l’aspect des vésicules et leur disposition dans les nucléoles ou a
l'état libre . . . portent a croire a des vacuoles a contenu spécial
formées dans le nucléole et finalement éliminées’’).
Morgan (96) studied Echinoderm eggs placed in artificial
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 367
media: immature ova of Sphaerechinus, placed in sea water to
which 1.5 gr. NaCl had been added, show artefacts in the
nucleolus: “Each [body] consists of an outer darker shell,
which is filled with a clear fluid, and the center of each sphere
is occupied by a small black granule’’; several of these struc-
tures are usually found on each section through the nucleolus.
(For previous descriptions of somewhat similar productions, cf.
Ransom (67), Leydig (88), and O, Schultze (87). The upper
of the two figures numbered “ 24” in Morgan’s plate should
be “23,” since it refers to the nucleolus.)
Rohde (96), ganglion cells of Doris and Pleurobranchus : the
nucleoli wander out of the nucleus and finally into the neuroglia,
and there acquiring an envelope (derived from the neuroglia)
form new cells. [Judging from his figures, however, these
supposed nucleoli would seem to be myelin drops. |
Wagner (96a), spermatogenesis of Avachnids: “Bei der
ersten Spermatocytentheilung theilt sich der Nucleolus ent-
weder in der Ebene der Aequatorialplatte mit den Chromo-
somen zusammen, oder ausserhalb derselben neben einem der
Spindelpole. Im letzteren Falle tritt er nach dem Ver-
schwinden der Kernhiille . . . aus dem Kerne heraus.”
Wheeler (96) gives no description of the nucleoli in the text,
but he figures several stages of the development in eggs of
Myzostoma (Figs. 9, 10-15, MW. cerriferum ; Figs. 23, 52-54, 56,
M. glabrum). In M. cirriferum (Figs. 12-15) is figured, in
addition to the single large nucleolus, also one smaller nucleolus.
E. B. Wilson (96) states of the true nucleoli or plasmo-
somes : ‘‘ There is strong evidence that the true nucleoli are
relatively passive bodies that represent accumulations of reserve-
substance or by-products, and play no direct part in the nuclear
activity.” In germinal vesicles he assumes that the “ principal
nucleolus” is chemically different from the nucleoli of somatic
cells ; but that the “accessory nucleoli” of the former corre-
spond to the nucleoli of the latter. He concludes that “we can
hardly doubt the conclusion of Hacker, that the nucleoli of the
germ-cells are accumulations of by-products of the nuclear action,
derived from the chromatin either by direct transformation of
its substance, or as chemical cleavage-products or secretions.”’
368 MONTGOMERY. [VoL. XV.
1897.
Toyama, cited by R. Hertwig (96), holds that the nucleoli
become centrosomes in the spermatogenesis of Bombyx.
Van Bambeke (97a), ovocyte of Pholcus: there is usually a
single large nucleolus, rarely also accessory ones ; the nucleolus
is vacuolated, “les vacuoles . . . faisant fréquemment saillie a
sa [tache germinative] surface; dans certains vacuoles, on
découvre des granules safraninophiles.’”’ At a later stage the
nucleolus retains much the same appearance, “mais fréquem-
ment le contour net, safraninophile, qui la délimitait, a disparu
en tout ou en partie, et l’on remarque parfois une solution
decontinuité au niveau de laquelle le contenu de la tache
s’épanche dans le reste du contenu nucléaire. Cette sorte
d’évacuation ne doit pas étre confondue avec la rupture de
vacuoles nucléolaires, laquelle peut s’observer a tous les stades.”
(‘97b, the same, with figures.)
Bouin (97), giant spermatogonia of Cavza: the accessory
part (‘corps juxtanucléolaire”’) of the double nucleolus stains
red in safranine and blue in haematoxylin (in opposition to
Hermann), though less deeply than the spherical portion of the
nucleolus, and is sometimes hemispherical in form. This part
is single, and appears to consist of a mass of very fine granules.
In degenerating cells, “les uns nous montrent deux nucléoles
flanqués chacun d’une ou de plusieurs petites masses hémi-
sphériques, réfringentes et teintées en rose pale; lors des
mouvements intranucléaires, les corps juxtanucléolaires con-
tractent des rapports plus intimes avec les vrais nucléoles,
deviennent plus réfringents et moins colorables, s’accolent a
leur substance, se divisent a leur suite, et les accompagnent
dans leurs migrations. Aprés plusieurs divisions répétées, ces
noyaux contiennent un certain nombre de nucléoles, cing ou six
généralement.” In the process of formation of the cells of
Sertolli the nucleoli fuse successively with one another.
Braem (97), Plumatella: in the egg of .o13 mm. diameter
the nucleolus contains one to four vacuoles: “Sie sind allem
Anschein nach Fliissigkeitsblaschen, welche im Nucleolus auf-
treten und auf dem Hohepunkt ihrer Entwickelung an die
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 309
Peripherie riicken, um da ihren Inhalt nach aussen zu ent-
leeren.”” The nucleolus becomes ovoid, and its substance
paler at its smaller end; the vacuoles are usually, but not
always, at the paler end. “ Zuweilen ist der Gegensatz der
beiden Nucleolus-Halften lediglich in der verschiedenen Farb-
barkeit derselben ausgesprochen. In anderen Fallen wird er
durch eine Einschniirung bezeichnet, die den Nucleolus in
einen grésseren, dunkeln und einen kleineren, hellen Abschnitt
zerlegt.... Die Einschniirung kann nun zu einer volligen
Abschniirung fiihren, so dass der Nucleolus doppelt erscheint
und von zwei neben einander liegenden Kugeln gebildet wird,
oder bei gegenseitiger Entfernung der Theilstiicke in zwei
raumlich getrennte Nucleoli zerfallt.... Selten ist der Nucle-
olus dreitheilig ..., wo das mittelste Stiick dunkler ist als die
beiden seitlichen.... Dies lasst vermuthen, dass der Keimfleck
im Stande ist, unabhangig vom Wachsthum des Eies seine
Gestalt zu verandern, und dass die Zweitheiligkeit auf der
Bildung eines pseudopodienartigen Fortsatzes beruht, der sich
bald mehr, bald weniger deutlich vom Hauptkorper abgegliedert
und auch hinsichtlich seiner Substanz bald mehr, bald weniger
von demselben verschieden ist.”
De Bruyne (97), double cells of the ovarian follicle of Mefa,
Periplaneta, Meconema, and Aeschna: in the amitotic division
of the nucleus the nucleolus divides first. (Since the cytoplasm
does not divide, each such cell finally receives two nuclei.)
Carnoy and Lebrun (97a) (an abstract of this paper may also
be found in the American Naturalist for July, 1897). This con-
tribution deals particularly with the relations of the nucleoli in
the growth period of the ovum of Salamandra and Pleurodeles.
In the youngest nuclei observed there is a nuclein filament,
but no nucleoli; the first nucleoli arise as buds from the fila-
ment, and these are termed “nucléoles primitifs.” Then the
nuclear filament becomes changed into an amorphous magma,
composed of irregular granules, and the latter then subse-
quently disappear, so that all trace of the original filament
becomes lost. All further changes within the nucleus are
of nucleolar character. From the “nucléoles primitifs”’ are
derived the “nucléoles secondaires’”’ which “sont dis a des
370 MONTGOMERY. [VoL. XV.
associations de granules provenant de la désagrégation de
l’élément nucléinien”’ ; and then follow the ‘“nucléoles ter-
tiaires,’’ which differ from the nucleoli of the preceding two
generations in that they do not come from degenerating gran-
ules of preceding generations, but are detached from them in
the form of spherules. Each nucleolus of each generation
arises, increases in size, becomes more complex in structure,
and then passes through a polymorphic “ figure de résolution ”’ ;
the form of these figures varies according to the particular
generation, and also according to particular ova. The greater
part of the “figure de résolution” then disappears, except a few
granules which serve as the starting point for the next genera-
tion ; that portion of the substance which disappears serves as
nourishment for the egg. So all the generations of the second-
ary and tertiary nucleoli arise “a l’aide des produits de la
résolution antérieure.” After each “résolution’’ new nucleoli
arise, and the number of these generations is large, continuing
through a length of three years. The number of primary
nucleoli is usually from two to six ; of secondary, from 400 to
500 ; of tertiary, from 500 to 1000; the number varying in
different ova. Fusions of nucleoli are of normal occurrence :
“cette attraction des masses nucléiniens rappelle a l’esprit ce
qui se passe au sein de ]’ceuf entre les noyaux de conjugaison.”
In the radiation exerted by each nucleolus upon the surrounding
caryoplasm “nous voyons ... la confirmation d’une thése
soutenue dans la ‘ Cytodiérése,’ a savoir : que c’est sous ]’influ-
ence du noyau que se forment les asters de division.” The
chromatin filament does not reappear, but there is a “grand
nombre de générations nucléolaires et filamenteuses qui naitront
et disparaitront tour a tour, l’une aprés l’autre, jusqu’a l’époque
des globules polaires.”” The authors necessarily regard all the
previous observations on the amphibian ovum as erroneous.
General conclusions for all kinds of cells, based in part on
previous observations : there may be distinguished “ nucléoles
plasmatiques,”’ “nucléoles nucléiniens,” and ‘‘nucléoles mixtes”’
(‘qui sont rare’’), Plasmatic nucleoli consist of at least two
substances, ‘une plastine et une globuline digestible.” All
nucleoli, ‘lorsque leur formation est achevée . . . représentent
”
No.2.) COMPARATIVE CYTOLOGICAL STUDIES. Byfl
la totalité de l’élément filamenteux d’un noyau ordinaire;.. .
dans bien de cas —aujourd’hui nous pourrions peut-étre dire
dans tous —on constate dans ces corps la présence d’un véri-
table appareil filamenteux, tortillé sur lui-méme, comme dans
un noyau ordinaire, et présentant les mémes proprietés que
dans ces derniers. C’est que ]’on voit surtout dans les nucleé-
oles-noyaux, c’est-a-dire dans les nucléoles nucléiniens uniques,
qui ont absorbé tout l’élément filamenteux primitif.” All
nucleoli develop from the chromatin filament ; and chromo-
somes are derived from “nucléoles noyaux.” The chemistry
of nucleoli is also considered.
Carnoy and Lebrun (97b), fecundation of the ovum of
Ascaris megalocephala:; the centrosomes are “nucléoles plas-
’ which have left the nucleus at the
commencement of mitosis ; one is derived from the male, the
other from the female, nucleus. They totally disappear after
mitosis, and neither reénter the nuclei nor divide to produce the
centrosomes of the subsequent division.
Cunningham (97) : ‘“ There are indications in the ova of the
turbot that the substance of the nucleoli is absorbed into
the central fibrils to form the chromosomes of the polar mito-
ses, but the actual formation of these chromosomes was not
followed.”
v. Erlanger (97), a brief mention of certain recent views
upon the nucleolus : “Als echte Nucleolen waren allein solche
Korper zu bezeichnen, welche sich durch ihr Verhalten gegen
Chemikalien . . . scharf von dem Chromatin unterscheiden.
Vorderhand bleibt also die Bedeutung der echten Nucle-
olen ratselhaft, falls man diese Gebilde nicht mit Hacker als
eine Sekretion des Kernes beurteilen will.” They bear no
relation to centrosomes.
Fauvel (97), ovogenesis of Ampharete: ovarial ova of 30u
diameter, and at this stage only, contain two nucleoli. “On
rencontre toutes les modifications : nucléole simple, nucléole
étranglé par le milieu, deux nucléoles accolés, et enfin deux
nucléoles bien nettement séparés. ... Nous n’en avons
jamais rencontré deux dans l’ceuf mfr, ni dans l’ceuf non
détaché de l’ovaire.” The nearly mature ovum contains one
matiques ou achromatiques’
372 MONTGOMERY. [Vou. XV.
large nucleolus, with a large vacuole ; he believes that subse-
quent to the two-nucleolus stage one of the nucleoli is extruded
from the nucleus. Two nucleoli were observed also in the ova
of Amphictets, Sanytha, and Melinna.
Flemming (97) recurs to the controversy between Korschelt
(96) and Meves (97), and agrees with Meves that the macro-
somes are nucleoli, and the microsomes chromatin granules.
He also mentions the following observation on the ovum of
Ascidia cantina: here there is one “ Nucleolus”’ and one much
smaller ‘“‘Kernk6érper” ; ‘ beobachtet man ihn [Kernkérper]
am lebend entnommenen Ei, so findet man ihn so gut wie
stets in Molekularbewegung, und zwar oft in recht grossen
Exkursionen.”’
Hacker (97a) (‘96 is a preliminary communication), cleavage
stages of Cyclops brevicornis. This paper deals particularly with
the “intraspharale,” ‘ extranucleadre,” or ‘ Aussen-Koérnchen
(Ektosomen)”’ found in certain of the astrospheres of the
cleaving ovum. These ectosomes are small spherical bodies of
various size, which stain like the nucleoli, but somewhat more
intensely. In the resting stage of the cell there are several
nucleoli in the nucleus, and no ectosomes outside of it ; when
the nucleus enters on the aster stage, the nucleoli have disap-
peared and ectosomes are present in one of the astrospheres,
at first at the base of, subsequently on the whole periphery of,
the latter ; towards the close of the metakinesis there appear
in the place of the ectosomes larger clumps of red-staining
substance. He concludes : “So glaube ich es denn mit Sicher-
heit aussprechen zu diirfen, dass diese gréberen Brocken auch
genetisch mit den Kornchen [Ektosomen] zusammenhangen,
sei es, dass sie direkte Umwandlungsprodukte derselben, sei es,
dass sie Neubildungen sind, welche dem namlichen Process
ihre Entstehung verdanken, aber in Folge der wahrend der
Theilung eintretenden Zustandsanderungen der Zelle eine etwas
andere Beschaffenheit, einen anderen Aggregatzustand ange-
nommen haben. Wie ich gleich hier hinzufiigen méchte, ver-
schwindet die Erscheinung, sowohl im Zweizellenstadium als in
den spateren Stadien, wahrend der eigentlichen Ruhepause
vollstandig, indem vermuthlich jene Massen einer Resorbtion
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 373
oder chemischen Umwandlung anheimfallen.” In only one
astrosphere of only one cell in each of the following cleavage
generations this process is repeated, and the line of these par-
ticular cells (‘‘ Kornchen-Zellen’’) constitutes the line of devel-
opment of the sexual cells ; but the ectosomes are present in
these particular cells only during mitosis, and in the resting
stages are absent, while nucleoli occur in the nuclei ; this proc-
ess was observed from the first through the ninth cleavage
stages. He concludes that in each generation there is a produc-
tion de xovo and a subsequent solution (Auflésung”’) of the
ectosomes. The first appearance of the latter coincides in point
of time approximately with the disappearance of the nucleolar
substance in the nucleus ; from this and certain other factors
he concludes : “So . . . wiirde also der Annahme kaum etwas
im Wege stehen, dass die zu Beginn der Mitose noch vorhan-
denen oder neugebildeten Nucleolen aus dem Kernraum in der
Richtung der ezzex Spahre auswandern und sich hier in die
Aussenkérnchen umwandeln. . . . Fiir die Kerne der Kérn-
chenzellen ist dann allerdings, in Gegensatz zu den ibrigen
Embryonal-Elementen, eine besonders reichliche Produktion
der Nucleolarsubstanz und demnach eine besonders intensive
vegetative Thatigkeit [cf '95] anzunehmen.” The explanation
for the arrangement of the ectosomes in only one of the
astrospheres he finds in the assumption “dass die beiden
Centrosomen einen verschiedenen (vielleicht einen verschieden
‘kraftigen’) Einfluss auf das umgebende Plasma, beziehungs-
weise auf die beweglichen Inhaltskérper desselben ausiiben.”
Hacker ('97b) finds that in germ cells of both animals and
plants there is to be noted “das Auftreten eines einzigen,
vacuolenhaltigen, dunkel tingierbaren ‘“ Hauptnucleolus” in
den jiingeren Stadien, das Hinzutreten von blasseren adventiven
oder “Neben-Nucleolen”’ in einer friiheren oder spateren
Phase.” Nucleolar substance arises during one or several stages
of nuclear activity as a by-product of metabolism, possibly also
as chromatin substance which has become structureless and
chemically changed; and, finally when the nucleus begins to
divide, is removed out of the latter. He confirms Wheeler’s
(96) observations on the ovum of J/yzostoma, that the nucleolus
374 MONTGOMERY. [VoOL. XV.
wanders out of the nucleus into the cytoplasm, where it slowly
decreases in size,
Hermann ('97) figures (Fig. 20) a spermatogonium nucleus of
Scyllium containing a single and a double nucleolus ; the latter
consists of two apposed spheres, which differ chemically and
dimensionally.
Korschelt ('97) maintains his previous opinion (96) of the
chromatin nature of the macrosomes of the nuclei in the
spinning glands of caterpillars, in answer to the criticism of
Meves ('97) (reviewed immediately below). Korschelt employed
the Ehrlich-Biondi stain with increased strength of the methyl
green, and thereby obtained a coloration of the macrosomes
and microsomes the very opposite of that procured by Meves.
‘‘Ob man iiberhaupt achromatische, chromatische Substanz und
Nucleolen in allen Kernen so scharf auseinanderhalten kann,
wie dies vielfach geschieht, ist mir héchst zweifelhaft. Wenn
man in verschiedenen Zustanden der Kerne Nucleolen auf-
treten und wieder verschwinden sieht, wird man annehmen
miissen, dass sie sich aus den sogenannten achromatischen oder
chromatischen Substanzen des Kerns, vielleicht aus beiden
herausbilden. So kénnen sich moéglicher Weise auch die von
mir als Makrosomen bezeichneten Theile in Nucleolen umbilden
und das von Meves angegebene Auftreten von Vacuolen in
ihnen wiirde damit seine Erklarung finden.”
Meves (97) contends that the microsomes in the spinning
glands of caterpillars, which Korschelt regarded ('96) as lanthanin
granules, are chromatin ; and what Korschelt regarded as chro-
matin granules (7.¢, the macrosomes) are nucleoli. Meves
employed the usual formula of the Ehrlich-Biondi stain (Heiden-
hain’s receipt), and finding that the macrosomes thereby become
stained red, concludes from this reaction their chromatin nature.
Stauffacher (97) finds in the aster stage of the mitosis of one
of the pronephral cells of Cyc/as, that the nucleolus still persists
intact in apposition to the spindle fibers.
Wheeler (97), maturation of the ovum of Jyzostoma: this
object, previously described by the author (95), is here more
fully described with the addition of figures. A remarkable
mode of formation of nucleoli in the pronuclei is described ;
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 375
each “chromosome”’ consists of ‘two granules, at first of the
same size [which] grow very unequally, so that one is often
considerably larger than the other. Hereupon some, but not
all, of these granules break down to form irregular strings of
minute karyomicrosomes which are distributed along the fibers
of the achromatic reticulum. ... The large chromatin granules
which do not break down become the nucleoli of the pronuclei.
I am unable to state positively that in each Diplococcus-shaped
chromosome one of the granules breaks down to form a chain
of minute karyosomes while the other persists as a nucleolus,
but I am very strongly inclined to believe that this is the case.”
These nucleoli are cast out into the cytoplasm when the first
cleavage spindle is formed, and there rapidly dissolve. Wheeler
accepts “ Hacker’s view of the secretory nature of the nucleo-
lus, at least so far as the germinal vesicle is concerned.”
Bancroft ('98), germinal vesicle of Dzstaplia: the nucleolus
“does not form the stellate body found in the old ova, as
Davidoff maintained, but is found within this body, which is
itself the remains of the germinal vesicle. The nucleolus at
this stage is quite complex, consisting of a homogeneous cortex,
an excentric finely granular medulla, and within the latter
several very highly refractive bodies, the largest of which may
have a granular appearance. During the greater part of the
growing period these refractive bodies are the only substance
in the germinal vesicle that takes the chromatin stain with a
methyl green and acid fuchsine combination.”
1898.
Kostanecki ('98) confirms the observations of Wheeler ('95,
97) in regard to the casting out of the nucleolus into the
cytoplasm, in the maturation of the ovum of J/yzostoma.
B. BoranicaL LITERATURE.
Schleiden ('38) is the discoverer of the nucleolus in plants, but
he gives it no name: “einen kleinen, sich scharf abgren-
zenden Ko6rper, der, nach dem Schatten zu urtheilen, einen
376 MONTGOMERY. [VoL. XV.
dicken Ring oder ein dickwandiges hohles Kiigelchen darzu-
stellen scheint’; while in other cases it may be a simple spot,
or may be wholly absent. ‘Aus meinen Beobachtungen an
allen Pflanzen, die eine vollstandige Verfolgung des ganzen
Bildungsprocesses erlaubten, geht hervor, dass dieser kleine
Korper selbst friiher sich bildet, als der Cytoblast [Nucleus].”
Macfarlane (’81) examined various plant cells, in all of which
he found one or several bodies (‘‘nucleolo-nuclei’’) within the
nucleolus. The nucleolus of Sfzvogyra has a distinct membrane,
which disappears at the period of the nucleolar division ; the
karyokinesis results in the formation of a “nuclear barrel,” at
each end of which is a mass of nucleoplasm, these two masses
being connected by fibers with the nucleolus which lies between
them. The nucleolus then divides, preceded by a division of
the nucleolo-nucleus, so that each daughter-nucleolus receives
a daughter nucleolo-nucleus, and the daughter-nucleoli then
wander apart to the nearest masses of nucleoplasm, ‘as they
retreat from each other they drive the polar masses before them,
thereby elongating the nuclear barrel. . . . The nucleoli at
length advance to the polar masses and bury themselves in
the nucleoplasm of these.” From these and numerous other
observations, Macfarlane concludes: ‘that the nucleolus, or
more probably the nucleolo-nucleus, is the center of germinal
activity, and that as we pass outwards to the periphery of the
cell, this reproductive activity becomes less and less. In no
other way, to my mind, can the number of nucleoli and nucleolo-
nuclei at different ages in the cells of any plant be explained.”
Strasburger (82a) gives reviews of previous observations on
the chemical constituency of nucleoli.
Strasburger (2p) studied nuclear division in various plant
cells (Fritillaria, Lilium, Hemerocallis, T; vadescantia, Galanthus,
Dicotyledons). ‘Pollenmutterzelle” of Frztillaria: between
the nucleus and its membrane collects a homogeneous, refrac-
tive, lens-shaped mass of substance; “sie geht nicht unmittelbar
aus den Kernkorperchen hervor, die ja schon auf vorausgehenden
Stadien verschwunden waren, vielmehr reprasentirt sie, allem
Anschein nach, ein Secret”; this body he terms ‘ Secretkor-
perchen.” At first it stains deeply with methylen green ; but
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 377
subsequently it ceases to stain, vacuoles arise in it, and it
decreases in size, until at the time of the spindle formation it
disappears. ‘Sie [Secretkorperchen] treten erst auf, nachdem
das Kernkorperchen oder die Kernkorperchen in dem Faden-
knauel des Kerns Aufnahme gefunden. Ihre Entwicklungsge-
schichte unterscheidet sich auch von derjenigen echter Nucle-
olen, denn sie treten nicht im Verlauf der Fadenwindungen auf,
vielmehr ausserhalb derselben, stets an der Wand der Zelle.
Ausgeschlossen ist ja nicht, dass in der so ausgesonderten
Substanz die Substanz friiher Kernkorperchen vertreten sei,
aber erweisen lasst sich dies nicht.” So he concludes that
before the mitosis of the spores and “ Pollenmutterzellen” a
certain change occurs in the nucleoplasma, in connection with
the formation of the “Secretkérperchen.” The nucleoli of
many plant cells contain vacuoles. In the embryo sac of
Galanthus a division of the large nucleolus takes place, which
division is probably passive, caused merely by the tension of the
cytoplasm. Gradations are to be found between the nucleoplas-
mic-microsomic substance and the substance of the nucleoli :
“ob die Nucleolen-Substanz trotzdem nur eine Modification
der Microsomen-Substanz sei und aus dieser hervorgehe, will
ich dahingestellt bleiben lassen. Wahrscheinlich ist mir aber
das letztere, wenn ich bedenke, dass bei Eintritt in die Thei-
lungsvorgiange selbst die stark modificirte Nucleolen-Substanz
in das Kerngeriist findet und*sich in demselben nicht anders
als wie die Mikrosomen-Substanz verhalt. Man konnte die
Nucleolen-Substanz vielleicht als einen Reservestoff des Zell-
kerns auffassen, als eine momentan ausser Aktion gesetzte
Substanz.”’
Tang] ('82) studied the nuclear division of three species of
plants. Hemerocallis fulva, flower buds: the “ Pollenmutter-
zelle” contains at first three or five nucleoli, which are homo-
geneous. ‘ Mit fortschreitender Entwicklung der Mutterzellen
verringert sich die Anzahl der Nucleolen,” until only one is to be
found; this one is always peripheral in position, never in contact
with the central ““ Kérnermasse.” Later, vacuoles appear in the
nucleolus (he believes these to be the results of reagents), and
while it still stains with carmine it no longer does with acidified
378 MONTGOMERY. [VoL. XV.
methylen green. In mitosis, when the nucleus is uninucleolar,
the substance of this nucleolus becomes dissolved in the nucleus;
when multinucleolar, however, one of the nucleoli may pass
out into the cytoplasm. Hesperus, “ Pollenmutterzelle”’: here
there is one nucleolus, which stains with methylen green, as does
the chromatic filament, and disappears in mitosis. Pzszum,
same cells: here there is one hat-shaped nucleolus, which stands
in no connection with the “ Fadenknauel”’; “Sehr eigenthiim-
lich ist das Verhalten des Nucleolus in den die Kerntheilung
vorbereitenden Stadien. Anfanglich besteht derselbe aus
homogener, stark lichtbrechender Substanz. Spater sind am
Nucleolus eine dichte dussere und eine innere, bedeutend
schwacher lichtbrechende Schichte unterscheidbar. Endlich
findet man Stadien, auf denen neben dem noch unveranderten
Fadenknauel ein sehr schwach lichtbrechender Korper gefunden
wird, dessen Umrisse vollkommen demjenigen des urspriing-
lichen Nucleolus entsprechen”’; finally even this disappears.
Zacharias (’g2) studied the epidermis cells of Pajus, and
concludes that the nucleoli (one or two in number) consist of
plastin. They do not dissolve in distilled water; swell with
.1% nitric acid; do not stain with methylen green; and
dissolve in weak “ Kalilaugelosung.”
Heuser (84) studied the mitoses in the embryo sac of /77¢z/-
lavia imperialis. In the resting nucleus there are from five
to nine nucleoli: “ Dieselben sind intensiv gefarbt und stehen
in deutlich wahrnehmbaren Zusammenhang mit dem Nucleo-
Hyaloplasma.”’ In the prophase of the mitosis they lose their
staining power and apply themselves to the chromatin threads.
He considers them, with Strasburger, “als Reserve-Behalter
der Kernsubstanz”’ (using the term ‘“ Kernsubstanz”’ as equiva-
lent to “Chromatin”’); their ground substance consists of
plastin, permeated with chromatin. In /77¢z//aria, as well as
in Galanthus and Leucojum, “fliesst das gesammte Kernkor-
perchen in die Kernelemente iiber, wahrend in anderen Fallen
ein Ueberschuss an Plastin als ‘Secretkérper’ ausgeschieden
werden mag.”
Strasburger ('84), nuclei in the embryo sac of F77¢z//aria - in
the spirem stage the large nucleoli disappear, ‘ wobei sich um
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 379
dieselben der Kernsaft wieder zu farben beginnt.’’ He con-
cludes that the nucleoli are not immediately taken up into the
chromatic thread, but dissolve in the caryolymph; “auch ist
hiermit wohl sicher der Nachweis gegeben, dass sie nicht iden-
tisch mit den Mikrosomen sein konnen.” The nucleoli arise
in the meshes of the chromatin network. Strasburger agrees
with Flemming that they represent a substance distinct from
the chromatin and nuclear sap, but does not consider it to be
a living substance, but rather a reserve stuff.
Guignard (85) investigated nuclear division in several species
of plants. Lz/¢wm, young embryo sac: the nucleus usually
contains a single nucleolus, which is very large, finely granular
in structure, and situated excentrically between the strands of
the chromatin network; with the double stain, methylen green
and fuchsine, it stains red, while the chromatin stains green.
At the time of the longitudinal division of the chromatin
filament, the nucleolus commences to stain less intensely,
vacuoles arise in it, and it finally fragments into small pieces
which subsequently disappear; the fine granules appearing in
the nuclear sap at this time are not derivatives of the nucleolus,
but originate from the cytoplasm when the nuclear membrane
vanishes. “Dans le Lzdiwm... rien ne fournit la preuve d’un
apport direct effectué dans la formation du fuseau par le nucléole,
dont la substance se dissout dans le suc nucléolaire, pour
s’incorporer et se mélanger, . . . aux autres éléments figurés
qui contiennent la chromatine.” In each daughter-nucleolus
there are several nucleoli of unequal size; these disappear also
in the subsequent mitosis. Clematis, embryo sac: the nucleoli
in karyokinesis gradually decrease in size, and it seems “ comme
si la plus grande partie de leur substance était absorbée par les
segments [chromatiques].” Vorthoscordum: here there are
several large nucleoli which disappear when the spindle is
produced, their substance being possibly incorporated in the
chromosomes. In the metaphasic spirem they reappear in
contact with the chromatin: ‘leur aspect général fait supposer
qu’ils naissent 1a ot on les apercoit dans les jeunes noyaux.. .
il est a croire que les nucléoles tirent une partie de leur
substance, tout ou moins, du filament nucléaire auparavant
350 MONTGOMERY. [VoL. XV.
homogeéne. Ils se nourrissent ensuite dans le suc nucléaire. . . .
Les nucléoles peuvent étre considérés comme une substance
de réserve que se sépare 4 un moment donnée de la charpente
nucléaire pour étre reprise par elle ultérieurement”’; he assumes
that Strasburger’s “corpuscule du sécrétion”’ is a true nucleolus.
“Dans le Lilium et dans l’autres plantes, les noyaux filles
n’offrent pas de nucléole avant d’entrer en division; en outre,
leur aspect général au début du phénoméne est bien different
de celui du noyau mére.... Le fait qu’ils se séparent du
filament dés que le noyau .. . arrive a l'état de repos, pour
étre repris par lui aux premiers stades de la division, permet
de les considérer, avec M. Strasburger, comme une sorte de
réserve.”
Macfarlane (85) studied nuclear division in Chara fragilis
(fixation with osmic acid): the nucleus of the apical cell contains
one nucleolus, in which lies an ‘‘endonucleolus”’ (a term here
substituted for his earlier term “nucleolo-nucleus”’). At the
commencement of all cell divisions this part of the nucleolus
first divides, then the nucleolus, last of all the nucleus. After
this division of the apical cell a nodal and internodal cell
are produced, and the former “continues to divide regularly,
forming cells each with one nucleus and nucleolus. In the
internodal complete cell division is henceforward absolutely
arrested: but the earlier steps are taken ; for while the nodal
cell has divided into three or four, the nucleolus of the inter-
nodal has divided and redivided, so that four nucleoli are present
in the nucleus of it. The internodal cell then increases rapidly
in length, the four nucleoli meanwhile continuing to proliferate,
so that in internodal cells, such as in the third removed from
the apex, we soon get a large nucleus with many little dark
nucleoli. The nucleus then divides in the simple manner
figured by Johow, so that in the fourth internodal cell there
may be two nuclei, each with many nucleoli, in the fifth, three
or four nuclei, and so on, so that the internodal cells soon
become multinuclear, and their nuclei multinucleolar.” The
cortical nodal cells do not divide further, but ‘their nucleoli
follow the example of that of the internode . . . the consequence
being that the cortical nodal, and soon after the cortical
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 381
internodal cells, become multinucleolar”’; the nodal leaf cells
proceed in the same way. From these observations Macfarlane
concludes : “in every active embryonic cell one nucleolus only
is present in the resting state”; in some cases a fluid globule
is present in the nucleolus, and this probably represents a
“degradation of the endonucleolus.” “The nucleolus, or more
probably the nucleolo-nucleus, is the center of germinal activity,
and that as we pass outwards to the periphery of the cell, this
reproductive activity becomes less and less.... The result is
that in all plants thus examined, after cell division has ceased,
continued division of the cell contents from the endonucleolus
outwards goes on... . I venture, therefore, to regard it as a
general principle that after cell formation has ceased, the cell
contents (especially the endonucleolus and nucleolus) persist
in their activity for a shorter or longer period; ... the most
exalted type of cell is one with abundant protoplasm containing
a single nucleus, nucleolus, and endonucleolus;...a cell with
vacuolated protoplasm, one nucleus and two to four nucleoli
is less exalted, while the multinuclear state is the most degraded
form of cell.”
Zacharias (85) gives critical reviews of numerous preceding
papers on nucleoli, besides observations of his own on various
cells of plants. Galanthus nivalis, cells of the inner layer of
the “ Fruchtknotenwand’’: the single nucleolus is about 2, the
size of the nucleus; examined in water it is homogeneous; after
the action of absolute alcohol it appears to be composed of
granules of various indices of refraction. Bast cells of Cucurbita
pepo: the nucleoli, when stained with “ Blutlaugensalz-Eisen-
chlorid,” become very intensely colored, while the remaining
nuclear substance stains only faintly. In the cells of Spzrogyra
and of the asci of Lichens he finds that there are no “ nucléoles-
noyaux,” such as Carnoy described. ‘Alle Autoren stimmen
gegenwartig darin iiberein, dass die Nucleolen bei der Kern-
theilung verschwinden.” In opposition to Strasburger he
contends that during the mitosis the dissolved nucleolar sub-
stance might as probably enter into the formation of the spindle
fibers as of the chromosomes. In Chara (observed living) each
nucleus contains one large nucleolus, with vacuoles: ‘ Naht
382 MONTGOMERY. [VoL. XV.
die Kerntheilung heran, so verliert der Nucleolus an Deutlich-
keit, er erfahrt langsame Gestaltsveranderungen, die schliesslich
einen amodboiden Charakter annehmen,” and the nucleolus
gradually disappears (this process lasting a half hour); “13%
Stunde spater wurden in jedem Tochterkern vier kleine Nucle-
olen bemerkt, nach 3% Stunden waren nur noch je zwei
Nucleolen vorhanden und nach weiteren 1% Stunden nur noch
je einer... . Bei der Verschmelzung bilden die Nucleolen
zunachst einen bisquitformigen Korper, der sich dann spater
kugelig abrundet. Die Deutlichkeit der Nucleolen nimmt
wahrend des Vorganges der Verschmelzung stark ab, um spater
wieder zu steigen.” Contrary to Strasburger and Tangl, he
believes that no ‘ Paranucleolen’’ wander out of the nucleus,
but that where such have been observed, it has been due to the
method of fixation. He notes that while the egg cells always
contain nucleoli they are frequently absent in the male cells.
“Tn alternden Zellen sind Gestaltsveranderungen, Kleiner-
werden und Schwinden des Nucleolus beobachtet worden. ...
Mir scheint es nicht begriindet zu sein, den Nucleolus als eine
Ablagerung von Reservestoffen zu betrachten. Wesshalb
sollte er nicht ein Organ der Zelle sein, wie es Flemming
annimmt?...” Against Strasburger’s views “ist zu erwidern,
dass wir iiber das active oder passive Verhalten der Nucleolen
im ruhenden Zustande oder dem der Theilung iiberhaupt gar
nichts wissen, und das Bestehen einer Organisation fiir die
Nucleolen ebenso gut angenommen werden kann wie fiir irgend
einen anderen Theil der Zelle.”
Meunier (86), Spirogyra; the single large nucleolus has a
limiting membrane, and in the fresh state contains no vacuoles,
vacuoles only appearing in the dying cell, and then are probably
introduced drops of water. It stains with methylen green
more intensely than any other structures of the nucleus, and
also stains with acid picrocarmine, alkaline carmine, and haema-
toxylin ; “ainsi ...on constate que les matiéres colorantes,
reputées spécifiques de la nucléine, limitent uniquement leur
action efficace et significative au corps réfringent et apparem-
ment réticulé du nucléole.’’ After the action of nitric acid of
from 2% to 4% a reticulation is found in the nucleolus; a 10%
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 383
or 12% solution of the same acid dissolves this reticulation and
only preserves the clear, non-refractive stroma; 2% or 4%
hydrochloric acid solution shows the reticulation of the nucle-
olus to be ‘un boyau continu et pelotonné. ... Le filament
chromatique du nucléole ne se digére pas dans la liquer digestive
[suc gastrique]. ... Nous ne craignons pas d’affirmer que
le nucléole des Spzvegyva reproduit fidélement, dans ses traits
essentiels, la structure des noyaux les plus parfaits. Ila une
membrane propre, probablement une partie protoplasmatique,
quoique fort réduite ; il renferme toute la nucléine du noyau, et
celle-ci est exclusivement confinée dans un étui de plastine,
qu’elle remplit plus ou moins complétement. . . . Quoi qu'il
en soit, nucléole par position, noyau par nature, on ne peut lui
refuser le nom de nucléole-noyau, dans le sens attaché a ce mot
par J. B. Carnoy.”
Schwarz ('87) studied the microchemistry of plant cells. He
distinguishes the following substances in the nucleus: “ chro-
matin,” “pyrenin” (nucleolar substance), amphipyrenin”’ (sub-
stance of the cell membrane), “linin”’ (achromatic fibrils), and
“paralinin” (nuclear sap). The pyrenin and amphipyrenin
“‘stimmen in fast allen Reactionen iiberein, sie unterscheiden
sich jedoch durch ihre Tingirbarkeit, indem das Pyrenin der
Kernkérperchen Farbstoffe fast immer sehr leicht aufnimmt
und festhalt, wahrend das Amphipyrenin nur wenig oder gar
nicht tingirt wird. ... In den weitaus meisten Fallen liegt
das Maximum des Nucleolusvolumens vor der Zone, in welcher
der Kern sein Maximum erreicht, und in vielen Fallen tritt
gerade dann die bedeutendste Verkleinerung des Nucleolus-
volumens ein, wenn der Kern sein Volumen am stirksten
vergrossert. Es scheint mir demnach wahrscheinlich, dass ein
Theil der Kernkérperchensubstanz direkt bei der Neubildung
der iibrigen Kernsubstanz verbraucht wird.”
Went ('87), mitosis in various cells of plants. Leucojum,
embryo sac: at the commencement of the prophasis there are
two large nucleoli, which lie between the fibers of the chromatin
network ; later they become apposed to these fibers, and he
notes how ‘die Masse des Nucleolus langsam in die des Kern-
fadens iibergeht.... Im Wandbelege des Embryosackes von
384 MONTGOMERY. [Vou. XV.
Helleborus viridis scheinen die Nucleolen auch im Kernfaden
aufgenommen zu werden’’; and there is apparently the same
process in /ri¢zllaria imperialis. ‘ Bei den Kernen im Wand-
belege des Embryosackes von Narcissus pseudonarcissus findet
die Aufnahme des Nucleolus ungefahr wie bei Galanthus statt ;
er wird also von allen Seiten vom Kernfaden umwunden ;
allmahlich windet dieser sich wieder los. Oft ist dann der
Nucleolus schon ganz aufgenommen, zuweilen aber werden
noch Theile davon vom Kernfaden fortgeschleppt und bleiben
dann wohl einmal sichtbar bis zum Anfang der Metaphase.
Wenn man Praparate mit diamantfuchsin-jodgriin tingirt hat,
sieht man, dass die Farbe des Kernfadens vor der Aufnahme
des Nucleolus blaugriin ist, wahrend dieser letztere roth gefarbt
ist; nach der Aufnahme des Nucleolus und wahrend der ganzen
Meta- und Anaphase ist die Farbe des Fadens deutlich violett
geworden, was naturgemiss verursacht ist durch die Aufnahme
des Nucleolus”; also during the mitosis of similar cells in
Hyacinthus and Tulipa nucleolar substance is taken up by the
nuclear filament. “Ich glaube aus den hier mitgetheilten
Thatsachen wohl den Schluss ziehen zu diirfen, dass in vielen
Fallen wenigstens der Nucleolus beim Anfang der Kerntheilung
im Kernfaden aufgenommen wird. ... Am wahrscheinlichsten
ist es wohl, dass, wo der Nucleolus vor der Theilung im Kern-
faden aufgenommen wird, er sich nach der Theilung auch
wieder daraus bildet.”’
Strasburger (gs) studied nuclear division in Spzrogyra poly-
taeniata. In the resting nucleus there is usually one large
nucleolus, which disappears immediately before the formation
of the nuclear filaments, and by dissolving in the nuclear sap
causes the latter to stain more intensely : ‘‘ Als wahrscheinlich
stellte ich [84] es aber hin, dass die im Kernsaft geldste
Nucleolussubstanz den Kernfaden als Nahrung diene.... Auf
Grund meiner neueren Erfahrungen erscheint es mir iiberhaupt
unwahrscheinlich, dass die Nucleolarsubstanz, auch nach ihrer
Auflésung im Kernsafte, den Kernfaden als Nahrung dienen
sollte.” In each daughter-nucleus several nucleoli arise, and
these have the same number, position, and size in the two nuclei;
later the several nucleoli of each daughter-nucleus unite to form
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 385
a single large nucleolus, and during this process the nuclear
sap gradually loses its staining power. He shows that when
the nucleolar substance is dissolved in the nuclear sap, and
after the cell division, a portion of this substance plays a part
in the production of the cellulose walls of the daughter-cells ;
but he holds that not all of it is thus consumed, but that the
nucleoli have probably some other, as yet unknown, function.
Mann ('91) introduces a new method of differential nuclear
staining: when plant tissues are stained for ten minutes in
saturated solution of heliocin in 50% alcohol, and then from ten
to fifteen minutes in a saturated aqueous solution of aniline blue,
the nucleolus is red, the rest of the nucleus and the cell blue.
Macfarlane (92) constructs the following hypothesis, based
on previous observations of his own and of Mann: ‘We would
consider, then, that the nucleolus is the special chromatic and
cell center ; that it sends out fine radiating processes — the
intranuclear network — which partially fuse externally to con-
stitute the nuclear membrane, the interspaces of the network
being occupied by nucleoplasm concerned in metabolic change;
that radiating continuations of the chromatic substance pass
out beyond the nuclear membrane and form a network in the
protoplasm, while we would suggest for future proof or disproof
that they further may be continued through wall pores to form
an intercellular chromatic connection. . .. We would thus
view a plant as a group of connected hermaphrodite cells, ...
bound together by a fine chromatic ramification, in the center
of which in each cell is the nucleolus.”
Mann ('92) studied the cells of the embryo sac of Myosurus
minimus. At the commencement of the conjugation of the two
nuclei resulting in the formation of the primary endosperm
nucleus, each nucleus contains ‘a large deeply stained nucleolus
enclosed by a very faintly stained nucleolar membrane,” and
in each nucleus are also one or two smaller globules, which
“seem to originate thus: when the nuclei about to conjugate
have come in contact, one or two small nucleoli arise by the
unequal division of the primary nucleolus. ... These secondary
nucleoli seem to have at first the power of division, but
gradually they lose this power and their property of becoming
386 MONTGOMERY. [Vou. XV.
deeply stained, and change into globular colloid-looking masses
with a central more deeply stained spot. I propose to call these
bodies paranucleoli, because of their origin they may always be
found in the micropylar nucleus and occasionally also in the
antipodal nucleus.’ When these nuclei begin to conjugate,
the large nucleoli of both fuse to form the single nucleolus of
the primary endosperm nucleus ; at the same time a new struc-
ture makes its appearance, in close contact with the nuclear
membrane of the primary endosperm nucleus : “This body...
corresponds, I believe, to the nucleolar membrane of the
antipodal nucleus”; it is at first granular, later homogeneous.
Still other, smaller spherical bodies later appear in the nucleus,
which may have some connection with the paranucleoli. Finer
structure of the nucleolus: in the nucleolar membrane “a
number of very minute dark radially placed pores or striae can
be observed, and . . . these striae are continued into very
delicate cilia-like fibrils radiating out from the nucleolar mem-
brane into the nuclear hyaloplasm. ... The nucleolus is
differentiated into an outer zone and an inner zone. The outer
zone is less deeply stained, and on careful examination is found
to be made up of a circle of peripheral endonucleoli, which are
slightly elongated radially. The inner zone of the nucleolus
is very darkly stained, and shows a number of large and irregu-
larly disposed endonucleoli.” The structure of the nucleolus
may be somewhat different in other stages of its development,
thus it may be composed of “(1) A thin unstained nucleolar
membrane ; (2) a great number of peripheral endonucleoli ; (3)
a deeply stained, apparently structureless, layer; (4) a corona
of minute, slightly elongated, endonucleoli surrounding (5) a
large central endonucleolus.... In a resting cell, . . . the
center of the nucleolus is occupied by a large endonucleolus,
which sends out minute fibrils through the nucleolar sub-
stance.... I believe the endonucleolar fibrils probably to pass
through the finer pores in the nuclear membrane”; and Mann
conjectures that “the endonucleolar filaments constitute the
linin element of the chromosomes.” Functions of the nucle-
olus: it is “concerned in the assimilation of food-material.”
He holds “the nuclear chromatin to be less highly elaborated
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 387
and less assimilative albuminoid material than the nucleolar
chromatin. On the assumption just stated, we could explain
also why we find. . . at the time of maturation portions of
nucleolar matter detaching themselves from the main nucleolus
to undergo a peculiar gelatinous change. The gelatinous change
would correspond to a conversion of the assimilative material
into achromatic elements, an explanation which would also
explain the disappearance of nucleoli during the division of a
cell... I believe the hypothesis that the nuclear chromatin-
segments and perhaps the nucleoli are organs for the conversion
of assimilated material into material directly available for the
achromatic elements of the cell to be not quite erroneous.” In
the mechanism of cell conjugation: ‘The endonucleolar fibers
running through the body-plasm of the two sexual cells .. . are
brought into contact with one another whenever the pseudopo-
dial processes of the two cells have met. As soon as an union
of fibrils has taken place, each fibril will commence to contract
similarly to a muscular fibril,’ which results in drawing the two
nuclei, afterwards also the two nucleoli, together; thus the
endonucleolus is the “tropic center” of the cell.
Rosen ('92a) studied the differential staining of the nuclear
elements in plants. Flowers of Scz//a: in the nuclei of the
« Biindelparenchym”’ are numerous large nucleoli, which differ
in form and size; the one or two larger ones, ‘‘ Eunucleoli,” are
each surrounded by a clear space, but none is present around
the smaller “ Pseudonucleolen.’’ With the double stain, Alt-
mann’s acid fuchsine and methylen blue, the Eunucleoli stain
red and the Pseudonucleoli blue, or vzce versa. Similar cells
of Hyacinthus : by the application of the double stain, aqueous
solutions of fuchsine and methylen blue respectively, the
Eunucleoli stain red, the Pseudonucleoli blue ; but when these
stains are applied in the reverse order, the nucleoli stain
reversely. He considers, following Auerbach ('90), that the
Eunucleolus is erythrophilic, the Pseudonucleoli kyanophilic,
the latter staining as does the chromatin network. “Meine
Pseudonucleolen aber sind eben offenbar weiter nichts, als
besonders selbstandig ausgebildete Bestandtheile des chroma-
tischen Kerngeriistes und sind wie dieses und sein Produkt, der
388 MONTGOMERY. [VoL. XV.
Kernfaden, kyanophil”’; these disappear before the mitosis,
while the Eunucleoli remain until about the end of the spirem
stage. Vacuoles arise only in the Eunucleoli.
Rosen in a second paper ('92b) presents further observations
upon nucleoli. J/yxomycetes ; the spore nucleus contains one
large nucleolus. Fuligo septa, plasmodium: one large, cyano-
philic nucleolus, which he terms ‘“ Mittelkérperchen,” since in
the atypical mitosis this body lies in the middle of the pole
plate, and disappears at the end of the nuclear division. Syz-
chrytrium : one large nucleolus with several vacuoles ; in the
first mitosis the division of this nucleolus precedes that of
the nucleus, but during subsequent divisions the nucleoli
vanish. In Cystopus there is no nucleolus.
Schottlander ('92), cells of cryptogams : the nucleus consists of
a blue-staining substance (network), and a red-staining (nuclear
membrane, nucleoli). Egg cell of Gymnogramme chrysophylla ;
here are one or several large nucleoli, each surrounded by a
vacuole; in the ripe egg the nucleoli are filled with small glob-
ules. Egg cell of Chava: the nucleoli contain vacuoles, which
later become so large in the largest nucleoli that they become
polygonally flattened against one another, and their thin walls
then present the appearance of a network within the nucleolus.
Demoor ('93), mitosis of 7vadescantia ; the nucleoli gradually
disappear during the prophase.
Gjurasin (93) investigated the nuclear division of Pezszza.
In the nucleus is one large, excentric nucleolus, which stains
red with Flemming’s triple stain, while in it as many as six
granules may occur, and these stain violet. In the mitosis
these granules disappear, but otherwise the nucleolus does not
change at first, but occupies its original position within the cell,
though now in the cytoplasm; eventually it disappears gradually.
In each daughter-nucleus a new nucleolus arises, which appar-
ently has no genetic connection with the mother-nucleolus (now
vanished). ‘Ich bin der Ansicht, dass. . . das Kernk6rper-
chen nicht eine Art von Reservestoff darstellt, sondern ein
specifisches Organ des Zellkernes ist.”
Karsten (93), nuclear division of Psz/otwm: in the resting
nucleus are two or three nucleoli, which are homogeneous, oval
No.2.) COMPARATIVE CYTOLOGICAL STUDIES. 389
or spherical, and after haematoxylin-eosin, stain a rose color,
while the chromatin is blue. At the time of the appearance of
the chromosomes, ‘‘treten die Nucleolen aus den sich zusam-
menordnenden Plasma und lassen sich hier in Form scharf
umschriebener, homogener, roth gefarbter Kiigelchen nach-
weisen.” Usually two nucleoli wander out, at least never more
than two were found outside of the nucleus. These two come
to lie at opposite poles of the nucleus, occupying the positions
of centrosomes; and when the longitudinal splitting of the
chromosomes takes place, each of the nucleoli also divides into
two. Karsten believes these nucleoli are identical with the
centrosomes of Guignard; but he does not explain what becomes
of the third nucleolus during the division.
Lauterborn (93), quoted by Karsten (93), diatoms: there
is a centrosome lying in a concavity of the nucleus; he noticed,
further, “beim Beginn der Theilung aber zwischen Kern und
Centrosom noch ein anderes Gebilde —, welches im spiateren
Verlauf der Karyokinese eine sehr bedeutsame Rolle spielt,
namlich die Anlage der Centralspindel”’; this body must be
derived either from the nucleus or the centrosome (I mention
it here since it may in the future be found to have some con-
nection with a nucleolus).
Moll (93) studied karyokinesis in Spzvogyra. There are one
or two nucleoli, which stain more intensely with gentian violet
than any other portion of the nucleus. They may be vacuolar
in structure, or contain a skein of chromatin; they appear
homogeneous only when too deeply stained. The skein struc-
ture (the skein itself staining as chromatin) is found in resting
nuclei, as well as in the prophases of mitosis, and at the same
time vacuoles may be present. Heassumes that the thread in
the nucleolus contains all the chromatin of the resting nucleus,
and “that by the nucleolus the chromatin substance for the
segments [chromosomes] is furnished’’; this chromatin leaves
the nucleolus in mitosis, and “it seems as if the chromatic sub-
stance were squeezed from the nucleolus by an aperture.” After
the chromatin skein has left the nucleolus, the latter disappears.
(Strasburger’s paper, '93, was reviewed under the head of
zoological literature.)
390 MONTGOMERY. [VoL. XV.
Wager (93), nuclear division in Hymenomycetes agaricus :
each nucleus of a basidium contains one large nucleolus, besides
the nuclear network. The two nuclei of the basidium fuse
together and form one nucleus, in which afterwards the two
nucleoli later fuse to form one nucleolus. This latter is
often vesicular in structure. In the mitosis it lies close to the
nuclear membrane, it gradually loses its staining intensity,
decreases in size, and finally disappears; at the same time the
cytoplasm in its neighborhood stains more deeply. But some-
times it persists until the diaster stage. ‘From the fact that
the chromosomes begin to stain red at the time of the disappear-
ance of the nucleoli, it would further appear that the former
can take up nucleolar substance from the nuclear sap, and as
fast as the nucleoli disappear the chromatic elements become
more deeply stained red.” In A. stevcorarius, in the daughter-
nucleus, ‘“‘the chromatin mass appears to be transformed at
once into the nucleolus,” and only later a chromatin network
appears. ‘I would suggest that the nuclear threads take up
the dissolved nucleolar substance at some period during the
division, and carry it over into the daughter-nuclei, to be given
up again later as the nucleoli of the latter.... But a certain
quantity of the dissolved nucleolar substance probably escapes
into the cytoplasm when the nuclear membrane disappears, and
this would be taken up at a later stage into the daughter-nuclei,
as is shown by the increase in size of the nucleoli, and by the
decrease in the capacity of the protoplasm for taking up stains.”
Zacharias (93) finds in plants that the nucleolus and cyto-
plasm are erythrophilic, the nuclein (chromatin) network is
cyanophilic.
Belajeff (94), “ Pollenmutterzellen” of Larix: after the
disappearance of the nuclear membrane in mitosis the nucleolus
becomes gradually smaller and then disappears; several nucleoli
reappear within each of the daughter-nuclei. “Es ist zu
bemerken, dass nach der Auflésung der Nucleolen der Mutter-
zelle im Zellplasma eine gewisse Anzahl grober Kérnchen
erscheint, welche mit Safranin farbbar sind. Mit dem Beginn
der Nucleolenbildung in den Téchterzellen verschwinden die
Kornchen vollkommen. ... Ich erklarte mir die Ergebnisse
e
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 391
meiner Beobachtungen derart, als losten sich die Nucleolen, nach
vorausgegangener Auflosung der Kernmembran, unter der Ein-
wirkung der in die Kernhohle aus dem Zellplasma gedrungener
Substanzen, ganzlich auf, um spater durch den Einfluss des
Kernsaftes, der die ganze Zelle durchdrungen, wieder hergestellt
zu werden, indem der Kernsaft die Nucleolensubstanz im Zell-
plasma so zu sagen gerinnen macht. Nach der Bildung der
Tochterkerne, welche ihren Kernsaft aus dem Zellplasma
absorbiren, werden die Kornchen abermals vom Zellplasma auf-
gelost, um zum zweitenmal im Inneren der jungen Kerne
(Téchterkerne) in der Gestalt von Nucleolen zu erscheinen.”
In Fritillaria and Lilium also the nucleolus is dissolved after
the disappearance of the nuclear membrane.
Humphrey (94) studied the “ Pollen-” and ‘“ Sporenmutter-
zellen”’ of Convalleria, Ceratozamia, Osmunda, and Psilotum,
and cells from the apex of the root of Vzcza and Hyacinthus.
The nucleolar substance is usually not to -be found in the
cytoplasm during mitosis. The nucleoli are “keine indivi-
duellen Bestandtheile, sondern unbestimmte Massen von Nucle-
olarsubstanz, und ihr Vorkommen im Cytoplasma hat keine
weitere Bedeutung als zu zeigen, dass eine Communication
zwischen Kernhohle und Cytoplasma bisweilen, wenn auch
nicht immer, sich herstellen kann und dass entweder die Nucle-
olen in einigen Fallen aus der Kernhohle, bevor sie von den
karyokinetischen Kraften angegriffen werden, austreten konnen,
oder dass die Menge der Nucleolarsubstanz in einem Kerne
grésser sein kann, als diese Krafte zu losen oder zu verbreiten
vermégen. . . . Die ‘Vacuolen’ der Nucleolen scheinen mir
das natiirliche Resultat der nachherigen Trennung der fliissigeren
von den festeren Theilen der Nucleolarsubstanz zu sein... .
Wenn also Zimmermann ['93] den Satz aufstellt ‘Omnis
nucleolus e nucleolo,’ so kommt er zu einer Verallgemeinerung,
die nicht zulassig und derjenigen ‘Omnis nucleus e nucleo’
nicht eleichwerthig ist.” In every nucleus of the “ Pollensacke”’
of Ceratozamia there is a large, peripherally placed paranucle-
olus (Strasburger): “In extremen Fallen kann die Anhaufung
von Substanz eine so grosse sein, dass die Kernmembran
hier bedeutend hinausgestossen wird... . Auf der Fuchsin-
392 MONTGOMERY. [VoL. XV.
Jodgriin tingirten Schnitten werden die Paranucleolen weder
reinroth wie die Nucleolen, noch blaugriin wie die chromatische
Substanz gefarbt, vielmehr nehmen sie eine Zwischennuance,
welche mehr der des Chromatins als der der Nucleolen ahnelt,
an”’; he believes these paranucleoli to be artefacts. In con-
tradiction to Karsten (93) he found no body in Pszlotum
comparable to a Nucleo-Centrosoma.
Zacharias (94) concludes, from numerous observations on
cells of plants that as the size of the nucleus increases (or
decreases) with the size of the cell, so also that of the nucleolus
increases (or decreases) with the size of the nucleus.
In Rosen’s (95) contribution a large number of new facts
are recorded, which may be briefly mentioned. The kyanophilic
nucleoli of Auerbach ‘sind eben keine Nucleolen und bediirfen
als wenig constante Theile des Chromatingeriistes iiberhaupt
keines besonderen Namens.” //yacinthus : in meristem nuclei
all the nucleoli except the smallest lie in special clear spaces,
and though fibrils are rarely found in connection with them,
“gleichwohl muss das Kernkorperchen in seiner scheinbar
schwebenden Lage wohlbefestigt sein, da es. . . stets seine
Lage im Centrum seines Hofes bewahrt.” The large nucleoli
of the “ Gefasszellen”’ become vacuolar as they increase in size.
In mitosis of root cells the nucleoli become gradually dissolved
within the nucleus in some species, in others they are extruded
into the cytoplasm; in the latter cases “ erfolgte die Zerkliftung
und Auflésung des Nucleolus viel langsamer, sodass bei dem
Schwinden der Kernmembran noch bedeutende Nucleolarreste
vorhanden waren.” The nucleoli reappear in the dispirem
stage before the daughter-nuclei have produced membranes,
and the new nucleoli stain from the commencement intensely ;
from which the general conclusions are drawn : in the prophase
the diminishing nucleolar substance penetrates, perhaps as a
micellar solution, into the cytoplasm, and this process may
cease before the nuclear membrane has disappeared. In some
cases larger particles of nucleolar substance may penetrate
into the cytoplasm, but only after the nuclear membrane has
disappeared, and these particles become subsequently dissolved
in the cytoplasm; in either case “das losende Agens muss wohl
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 393
der Kernsaft sein, vielleicht unter Mitwirkung eines nur
wahrend der Prophasen gebildeten Enzyms. Wahrend der
Anaphasen wandert die Nucleolarlosung als solche in den Raum
der Tochterkerne ein, und hier wird die Nucleolarmasse wieder
fest. Bei der Hyacinthe — und anderen Objekten — erfolgt
die Rekonstituirung der Nucleolen auch ausserhalb der Dis-
piremfigur. Die derart im Cytoplasma entstandenen Nucleolen
wandern, wie ich glauben méchte, in die Tochterkerne ein, ehe
sich diese mit einer Kernmembran umhiillen; wenn letzteres
geschehen ist, so findet man anscheinend niemals mehr Nucle-
olen im Cytoplasma, die, wenn iiberhaupt, auch wohl nur nach
nochmaliger Auflosung in den Kernraum gelangen kénnten.
Nicht ganz unméglich scheint es mir, dass die Nucleolen, die
man an fixirten Praparaten ... im Cytoplasma auffindet, doch
durch die coagulirende Wirkung des Fixirungsmittels ent-
standen sind. Ich glaube aber, dass dies von keiner grossen
Bedeutung ist, denn an den Stellen, wo wir extranucleare
Nucleolen vorfinden, muss dann die Masse der Kernkérperchen
als Lésung angesammelt gewesen sein.” Also in the mitosis
of root cells of Aspidistra, are nucleolar fragments seen in the
achromatic spindle. Root cells of Phaseolus: in the resting
stage there is a single nucleolus; in the mitotic prophase
it becomes first lobular, then lengthened in the direction of
the spindle, while at the same time it is undergoing a slow
dissolution; “‘wenn die Spindel gebildet und die Kernwandung
verschwunden ist, sieht man fast stets inmitten der zur Kern-
platte angeordneten Chromosomen einen mehr oder minder
ansehnlichen Nucleolarrest, welcher in derselben Richtung wie
die Chromosomen und die Spindelfaden gestreckt ist. Dieser
Nucleolarrest wird nun in der Mitte eingeschniirt, sodass er
Hantelform erhalt ; die beiden Halften reissen schliesslich von
einander und gelangen an die Spindelpole. In anderen Kernen
wird der Nucleolarrest einseitig aus der Kernplatte herausge-
drangt oder auch doppelt getheilt ; endlich findet sich meist an
einem oder an beiden Spindelpolen ein Restchen des Nucleolus;
seltener liegt ein solches neben der Spindel. Die Auflésung
ist nun meist bald beendigt’’; and only exceptionally is there
a minute nucleolar remnant in the cytoplasm at the end of
394 MONTGOMERY. [Vov. XV.
mitosis. ‘ Unzweifelhaft sind auch bei Phaseolus multiflorus
die Nucleolen der Tochterkerne Neubildungen. Wenn auch
die Nucleolarsubstanz méglicherweise bei der Karyolyse er-
halten bleibt und sich in den Tochterkernen nur wieder auf
Neue sammelt, so besteht doch keine von Generation zu
Generation sich fort spinnende Continuitaét in den Nucleolen
als solchen und von einem ‘omnis nucleolus e nucleolo’
[Zimmermann] kann keine Rede sein.” Root cells of Vicia
faba; the nucleolar mass diminishes as the cell degenerates ;
“dieselbe stellt das erste Zeichen der Kerndegeneration . . .
dar und ist, wie sonst, mit einer Zertheilung des Nucleolus
verbunden,” while a large nucleolus surrounded by a clear
space is an embryonic condition. In the mitosis of these cells
no nucleolar fragments pass into the cytoplasm, and in each
daughter-nucleus two nucleoli arise which subsequently fuse
into one. In opposition to Lavdowsky ('94), he contends that
the centrosomes have no genetic connection with nucleoli, and
that the nucleolar substance does not serve as nourishment for
the chromosomes ; ‘nichtsdestoweniger ware es voreilig zu
behaupten, dass von der Substanz der Nucleolen nichts in die
Fadensegmente gelangen kénne. . . . Die Violettfarbung der
Segmente in den spateren Phasen der Karyokinese . . . konnte
auf eine Einlagerung erythrophiler Nucleolarsubstanz in den
kyanophilen Kernfaden schliessen lassen.” In buds of Pse/o-
tum triquetrum the nucleoli are excentric, while in most plants
they have a central position. In the mitosis nucleolar frag-
ments are extruded into the cytoplasm (in agreement with
Zimmermann, in opposition to Karsten and Humphrey), and
none of the extruded masses can be regarded as centrosomes
(against the view of Karsten). Three nucleoli usually arise in
each daughter-nucleus : “Sie entstehen nahe der Peripherie
des jungen Kerns, oft in Contakt mit dem Cytoplasma, bevor
die Tochterkerne sich mit einer Membran umschliessen und
verschmelzen spater nicht miteinander.’’ In the mitosis of
sporangia the nucleoli are usually “aus den karyokinetischen
Figuren ausgestossen” ; and the “Secretkérperchen”’ of
Strasburger is a true extruded nucleolus.
Strasburger ('95, cited by Lauterborn, Zoo0/. Centralbl., 1896)
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 395
concludes that the nucleolar substance, dissolved in the nuclear
sap, may be used in the production of the spindle fibers.
Koernicke (96), study of mitosis on 77zticum: in the devel-
opment of the embryo sac when the two pole nuclei fuse
together, the two nucleoli also join to form one. In the mitosis
of the pollen the nucleolus always disappears before the forma-
tion of the spindle, but it could not be determined whether it
takes any part in the formation of the latter.
Lauterborn (96), nuclei of diatoms: there are several nucleoli
present; in the spirem stage of division they commence to
gradually disappear ; “es scheint mir ziemlich sicher, dass ihre
Substanz mit derjenigen der Chromatinkérnchen und des
Liningeriistes zur Bildung der Knauelfaden verbraucht wird.”
It is important to note that the central spindle arises outside
of the nucleus, before the nucleoli begin to disappear, so that
there can be no genetic connection between the two.
Poirault and Raciborski (96), binucleated (‘‘conjugate’’)
Uredineae during the production of the ascidiospore generation :
in the mitosis the nucleolus becomes extruded into the cyto-
plasm, almost always in the equatorial plane. ‘Bei manchen
Arten bleiben sie sehr lange erhalten so z. B. bei Pertdermium
Pini acicola, wo neben den langst ruhenden, mit neuen Nukleo-
len versehenen Kernen noch in den Plasma, die alten Kern-
korperchen der Elternkerne herumirren. Mit den Centrosomen
‘haben somit diese extranukleoldéren, vakuolirten Nukleolen
nichts zu thun.”
Zimmermann (96), a general critical summary upon the
vegetable nucleolus, with consideration of a part of the previous
literature. Nucleoli are almost always present in the cells of
the higher plants, and are of wide occurrence also in the lower
forms ; double staining serves to differentiate them from the
chromatin. There are usually from one to three to a nucleus,
but in the embryo sac of Lzdéwm martagnon there are from
twenty to thirty. In Chara the older nuclei show the nucleolar
substance in the form of very numerous, irregular fragments.
The distinction of “ Hauptnucleolus” and “ Nebennucleolus ”
is not tenable, since the latter may be possibly chromatin
globules. ‘Mit dem Chromatingeriist scheinen die Nukleolen
396 MONTGOMERY. [VoL. XV.
innerhalb der ruhenden Kerne in keinem Falle in direkter
Verbindung zu stehen.” The space frequently observed around
the nucleolus is probably not an artefact. Its substance is
probably homogeneous; ‘als die alleinigen mit Sicherheit
nachgewiesenen Einschliisse derselben konnen Vakuolen ange-
fiihrt werden... . Diese Vakuolen sind dem gewohnlichen
Einschluss in Kanadabalsam haufig ganz oder teilweise mit
Luft erfiillt oder stellen luftleere Raume dar. Sie erscheinen
dann bei hoherer Einstellung schwarz, bei niederer etwas rot-
lich, und es diirften wohl die namentlich in der die Kerne
beilaufig behandelnden Litteratur vorliegenden Angaben uber
stark lichtbrechende Einschliisse der Nukleolen zum Teil auf
derartige Bilder zuriickzufiihren sein” (e.g., the “« endonucleoli”
described by Mann). During mitosis nucleolar bodies are
often found in the cytoplasm, and such are probably extruded
nucleolar fragments ; “immerhin muss aber die allgemeine
Giltigkeit des friiher von mir als moglich hingestellten Satzes
omnis nucleolus e nucleolo nach den neueren Untersuchungen
als nicht sehr wahrscheinlich angesehen werden.” In the Pol-
lenmutterzellen of Lelia martagnon the nucleoli “ zerfallen
. in sehr zahlreiche kleine Kugeln, die... im Aster-
stadium ungefahr gleichmassig iiber den gesammten Zellinhalt
zerstreut sind.”” He made similar observations also on Aya-
cinthus candicans, Fritillaria tmperialis, young sporangia of
Equisetum and Psilotum, cells of the root apex of Vicza, and
stem apex of Phaseolus and Psilotum. There is also an extru-
sion of nucleolar substance in Chava, but it is doubtful whether
this process occurs in other low forms. This extruded sub-
stance may in some cases, but perhaps not as a rule, return
into the daughter-nuclei. That in mitosis the nucleolar sub-
stance may be incorporated into the chromosomes, “sei noch
erwahnt, dass ich neuerdings an den Kernteilungsfiguren des
Embryosack-Wandbelags von Lilium martagnon nach der
Fixierung mit Chromsaure und Platinchlorid und Farbung mit
Fuchsin und Jodgriin in den Endstadien des Spirems beobachten
konnte, dass einzelne rote Kugeln, die ausserdem auch in
grosser Zahl in der Umgebung der betreffenden Kerne zu
beobachten waren, den violettgefarbten Chromosomen teils
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 397
seitlich ansassen, teils auch ganz von denselben aufgenommen
waren, so dass sie ... kleine Auftreibungen an denselben
bildeten.” It is doubtful whether the nucleoli have any genetic
connection with either the centrosome or the nuclear membrane.
In the synapsis (Moore, '95) of the nucleus the nucleolus
becomes flattened against the nuclear membrane in most
Angiospermia, having thus on section a sickle shape (‘“ Sichel-
stadium’’) ; and the coincidence of this form of the nucleolus
with the synaptic stage “macht es jedenfalls sehr wahrschein-
lich, dass die im Sichelstadium eintretenden Metamorphosen
den Nukleolus eine gewisse Bedeutung besitzen.”’
Debski ('97), Chava: the space surrounding the large nucleo-
lus is caused by shrinkage of the latter, due to the fixing fluids,
and is not present in life. In the nucleolus are numerous
vacuoles which may become confluent. Within the cytoplasm
occur extranuclear nucleoli, which stain like the others. In
the mitotic prophase the nucleolus usually divides into two,
and the latter either gradually diminish in size and finally dis-
appear or else they persist for a while after the disappearance
of the nuclear membrane. Then the extranuclear nucleoli
collect at the poles of the spindle and “ bewegen sich wahrend
der Metakinese von beiden Seiten her gegen den Ort der
spateren Zellplattenbildung und verschmelzen dabei nicht
selten wahrend des Diasterstadiums miteinander zu unregel-
massigen Kugeln, Klumpen und Faden . . . die nucleolenar-
tigen Korper sind spater, nach der Bildung der Zellplatte und
der Membran, nicht mehr dort zu sehen; es finden sich alsdann
nur noch wenige durch das ganze Plasma der Zelle zerstreut,
oder sie fehlen, besonders in den 4lteren Zellen ganzlich.
Einige, wahrscheinlich solche, welche wahrend des Diasters
nicht in die Zellplattenebene geriickt sind, finden sich wahrend
des Dispirems in der Nahe der Tochterkerne ein; spater sind
sie zwischen den Faden des Kerngeriistes zu sehen ; in spateren
Stadien findet man an ihrer Stelle einige kleine Nucleolen,
deren Zahl immer mehr beschrankt wird, so dass sich schliess-
lich gewohnlich in jedem Kern ein einziger grosser Nucleolus
befindet.”
Fairchild (97), Bastdiobolus: ‘Das Verschwinden des
398 MONTGOMERY. [VoL. XV.
Kernkorperchens . . . spricht entschieden fiir Strasburgers
Annahme, dass es zur Bildung der Spindelfasern benutzt
werde.”
Harper (97), ascus of Evysiphe: the nucleolus and the cen-
trosphere stain in the same way, and ‘die achromatischen
Fasern, aus welchen diese intranucleadren Strahlenkegel gebildet
werden, entstehen wahrscheinlich grésstentheils auf Kosten
der Kernkorperchensubstanz, die zu dieser Zeit regelmassig
verschwindet.”
Huie ('97), cells of Drosera: the nucleoli (« nucleolar chromo-
somes’’) are spherical and usually central; ‘“endonucleoli”
are enclosed spaces, not granules. During the process of food
assimilation by the nucleus the nucleolus becomes smaller, and
its vacuoles less apparent.
Lidforss ('97) gives a thorough review of the “ Sichelstadium ”
(Strasburger’s “ Sekretkérperchen’’) of the nucleolus in plant
cells, as also the results of observations of his own on the
embryo sac. Zz/ipa: at first there are several small nucleoli
within the nuclear cavity, which later by their fusion produce
a large one which becomes flattened against the nuclear mem-
brane (the process is essentially the same in Fritillaria, Anthe-
vicum, and Lilium). Gagea: the nucleolar changes are as in
the preceding forms, except that when the nucleolus reaches
the periphery it remains spherical; this is also the case in
Ornithogalum. Ocnothera: in the youngest cells there is one
central nucleolus ; subsequently this flattens against the nuclear
membrane, but finally wanders back to the center and becomes
spherical. He concludes that in the angiosperms the sickle
stage of the nucleolus is a normal phenomenon, as is also its
excentric position. In male and female germ cells these meta-
morphoses occur at corresponding stages, namely, when the
reduction of the chromatin takes place; “indessen bleiben
vorlaufig alle Speculationen tiber die Bedeutung des Sichel-
stadiums von problematischen Werth. . . .”
Mottier (97), cells of Podophyllum and Lilium: in mitosis,
at the time of disappearance of the nuclear membrane, the
nucleolus breaks into fragments of various size. “ Bei der
Anlage der vielpoligen Spindel nun treten im Cytoplasma
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 399
kleinere, dem Nucleolus ahnlich tingirte Korper auf. ... Es
unterliegt keinem Zweifel, dass dieses die zerfallene Kernk6r-
perchensubstanz darstellt . . . nachdem der Tochterkern mit
einer Wandung versehen wurde und in ihm Kernkorperchen
zum Vorschein kamen, sind oft noch extranucleare Nucleolen in
dem Cytoplasma zu sehen. Dasselbe gilt fiir die zweite
Theilung. Ob die in dem Tochterkern zum Vorschein kom-
menden Kernkorperchen aus den im Cytoplasma liegenden
Korperchen entstehen, lasst sich nicht feststellen. Hingegen
ware hier hervorzuheben, dass die im Kern wieder entstehenden
Kernk6rperchen stets in Contakt mit den Kernfaden sich befin-
den . . . meine Ansicht geht aber dahin, dass in den Kern-
korperchen ein Kraftvorrath gegeben ist, welcher der Zelle
nach Bedarf zur Verfiigung steht.”
Pennington (97), cells of Spzvogyra treated with .1478%
palladious chloride : “ The nucleolus showed a dark bounding
layer of double contour. ... The dark layer is undoubtedly
a true membrane dividing the nucleolus from the nucleus.”
Strasburger ('97) reiterates his view ('95) that in plant
mitoses the achromatic spindle is formed from nucleolar sub-
stance, and that also the “Zellplatte”’ and ‘“ Centralspindel-
korperchen”’ of animal cells must be of nucleolar origin.
Swingle (97), algae (Sphacelariaceae) : the vacuolization of
the nucleoli occurs simultaneously with the separation of the
two centrosomes, and probably at the same time that the differ-
entiation of the chromosomes occurs. Though ‘die schnelle
und vollstandige Auflésung der iibrigen Substanz des stark
vacuolisirten Kernk6rperchens findet statt, wenn die Spindel-
fasern an den Polen einzutreten beginnen,”’ there yet seems to
be no direct proof that these fibers have their origin in nucleolar
substance. ‘‘ Kénnte er [Nucleolus] nicht eher einen speciellen
Vorrath organischer Nahrung zur ETRE des LSE
wahrend der Karyokinese vorstellen ?’
C. SyNonyMS OF THE TERM NUCLEOLUS.
Since there are quite a large number of synonyms of the
nucleolus, they may for convenience’ sake be classified together
400 MONTGOMERY. [VoL. XV.
at this place. Certain of the following terms, however, apply
not to the true nucleoli but to the Cavyosomata.
German writers. —Nucleolus (Valentin) Keimfleck, Keimkern,
macula germinativa (Wagner) ; Kernkérper (chen) (Schwann, Val-
entin); Keimkorper (chen); Wagner’scher Fleck ; Binnenkorper
(Rhumbler); Hauptnucleolus, Nebennucleolus (Flemming) ;
Metanucleolus (Hacker) ; Plasmosoma (Ogata) ; Formations-
nucleolus (Marshall) ; Kernfleck, Nucleolide, Morulit (Frenzel) ;
Nucleolo-Centrosoma (Keuten) ; Mittelkérperchen, Eunucleolus
(Rosen); Nucleolkérperchen (Lonnberg); Stammnucleolus,
Nebenkiigelchen (Auerbach); Hauptkeimfleck, Nebenkeimfleck
(Leydig) ; Chromatin-Nucleolus, Paranucleolus (R. Hertwig).
English and American writers. — Wagnerian vesicle, ento-
blast (Agassiz) ; pronucleolus (Mark); nucleole, germinal spot,
germinal dot, principal nucleolus, accessory nucleolus, proto-
macrosome (Greenwood).
French writers. —Nucléole, tache germinative ; pseudonucléole
(Van Beneden); tache de Wagner, nucléole plasmatique, n.
mixte, n. nucléinien, nucléole-noyau (Carnoy) ; nucléole adventif
(Roule) ; corps nucléolaire, nucléolite (A. Schneider) ; nucléole
primitif et secondaire (Carnoy and Lebrun) ; corpuscule ger-
minatif (Van Beneden).
Italian writers. — Macchia germinativa, macchia germinativa
principale, m. g. laterale, m. g. accessoria.
Synonyms of the nucleolinus.—Nucleololus, Nucleollolus
(Frenzel) ; Schrén’scher Korn, Valentinian vesicle, entostho-
blast (Agassiz) ; Centrosoma (Lavdowsky); nucleolo-nucleus,
endonucleolus (Macfarlane) ; Nucleolinus, Keimpunkt, punctum
germinativum (Haeckel).
III. OBSERVATIONS.
A. MeEtuHops oF Stupy.
The following observations have been made upon material
collected, fixed, stained, and sectioned by myself, with the
exception of the preparations of the ova of Rodalia, which
were kindly loaned to me by Dr. E. G. Conklin. In no case
were observations made upon the living tissue ; however, but
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 401
little could be gained from a study of the living cells, in regard
to the minute structures with which we are chiefly engaged.
With only few exceptions (Rodalia and the two gregarines
examined) no cells were studied which had not been preserved
with at least three fixing reagents, and in some cases at least
half a dozen different fixatives were used. The preserving
reagents employed were the following: saturated solutions of
corrosive sublimate in distilled water (this being the only fluid
used hot), sat. sol. of the same in 50% or 35% alcohol, Flem-
ming’s stronger fluid (chromo-aceto-osmic acid), Hermann’s
fluid (platinum chloride, acetic acid, osmic acid), sat. sol. of
picric acid in 50% alcohol, Perenyi’s fluid (chromo-nitric acid),
2% aqueous sol. of chromic acid, absolute alcohol, picro-nitro-
osmic acid. Those reagents which gave the best general results
were the fluids of Flemming and Hermann, and the alcoholic
solution of corrosive sublimate ; though the particular reagent
demanded depends both upon the object of study, as well as
upon the method of staining which is to follow. It is hardly
necessary to state that a structure found after the use of a
given fluid, but not apparent on material treated in a different
manner, was either regarded as an artefact, or doubts were
expressed as to its naturalness ; that is, only when a structure
was found to present itself to the eye in more or less the same
manner, after various methods of preservation had been
employed, have I regarded it as a natural appearance and not
as a result of the fixatives used. Thin serial sections were cut
of objects imbedded in paraffin, in the usual way. All staining
done was upon the sections on the slide, and the stains employed
were as follows : Ehrlich’s or Delafield’s haematoxylin followed
by eosin (sat. sol. in distilled water), nigrosine (a sat. sol. in water
diluted by six vols. water), sat. sol. of acid fuchsine in 50% alco-
hol, the triple stain of Ehrlich-Biondi-Heidenhain (as prepared
by Griibler, Leipzig), Flemming’s triple stain (safranin, gentian
violet, and orange G.), Lyons blue (sat. sol. in 50% alcohol),
gentian violet (sat. aqueous sol.), methylen blue (sat. aq. sol.),
brasilin (sat. sols. in water and in 35% alcohol), Mayer’s acid
carmine, cochineal (sat. sol. in 70% alcohol) ; while Grenacher’s
borax carmine and alum carmine, Heidenhain’s iron haematoxy-
402 MONTGOMERY. [VoL. XV.
lin, indigo-borax carmine (Norris and Shakespere), and certain
others were tried, but proved unsatisfactory. With the excep-
tion of the three triple stains mentioned, the others were used
in various combinations as double stains ; worthy of recommen-
dation are (with especial regard to the differentiation of the nucle-
olus) Delafield’s, or better, Ehrlich’s haematoxylin followed by
eosin ; acid carmine followed by nigrosine ; methylen blue fol-
lowed by brasilin. Other combinations were also used, but it is
not necessary to mention these here, nor to speak of the dura-
tion of the staining baths, since in the explanation of the figures
these data are given for each case separately.
For the study of the finer structural details, the 7,th homo-
geneous immersion lens of Zeiss was used, in combination with
oculars 2 and 4. I would emphasize the fact that the drawings
from the preparations were made gradually, as I proceeded in
the study of each particular cell, and were not postponed until
the end of the particular investigation, so that almost all were
made before I had arrived at any views upon the nature of the
nucleolus ; and I have pursued this method in order to elimi-
nate from the figures as much as possible of the subjective ele-
ment. In other words, I have made as close copies as possible
of the preparations, drawing every cell or structure present-
ing some appearance with which I had not as yet become
acquainted, or rather the significance of which I had not
learned, and then from the figures so made I have endeavored to
learn the nature of the phenomena there presented, at the
same time recurring to the preparations themselves. This
method of study is the one employed by many investigators,
though it can scarcely be termed the one most in vogue. The
colors of the original figures have on the whole been most excel-
lently reproduced by the lithographs of Werner and Winter.
B. PROTOZOA.
1. Gregarine from Lineus gesserensis (O. F. Mil).
(Plate 21, Figs. 1-19.)
(Description of the animal. — The largest individuals are just
visible to the naked eye, and are of a whitish color. No
synzigia were observed among the thirty individuals exam-
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 403
ined. Form: elongate, slightly larger at one end than the
other, the thinner end sometimes flattened, slightly curved or
sickle-shaped ; the greatest diameter is found in the region of
the nucleus, which is situated nearer to the larger than to the
smaller end ; both ends of the animal are rounded. In one
individual (Fig. 2) the surface of the body was slightly furrowed
in a spiral direction. Nucleus large, with a very thick mem-
brane, and seldom oval, usually irregular in outline. Ina single
case (Fig. 1) two nuclei were present in one gregarine (the
youngest individual seen), the two nuclei were of unequal size,
though each contained a single nucleolus. Kdolliker (49) has
described a gregarine with two nuclei ; Iam unacquainted with
any other cases. Sporocysts were not observed ; but in one
case the cytoplasm was quite densely filled with minute spher-
ical and oval bodies, which stained lightly with eosin, and in
each occurred a small granule (this staining with haematoxy-
lin) ; in the same individual a normal nucleus was also present
(Fig. 4). These small bodies cannot be other than spores, even
though they occur in the endoplasm of a gregarine in which
a nucleus occurred at the same time; this observation stands in
no accord with what has thus far been described of the sporu-
lation among gregarines, and I am thoroughly at a loss to
explain the phenomenon. These gregarines occurred only in
the posterior intestine of Lzweus, but were not present in all
the individuals of this nemertean sectioned. The absence of
synzigia, the transverse furrows of the body, and the oval-shaped
spores would relegate this form to the neighborhood of the
genus Gonxospora of Schneider.)
In the smallest nuclei found (the size of the nucleus stands
in some degree in proportion to that of the animal) only one
nucleolus was present (Figs. 3 and 5) ; in all the larger nuclei
their number varied from two to four, though since four nucleoli
were found in only two cases, two or three nucleoli may be
regarded as the usual number in the larger individuals. As
an inspection of Figs. 3-19 shows, the comparative size of
the nucleoli within the same nucleus is very variable, and the
nucleoli of one nucleus are always of unequal size. When only
two nucleoli occur, one is about one-half or three-quarters the
404 MONTGOMERY. (VoL. XV.
size of the other; but when three nucleoli are present, either
(1) one is particularly large, and the other two small; or (2) two
are large, and the third is much smaller than either ; or (3) all
three are Jarge, the smallest being about one-half the size of the
largest. In the two cases of nuclei with four nucleoli apiece, in
the one there were two larger and two smaller nucleoli, in the
other one large and three small ones.
The nucleoli vary from a spherical to an oval shape. In the
smallest usually no vacuoles (z. Vac.) are to be seen, but such
vacuoles are always to be found in the larger nucleoli. In the
largest there is usually a large excentric vacuole, while small
ones may or may not be present in other portions of the nucle-
olus. In nucleoli of medium size it is most usual to find a
number of small vacuoles. These vacuoles have already been
noticed in numerous other gregarines, but I would call especial
attention to a remarkable polarity of the nucleolus with regard
to their position. In all those nucleoli in which vacuoles
occurred, with the exception of not more than five or six, the
single large vacuole, or the group of smaller ones, was situated
at that pole of the nucleolus nearest the nuclear membrane
(Figs. 7-9, 16, 17-19). There are almost no exceptions to
this phenomenon in the smaller nucleoli, those, namely, in which
only a single small vacuole or a few small ones are present.
Accordingly, it would seem to be the rule that the vacuoles
first appear in that portion of the nucleolus which approaches
nearest to the nuclear membrane. The number and size of
these vacuoles increase with the size of the nucleolus ; or, as
is more usually the case, as the nucleolus increases in size they
gradually fuse together to form a single large vacuole, which
may occupy the greater part of the nucleolus (Fig. 15). Thus
the vacuoles first arise at one point in the nucleolus, so that
here one can speak of a polarity of the nucleolus ; but as the
vacuoles increase in number and commence to fuse together
the fluid substance of them begins to diffuse more widely
throughout the nucleolus, so that evidences of this primitive
polarity gradually become obliterated.
The ground substance of the nucleoli is very finely granular,
and stains deeply red with eosin, and brownish red with the
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 405
Ehrlich-Biondi stain. The vacuoles are filled with a structure-
less fluid, which stains but lightly. But in four nuclei, the
sections of which were stained in aqueous solution of methylen
blue followed by brasilin, a differential stain of the ground sub-
stance was acquired: that pole of the nucleolus which con-
tained vacuoles was stained a bluish green (methylen blue), the
opposite pole, where no vacuoles could be seen, being of a light
pinkish color (brasilin), the vacuoles themselves appearing as
clear unstained spaces (Figs. 17-19). In one nucleus, in which
two minute nucleoli were present, the one without, the other
with, a single small vacuole, both nucleoli stained a bluish
green throughout (Fig. 18). Further, in an unstained nucleus
fixed with Flemming’s fluid a somewhat similar differentiation
was visible in the two larger nucleoli (neither of which con-
tained vacuoles), the pole of each nucleolus nearest the nuclear
membrane being of a deeper color than the opposite pole (Fig.
11). This differentiation produced by staining would show that
the ground substance of the smallest nucleoli is homogeneous,
but that in the larger ones a chemical change takes place in it,
whereby that portion of the substance opposite the pole where
the vacuoles first appear differentiates itself chemically from
that portion of the ground substance lying at the latter pole.
Unfortunately I had too little material to carry further the
study of this differentiation.
In the nucleus is a faintly staining nuclear sap, in which
irregular granules of various size are massed together espe-
cially near the center of the nucleus ; they do not come into
contact with the nucleoli, usually leaving a clear space around
each of the nucleoli (Figs. 7, 8, 11, 14, 17-19). These do not
stain with haematoxylin or with methylen green, but stain
red with eosin and brownish red with the Ehrlich-Biondi mix-
ture, in their staining differing little from the substance of the
nucleoli. With the methylen-blue-brasilin stain mentioned
above they stain pink, a little more deeply than does the inner
pole of each of the larger nucleoli (Figs. 17-19). Whether they
represent physiologically chromatin, or whether they are
masses of (perhaps nutritive) substance taken into the nucleus
from the cytoplasm, which might be chemically and genetically
406 MONTGOMERY. (VoL. XV.
akin to part of the substance of the nucleoli, I am unable
to decide. I am also unable to determine from the prepara-
tions at hand whether the nucleoli themselves are partially
composed of chromatin ; but the usual diagnostic stains for
chromatin do not show the presence of this substance within
the nucleus.?
To revert again to the polarity of the nucleoli. The fact
that the vacuoles first arise in that portion of the nucleolus
nearest the nuclear membrane would seem to prove that the
substance of these vacuoles is extranuclear in origin, or else is
secreted in the peripheral portion of the nucleus. But since it
would be obscure how the peripheral portion of the nucleus
should secrete a substance, and the central portion should not,
I incline to the former explanation, namely, that the substance
of the vacuoles is first produced in the cytoplasm, and then this
substance penetrating through the nuclear membrane, it, or a
part of it, arrives at that pole of the nucleolus nearest the
nuclear membrane, and then is taken into the nucleolus at
this pole. The size of the vacuoles stands in a more or less
direct ratio to the size of the nucleolus itself; at the same time
the ground substance of the nucleolus also increases in amount,
though apparently not as rapidly as the amount of the vacuolar
fluid.
2. Gregarine from Carinella annulata.
(Plate 21, Figs. 20-35.)
(Description of the animal.—Monocystid gregarines occurring
in the body cavity of this nemertean. No synzigia observed.
Form : elongate, though not attenuate, the end in which the
nucleus lies being broader and terminally more obtuse than the
opposite end (Figs. 20 and 21). The longitudinal axis is never
perfectly straight, and the cuticula shows no transverse fur-
rows. The single nucleus is usually spherical or oval, rarely
lobular in outline. In the entosarc of many individuals occur
numerous minute, refractive granules. Neither cysts nor spores
having been observed, I was unable to determine the genus of
1 However, the chromatin here might exist in the state in which it is found in
the growth period of ovocytes, namely, commingled with plastin.
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 407
this form. Only two individuals of Carznella were examined
(both from Bergen, Norway) ; in the one all the gregarines were
large, in the other of a smaller size.)
The nucleoli are nearly always more numerous than in the
preceding species of gregarine, the number varying from four to
about twenty-six, in those stages found (Figs. 22-35). In the
larger nuclei they are usually more numerous than in the smaller
ones, but exceptions to this rule are quite frequent. In the
same nucleus some are nearly or quite spherical, others very
irregularly lobular in outline. Their size within a given nucleus
is also very variable, though as a rule they are unequal in their
dimensions. In the larger nuclei the nucleoli are larger (or at
least some of them are) than in the smaller nuclei. In a given
nucleus there may be either (1) from two to four larger nucleoli
and a number of smaller ones ; or (2) a single large nucleolus
and several much smaller ones. In the smaller nuclei the
nucleoli are more equal in size than in the larger ones. The
largest nucleoli in a nucleus are as a rule of oval or spherical
form, with regular contour (an exception is seen in Fig. 26) ;
the irregularly lobular nucleoli (Figs. 23, 25, 27, 28, 33) are
usually of medium or small size. There is no apparent regu-
larity with regard to their distribution in the nucleus. None of
the nucleoli appear to have limiting membranes.
All these gregarines were fixed with alcoholic solution of
corrosive sublimate. With the double stain, haematoxylin and
eosin, the larger nucleoli were stained with a deep blackish
red, the smaller ones either of the same color or a clearer red ;
all became stained so intensely by this method that the vacu-
oles in them were greatly obscured (Figs. 27 and 28).
The Ehrlich-Biondi method produces a yellowish brown or
reddish stain of the nucleoli, differences of stain being observ-
able in the different nucleoli of the same nucleus (Figs. 26,
31-35). This staining method brings out very clearly the
vacuoles in the homogeneous (?) ground substance of the
nucleolus ; the structureless substance of these vacuoles
stains less intensely than the enveloping substance. Vacu-
oles are absent in the smallest nucleoli, as well as in those of
irregular form ; in the larger ones they are almost invariably
408 MONTGOMERY. [VoL. XV.
present, though variable in size and number. They do not
regularly arise at one particular part of the nucleolus, as we
found to be the case in the preceding species. Further, there
is rarely in this species a single large excentric vacuole ; but
as the figures show, usually a number are present, either
arranged in a circular row near the periphery, or in a row
around a larger central vacuole, or grouped together at one
point in the nucleolus. There can be no doubt that the larger
vacuoles are produced by the fusion of smaller ones, since two
or three smaller ones are frequently found in close contact
with each other.
The double stain, haematoxylin and alum carmine, gives
different results from the preceding stains, in that by it not
only the different nucleoli within a nucleus become colored
differently, but also in some cases different stains of the
different portions of the same nucleolus are attained (Figs.
22-25). It is only the larger nucleoli, those with regular con-
tours, which become differentially stained in this manner. In
such a large nucleolus a portion of its substance stains a deep
blue (haematoxylin), another portion or portions purplish or
reddish (alum carmine) ; the part stained blue is usually central
in position, and encircling it is a zone of red-stained substance.
In one case (Fig. 22) the two opposite poles of the nucleolus
were reddish, the intermediate part being a deep blue. The
medium-sized, irregular nucleoli always stain blue throughout,
the smaller ones usually red, but sometimes blue. This stain,
accordingly, shows that in this gregarine some of the larger
nucleoli are composed of two different substances similarly as
we had found two substances in the preceding species, though
there by using the methylen-blue-brasilin stain.
With all three staining methods employed, a mass of irregular
granules is present in each nucleus, which stain less intensely
than the nucleoli. In the smallest nuclei (Figs. 22-25) these
granules are more or less regularly distributed through the
nucleus, but in the larger ones (Figs. 28, 31-35) they com-
pose a dense mass around the nucleoli or around the largest
nucleolus, while the peripheral portion of the nucleus remains
nearly free of them. Delicate, faintly stained fibers transverse
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 409
this peripheral part of the nucleus, which may be radially dis-
posed or else form a loose network. The size of the granules,
their abundance and staining intensity vary in different nuclei
of the same size, and there is no sharp distinction between the
smallest nucleoli and the largest of these granules. In this
species, as in the preceding, I was unable to detect any sub-
stance which stained like chromatin.
I have been unable to determine the origin and ultimate fate
of these nucleoli, owing to lack of material ; but a few justifi-
able conclusions may be drawn from the facts at hand. Thus
the number and size of the nuclecli stand, as a rule, in a direct
ratio to the size of thenucleus. Further, those irregularly lobular
nucleoli described above probably represent amoeboid changes
of the nucleolus, such as have been seen in life by previous
investigators, though it is strange that these nucleoli differ from
all others in consisting of a single substance and in containing
no vacuoles. Lastly, the number and size of the vacuoles
increase, as a rule, with the size of the nucleus.
It is worthy of mention that usually there are a larger num-
ber of very small nucleoli in the larger nuclei than there are
in the smaller nuclei, although the largest nucleoli of the
former are much larger than the largest nucleoli of the latter
nuclei. We must conclude, then, that though the size of the
nucleoli increases as a rule with that of the nucleus, new
nucleoli are also being formed as the nucleus grows larger.
Now some of these new small nucleoli found in the largest
nuclei have undoubtedly been produced by division from some
of the larger ones : thus I have frequently observed irregular
(amoeboid) nucleoli with oval prolongations, or with small
nucleoli closely apposed to their surfaces, and it probably is
correct to conclude that such small nucleoli are in process of
division from the larger ones (Figs. 23, 25, 27, 28, 33).
Whether all the small nucleoli of the larger nuclei have
had such a formation is difficult to determine, since in some
of the largest nuclei most of the smallest nucleoli may be
peripheral in position, close to the nuclear membrane, and
far removed from the larger nucleoli, so that it might seem
that the substance of these was extranuclear in origin. The
410 MONTGOMERY. (VOL. XV.
mass of irregular granules within the nucleus appears to stand
in some relation to the growth of the nucleoli, at least there is
a relatively greater amount of this substance in the larger
nuclei ; it envelops the largest nucleoli and imbibes the same
stains, though more faintly, with which the nucleoli become
stained. Now as the gregarine grows, at the same time both
nucleus and the total mass of nucleolar substance increase in
size ; but the nucleus cannot grow without the addition of a
substance or substances to it, which have been derived from
without. Accordingly, I suppose that the substance of these
granules has an extranuclear origin, a substance, z.e., which, hav-
ing penetrated the nucleus from the cytoplasm, undergoes a
chemical change in the nucleus and there becomes precipitated
in the form of granules, for no such substance occurs in granu-
lar form in the cytoplasm. The growth of the nucleoli might
then be explained on the assumption of the intussusception of
this substance by the nucleoli. This explanation is offered
merely as a hypothesis, since I cannot prove its correctness
with the limited material at my disposal. Since no chromatin
was demonstrable in these nuclei, it remains for future workers
to show whether the chromatin is in these stages commingled
with the nucleolar substance, or whether it is represented by
one of the two substances of which some of the nucleoli are
composed ; and if so, whether all, or whether only a certain
number, of the nucleoli are thus partially constituted of
chromatin.}
C. METAZOA.
a. Egg Cells.
1. Montagua pilata (Verr.).
(Plate 22, Figs. 57-63, 65-87.)
In the germinal vesicles of this mollusc two kinds of nucleo-
lar structures occur : the true nucleolus, which is of large size
and almost invariably single ; and certain secondary structures,
1 For observations of other authors on nucleoli in Gregarinida, cf. the reviews
of Minchin (’'93), Van Beneden (’69), Marshall (’92), Frenzel (’93), Koelliker
(49), A. Schneider ('75, ’83), Wolters ('91), Carmoy ('84).
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 411
which appear at only a certain stage of the cell. The true
nucleolus may be considered first, then these other structures,
or “ pseudonucleoli.”’
There is always one true nucleolus to each nucleus, and in
only two cases out of hundreds of ova examined have I seen
two nucleoli (Figs. 57 and 61). The position of the nucleolus
within the nucleus is in most cases excentric, seldom central,
and never apposed to the nuclear membrane; it apparently lies
free in the caryolymph, and is not supported by the chromatin
threads. In the youngest, most immature germinal vesicles
(I have not studied it in the ovogonia) it is apparently wholly
homogeneous, dense, not noticeably refractive, and usually
spherical (Figs. 57-61) ; sometimes, however, it shows an oval
or more elongate form, and in the latter case its long axis
usually coincides with that of the nucleus (Fig. 58) ; it is
never irregular in outline.
The nucleolus always colors differently from the chromatin,
when treated with double stains, as follows :
STAIN. NucLeovus. CHROMATIN.
Ehrlich-Biondi —. . maroon . > . green.
Haematoxylin, eosin . orange red : . blue.
Acid carmine, nigrosine blue . : - eeeted.
Haematoxylin, fuchsine _ purple ‘ : . blue.
Flemming’s stain . . yellow 2 : - violet.
With the increase in size of the nucleus the nucleolus
enlarges, and in such a way that the size of the latter usually
preserves its proportion to that of the former; but as the
figures show, this proportion is quite frequently not preserved.
What may be termed the first stage of this nucleolar growth
consists merely in an increase in the amount of the homogene-
ous substance, and between the largest homogeneous nucleoli
(Fig. 65) and the smallest (Fig. 57) there is no difference
except one of size.
The second period of nucleolar growth is introduced when
vacuoles commence to appear in the substance of the nucleolus
(Fig. 62). Since my observations show that these nucleolar
vacuoles are derived from small fluid globules which first appear
412 MONTGOMERY. [Vor. XV.
in the nuclear sap, these globules may best be treated first.
In the nuclear sap, at a certain stage in the growth period of
the germinal vesicle, small globules of varying size occur ;
there are usually one or two of them in a given nucleus, but
sometimes they are quite numerous (Ww. G/. in Figs. 62, 63,
69-71, 73, 75, 81). When I first noticed these structures I
conjectured that they might represent centrosomes such
as have been found within nuclei at stages previous to mito-
_sis (by Brauer in the spermatocytes of Ascaris); but further
investigation shows that they have no kind of relation to cen-
trosomes, since they vary in number and size, and further they
readily imbibe stains, which centrosomes do not. They have
a close resemblance to the smallest yolk granules found in the
cytoplasm in point of form, size, and manner of staining. How-
ever, sometimes one or two of these bodies may be found in
the nucleus when there is no evidence of yolk in the cytoplasm.
Accordingly, they would seem to consist of a substance very
similar to the young yolk at the time of its first formation.
And since they may arise in the nucleus before yolk spherules
appear in the cytoplasm they are probably not always taken up
by the nucleus from the cytoplasm in the form of globules, but
acquire this spherical form first in the nucleus. In other words,
we may consider that the nucleus assimilates from the cyto-
plasm a thin fluid, similar to, if not identical with, that from
which the yolk spherules themselves are ultimately formed, and
that in the nucleus this substance becomes deposited in the
form of globules, perhaps after having undergone a chemical
change within the nucleus. Further, this substance must be
regarded as having a nutritive value, on account of its similarity
to the substance of the yolk, which certainly is nutritive in
function. In the more mature, larger germinal vesicles (Fig.
78) large yolk globules are usually found, and are wholly simi-
lar to those in the cytoplasm in these stages ; as can be easily
determined, their position within the nucleus is not due to
removal by the knife in sectioning, so that as the nucleus
becomes larger it regularly takes up large yolk globules from
the cytoplasm, and from these probably derives the greater
part of the nourishment necessary for its rapid growth. We
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 413
may conclude, then, that when the nucleus is comparatively
small, and when no yolk or only small yolk globules are pres-
ent in the cytoplasm, the nucleus derives a nutritive substance
from the cytoplasm, which is closely similar to that composing
the youngest yolk globules ; but when the nucleus has grown
large, and the cytoplasm is packed with large yolk globules, it
has the power to take up these larger globules also.!
To return, then, to the second stage of nucleolar differentia-
tion. This stage does not commence when the nucleolus has
attained a certain size, but may commence in some nucleoli
earlier than in others; and again it is not marked by a particular
stage of development of the yolk in the cytoplasm. The fluid
vacuoles probably stand in a genetic relation to the small
nutritive globules found in the nucleus, which have been
just described. That is, these globules of the nucleus pene-
trate into the nucleolus and then constitute the fluid vacuoles
of the latter structure. I have reached this conclusion after
observing that the vacuoles of the nucleolus and the small
nutritive globules within the nucleus always stain in exactly
the same way. This assumption is further strengthened by
the fact that, when the nutritive globules lie in the nuclear
sap at some distance from the nucleolus, they have invariably
a spherical form ; but in those numerous cases where they may
be seen apposed to the outer surface of the nucleolus they
become flattened against the surface of the latter, as if the
nucleolus were (figuratively speaking) a loadstone which
attracts them to itself (Figs. 63, 69, 75). If this origin of
the vacuoles of the nucleolus were not the true one it would
be difficult to explain their mode of genesis, since there appears
to be no other substance within the nucleolus from which they
could be derived, and there is no reason for supposing that the
1 The intensity in the staining of the yolk globules increases with their size, and
the largest stain much more deeply than does the nucleolus. During all the
earlier growth stages the nuclear membrane is retained, and it is seldom, and
then only slightly, irregular in outline ; therefore the yolk cannot be taken up by
the mechanical aid of amoeboid processes of the nucleus, but its substance must
osmotically penetrate the nuclear membrane. And as I mentioned above, it does
not seem probable that the yolk globules retain their shape while penetrating this
membrane, but diffuse through it in the form of an irregular fluid mass, and then
in the nucleus this fluid becomes re-formed into globules.
414 MONTGOMERY. [VoL. XV.
substance of these vacuoles is a differentiation of the nucleolar
ground substance. We may assume, then, that this explana-
tion of the genesis of the nucleolar vacuoles is the correct one,
and now proceed to explain the changes in the nucleolus dur-
ing the successive development of its vacuoles. If we take the
size of the nucleolus as a general criterion (though it is not an
infallible one, since there are considerable individual differences
in different nucleoli (cf Figs. 62, 65, 80)) of the stage of
the nucleolus, the process of assimilation of the nutritive glob-
ules from the nucleus by the nucleolus seems to be in general
as follows : first, one or two globules are taken into the nucleo-
lus, and later when others (apparently a varying number) are
also taken up into it, we reach a stage when the nucleolus
contains a number of fluid vacuoles (the assimilated nutritive
globules) (Figs. 64 and 70). Then these vacuoles commence to
fuse together (Figs. 63, 66, 72), finally by their fusion giving
rise to one large vacuole, which fills about three-quarters of the
space of the nucleolus, and always lies excentrically within the
nucleolus (Figs. 68, 69, 73, 77, 79). The nucleolus has now
attained its greatest dimension and is either perfectly spher-
ical, or more usually ovoid in shape. Its large excentric vacuole
is encircled by a peripheral layer of the primitive homogeneous
ground substance of the nucleolus, which has undergone no
structural or chemical change. This layer of ground sub-
stance becomes necessarily thinner as the vacuole becomes
larger, z.e., as the pressure from within becomes greater. But
since the large vacuole lies peripherally, the peripheral sub-
stance of the nucleolus remains thickened at that point opposite
the vacuole, and this thickened portion of the nucleolar wall
has most frequently the form of a concavo-convex lens (or on
a cross-section, of a half moon), the concave side of which
borders upon the vacuole. This thickened part, as the remain-
ing portion of the peripheral layer of the nucleolus at this stage,
is in every respect identical with the ground substance of the
nucleolus in earlier stages, before vacuoles had made their
appearance in it ; and the total amount of the substance of the
peripheral layer seems to be equal to the amount of the homo-
geneous substance of the nucleolus at the end of the preceding
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 415
stage. Accordingly, in this second period of the nucleolar growth
there appears to be no increase in the amount of the true
nucleolar substance, but merely an increase in the amount of
the vacuolar substance. The thickened portion of the periph-
eral layer of the nucleolus is at first biconvex, but as the large
vacuole grows larger the pressure of the latter causes it to
gradually assume a concavo-convex form (Figs. 84-86). Thus
the shape of the large vacuole is at first concavo-convex, and
later spherical or oval. This thickened portion of the outer
layer of the nucleolus is usually homogeneous in structure, as
is the remainder of the true nucleolar substance which envelops
the vacuole ; but sometimes small vacuoles may occur within
it also (Fig. 71).
Two poles may be distinguished in the nucleolus at this
second stage of its differentiation : (1) the pole at which the
large vacuole lies ; and (2) the pole at which the thickened mass
of the true peripheral substance is situated. From the study
of a large number of nuclei at this period I find that in about
75% of them the second of these poles is directed towards the
nuclear membrane, the first pole towards the center of the
nucleus ; at this stage, as in the preceding, the nucleolus lies
usually excentrically within the nucleus.
The later differentiation of the nucleolus consists, accord-
ingly, in the accumulation in it of fluid vacuoles (their substance
identical with that of the nutritive globules of the nucleus),
but the true nucleolar substance undergoes no change whatever,
as far as can be determined from differential staining. There
is no chemical union of the vacuolar with the true nucleolar
substance, but the fluid vacuoles simply push aside this sub-
stance, so that, after these numerous smaller vacuoles have
united to form a single large vacuole, the true nucleolar sub-
stance remains unchanged as a peripheral layer around this
vacuole. The substance of the vacuoles becomes colored with
the same stains, though always more lightly, as does the true
nucleolar substance, so that we find in this stage a more deeply
staining envelope of substance around a less deeply stained
portion. This difference of staining between these two parts
of the nucleolus is best shown by employing haematoxylin
416 MONTGOMERY. [Vor. XV.
and eosin (Figs. 68 and 69). With the Ehrlich-Biondi method
this difference is not quite so clearly demonstrable. The latter
stain is peculiar and differs from all other stains used by me
for these cells, in that it very often gives to the smaller vacuoles
of the nucleolus the appearance of black, refractive granules ;
but a careful focusing of these supposed granules shows them
without doubt to be vacuoles, their apparent solidarity being
probably due to the refraction of light by the enveloping
nucleolar substance.
The chief result derived from the foregoing observations is
that the nucleolus takes up some or all of those nutritive
globules which lie in the caryolymph, and whose substance had
been probably derived from the cytoplasm. Some of these glob-
ules then become collected within the nucleolus, representing
its fluid vacuoles ; and these globules, increasing in number at
the same time, gradually fuse together and thus give rise to
a single large excentric vacuole, which is enveloped by the
unchanged true nucleolar substance. Since the substance of
these small globules is probably nutritive in function, the
nucleolus in thus collecting some or all of them would appear
to act as a reservoir for nutritive substance, or as a reservoir
for that portion of the nutritive substance accumulated in the
nucleus, for which the nucleus may have no use. Of course
it is not a priori impossible that these globules may represent
waste products of a nutritive substance, so that the nucleolus
might here fulfill the office of an excretory organ. But the
function of these nucleoli can only be decided when the
behavior of the nucleolus during the pole-body mitosis is
known ; I had no ova showing pole-spindle formations.
Finally, the true nucleolus appears not to be bounded by a
special membrane ; after staining with acid carmine and nigro-
sine the nuclear substance appears bluish green and a red
membrane seems to envelop it (Fig. 80), but this appearance is
probably due to the refraction of light, since nothing of the
kind can be found after the use of other staining methods.
We now come to speak of what I have called the “ pseudo-
nucleoli,” but merely in order to distinguish them from the
true nucleolus, and without wishing to express by the use of
No. 2.}] COMPARATIVE CYTOLOGICAL STUDIES. 417
this term any particular significance of these bodies. In eight
individuals of M/ontagua which were sectioned, and which were
of slightly different sizes, though the various growth stages of
the ova were more or less the same in all, in only four were
pseudonucleoli to be seen, and in only one of these four
were they quite abundant, occurring in about 30% of the larger
germinal vesicles. There are never more than from one to
three ina nucleus. They are usually irregularly spherical and
sometimes even angular in form (Ps. z. in Figs. 72-77, 79). The
largest attained about three-quarters the size of the true nucle-
olus (of the same nucleus), though this size was attained by
few, since they are, as a rule, but little larger than the nutritive
globules which are observed in the caryolymph. Each pseudo-
nucleolus consists of a denser, more deeply staining layer
surrounding a less dense, more faintly staining core. The
denser outer layer is homogeneous, somewhat refractive, and
stains in the same manner as the ground substance of the true
nucleolus. In smaller pseudonucleoli this outer portion appears
on cross-section as a deeply staining ring, with regular out-
lines, but in the larger ones small, irregular prominences may
often be seen on its inner surface. The peripheral layer or
ring, further, shows a double contour, but I am unable to deter-
mine whether it is bounded by an outer membrane. It increases
slightly in thickness with the growth of the pseudonucleolus,
and in one case (Fig. 77) it was noticeably thickened at one pole,
which gave to it somewhat the appearance of the ‘out ensemble
of atrue nucleolus. This peripheral layer surrounds a homo-
geneous, non-refractive, probably fluid mass, which either stains
not at all or else only faintly ; when it stains, it is either in the
manner of the caryolymph or of the vacuoles of the true
nucleolus. I have never noticed that the nutritive globules of
the nuclear sap were apposed to these pseudonucleoli. What
their origin is, and what their relation to the true nucleolus, I
do not know. They are never found in contact with a true
nucleolus and so are probably not buds from one. It is curious
that they were frequent in the ova of only one mollusc, and in
the same stages of the eggs of three other individuals were
present in only a few cells, and in four other individuals were
418 MONTGOMERY. [VoL. XV.
present in none of the ova, though here the same stages of
the ova were present as in the first individual. When they
occur it is only in the larger germinal vesicles. They are
apparently structures saz generis, and I have only the sugges-
tion to offer, that they might be characteristic of a particular
generation of egg cells, as their absence in the ova of some of
the individuals of the mollusc would render probable (compare
the observations of Hacker, 98a, where nucleolar differences
were found in the ova of primiparous and multiparous individ-
uals of Cyclops strenuus).
In Fig. 70 is a remarkable case depicted, namely, two small
nuclei lying within a larger germinal vesicle, the former having
apparently been assimilated by the latter.
2. Doto.
(Plate 22, Figs. 64, 68, 69.)
The nucleolar differentiation of these ova is essentially as in
Montagua, so that no detailed description of the process need
be given here. But in the five individuals of Doto which were
sectioned, no traces of pseudonucleoli were seen, and the nutri-
tive globules within the nuclear sap are usually smaller and
much more numerous than in A/ontagua. The yolk globules
also have different shapes in these two genera.!
3. Amphiporus glutinosus (Verr.)
(Plate 24, Figs. 140-158.)
(For descriptions of the connective-tissue elements of the
nemerteans, from which the genital products are derived, cf
my previous paper '96.)
In the nuclei of the connective elements, by a differentia-
tion of which the ova are produced (without any intervening
1 For the observations of other authors on molluscan germinal vesicles, cf. the
reviews of the papers of Wagner ('35, '39), Flemming ('74), O. Hertwig ('78b),
Lonnberg (92), Balbiani (’'65b), Platner ('86), Leydig ('55a, '50), Stauffacher
(93, '97), Stepanoff ('65), Lovén (’49), Mark (81), List ('96), Blochmann ('82),
Trinchese ('80), Heuscher ('93), Hubrecht ('81), Carnoy ('84, '85), Wirén ('92),
Fol (89), Lacaze-Duthiers ('57), Quatrefages (’49).
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 419
mitosis), I could find no nucleoli ; but one or two small minute
nucleoli might nevertheless be present within these nuclei, but
escape detection, owing to their small size and to the compara-
tively great amount of chromatin. These nuclei are usually
elongated and irregular in form (Figs. 144 and 145, C. 7. W.).
The smallest germinal vesicles, which are recognizable as
such by slightly larger dimensions and more regular, spherical
shape, show likewise no recognizable nucleoli.
In what may be termed the first nucleolar stage, the nuclei
have grown still larger, and in them are to be seen from one to
about twelve small nucleoli. These are all peripheral in posi-
tion, being flattened against the inner surface of the nuclear
membrane, which results in their not being spherical, but more
or less flattened, lens-shaped, or hemispherical (Figs. 140 and
141).
Second nucleolar stage. — The peripheral nucleoli commence
to wander towards the center of the nucleus, at the same time
growing larger and increasing in number (Figs. 142-145, 152).
This process goes on until a considerable number of quite large
nucleoli are present, none of which are any longer in contact
with the nuclear membrane. As a rule they are not evenly
distributed throughout the nucleus, but groups of them occur
at different points in the nucleus (Figs. 153, 146-150). This
period of differentiation, then, consists in the grouping of most
or all of the nucleoli at or near the center of the nucleus,
accompanied by their increase in size. There is no ground for
supposing that at this stage they fragment into smaller nucleoli ;
but very frequently groups of two or three nucleoli may be seen
in close contact with one another, and these would represent
states of fusion rather than of division, since they are found to
be flattened at the point of contact, and not attenuated. Thus
the increase in the size of the nucleoli would be due, in part at
least, to fusion of contiguous ones. While some of the nucleoli
have left the periphery of the nucleus, others are at the same
time forming there, which in their turn eventually reach the
center, so that a continual process of formation of nucleoli, and
wandering of those already formed towards the center, takes
place at this stage.
420 MONTGOMERY. [VoL. XV.
Third nucleolar stage. — The nucleoli increase in number, but
gradually become smaller and wander towards the periphery
of the nucleus (Figs. 154 and 155), until they all lie close to
the inner surface of the nuclear membrane. In this stage
they attain their maximum staining intensity, as is well seen
after the use of Heidenhain’s iron haematoxylin, by which
they become colored a greenish blue (Fig. 157), while in the
previous stages they are brownish yellow, unstained by the
haematoxylin.
Fourth nucleolar stage. — Vacuoles of varying size arise in the
nucleoli, and become somewhat irregular (instead of spherical)
in outline (Figs. 156 and 158). In numerous nuclei it may
be noticed that all the nucleoli lie close to the nuclear mem-
brane, except a single one, which is placed nearer the center
and differs from the others in not staining with haematoxylin,
though it usually contains vacuoles ; it may be a nucleolus
which has not developed as fast as the others have (Fig.
156).
All nucleoli in the third and fourth stages are very uniform
in size, and smaller and much more numerous than in the
second ; since there are no facts which permit us to conclude
that new nucleoli are being formed in the last two stages we
must consider that in them a division of the nucleoli must take
place, and this would explain their increase in number and
concomitant decrease in size. The fourth stage would seem to
be characterized by the commencement of a degeneration of
the nucleoli, if the presence of vacuoles and the irregularity
of form may be taken as a criterion of degeneration. Neither
in this species nor in the other nemerteans examined have I
seen stages showing the formation of the pole spindle, so that
I cannot describe the ultimate fate of the nucleoli. But the
observations of those who have studied these divisions seem
to show that they all disappear before the pole spindles are pro-
duced; and accordingly the phenomena characteristic of our
fourth nucleolar period might represent the commencement of
these degenerative processes.
The method of formation of the yolk may next be considered,
since the yolk stands in a certain relation to the genesis of the
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 421
nucleoli. The cytoplasm, when the yolk first arises in it,
stains with haematoxylin (with the double stain of this and
eosin); this blue stain of the cytoplasm I have noticed to be
characteristic for the cytoplasm of many immature ova, while
the cytoplasm of somatic cells usually stains with eosin. The
yolk first appears in the form of large yolk balls (Figs. 144 and
145, Yk. Bl), as they may be termed; the number of these
balls varies in cells of the same size, as well as in those of
different dimensions, and they appear to be produced succes-
sively in a cell, until at the end of the third nucleolar stage they
all have disappeared, having given place to the mature yolk
spherules. They arise in the cytoplasm at no fixed point,
though usually at some distance from the nucleus ; it is hardly
necessary to state that they stand in no genetic relation to the
nucleus, either in this or in the other nemerteans studied.
The yolk balls are at first dense and homogeneous, and stain
intensely with eosin ; the size that they may attain while still
homogeneous is very variable. Subsequently they become
vacuolated, even sometimes granular, and different portions of
the same ball may stain differently, which shows that both a
chemical and a physical change takes place in their substance.
Finally, they fragment into unequal sized granules, which stain
less deeply, and then these latter split up further, until the
ultimate yolk spherules (Y%. G/.) are produced. In the largest
ovarial eggs all the yolk balls have disappeared (they linger
longest at the periphery of the cell), the cytoplasm being
densely filled with the yolk spherules. In some cases yolk
balls lie in the cavity of the gonad (Fig. 155), and these are
probably derived from degenerated ova.
The following facts show, I think, that the nucleoli stand in
a genetic connection with the yolk substance. The nucleoli
stain in the same way and have in other respects the same
appearance as the smaller fragments of the yolk balls and as
the mature yolk spherules (Figs. 144-146). Fragments of
yolk balls occur frequently in close contact with the outer sur-
face of the nuclear membrane. Now since the nucleoli first
appear in contact with the inner surface of this membrane, the
conclusion is plausible that the nucleoli represent portions of
422 MONTGOMERY. [Vou. XV.
a yolk substance, either of the yolk-ball fragments or a sub-
stance equivalent to that out of which the latter are differen-
tiated, and this substance, then penetrating osmotically the
nuclear membrane, becomes deposited or precipitated in the
nucleus in the form of spherical globules, which are the nucleoli.
From this yolk substance taken into the nucleus the chroma-
tin, linin, and nuclear sap might derive the nourishment neces-
sary for their growth, and those nucleoli which remain through
the fourth nucleolar stage might represent either a reserve supply
of this nourishment, or chemically changed portions of it, from
which all nutritive substances have been extracted ; the latter
view would seem substantiated by the fact that the nucleoli
stain somewhat differently in the third and fourth stages.
The nuclear membrane is present during all these stages.
The nucleus is always regular in outline, usually oval, except
during the third stage, when it may become slightly irregular,
though it never becomes noticeably lobose or amoeboid.
In the first nucleolar stage (Figs. 140 and 141) the chromatin
appears as a network of delicate fibers, which stain with haema-
toxylin. Towards the end of the second stage (Figs. 146-150)
it assumes the form of irregular masses, and the fibers become
less numerous. In the largest ovarial nuclei (Figs. 154 and
157) it is finely distributed throughout the nucleus in the form
of minute microsomes ; traces of fibers may be found only at
the periphery of the nucleus, though I have not determined
whether these are fibers now for the first time forming, as is
the case in the other nemerteans. The nucleoli are never
suspended by the chromatin fibers.
This species is characterized by the formation of a mem-
branous structure in the cytoplasm, during the second and
third nucleolar stages, which is present in none of the other
nemerteans. This is a membrane within the cytoplasm,
separated from the nucleus, as well as from the cell membrane
by cytoplasm; it lies close to the nucleus (Figs. 146 and 155,
fv. Mob.). Wt is thicker than the nuclear membrane, though
not so dense, and differs in no wise structurally from the
cytoplasm, except in its greater density, the cytoplasmic gran-
ules in it lying closer together (these granules appear to be
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 423
the nodal points of a “‘ Wabenwerk”’ in the sense of Biitschli).
This intracellular membrane is not open at any point, and
a longitudinal section of it shows it to be not spherical but
oval in outline, the apices of the oval being furthest removed
from the nucleus. It is present only in the second stage of the
nucleolus, and between it and the nucleus no yolk balls occur.
I have never seen such a structure in any other egg cells except
in the ova of Gryllus abbreviatus ; a similar structure was found
by van Bambeke ('83, eggs of Leuczscus, Lota), Shafer ('g0, egg
of Lepus), and Gerould (96, Caudzna egg).
4. Tetrastemma catenulatum (Verr.) Montg.
(Plate 23, Figs. 103-133; Plate 24, Figs. 137-139.)
The formation of the yolk may be spoken of first, then the
nucleoli proper, and afterwards certain large nuclear structures
which may or may not represent nucleoli of another kind.
The yolk first appears in the form of one or two yolk balls
(Yk. Bl. Figs. 107, 108, 112, 114-116) in the cytoplasm ;
the larger ones are regularly oval as a rule, and the smaller
ones spherical. A number of these yolk balls are produced
successively in each cell, and by their fragmentation the ulti-
mate yoke spherules (Y. GZ) are evolved. Each such ball is at
first smaller than the nucleus of the cell in which it occurs, but
gradually increases in size, though the maximum size which it
may attain is not a fixed quantity, but is quite variable. As it
increases in size it also gradually becomes more deeply stained,
attaining its most intense staining when it has attained the
limit of size. The substance of these balls is dense, finely
granular, not brittle, somewhat refractive ; in the youngest
stages of their formation they often appear nearly homogeneous.
About the time a ball has reached its maximum size it com-
mences to change both structurally and chemically, vacuoles
appear in it, it begins to stain less intensely, and becomes
irregular in outline. Thus it becomes either coarsely granular,
or else unstaining vacuoles appear scattered through it, and
with eosin stains no longer a deep red, but a light red or even
yellowish. Next it breaks into a number of pieces, whereby
424 MONTGOMERY. [VoL. XV.
the primitive yolk ball may break either into two fragments
(which are usually unequal in dimensions), each of which then
fragments further, or it breaks at once into a considerable
number of larger granules. The final stage in this process of
division shows the daughter yoke balls fragmenting to form
the ultimate yolk spherules (Fig. 118) ; the latter stain an
orange red with eosin, are homogeneous in appearance, and
usually oval or spherical in form, seldom irregular. Two main
stages may accordingly be distinguished in the formation of the
yolk : (1) the formation of a large, regularly shaped yolk ball ;
and (2) the successive fragmentation of this ball, accompanied
by a gradually lessening affinity for stains, resulting in the
evolution of the mature, small yolk spherules, the cytoplasm
of the ripe egg being thickly filled with the latter. It is usu-
ally the case that the yolk ball attains its greatest size at the
end of the first stage. In cells of medium size all the various
stages of yolk formation may be found, which shows that the
yolk balls are being successively produced and are successively
fragmenting ; quite a number of these balls need to be pro-
duced in order to furnish the large quantity of yolk globules of
the mature egg. The time when the yolk balls first appear,
the size they reach, and the manner in which they segment,
seem to vary much in individual cells.
I have not been able to determine the manner of the first
differentiation of the yolk substance in the cytoplasm. Two
possible explanations suggest themselves : (1) either a certain
portion or constituent of the cytoplasm changes into yolk sub-
stance ; or (2) the yolk balls may represent a nutritive
substance accumulated in the cytoplasm, which may have been
derived from the blood or from some neighboring tissue, if not
directly from the posterior intestine. But it is without doubt
that this substance is not of nuclear origin, for the yolk balls at
their first appearance are not in contact with the nucleus, but
usually at some distance from it ; and also during the earlier
stages of the yolk formation the nucleus is irregular in outline,
with short, blunt processes, which would show that it is tak-
ing up substances from the cytoplasm, rather than excreting
substances.
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 425
The cycle of the formation of the nucleoli may here also be
divided into three stages, which do not quite correspond to the
four of Amphiporus gelatinosus.
First nucleolar stage. —In the smallest germinal vesicles
found one or two relatively very large nucleoli were present,
one of them often in the center of the nucleus, the other
more excentric or even against the nuclear membrane (Figs.
103, 114, 115). The nucleoli in these smallest nuclei are
as large or nearly as large as in any of the following stages.
In germinal vesicles of slightly greater dimensions three or four
nucleoli may be present, and some of these may have increased
a little in size ; the amount of nucleolar substance at this stage
is often so great as to occupy a fifth of the nucleus. They
now increase in number, until at the close of this period we
find a considerable number of mostly large nucleoli quite evenly
distributed through the nucleus (Figs. 104-106, 109, 110, 116),
but often they are at one of its poles more numerous than at
other points. This stage would seem to correspond to the first
and second of Amphiporus glutinosus.
Second nucleolar stage. — The nucleoli continue to increase
in number but now decrease in size and commence to pass to
the periphery of the nucleus, until at the end of this period
they all lie close to the nuclear membrane, are regular in out-
line, and adequal in size (Figs. 107, 119, 122, 124-126, 130, 131).
At the beginning of this stage numbers of nucleoli may be
found arranged in chain-like rows, as is to be seen in Fig. 111.
This would correspond to the third stage of Amphiporus.
Third nucleolar stage. — Nearly all the nucleoli are close to
the nuclear membrane, often flattened against it (Figs. 117, 120,
127, 129, 137, 138). They show signs of degeneration; thus
they stain less intensely, are irregular in outline, and have a
vacuolar or granular structure. In the largest germinal vesicles
their number has apparently decreased and small non-coherent
masses of granules may be seen, which are probably degen-
erated nucleoli. Sometimes a nucleus may be found in this
stage in which almost all of the nucleoli contain each one
large, excentric, lightly stained globule or vacuole (Fig. 117).
Staining of the nucleoii.— The natural color would appear
426 MONTGOMERY. [VoL. XV.
to be a light yellow. In a preparation stained with haematoxy-
lin and eosin, though not very thoroughly colored with the
latter stain, the large nucleoli of the first nucleolar stage were
of a light-yellow color, apparently stained only slightly with the
eosin ; those of the end of the second stage were mostly stained
red, and those of the third stage were stained red, except those
which had broken into granules, these latter being stained very
little. In another preparation, in which the eosin had acted
for one or two minutes longer than in the preceding prepara-
tion, the nucleoli in the first stage were stained orange, those of
the second stage red, and those of the third stage very slightly
or not at all stained. Accordingly, they stain more lightly at the
commencement of the first and at the end of the third stage than
during the second stage; these differences of stain are probably
due to chemical differences in the nucleoli at different stages.
The chief differences between the nucleoli of this species and
those of Amphiporus glutinosus are as follows: in the former
there is no stage which exactly corresponds to the first stage of
the latter, where we found a number of small peripheral nucle-
oli; in 7. catenulatum there are at first one or two large
nucleoli which are not always peripheral in position. The
nucleoli in the third stage of 7. catenulatum are more irregular
in form and dimensions and stain less intensely than those of
the fourth stage of Amphzporus. But the most important differ-
ence between the two species is to be found in the fact that
in 7. catenulatum new nucleoli continue to be produced even in
the third stage. Thus there are at the periphery of the nucleus,
between the larger degenerating nucleoli which had their origin
during the first stage, also much smaller, newly formed nucleoli
arising while the former are disappearing. Such younger
nucleoli may be seen at the close of the third stage, when the
nuclei are largest and chromatin filaments appear in them,
arranged in contact with the chromatin threads or near to
them (Figs. 127, 137, 138). These smallest nucleoli of the third
stage always stain intensely red with eosin, while the much
larger ones of the first and second stages stain more of an orange
color with this stain. This difference of staining in these two
kinds of nucleoli might be explained thus :
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 427
As we had concluded for the preceding species, so also in the
present and in the species of nemerteans yet to be described,
the nucleoli are in all probability accumulations within the
nucleus of a substance taken up from the cytoplasm, this sub-
stance being related to that which constitutes the yolk balls.
In the least mature germinal vesicles of 7. catenulatum we
found one or two very large, lightly staining nucleoli; these stain
in the same way and show the same structure and degree of
refraction as do the daughter yolk balls (Figs. 107 and 116).
Further, I have noticed in the cytoplasm small yellowish spher-
ules (yolk-ball fragments) which are in every way similar to the
smaller nucleoli, and quite frequently I have observed one or two
of them pressed so close against the outer surface of the nuclear
membrane as to cause a depression of the latter (Figs. 112 and
118). In other words, it would seem that the substance of
some of the yolk-ball fragments is taken into the nucleus and
in the latter is re-formed into nucleoli. As long as yolk balls
or their fragments are found within the cytoplasm lightly
stained nucleoli of approximately the same dimensions as these
may be seen in the nucleus. I have never seen a pore in the
nuclear membrane through which a yolk-ball fragment could
penetrate, though this membrane sometimes appears to be
thinner at the point of contact with a yolk-ball fragment than
at other points in its circumference. But in the third stage,
when all yolk balls and their fragments have disappeared and
the whole cytoplasm is thickly filled with their derivatives, the
mature yolk spherules, large, faintly staining nucleoli, are no
longer present in the nucleus, but the smallest nucleoli present
at this time resemble in form, size, and stain, the yolk globules.
Therefore we must conclude that the young, small nucleoli
which first appear about the end of the third nucleolar stage
represent mature yolk spherules, or at least that the substance
of the two is equivalent. While the nucleoli of the first gener-
ation (formed in the first stage) are commencing to degenerate,
new nucleoli of a second generation begin to arise in the
nucleus, and the latter, which may serve as nourishment for
the chromatin threads, differ from the former genetically, in that
they are not assimilated portions of yolk-ball fragments, but
428 MONTGOMERY. [VoL. XV.
assimilated yolk spherules. Thus, as we find in the cytoplasm
first yolk balls, then their fragments, and finally the mature yolk
spherules, so in the nucleus the first generation of nucleoli
are assimilated yolk balls and their fragments, while the small
ones of the second generation are derived from the only yolk
elements then present in the cytoplasm, namely, yolk spherules.
The nucleoli of the first generation also differ from those of
the second, at the time of the first appearance of both, in
their manner of staining ; so that they would seem to differ
chemically from each other.
Nuclear structures of problematical significance. —In only
one out of the three individuals of this worm studied were the
following remarkable structures to be observed, though the fixa-
tion method of both of the other individuals was exactly the
same. These bodies first appear in ova of the second nucleolar
stage, but here show always the same typical structure, so that
I can say nothing as to the manner of their first formation. In
preparations stained with haematoxylin and eosin they are
colored by the former stain a little more deeply than the
nuclear chromosomes, so that they stand out sharply in the
nuclear substance (JV. Bd., Figs. 122-139). The smaller ones,
z.e., those of the younger germinal vesicles (Figs. 122-126),
are finely granular, though whether they each consist of a mass
of fine granules or of homogeneous ground substance in which
granules are distributed, I cannot determine. In the larger
nuclei they often appeared wholly homogeneous (Fig. 132).
In shape they are usually nearly spherical, with a sharp outline,
which may or may not represent a limiting membrane; the
larger ones are often more irregular in form (Figs. 132, 133,
139). In the smaller nuclei they are as a rule, but not always,
smaller than in the larger ones ; in the smallest nuclei in which
I have found them there is only one of these bodies to a
nucleus ; while in the larger nuclei they are not only larger,
but also there may be from one to four of them in each nucleus.
In only one small nucleus were three of them present (Fig.
128). In two cases, both larger nuclei, I found division stages
of these bodies : in the one case (Fig. 131) the body was ovoid
in outline, with a shallow constriction at right angles to its
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 429
longitudinal axis, at about its middle; in the other case
(Fig. 129) the body was plainly biscuit-shaped, with a well-
marked medial constriction : these would probably represent
respectively successive stages of division.
The various stages found would show the metamorphoses of
these structures to be as follows : in the medium-sized nuclei,
those in which they first appear, there is only one to a nucleus.
This one increases in size up to a variable point, when it begins
to divide, producing two daughter-bodies, which are not always
of equal size. One or both of these bodies may now divide
again, resulting in the formation of (respectively) three or four
bodies. Since, however, the four bodies sometimes found in
the larger nuclei are often quite unequal in size, we must
assume : (I) either that the divisions have been very unequal,
and each daughter-body had divided ; or (2) that after the first
division, which may or may not have resulted in unequal
daughter-bodies, only one of the latter divides further, and it
divides once, and one of its products divides once. It is to be
noted that the number, the size, and the time of the division
of these bodies stand in no regular relation to the size of the
nucleus. Thus in one small nucleus (Fig. 128) three were
already present, so that here two divisions must have taken
place; while in some much larger nuclei (Figs. 130 and 133) a
single, much larger one was present, which showed no signs
of division. In the larger nuclei these bodies are often quite
irregular in form; may this increasing irregularity portend
an on-coming dissolution or other degeneration? They were
found, as remarked above, in the ova of only one of the three
individuals of this species examined, though in all three indi-
viduals the stages of egg development were very much alike;
in the single individual in which they occurred they were not
present in all the larger eggs. Their whole appearance and con-
sistency show that they are not artefacts (the fixation was with
hot aqueous corrosive sublimate), and they have no resem-
blance to any parasitic organisms, as e.g., Protozoa, with which
I am acquainted. Nor can they be centrosomes nor true nucle-
oli, and stand in no apparent relation to the nucleoli. In a
single case I found two nucleoli enclosed by one of these bodies ;
430 MONTGOMERY. [VoL. XV.
but in no other cases were these structures in contact with
nucleoli. They are also never in contact’ with the nuclear
membrane. Male pronuclei they cannot be, since the fecunda-
tion takes place in later stages than-those which I have had
opportunity to observe. I must conclude, though with reserve,
that they are either parasitic Protozoa, or, more probably per-
haps, structures which characterize ova of a certain generation.
(Compare my remarks on the “pseudonucleoli” of JMJontagua.
The structure figured by Henneguy ('93), in the immature ger-
minal vesicles of Sygnathus may have some connection with
these bodies.)
Chromatin. —The chromatin in the youngest germinal vesi-
cles (Figs. 103-105, 112-114) is distributed throughout the
nuclear sap in the form of minute microsomes. In the second
and sometimes the first nucleolar stage such microsomes can
often not be detected, but the whole nuclear substance, with
the exception of the nucleoli, appears homogeneous and stains
with eosin a yellowish red (Fig. 115). This peculiar coloration
might be accounted for on the ground that in these stages there
is a diffusion of nucleolar substance throughout the nucleus.
Towards the conclusion of the second and the commencement
of the third nucleolar stage, the minute chromatin microsomes
again become evident (Figs. 118 and 130). At the end of the
third stage a few chromatin threads begin to arise in the
nucleus (Fig. 127), and these stain slightly with haematoxylin
in the same manner as the microsomes do; they appear to
arise separately and at different points in the nucleus, and are
at first short, but gradually increase in length. As noted
above, the small nucleoli of the second generation are often
apposed to these threads, and sometimes lie inthe meshes of
them.
Nucleus.—In the first and second nucleolar stages the
nucleus has often short, lobular processes, which may be amoe-
boid in life (Figs. 109, 112, 114, 116, 125); these changes in
the form of the nucleus no doubt stand in a direct relation to the
assimilation of yolk substance from the cytoplasm. Towards
the end of the third stage the nucleus becomes regular in out-
line, with no traces of amoeboid processes; at this stage also
No.2.] COMPARATIVE CYTOLOGICAL STUDIES. 431
the nuclear membrane has attained its greatest thickness. The
thinness of this membrane in previous stages would allow the
penetration of nutritive substances into the nucleus from
the cytoplasm. The small nuclei from which the germinal vesi-
cles are directly derived, without any intervening mitoses, are
irregular in shape, and no nucleoli are to be seen in them
(Figs. 108 and 112, C. 7. M.).
5. Tetrastemma elegans (Verr.).
(Plate 28, Figs. 282-299.)
Having only two mature individuals of this worm for study,
I am unable to give as thorough a description of the nuclear
metamorphoses of the egg as was possible for the other nemer-
teans ; one preparation was fixed with Hermann’s fluid, the
other with aqueous solution of corrosive sublimate, but the
latter had been too deeply stained (haematoxylin, eosin) to
allow the study of certain details, as e.g., the cytoplasmic
changes leading to the formation of the yolk. Yolk balls were
observed in only a few ova, and are much less numerous than
in 7. catenulatum ; it is possible that the development of the
yolk in the present species may be as in Zygonemertes, that is,
the mature yolk spherules may as a rule be directly formed
without the interpolation of a yolk-ball stage.
First nucleolar stage. — The youngest germinal vesicle, recog-
nizable as such, showed a large nucleolus close to the nuclear
membrane (Fig. 282) ; I have seen no smaller nuclei than this
one, but would conclude by analogy from the facts in the other
metanemerteans that also here all the nucleoli have an extra-
nuclear origin. In slightly larger nuclei (Figs. 283-287) there
are from one to three nucleoli, whose size varies considerably
with regard to that of the nucleus, as well as to the size of one
another. In such cases (Fig. 283) where only two nucleoli are
present, one near the center of the nucleus, the other close to
the nuclear membrane, the former is probably the older and
has left the periphery for the center of the nucleus, while the
other is younger and is still in process of formation. These
first-formed nucleoli are usually rather large in proportion to
432 MONTGOMERY. [VoL. XV.
the size of the nucleus, seldom small. It is the rule that in
one, sometimes in all the nucleoli, a large unstaining globule
is present, which has the appearance of a vacuole (Figs. 284—
287, 298); no nucleolus has more than one such globule. Quite
often there is only a single large vacuole-containing nucleolus
in a nucleus ; or there may be from one to six nucleoli, only
one of which contains a vacuole, and then the latter is usually
the largest ; or again, there may be two or three large nucleoli,
nearly equal in point of size, each of which contains a vacuole
(of course numerous intermediate stages may be found). There
is certainly a successive production of nucleoli, but it is diffi-
cult to decide whether some of these after leaving the periphery
of the nucleus fuse together, or whether some divide into smaller
nucleoli. Now it seems probable that those nucleoli which are
formed first are usually unequal in size, both in the same nucleus
and in different nuclei, as a comparison of the figures shows.
And though a gradual fusion of the nucleoli might play some
part in the youngest germinal vesicles, nevertheless it would
seem more probable that we have to do in these early stages
with divisions of the nucleoli, especially since in the following
stage they are much more numerous, as well as smaller. Fig.
287, in which three apposed nucleoli are to be seen, may thus
represent a division of a single nucleolus. It is not unlikely
that the unstaining globule within a nucleolus might aid, if it
is not the direct mechanical cause of, such division. This first
nucleolar stage is then characterized by the successive forma-
tion of a few comparatively large nucleoli at the periphery of
the nucleus, and the migration of these towards the center ;
the presence of large vacuoles within some of the nucleoli is
also a criterion of this period.
Second nucleolar stage. — We find a group of numerous
nucleoli near the center of the nucleus, which are frequently
more numerous than in our Fig. 292. At this stage they
attain their smallest dimensions, and are approximately equal
in size; they are completely homogeneous and contain no
vacuoles. The total number of the nucleoli is apparently
greater at this stage than at any other.
Third nucleolar stage. — This is characterized by an increase
No.2.] COMPARATIVE CYTOLOGICAL STUDIES. 433
in the size of the nucleoli, a decrease in their number, and the
gradual migration of them towards the periphery of the nucleus.
At the beginning of this period (Figs. 290, 291, 293, 294), the
nucleoli are quite evenly distributed throughout the nucleus; at
its close they are mainly peripheral in position, near the nuclear
membrane (Fig. 297). The increase in the size of the nucleoli
is due, in some part at least, to the coalescence of every two or
three neighboring ones, and such juxtapposed groups of two
or three nucleoli may be often found (Fig. 294). None of the
nucleoli contain vacuoles.
Fourth nucleolar stage. — Now we find unstaining globules
or vacuoles reappearing in the nucleoli, and there may be either
a single large one to each nucleolus, or a number of smaller
ones ; the large one is probably formed by the coalescence of
smaller ones. Almost all the nucleoli are in contact with the
nuclear membrane, often flattened against it (Fig. 299). They
have become larger than in any preceding stage, and less
numerous, but are now quite unequal in size. This stage may
mark the commencing degeneration of the nucleoli, though I
have observed no evidences of a commencing fragmentation.
At the beginning of the first stage the nuclear sap never
stains ; but at the end of this period, when the nucleoli have
become more numerous, it stains very noticeably with eosin
(Fig. 286), which would point toa solution of nucleolar sub-
stance in the nuclear sap.
6. Zygonemertes virescens (Verr.) Montg.
(Plate 27, Figs. 236-248.)
Yolk.—In only two cases out of the numerous egg cells
examined (three individuals of this worm were sectioned) have
I seen yolk balls, so that the formation of yolk balls must be
regarded as abnormal, if not pathological ; in this species the
yolk arises as minute yolk spherules in the cytoplasm (Fig.
246), without (except in the cases noted) a yolk-ball stage
being passed through. These minute globules stain at first
very faintly, and when they first appear are isolated from one
another. There is no given point in the cytoplasm where they
434 MONTGOMERY. [Vov. XV.
are first produced, but a varying number are formed simultane-
ously and at different parts of the cell; it is usually, though
not always, the case that they first arise at the periphery of the
cell at some distance from the nucleus. The mature yolk
globules are slightly larger than these and stain somewhat
more intensely, which shows that they gradually become denser
as they increase in size ; in the largest ova these spherules
are so abundant that the true cytoplasm is quite obscured
(Fig. 247).
First nucleolar stage. — In the smallest nuclei found there is
a peripheral group of several nucleoli lying close to the nuclear
membrane, which are spherical in form (Figs. 236-238).
Second nucleolar stage. — The nucleoli have increased in num-
ber, and, departing from their original peripheral position, now
occupy the center of the nucleus (Figs. 239 and 240). So small
are they, and so densely grouped may they become, that at
first sight one might be led to suppose that each group of
numerous nucleoli was a single nucleolus. In those cases
where the nucleus is oval or elongated in form, instead of
spherical (the usual case), in the place of a single cluster two
are commonly present, or else the single mass or cluster of
nucleoli is elongate in shape, its outline being more or less par-
allel to the contour of the nucleus. The nucleoli in this stage
are always more numerous and usually also smaller than those
of the previous period ; their increase in number might thus
be brought about, in part at least, by divisions of the earlier
nucleoli.
Third nucleolar stage. —The nucleus now is much larger,
and the nucleoli begin to wander apart towards the periphery
of the nucleus (Figs. 241, 243, 246, 247). I have observed all
stages between nuclei containing centrally grouped, small
nucleoli and those in which they have come to lie close to the
nuclear membrane. In this stage, as in the preceding one,
the nucleoli are perfectly homogeneous without vacuoles, and
spherical in form. In a few nuclei, however, they appear
greatly vacuolated, but these cases are so rare that they must
be considered abnormal. At the end of this period they attain
their greatest dimensions, though they thereby become some-
No.2.) COMPARATIVE CYTOLOGICAL STUDIES. 435
what unequal in size. In this stage, accordingly, they increase
in size (perhaps by the fusion of contiguous ones (Fig. 242),
and decrease in number, whereas in the preceding one the
reverse process took place.
Fourth nucleolar stage. — Almost all the nucleoli are flattened
against the nuclear membrane (Figs. 245 and 248), and they com-
mence to show a vacuolated structure; these apparent vacuoles,
which are unstaining globules, when stained by the Ehrlich-
Biondi method, whereby only the ground substance of the
nucleolus is colored, appear as refractive granules (Fig. 248).
At the conclusion of this period the nucleoli become irregular
in shape, granular in appearance, stain less deeply, and each
finally breaks up into a mass of granules. In this manner they
decrease both in number and size.
During the third and fourth stages, while the nucleoli are
undergoing the metamorphoses described, a small number of
newly formed ones appear in the nucleus, which are of later form-
ation than the others (Figs. 245, 247, . 2). These may serve
as nourishment for the chromatic filaments, as in 7etrastemma
catenulatum ; but in the present species I have not observed
any distribution of them along these filaments, and further
they are numerically scarcer than in Jetrastemma.
No yolk is present in the cytoplasm in the first and second
nucleolar stages. This fact is easily proved by the use of the
Ehrlich-Biondi stain, by which the cytoplasm is stained green,
and the yolk substance, when present, a brownish maroon color.
Yolk first appears in the third nucleolar stage, and at the com-
mencement of the following stage the whole cytoplasm is nearly
filled with it. Further, the nucleoli stain differently from the
yolk globules by the use of the stain mentioned. These facts
show that the origin of the nucleolar substance is not to be found
in the yolk substance proper, but in a cytoplasmic substance
from which the latter may later be evolved. That the sub-
stance of the nucleoli is extranuclear in origin is shown by the
fact that the nucleoli at their first appearance lie in contact
with the nuclear membrane (Figs. 236-238), and only later do
they take a central position. Though I have seen no nuclei
smaller than those figured, which could without doubt be
436 MONTGOMERY. [VoL. XV.
classed as germinal vesicles, yet it seems so probable that the
substance out of which the nucleoli ate formed is extranuclear,
that I would conclude, a priorz, that no nucleoli are present
in stages of the germinal vesicle much earlier than those which
have been here described. Those small nucleoli of a second
generation, which are first produced in the third and fourth
nucleolar stages, may represent yolk globules assimilated by
the nucleus, since in these stages the cytoplasm is filled with
such globules.
On the other hand, the yolk cannot be considered as having
its origin in nucleoli which have wandered out of the nucleus,
since in none of these stages are nucleoli found in the cyto-
plasm. And if such were the case, one certainly should be
able to observe the large nucleoli of the third nucleolar stage
in the cell substance, for it is at this period that the yolk first
appears. I conclude that the yolk globules have their origin
in some substance contained in the cytoplasm, and that the
nucleolar substance also has its origin in some cytoplasmic
substance. But whether the primitive nutritive substance of
the yolk globules and that from which the nucleolar substance
is derived are identical, is of course open to question ; how-
ever, judging from the similarity in appearance, we might con-
clude that the primitive cytoplasmic substance was the same
in both cases, and especially if we consider, which seems plaus-
ible, the nucleoli to represent the nutritive substance of the
nucleus, as the yolk globules certainly represent that of the
cell body.
In the first nucleolar stage the nuclear membrane is usually
very thin, but always perceptible ; in the later stages it becomes
thicker. The nucleus is never noticeably irregular or amoe-
boid in outline. Might this be explained by the absence of
yolk balls in the cytoplasm ?
In the second and at the beginning of the third nucleolar
stages, the central mass of nucleoli is usually surrounded by a
clear space, in which space few or no chromatin microsomes
occur, though it may be transversed by a few achromatic fibers
(Figs. 239 and 240). This space was found in most of the egg
cells of this stage in the three individuals sectioned, though it
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 437
may have been produced by the action of the preserving fluid
(hot aqueous solution of corrosive sublimate).
7. Stichostemma etlhardi (Montg.).
(Plate 27, Figs. 213-235.)
The yolk changes may first be delineated, then those of the
nucleoli. In my paper on this fresh-water form (95), I have
described the ovogenesis to some extent, and here shall follow
it more in detail.
Yolk.— The yolk first appears in the cytoplasm in the form
of small, more or less spherical masses (Fig. 213, Yk. B/.),
which at first stain like the cytoplasm; but these youngest
recognizable yolk balls consist of a substance in which the fine
granules (or nodal points of an alveolar structure) are much
more densely grouped than in the surrounding cytoplasm.
Thus the young yolk ball may be distinguished from the
cytoplasm proper by its greater density. A number of these
yolk balls appear to arise simultaneously, though in these
earliest, as well as in the later stages of yolk formation, a
successive production and metamorphosis of yolk balls take
place, since in all but the earliest stages of their development
yolk balls occur in the cytoplasm in various stages of forma-
tion. There is no rule as to the part in the cell at which these
balls are destined to arise, for they may be found anywhere
between the nucleus and the periphery of the cell; the fact
that they first arise just as frequently at some distance from
the nucleus as in its immediate neighborhood shows that they
have no nuclear origin. An anabolic and a katabolic series of
changes of each yolk ball can be distinguished, and these series
of metamorphoses may be described in succession and termed
respectively the prophasis and metaphasis of the yolk balls.
Prophasis (Yk. Bl. in Figs. 217, 218, and the median ones of
Fig. 215). — The progressive or anabolic changes of the yolk
balls consist in (1) their absorbing protoplasmic stains with
great intensity, so that they stand in marked contrast to the
cytoplasm ; and (2) in their becoming quite homogeneous in
structure, this homogeneity probably explainable by supposing
438 MONTGOMERY. [VoL. XV.
that a dense condensation of the fine granules of which they
are composed takes place. They continue to increase in size,
and gradually stain deeper as they do so, until they attain about
the dimensions given in Fig. 217 ; but I am unable to deter-
mine whether they all reach exactly these dimensions before
the metaphasic changes commence. At the conclusion of this
period of their development they are large bodies, regularly
spherical or oval in outline, and apparently without a limiting
membrane ; especially characteristic is their homogeneity and
their intense staining.
Metaphasis. — These katabolic metamorphoses are intro-
duced when a few unstaining globules arise in the substance
of the yolk balls. These globules increase in number and size
until the yolk ball assumes a vacuolated appearance (Figs. 215,
217, 228). At the same time its ground substance loses its
staining power and no longer stains homogeneously. At the
commencement of these changes the yolk ball may even
increase somewhat in size, since the substance of the globules
is added to it. These changes continue until the yolk ball either
breaks up into the mature yolk globules (Y%. GZ, Fig. 235),
or first breaks into a varying number of larger pieces, and then
each of the latter divides into yolk globules. The yolk globules
are usually nearly spherical in shape, and though by no means
equal in size are always larger than those of the other nemer-
teans examined.
During the prophasis each yolk ball is enveloped by a clear,
structureless zone of cytoplasm ; but this surrounding zone is
usually not noticeable around the larger yolk-ball fragments,
and never around the mature yolk globules.
As to the cause of the fragmentation of the yolk balls, I can
find no sure explanation from the facts at hand. However, the
appearance of the colorless fluid globules within their substance
must have an important connection with these katabolic changes,
since they characterize the commencement of this period of
change. It would seem likely that these colorless globules
represent a fluid constituent of the cytoplasm which has
actively or passively been taken into the yolk ball, — perhaps
from the clear cytoplasmic zone enveloping each yolk ball,—
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 439
since the yolk balls increase in size at the beginning of the
metaphasis, though there appears to be no increase in their
own ground substance. These unstaining globules are at first
localized at different points in the yolk ball ; and it would seem
probable that their substance later mixes itself with the ground
substance of the yolk ball, since this supposition would account
for the lessening intensity of the stain of the yolk ball during
the metaphases. It would appear less probable that these
globules are metabolic products of the true substance of the
ball ; however, we have too few facts to enable us to deter-
mine which of these is the correct view.
Certain curious structures found in the cytoplasm of an
immature worm fixed with Lang’s fluid (aqueous corrosive
sublimate solution, NaCl, acetic acid) may be mentioned here.
In the cytoplasm of a number of ova, in none of which any
yolk was present, I found a few small, ring-shaped bodies,
which stained with haematoxylin much more intensely than
the surrounding cytoplasm (VY. B/.? Fig. 233). Each con-
sisted of a ring composed of a dense, homogeneous substance,
the inner surface of the ring being smooth, but the outer irregu-
larly jagged, this whole ring (as it appears on a section) enclos-
ing an unstaining vacuole or globule. In reality these bodies
are spheres, but my description applies to sections of them.
These structures vary considerably in size, and sometimes are
not spherical but oval, the larger ones usually staining more
deeply than the smaller ones. I found them only in some of
the ova of this one individual, and nothing of the kind was to
be seen in the ova of about twenty other individuals sectioned,
which had been variously preserved in picric,osmic, and chromic
acids, in simple aqueous solution of corrosive sublimate, and in
the fluids of Hermann and Flemming. Accordingly, they must
be regarded as artefacts, produced by the action of the acetic
acid, which I have long since found to be a very unreliable fixa-
tive for the cytoplasmic elements of the nemerteans. It is
most probable that they are young yolk balls, to which the
acetic acid has given an abnormal appearance. Or might they
represent multiple asters, such as have been recently described
by Mead in Chaetopterus ?
440 MONTGOMERY. [VoL. XV.
.
Germinal vesicles, nucleolz.—In this genus the earliest egg
stages are more favorable for study than in the other metane-
merteans. In the connective-tissue nuclei from which the ger-
minal vesicles are directly derived (with no intervening cell
generations) no nucleoli are present, though this conclusion was
possible only after much careful observation. These small
nuclei (Figs. 213, 217, 218, 220, 228, C. 7. WV.) are character-
ized by a relatively thick membrane and by chromatin which is
usually granular in distribution, but which may sometimes
occur in the form of granular fibers. These chromatin masses
might at first sight be confounded with nucleoli, but their small
size and irregular contours show that they are true chromatin
granules. Further, when these nuclei are stained by the
Ehrlich-Biondi method, these fibers and granules always stain
with methylen green (chromatin reaction) and not a single one
stains with fuchsine (which invariably stains any true nucleoli).
Accordingly, what could not be finally proved for the other
metanemerteans, though all observations pointed to its being
the case there, could be definitely settled for Szcchostemma,
namely, that these connective-tissue cells contain no nucleoli ;
in other words, nucleoli first arise in the definite germinal
vesicles.
Before proceeding to the description of the egg cells it may
be noted that not all the undifferentiated connective-tissue
cells within the gonad become germinal vesicles. I have previ-
ously (95) shown that the young gonad is a cell syncytium in
which numerous nuclei are unevenly scattered through a mass of
cytoplasm, but cell boundaries cannot be seen (Figs. 217 and 218).
A few of these nuclei increase in size and eventually become
germinal vesicles, and the latter reach maturity not simultane-
ously but in succession, so that no gonad contains more than one
large ovum at a given time. The numerous other nuclei which
do not become thus differentiated degenerate, and their sub-
stance is eventually absorbed by the gradually increasing mass of
cytoplasm of one of the growing egg cells. These regressive
processes are as follows (Fig. 218, C. 7. WV.) : the nuclei
increase a little in size, but become much clearer in appearance,
z.e., the relative amount of their chromatin appears to decrease ;
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 441
next the cell membrane gradually disappears; then the chro-
matin granules no longer become colored by any of the stains
employed, but become refractive and yellowish. All the chro-
matin granules do not lose their affinity for stains simulta-
neously, but two or three of them may often remain stained as
before, while the remaining granules of the same nucleus may
have entirely lost their stain. At this period in the nuclear
degeneration we find small masses of these unstaining, yellow-
ish granules in the cytoplasm, each mass still preserving the
form of a nucleus. Later these individual granules wander
apart, or those of several nuclei may partially fuse together to
produce a larger mass ; these larger masses of granules are
always enveloped by a clear zone of cytoplasm, sometimes of
considerable extent, so that they appear to be situated in
vacuoles of the cytoplasm. The degeneration stages of these
nuclei are most frequent in the cytoplasm, before yolk balls
begin to arise in it ; as the latter appear, the remnants of the
degenerated nuclei gradually vanish, so that when the cell is
filled with the yolk balls all vestiges of these nuclei have
vanished. We must suppose that they become assimilated by,
or dissolved in, the cytoplasm. These formations, the katabolic
changes of degenerating nuclei, can in no way be confounded
with stages of yolk development, since the small size, yellowish
color, and refrangibility of these granular masses serve to dis-
tinguish them sharply from any stage of the yolk balls, even
though both are often found in the immediate vicinity of each
other.
The nuclei which are destined to become germinal vesicles
increase in size to some extent before nucleoli appear in them ;
they now differ from the connective-tissue nuclei, apart from
their greater dimensions, in having a relatively greater amount
of chromatin and in being regularly spherical or oval in form.
The first nucleoli to arise always lie in close contact with the
inner surface of the nuclear membrane (Figs. 214, 216, 219,
220, 224, 225). They usually appear in the form of a thin
disc-shaped mass on the inner surface of the membrane, but
there is considerable irregularity in the form of this mass, which
may be angular or nearly spherical in outline. At the com-
442 MONTGOMERY. [VoL. XV.
mencement of this first nucleolar stage the nucleolar substance
appears at only one point in the periphery of the nucleus, and
always in the shape of an irregular mass.
Second nucleolar stage.— This period is characterized by
the formation of other nucleolar masses at various points in
the periphery of the nucleus, the successive detachment of all
of these from their connection with the nuclear membrane, and
their migration towards the center of the nucleus. The com-
mencement of this process is to be seen in very young nuclei,
where but a single peripheral nucleolar mass is present ; from
the inner side of this mass small particles become divided off
(Figs. 219, 224, 225), then each of these particles assumes a
more or less spherical shape and wanders to the center of the
nucleus ; this process continues until the whole mass of nucleo-
lar substance has reached the center in the form of separate
particles (Figs. 217, 218, 223, 227). The peripheral nucleolar
mass usually stains less intensely than the portions which have
already reached the center of the nucleus. While the first-formed
peripheral nucleolar mass is thus gradually wandering to the
center, other masses are successively forming at the periphery
of the nucleus, and their detached portions successively passing
to the center. When a considerable number of these nucleoli
have reached the center of the nucleus they naturally come
into mutual contact, and then a process of fusion sets in, which
results in the coalescence of neighboring groups of nucleoli, so
that a smaller number of larger ones are formed. Sometimes
this fusion may proceed to such an extent that one single,
enormous nucleolus results (Fig. 226), but usually several
large nucleoli are the result, these being unequal in size.
The irregularity both in the dimensions and the forms of the
nucleoli is particularly characteristic for this stage ; thus the
individual nucleoli often have elongated processes and angles,
and this irregularity is frequently so excessive that the nucleoli
within the nucleus appear like smears of ink upon a page (Figs.
226, 227, 230). I think that this irregularity in form may be
explained by the assumption that at this stage the substance
of the nucleoli is viscid in its consistency, while in the follow-
ing one, where the spherical form is the rule, its nature must
No.2.] COMPARATIVE CYTOLOGICAL STUDIES. 443
be more freely fluid. Further, at this period we usually find
vacuoles within some of the nucleoli of each germinal vesicle
(Figs. 217, 218, 226, 229-231); sometimes no vacuoles are
present in any of the nucleoli of a nucleus, but it is the rule that
at least one of them, and that usually the largest, contains one
or several vacuoles. Sometimes four or five of the nucleoli,
which may be very unequal in size, may each have vacuoles.
Occasionally a nucleolus contains only one vacuole, and in the
latter there may be one or several small solid bodies, which
stain like the ground substance of the nucleolus, and may be
termed nucleololi; one of the latter may be fused with-the inner
surface of the nucleolar ground substance (Figs. 217, 218, 230,
231). These nucleololi vary in number and size, and are
absent in the greater number of the vacuoles ; so no particular
significance should be attached to them, since they are probably
nothing more than portions of the ground substance of the
nucleolus which have become detached from the surrounding
substance and have come to lie within the vacuole. During
this period the nuclear membrane is thinner than at any other
stage, and the nucleus is very noticeably amoeboid in form, the
amoeboid processes being much more pronounced than in any of
the other nemerteans examined ; these processes in reality repre-
sent changes in the form of the nucleus, and are not artefacts,
since they are seen equally well after preservation in the most
diverse fixing fluids (Figs. 226, 227, 230, 232, 233). The nu-
clear membrane is always particularly thin around these nuclear
processes, but, as far as I could make out, never becomes broken.
Third nucleolar stage.—The large nucleoli which were
present at the end of the preceding stage now commence to
fragment into smaller nucleoli, which are more or less equal in
size, and then the latter wander towards the periphery of the
nucleus ; at the conclusion of this period, which must take
place in a very short time, since I found only a few germinal
vesicles exhibiting it, there are a large number of rather small
nucleoli close to the nuclear membrane (Fig. 234). At this
time the nucleoli attain their maximum staining intensity ; the
nucleus usually shows no traces of an amoeboid form, and its
membrane has increased in thickness. None of the nucleoli
444 MONTGOMERY. [Vor. XV.
contain vacuoles ; and in every respect the nucleolar changes
during this stage are the very reverse of the preceding.
Fourth nucleolar stage. — This is characterized by the gradual
degeneration and disappearance of the nuclei (Fig. 235). Small
vacuoles arise in them, and these increase numerically, while at
the same time the nucleolar substance stains less intensely.
Fusion of neighboring nucleoli is very frequent at this time, or
perhaps a little time before the nucleoli lose their staining
power ; accordingly, in the largest germinal vesicles it is the
rule to find a small number of large nucleoli. The nucleoli
are not evenly distributed along the periphery of the nucleus,
and are often flattened against the nuclear membrane. This
nucleolar stage is found only in the largest ovarial eggs, where
the nucleus is perfectly regular in outline, without amoeboid
processes, and its membrane has attained its greatest thickness.
Since this species is a protandric hermaphrodite, in which
male and female sexual products ripen successively in each
gonad, I found it at first difficult to determine whether a young
nucleus in a given gonad corresponded to a male or to a female
cell. But after comparing briefly the spermatogenesis of the
other metanemerteans mentioned in this paper, and finding in
them that no nucleus in any stage of spermatogenesis was larger
than any of the smallest germinal vesicles here figured, I con-
cluded that also in Stzchostemma no male nuclei can attain the
dimensions of even the smallest nuclei of our second nucleolar
stage, and hence that all these nuclei were correctly concluded to
be germinal vesicles, and not nuclei of spermato-genetic stages.
We notice in the succession of the nucleolar stages described
the rhythmic sequence in regard to (1) the position of the
nucleoli, (2) their states of fusion and division, and (3) the
absence and presence of vacuoles in them ; these successive
changes may be expressed as follows :
NvucLeou1.
(as ae S090, 0080 wy
Stage. Position. Vacuoles. Fusion, division.
First . . . peripherall . . . absent... . . fusion?
Second . . . central . + + present. . . division, then fusion.
Third . . . peripheral . . - absent ~ . . ~ ~ division:
Fourth . . . peripheral . . . present. . . fusion, then division.
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. AAS
There is without doubt in this genus, as in the other meta-
nemerteans, an extranuclear origin of the nucleolar substance.
This is proved (1) by the absence of nucleoli in the nuclei
from which the germinal vesicles are derived; (2) by the
nucleoli first appearing close to the nuclear membrane. And
since yolk globules do not arise in the cytoplasm until nearly
the close of the second nucleolar period,when most of the nucleoli
are near the center of the nucleus, to the yolk substance cannot
be attributed a nucleolar derivation, and other reasons, such as
the fact that the yolk balls usually appear at some distance
from the nucleus, would contradict such an assumption. The
nucleolar substance is apparently formed from an unstaining
fluid constituent of the cytoplasm, which after it is taken into
the nucleus undergoes a chemical change, since it stains there
and is deposited in the form of nucleoli. In the second nucleolar
stage, when the formation of nucleoli is at its height, the
nuclear sap stains more deeply than at any other period (Figs.
224-227, 233), so that it is probable that at this time nucleolar
substance is finely distributed throughout the nuclear sap, as
well as in the form of nucleoli. (This staining of the nuclear
sap is especially well seen on material fixed with Flemming’s
fluid and stained with alum carmine.)
In the third and fourth nucleolar stages a few yolk globules
are often found in a number of germinal vesicles (Figs. 234 and
235, Yk. G/.); these have probably been taken up by the nucleus
from the cytoplasm.
Chromatin. — In the nuclei of the first stage, the chromatin
is always demonstrable in the form of coarse granules (Figs.
214, 216, 219). In the beginning of the second it may usually
be found in the form of a reticulation (Figs 218, 229, 233), but
at the end of this stage it is not demonstrable (Fig. 227). In
the third and fourth stages it reappears, but now in the form
of fine microsomes (Fig. 235); and at the conclusion of the
‘fourth stage short chromatic filaments begin to arise, similar
to those described for Zetrastemma catenulatum.
446 MONTGOMERY. [VoL. XV.
8. Lineus gesserensis (O. F. M.).
(Plate 24, Figs. 159-177.)
Yolk. — The yolk first arises in the cytoplasm in the form of
irregular yolk balls, which are much smaller than in the other
nemerteans examined (Ys. 4/., Figs. 159, 160, 177); these
increase in number and size, the largest sometimes contain-
ing vacuoles. In the largest ovarial ova seen (though I had
only immature individuals of this species) yolk balls are no
longer present, but in their place smaller yolk globules, which
in all probability represent fragments of the earlier balls. The
yolk usually makes its first appearance in a zone of the cyto-
plasm, midway between the nucleus and the cell membrane,
which is characterized from the rest by a less dense structure
(Fig. 177). The extreme peripheral portion of the cytoplasm
retains its density longest, as is also the case in the other
species. The cytoplasm of the connective-tissue cells (Fig. 159),
from which the egg cells take their origin, stains very faintly,
while that of the young egg is dense and stains deeply.
Nucleoli. — Only three worms out of eighteen sectioned con-
tained ovogenetic stages, and since in these individuals only the
earlier stages of this development were found, I am able to
describe only the younger stages of nucleolar formation. The
egg cell of this heteronemertean contains a single nucleolus ;
apparent exceptions will be considered later.
In the smallest nuclei (Fig. 159) of the cell syncytium of the
gonads no nucleoli are to be seen ; we find nucleoli for the
first time in cells whose nuclei are a little larger and whose
cytoplasm commences to stain more intensely. These are the
earliest stages of the ovocytes.
Now in these youngest germinal vesicles (Figs. 159, 161,
164, 166) the nucleolus is very frequently peripheral in position,
close to the inner surface of the nuclear membrane ; while in
the later stages (certain mitotic stages excluded) it is almost
invariably never in contact with the nuclear membrane. Fur-
ther, yolk balls first appear in the cytoplasm when the nucleus
contains a nucleolus. These facts, being considered together
with the fact that nucleoli are absent in the nuclei of the con-
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 447
nective-tissue cells, lead to the conclusion that the nucleolus
first appears in the young germinal vesicle, and more particu-
larly, that the substance of the nucleolus is extranuclear in
origin, and stands in a genetic relation to the substance of the
young yolk balls. The substance of both is homogeneous and
stains identically; by fixation in Hermann’s fluid, followed by the
triple stain of Flemming, the nucleolus and the yolk balls stain
a brownish yellow (Fig. 160) ; by fixation in corrosive sublimate
and staining in haematoxylin and eosin both structures are
colored a yellowish red (Fig. 177). Still more conclusive is
the following observation: while the greater number of the
yolk balls may lie at some distance from the nucleus, one or
several are very frequently in close contact with the outer sur-
face of the latter, and yolk balls may even be found which are
halfway through the nuclear membrane, or which have com-
pletely transversed it and lie within the nucleus (Fig. 160).
Thus the nucleolus would seem to owe its origin to the sub-
stance of yolk balls which have been taken into the nucleus.
The very marked increase in the size of the nucleus and the
nucleolus is probably caused by a continued process of yolk-
ball assimilation on the part of the nucleus. This may be
observed in numerous cases where small globules of yolk-
ball substance lie within the nucleus, some at its periphery or
close to the nuclear membrane, others flattened against the
nucleolus (Figs. 160 and 177). By the use of the haematoxylin-
eosin stain the nucleolar substance usually stains a little more
intensely than the substance of the yolk balls (Fig. 177); this
would show that this substance, after being taken up by the
nucleus, undergoes a chemical change within the latter. Those
yolk balls which are not assimilated by the nucleus remain in
the cytoplasm and give rise to the yolk globules, as has been
described. Thus the nucleolus probably has an extranuclear
origin and represents a portion of the yolk-ball substance taken
into the nucleus; its rapid increase in size is due to the addition
to it of other similarly assimilated globules of substance.
In the largest germinal vesicles seen (though these were
not mature) the nucleolus is usually spherical in form, seldom
oval, and homogeneous in structure, except that it sometimes
448 MONTGOMERY. [Vou. XV.
contains a single large, unstaining globule, which appears as
a vacuole (Figs. 162, 175-177); or there may be from one
to three minute globules in it, which, when seen in their
entirety, present the optical appearance (due perhaps to refrac-
tion) of black granules, which might be mistaken for solid
bodies. The nucleolus has no limiting membrane. The largest
are relatively enormous and stain more intensely with eosin
than the smaller ones. There is no clear zone in the nucleus
around the nucleolus.
In Lineus the study of the metamorphoses of the nucleolus
is complicated by the occurrence of nuclei in various mitotic
stages. Karyokinetic figures were absent in the ovarial stages
of the other nemerteans examined, so that in those species
the connective-tissue nuclei and the egg nuclei both stand in
the same cell generation, and the germinal vesicle may either
be regarded as equivalent to an ovogonium or to a true ovocyte
of the first order. In those species no cell generation separates
the connective-tissue nucleus and the germinal vesicle, but the
latter is merely evolved from the former bya gradual process of
differentiation. But in Zzzews the germinal vesicle is separated
from the connective-tissue nucleus by at least one and prob-
ably by two or three generations (if the differences in the size
of the cells offer a sure criterion). Here, accordingly, the
indifferent connective-tissue cell represents an ovogonium, and
perhaps another generation of ovogonia may intervene before
the germinal vesicle, the ovocyte of the first order, is produced.
Of the two individuals on which these nuclear studies were
made, I found mitotic stages in only one individual, while
none were to be seen in the other individual, though here
these nuclei had reached nearly the same degree of devel-
opment. I have studied the mitosis merely with regard to the
behavior of the nucleolus. The most abundant stages were
those of the spirem and dispirem, asters and dyasters being
much less frequent (Figs. 163, 166, 169, 170-172) ; the time
duration of the latter stages may be less than that of the
former. In by far the greater number of the spirem stages one
nucleolus was present; it is probably present in each nucleus
of this stage, but sometimes may escape observation by being
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 449
covered by the chromatic filament or by lying in a part of the
nucleus outside of the plane of the section. In this stage,
further, two nucleoli are never present ; accordingly, in the
spirem there is neither a disappearance nor a division of the
nucleolus. In the dispirem stage each daughter-nucleus contains
one nucleolus (Fig. 171), the two nucleoli being, however, often
unequal in size. I found very few aster stages, and these were
either so unfavorably placed for study or the chromosomes so
densely entangled that I could not determine whether a nucleo-
lus is present in this stage and whether a division of it takes
place at this time. The facts determined are (1) that no divi-
sion of the nucleolus occurs in the typical spirem stage, since
here only one nucleolus is present ; and (2) that each nucleus
of the daughter-spirem has one nucleolus. But I cannot show
whether a division of the nucleolus occurs in the time between
these two stages or whether the original nucleolus passes over
into one of the daughter-nuclei, while in the other one a new
nucleolus is produced. In these various mitotic stages the
nucleolus usually lies at the periphery of the nucleus, and it is
most frequently the case that it is not in contact with the
chromatin filament ; it preserves its former shape and staining
intensity, and apparently does not decrease in size during the
mitosis. To be sure, in the karyokinetic stages under considera-
tion it usually appears small in proportion to the size of the
particular nucleus, but then it is usually the case in most
mitoses, and probably so here, that before the disappearance
of the nuclear membrane the volume of the nucleus greatly
increases.}
Two nucleoli, never quite equal in size, are frequently found
in certain small nuclei, which the distribution of the chromatin
would show to be in a stage at the commencement of the
prophasis of the mitosis or at the conclusion of the metaphasis
(Figs. 163, 164-167, 170,172). As the figures show, all these
nuclei which contain two nucleoli are more or less of the same
size. Nuclei which are a little smaller than these, as well as
those which are larger, invariably contain a single nucleolus.
1 The chromatin filament has considerable thickness and is apparently a con-
tinuous thread ; it is looped around the inner surface of the nuclear membrane.
450 MONTGOMERY. [VoL. XV.
It is probable that the two nucleoli of such nuclei have not
arisen by division from a single nucleolus, but are nucleoli
which have been developed at different points in the nucleus
and which are destined to fuse together later and form a single
one. This assumption was based upon the observation of
nuclei where two nucleoli lie at opposite poles of a nucleus
(Fig. 166) and each is apposed to the nuclear membrane, or
where only one occupies such a peripheral position, the other
being in the center of the nucleus (Fig. 164). In one figure
(Fig. 165) we see a nucleus in which the two nucleoli lie near
the center, close together, which might denote the beginning
of such a fusion. On a little reflection this explanation of the
presence of two nucleoli will appear quite allowable. In the
more usual mode of development a larger nucleolus is formed
at the periphery of the nucleus, wanders towards its center, and
then much smaller masses of nucleolar substance are similarly
formed and later fuse with the large nucleolus ; while in the
cases under consideration two nucleoli of nearly equal size are
produced, either simultaneously or in succession, and these
afterwards fuse together. These two nucleoli of nearly equal
size cannot be division products of a single primitive nucleolus,
since two nucleoli are never found in the larger germinal
vesicles.
The nuclear sap of the smaller germinal vesicles does not
stain at all; in the larger ones (Figs. 168, 173-175, 177) it
does, and the explanation for this staining may be given by the
assumption that there is a dissolution of nucleolar substance
throughout the whole nucleus, 7.¢., of that substance of the
assimilated yolk balls which does not enter into the formation
of the nucleoli. During the mitotic stages no constituents
of the nucleus stain except the nucleolus and the chromatin
filament, but these do not stain in the same manner.
At first sight the heteronemertean Lizeus seems to differ
markedly from all the metanemerteans here examined, in that
it contains a single, enormous germinal spot. But in Lzneus,
though a single large nucleolus is first formed, it nevertheless
grows by the addition to it of much smaller nucleolar globules
(Nut. Gl., Figs. 168, 174, 177) which have the same method of
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 451
formation and fuse with the former. Were these secondary
nucleolar globules in Lzwews as large as the first-formed
nucleolus, and were they all to remain separate from one
another, the nucleolar metamorphosis in this genus would
correspond to that of the metanemerteans ; accordingly, the
difference in the nucleolar production is not very important.
(For the nucleolar relations in the other nemerteans, ci my
reviews of the papers of v. Kennel, Hubrecht, Coe and Biirger.')
9. Siphonophore (Rodalia ?).
(Plate 26, Figs. 204-212.)
(Dr. Conklin kindly loaned me the preparations on which his
earlier studies were based ('91); these were preserved in alcohol
and stained with haematoxylin.)
There were no very young stages of the ovogenesis in this
specimen ; I have studied the ova in the egg pouches and in
the gonophores, each gonophore containing a single large ovum
(as shown by Conklin and Brooks), while in the egg pouches a
number of smaller ova may be present.
A single large nucleolus is contained in each germinal
vesicle. This is not only large in relation to the size of the
nucleus, but is also absolutely probably one of the largest
nucleoli ever described in animal cells (Fig. 212). It is always
excentric in position, though seldom close to the nuclear mem-
brane. In those younger stages where the nucleus is still near
the center of the egg (Fig. 205, and the dorsal cell of Fig. 211)
1 The only other observations of the yolk development in the nemerteans are
those of Biirger ('90) on Drefanophorus. Near the young germinal vesicle lies in
the cytoplasm a homogeneous, deeply staining body, of smaller size than the
nucleus, which Biirger assumes may correspond to a yolk nucleus. This body
disappears, ‘und es sammeln sich namlich, dem Keimblaschen anliegend, in jenem
[Plasmahiigel] kuglige oder langliche, tropfchenahnliche Gebilde an, erst sparlich
ein einziges, zwei und mehrere, spater aber mit dem immer noch fortschreitenden
Wachstum des Keimblaschens sich zahlreich vermehrend in grésster Menge. Sie
sind durchaus homogen, von mattem Glanze und ausserst tinktionsfahig. . . .
Erst nach der Entwicklung des Keimblaschens geht die des Deutoplasmas vor
sich und zwar nun auf Kosten der glanzenden Dotterballen, welche aufgebraucht
werden und so im reifen Ei verschwinden.” In the ripe egg the cytoplasm is
granular and stains lightly.
452 MONTGOMERY. [VoL. XV.
the nucleolus is usually nearer the center of the nucleus than
in those more mature stages where the nucleus lies near the
periphery of the cell. But in the more mature stages the
nucleolus may lie at the animal pole, or the vegetal pole, or
at one side of the nucleus, so that no coincidence between
the position of the nucleolus and the age of the nucleus can be
determined. Thus the nucleolus stands, ¢.g., in no relation to
the animal pole of the more mature nucleus, that pole where
amoeboid processes are produced (Figs. 204 and 209). The
ground substance of the nucleolus is dense and homogeneous,
and stains quite deeply; the nucleoli of the smaller germinal
vesicles stain, as a rule, less intensely. In the ground sub-
stance of all the nucleoli more or less numerous fluid globules
occur, which stain very faintly or not at all, and their presence
gives a vacuolated appearance to the nucleolus ; those within
the same nucleolus are of unequal size, and among them two
or three usually occur which far exceed the others in size.
Occasionally there is one large central vacuole (Fig. 206), but
as a rule the larger ones are peripheral, and may produce prom-
inences of the surface of the otherwise perfectly smooth and
spherical nucleolus (Figs. 209 and 212). In one large vacuole
(Fig. 212) a finely granular mass was found, though this may
have been an artefact. Since in the smaller nucleoli these
vacuoles are less numerous and smaller in size, it would seem
probable that in stages antecedent to those found by me the
nucleolus may be wholly devoid of such vacuoles. The nucleo-
lus has no enveloping membrane, for what at first view appears
to be such a structure careful study shows to be merely the
result of refraction.
In addition to the single large nucleolus described, there are in
the most mature nuclei also from about one to five minute nuclei
(Fig. 209). These vary somewhat in size, are perfectly spher-
ical and homogeneous, without vacuoles, and stain more deeply
than the larger one. Sometimes they are found in close con-
tact with the nuclear filaments (cf the nucleoli of the second
generation in Zetrastemma catenulatum and the observations
of Riickert (92) on the germinal vesicles of Se/achiz). These
probably have no genetic relation to the large nucleolus, since
No.2.) COMPARATIVE CYTOLOGICAL STUDIES. 453
they never lie in contact with the latter and are frequently
situated at some distance from it. Were they buds from the
large one, one would expect to find in them vacuoles such as
occur in the large nucleolus, but they never contain vacuoles.
In one nucleus (Fig. 207) I saw a disc-shaped mass apposed to
the inner surface of the nuclear membrane, which stained more
intensely than the chromatin. Such a peripheral mass may
be regarded as a substance taken up from the cytoplasm by
the nucleus, which, after passing through the nuclear mem-
brane, undergoes a chemical change to such an extent that it
stains with haematoxylin. The minute nucleoli may stand in
a genetic connection with such a mass of substance, that is, be
portions of a substance assimilated by the nucleus and after-
wards scattered through the latter. They might serve as
nourishment for the chromatin threads with which they are
often in contact.
In seven nuclei out of about one hundred or more examined the
large nucleoli differed much from the ordinary type described
above. Inone egg pouch there was a smaller ovum apposed to
the animal pole of a larger one (Fig. 211); a normal nucleolus
was present in the nucleus of the smaller one. But in the larger
ovum two nuclei were present, in close contact with one another,
though separated by a membrane (coalesced nuclear membrane).
It is in each of these latter nuclei that an abnormal nucleolus
is present. Each of these nucleoli is finely granular, without
enclosed vacuoles, and stains faintly with haematoxylin ; the
one is regular in outline, but the other is jagged at one pole,
and a ring-shaped portion of its substance stains more deeply
than the remaining portion. In another ovum I also found two
nuclei, in each of which was a nucleolus similar to those just
described. In still another ovum two nuclei were found in
contact with each other, the nucleolus of one of which was
similar to those here described, but the nucleolus of the other
nucleus was intermediate in structure between these and the
ordinary type of nucleoli (Fig. 210). In only one case was
such an abnormal nucleolus present within an ovum contained
in a gonophore (Fig. 208); in the other six cases the abnormal
nucleoli were in ova of egg pouches.
454 MONTGOMERY. (VoL. XV.
Now what do these lightly staining, granular nucleoli repre-
sent? In all except the seven cases here mentioned the nucleo-
lus was always of the deeply staining, vacuolar type, irrespective
of its occurrence in ova of egg pouches and of gonophores.
The abnormal nucleoli, with one exception, were found in the
largest ova of the egg pouches. Types intermediate between
the two are represented in Fig. 210. Conklin and Brook’s
observations, which I can corroborate, show that a number of
ova are produced in an egg pouch, but that only one of these
passes into a gonophore, and there develops into the ripe ovum,
while the others remain behind in the egg pouch and do not
reach maturity, but degenerate. I would hold that the abnormal
nucleoli described by me are degenerating nucleoli of degener-
ating ova. All the facts seem to favor such an explanation.
The cytoplasm of the youngest egg cells appears finely granu-
lar (it may be an alveolar meshwork). In the largest it was
coarsely vacuolar, especially near the center of the cell; I find
no evidence of yolk. Conklin and Brooks evidently mistook
the vacuoles of the cytoplasm for yolk globules.
No chromatin threads were apparent in the smallest germinal
vesicles (Figs. 204-206), but only a fine granulation in the
nuclear sap ; chromatin threads make their appearance gradu-
ally in the larger ova (Figs. 207, 209, 211) and stain more
intensely as they increase in number and size. Each thread
often has the form of a chain of transversely placed discs ; or
sometimes it would seem to consist of a large number of short
fibrils, placed at right angles to a common longitudinal axis, as
is the structure of the chromosomes of the Selachian egg.
These threads usually make their first appearance in the neigh-
borhood of the nucleolus, from which they sometimes radiate
outwards ; only in the largest nuclei are they more generally
distributed throughout the nucleus. This fact might show a
physiological relation between these two structures. But there
is in all probability no genetic connection between the two ;
rather, the chromatin threads are built up of the minute micro-
somes found in the nuclear sap of the smaller ova. But the
formation of the chromatin threads must be determined by the
investigator who has more abundant material at his disposal,
No. 2:] COMPARATIVE CYTOLOGICAL STUDIES. 455
and material which has been more advantageously fixed and
stained.}
10. Polydora.
(Plate 28, Figs. 249-281.)
The egg cells of this form, as those of most Polychaeta, are
derived from the peritoneal cells of the body cavity, the latter
cells building pseudoepithelia around the intestine, as well as
occurring free in the body cavity. Those in the pseudoepi-
thelia (Fig. 249) are more or less flattened, disc-shaped, while
the free cells (Figs. 250-254) are oval in shape, with more
regular outlines. Their cytoplasm is not dense, and one or several
large vacuoles are frequently found at the periphery of the cell ;
a delicate cell membrane is present. The cytoplasm of these
sexually indifferent cells does not stain with haematoxylin. The
nucleus is small, irregular in outline, and contains a fewchromatin
granules ; very frequently the greater part of this substance lies
close to the nuclear membrane. I have never found more than
one minute nucleolus, and this is almost always close to, or in
actual contact with, the nuclear membrane (Figs. 251, 252, 254);
in many nuclei I failed to find nucleoli, though in these cases they
may have been obscured by the chromatin. I found one divi-
sion stage of a nucleus (Fig. 249) ; there were two daughter-
nuclei of the same size and form lying close together ; the
nucleolus of each was somewhat elongate in form (in all others of
these cells examined it is spherical), which might show that the
nucleoli had been produced by the division of a single one in the
mother-nucleus. In many of the smaller free peritoneal cells
a peculiar body often occurs (JV. P. Fig. 253). , This is always
smaller than the nucleus, more or less spherical, often homo-
geneous in appearance, and it may stain either deeply red with
eosin or faintly with haematoxylin, or in other cases it may not
stain at all, but appear as a light yellowish, refractive mass.
From the comparative study of a large number of cells contain-
ing these bodies it may be determined that they are degener-
ated nuclei or portions of nuclei. Thus in Fig. 250, which
1 For other observations on nucleoli of Siphonophora, cf, besides the paper by
Conklin and Brooks, the review of O. Hertwig ('78b).
456 MONTGOMERY. [VoL. XV.
probably represents the commencement of such a degeneration,
there lies close to the nucleus what seems to be a much smaller
nucleus, or a portion of one; and I have found all intermediate
stages between such a body, which is granular and stains with
haematoxylin, and the body reproduced in Fig. 253, which
appears nearly homogeneous and stains with eosin. These
bodies then seem to be degenerated or cast-off portions of
nuclei. We might conclude also that the cells in which these
structures are found, are themselves fated not to develop into
egg cells, even if they are not degenerating ; for no such bodies
are to be seen in the cytoplasm of the true egg cells.
These peritoneal cells have the morphological value of ovo-
gonia. Those which are destined to become ova seem to
become detached from the pseudoepithelial connection, but in
such a way that they do not become detached singly, but
portions, each of which is composed of a number of cells,
become loosened from the epithelium. Thus the earliest ovo-
genetic stages are to be found in strings of cells arranged
radially around a common longitudinal axis, each such string of
cells situated free in the body cavity (Fig. 270 represents a
portion of such a string). At the one end of such a cellular
string lie, densely grouped, the numerous mitoses of the
ovogonic stages, while the remaining portion of the string is
usually composed of young ova, sevsu strictiovi. I have never
found mitoses in cells which lie singly in the body cavity.
The first change noticeable in the ovogonium leading to the
formation of the ovum consists in (1) the increase in the size
of it and of its nucleus, and (2) in its cytoplasm gradually stain-
ing with haematoxylin. This deep blue staining of the cyto-
plasm, accompanied by its increasing density and the loss of the
vacuoles in it, continues from now on until yolk granules begin
to arise in it, when the cytoplasm commences to stain faintly
with eosin and loses its dense structure. At the conclusion of
the ovogonium rest stage the nucleolus has increased a little
in size, accompanying the growth of the nucleus.
The next stage is a mitosis. Whether there is more than
one mitotic generation separating the ovogonium from the
ovum I have not been able to determine; the slight differences
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 457
in the size of the mitoses hardly afford a satisfactory criterion
for deciding this point (Figs. 255-261). All the typical stages
of the prophase and metaphase are to be found, though only in
the arrangement of the chromatin, for I have been unable to
find either centrosomes or achromatic spindle. After careful
study of a large number of these dividing nuclei I find the
nucleolus to persist in the nucleus throughout the mitosis.
Further, it appears to retain its original size throughout this
process, without any diminution in volume. Thus the nucleolus
seems to be retained without change in the spirem and aster
stages of the prophasis. In the dyaster stage (Fig. 258) each
pole of the nucleus usually contains a nucleolus, so that the
nucleus contains two nucleoli; and when the nuclear divi-
sion is completed, z.e., when in one and the same cell two
nuclei occur in close contact with each other, in the aster as
well as in the spirem of the metaphasis, each daughter-nucleus
has its own nucleolus (Fig. 257). Now the ovogonium contains
only one nucleolus, so that we must assume either (1) that a
division of the nucleolus has taken place during the mitosis, or
(2) that to one of the daughter-nuclei is allotted the whole
original nucleolus, while in the other nucleus a new one is pro-
duced. I have not seen any dividing nucleoli in these mitoses,
their small size being a great obstacle to their study. But I
should judge that such a division occurs, for these reasons :
(1) the nucleus of one or of both the daughter-nuclei has
sometimes a somewhat elongate form (Fig. 257); and (2) in
later stages of the ovum proper I have found dividing nucleoli,
and these cases would show that if such divisions take place in
stages subsequent to the mitosis they might also occur during
the mitosis. The two cases of division of the nucleolus found
are here figured (Figs. 264 and 265), and in each of the elongate
nuclei is a dumbbell-shaped nucleolus lying in the longitudinal
axis of the nucleus ; in these figures the two halves of each
nucleolus appear unequal in dimensions, but this is so because
neither of these nucleoli happened to lie wholly in the plane of
the section. I have found numerous other cases of elongated
nuclei, each with an elongate nucleolus without any median
constriction (Fig. 270). These facts would show that a division
458 MONTGOMERY. [Vou. XV.
of the nucleolus may take place during the mitosis, and
probably does so.
After the completion of the mitosis just described, each
daughter-nucleus, which now has the value of a germinal vesicle,
first passes through the spirem stage of the metaphasis and
then enters upon the stage of synapsis, namely, the nucleolus
has a more or less central position, and all the chromatin of the
nucleus becomes grouped immediately around it (Figs. 264-266,
270, 271, 278), the peripheral part of the nucleus being trans-
versed by only a few fine, unstaining strands of substance
(linin ?). All intermediate grades between this and the preced-
ing stage of the nucleus may be found. This is not an artificial
appearance caused by the use of a particular preservative, since
it is equally demonstrable on preparations fixed with aqueous or
alcoholic corrosive sublimate, sublimate with acetic acid, Flem-
ming’s fluid, and alcoholic solution of picric acid; only after
the use of Perenyi’s fluid is this arrangement of the chromatin
not found, but this fluid seems to be rather a poor one for most
cytological study. It cannot be an artefact, since this appear-
ance is found only in ova of a certain size but not in those
which are larger ; thus it cannot be produced by the resistance
offered by the cell membrane to the penetration of the fixa-
tives, since this membrane is much thicker in the larger ova.
This central arrangement of the chromatin then represents a
definite stage of the germinal vesicle concomitant with the
first appearance of yolk globules in the cytoplasm.!_ So at this
point we may briefly describe the yolk development and then
return to the changes of the nucleolus.
The yolk first arises in the cell during the stage just
described, that is, immediately after the conclusion of the spirem
stage of the metaphasis. It appears in the form of small glob-
ules (Y&. G/., Figs. 262-264, 266, 270, 271), most of which are
arranged close to the outer surface of the nuclear membrane,
the first globules rarely arising at a distance from the nucleus.
At this period they stain less deeply than later. The yolk
1 This stage of synapsis (Moore) appears to be characteristic of the anaphase
of the last spermatogonic and spermatocytic division in all the higher animals,
and no doubt can any longer be expressed of its representing an artefact,
No.2.] COMPARATIVE CYTOLOGICAL STUDIES. 459
rapidly increases in amount, spreading from the region of the
nucleus (which is central) to the cell periphery. In the largest
ovarial ova the cytoplasm is densely filled with larger and
smaller yolk globules; the larger ones appear homogeneous
when stained with eosin (Fig. 269), but the Ehrlich-Biondi stain
shows them to be composite masses of small globules.
The nucleolus rapidly increases in size, at a somewhat greater
proportionate rate than the nucleus itself. It is now large
enough for its structure to be clearly made out : it consists of
a homogeneous ground substance, which seems to stain more
deeply with eosin as it grows larger; a limiting membrane is
clearly demonstrable in the largest nucleoli (Figs. 271-277,
279-281) after staining by the Ehrlich-Biondi method or
after fixation with Flemming’s fluid, though it does not differ
chemically or in structure from the ground substance and is
only a thin layer of the latter in which vacuoles never occur.
At the close of the metaphasis of the mitosis small vacuoles
make their first appearance in the ground substance of the
nucleolus (Figs. 263 and 270). There are only a few of them at
the start, but their number rapidly increases as the nucleolus
grows larger, until there are large numbers of them in its center
(Figs. 268 and 269). They are always more numerous at the
center than at the periphery of the nucleolus, and usually first
appear at the former point. On preparations stained with
eosin the small vacuoles appear either as clear spaces or as
black granules, according to the focusing of the microscope ;
after the use of the Ehrlich-Biondi stain they become a light
grayish color (note the contrast, —that in the eggs of Doto and
Montagua the nucleoli appear as black granules only after
treatment with the latter stain) ; after fixation in the fluid of
Flemming the substance of these vacuoles is of a lighter color
than the ground substance. This vacuolar substance is homo-
geneous, and is probably of a thin, fluid nature. With the
growth of the nucleolus the number of the vacuoles becomes
very great, though their size does not seem to increase. In the
nucleoli of the largest germinal vesicles examined the vacuoles
no longer retain their original spherical form, but become mutu-
ally confluent to some degree, not in such a manner as to pro-
460 MONTGOMERY. [VoL. XV.
duce one or a few large vacuoles, but as to produce an irregular
canicular network of vacuolar substance in the nucleolus (Figs.
272-277, 279-281). This process often goes so far that in the
largest nucleoli the deeply staining ground substance may
appear in the form of a skein of threads, or merely of scattered
granules surrounded by vacuolar substance. Especially on
preparations stained by the Ehrlich-Biondi method is the skein-
like arrangement of the ground substance well marked. I
have no doubt that it was the observation of similar nucleoli in
like stages which led Carnoy to the assumption of a “ nucléole-
noyau,” that is, a nucleolus with a limiting membrane, and
containing a wound thread of chromatin ; it is probable that
Carnoy mistook the reticulum of the true ground substance of
the nucleolus for chromatin, and considered what is really vacuo-
lar substance to be the original ground substance; only
studies on the genesis of a nucleolus can explain its various
components. ;
In the largest ova found in the body cavity the nucleolus
reaches its maximum size (Figs. 279-281). It contains a greater
amount of vacuolar than of ground substance, and instead of
being regularly oval, as it was before, is often quite irregular
in form, and very frequently apposed to the nuclear membrane
(a position not noticed in any of the preceding stages). Whether
this irregularity of form denotes the commencement of a degen-
eration of the nucleolus I cannot say, since I had no prepara-
tions of the stages of reduction.
Two nucleoli were found in only two germinal vesicles (Figs.
262 and 266), and in a spirem stage of an ovogonium three small
nucleoli were present in one nucleus (Fig. 261). In the hun-
dreds of other resting nuclei examined a single nucleolus was
invariably present. These exceptional cases must, therefore,
be considered abnormal, and not typical for certain stages of
the nucleus.
In the larger germinal vesicles there is a peculiar body in
contact with the nucleolus, which remains to be described.
This body (zx., Figs. 272, 274-277, 279, 281) is homogeneous,
somewhat refractive, and lies either in close contact with the
surface of the nucleolus, projecting beyond the periphery of the
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 461
latter, or (and this is the rule for the largest, irregular nucleoli)
it is imbedded in the peripheral portion of the nucleolus ; in
the former position it is concavo-convex, in the latter, bicon-
vex in outline, always being thickest in its median diameter.
With the Ehrlich-Biondi staining method it almost invariably
colors yellowish, and in only one or two cases did it stain
somewhat similarly to the ground snbstance of the nucleolus ;
after fixation in Flemming’s fluid, and staining with safranin,
gentian violet, and orange G., it always appeared yellowish,
while the ground substance remained wholly unstained. The
largest nucleoli, z.e, those of the largest germinal vesicles,
have always at least one of these bodies in contact with their
surface, but quite frequently two may be found on opposite
sides of the nucleolus, and in one case I found three (Fig. 277).
Those of different nucleoli vary slightly in their dimensions,
but my observations give no clue as to their origin. All that
can be said of their growth is that in the smaller nucleoli they
lie upon the surface of the latter, while they are sunk into the
peripheral portion of the larger nucleoli. It differs both chem-
ically and structurally from the ground substance of the nucleo-
lus, and from the vacuolar substance ; and it would seem to be
derived from some part of the nucleus outside of the nucleolus,
since it at first lies upon the surface of the nucleolus. This
body may be comparable to the ‘ Nebennucleolus ’’ described
by Flemming in the egg of Axodonta; but I have found no
structure in any of the other ova here examined which is
identical with it.
Yolk globules are assimilated by the nucleus from the cyto-
plasm, though without the production of amoeboid processes.
Such assimilated globules are usually of small size, but some-
times large, compound ones are taken into the nucleus (Figs.
267-269, 272, 274, 280); they occur most frequently singly or
in small masses close to the inner surface of the nuclear mem-
brane (Figs. 274 and 280) in almost all of the larger germinal
vesicles, and in a few cases some globules may be found near
the center of the nucleus. Careful observation shows that the
yolk globules really occur within the nucleus, and are not arti-
ficially removed there by the knife in sectioning. Usually these
462 MONTGOMERY. [Vou. XV.
stain in the same manner as those contained in the cytoplasm.
But occasionally from one to three of the larger globules (Fig.
267) in the nucleus stain much more intensely than the others,
though intermediate degrees of staining are to be found between
these largest, most deeply colored ones and the smaller, less
deeply stained ones; so that there can be no doubt of the
genetic relation of the two kinds. By staining with eosin
these largest yolk globules in the nucleus stain almost or quite
as deeply as the nucleolus itself, so that at first I mistook them
for nucleoli ; but that they are chemically metamorphosed yolk
globules and not nucleoli is shown, even leaving aside the fact
that all intermediate forms may be found between them and
the less deeply staining globules of the cytoplasm, by the fact
that vacuoles are never found within them. By the Ehrlich-
Biondi staining method no color differentiation was to be
obtained for the larger and smaller yolk globules of the nucleus.
But nevertheless I would think that these large yolk globules
(or accumulations of such globules) which have been taken
into the nucleus from the cytoplasm and there have undergone
some degree of chemical change, possibly stand in genetic
connection with that body which is apposed to the nucleolus
in the larger germinal vesicles, and which has been described
in the preceding paragraph.
Chromatin. — We found the chromatin in the primitive peri-
toneal cells and in the youngest ovogonia to be arranged in the
form of granules (Figs. 250-254). In the following mitoses it
is arranged in the form of a spirem, then of chromosomes, and
again of a spirem (Figs. 255-261). Just after the conclusion
of the spirem stage (of the metaphasis) it comes to lie in a
more or less dense mass around the nucleolus, this mass
appearing to be composed of a reticulum of short fibers (Figs.
263-266, 270, 271, 278). In all these stages the chromatin is
marked by its deep blue staining with haematoxylin. After
the last stage described it gradually departs from the close
vicinity of the nucleolus and becomes evenly distributed
throughout thenucleus. But when it has thus become diffused
it does not stain with haematoxylin as before, but appears in
the form of a very large number of minute microsomes, which
No. 2.) COMPARATIVE CYTOLOGICAL STUDIES. 463
appear not to stain at all, and of a few delicate fibers, which
stain a lilac color (Figs. 267-269). As the germinal vesicle
increases in size these chromatin fibers gradually become
thicker and more numerous, commence to stain more deeply
with haematoxylin, and gradually connect together to build
a chromatin reticulum; the minute, unstained microsomes
still occur between these fibers. Finally, in the largest nuclei
at my command, and ones which had been fixed with the fluid of
Flemming and stained by the triple stain of this cytologist, we
find, in addition to the abundant unstained microsomes, short,
rod-like masses of chromatin, which stain deeply with gentian
violet, and each appears to be formed of a row of granules or
thickened discs (Fig. 280). Whether the minute microsomes
are true chromatin or are lanthanin (oedematin) granules is
open to question ; the latter assumption might be the correct
one. We notice two remarkable phenomena in the chromatin
changes just depicted : (1) the grouping of the chromatin in
the center of the nucleus, around the nucleolus, at the comple-
tion of the mitotic stages ; and (2) immediately subsequent to
the preceding, the lilac stain of the chromatin after haematoxy-
lin. Now, concomitant with the former of these two phenomena,
the yolk makes its first appearance in the cytoplasm, and as we
have shown above, usually in the close vicinity of the nucleus.
It would be quite erroneous to conclude that the yolk globules
are in any way produced by the chromatin, as e.g., by a migra-
tion of chromatin particles out of the nucleus ; for in this stage
all the chromatin is removed from the periphery of the nucleus.
On the other hand, however, I would suggest the hypothesis
that the reason for the chromatin being removed from the
periphery of the nucleus is because at this period the peripheral
portion of the latter is chiefly concerned in the assimilation of
yolk substance from the cytoplasm. In support of this assump-
tion the fact may be recalled that in the following stage the
chromatin fibers are stained a lilac color, as if they were stained
with eosin, as well as haematoxylin, and not as before, simply
with the former stain ; this would show that during this period
there is an addition of a cytoplasmic substance to the chromatin
fibers, perhaps allied to the substance of the yolk globules, and
464 MONTGOMERY. [VoL. XV.
this substance would superinduce the lilac staining of the
chromatin threads. This addition of a probably nutritive
substance would seem necessary, in order that the amount of
the chromatin continue to increase as the nucleus itself grows
larger. Subsequently all that nutritive substance attached to
the chromatin threads would seem to become metamorphosed
into chromatin, since in the largest germinal vesicles these
threads again stain a deep blue. And as a matter of fact, the
quantity of the chromatin must increase with the growth of the
ovum, since it can easily be demonstrated that in the larger
nuclei there is an absolutely greater amount of this chromatin
present than in the nuclei of the primitive peritoneal cells.1
11. Péscicola rapax (Verr.) (=Pontobdella rapax Verr., which
Dr. Percy J. Moore assures me is a true Pisczcola).
(Plate 29, Figs. 300-316.)
(The ovary is a tubular, contorted sack; from its inner sur-
face numerous smaller, likewise tubular (round on cross-section),
acini project into its cavity, each acinus containing numerous
ovogenetic stages, the least mature of which lie at its proximal
end, the most mature at its distal. These several acini are
not continued as far as the external opening of the ovary, but
their distal ends apparently open into a large ovarial cavity,
and the ova drop into this cavity before they can arrive at the
external genital opening. Each single acinus of this leech may
be compared to either of the two whole ovaries of Ascaris.)
The youngest ovarial stages are small ovogonia in stages of
mitotic division (Fig. 300). In them no nucleoli were to be
seen; a minute nucleolus might be present in each of these
nuclei and be obscured by the dense mass of chromatin. In
all stages subsequent to these a single nucleolus is present in
the nucleus (now a germinal vesicle) until the pole spindle is
formed ; in the smaller nuclei the nucleolus is usually oval, in
the larger ones spherical. The growth of the nucleolus keeps
1 For the researches of other authors on germinal spots of polychaetous anne-
lids, cf. the reviews of the papers of Korschelt ('89, '95), Graff ('88), Giard ('81),
Vejdovsky ('82), Eisig ('87), Fraipont ('87), Mead ('95), Fauvel ('97), Michel
(96), and Carnoy ('84),
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 465
pace proportionally to that of the nucleus (Figs. 301-304).
Then vacuoles arise in the nucleolus, these being neither very
numerous nor very minute (Figs. 304-310, 312-316). The time
when these vacuoles first arise is very variable, though in the
majority of cases they do not appear before the nuclear sap
begins to stain red. The size of the nucleolus does not
always stand in the same proportion to that of the nucleus.
Its ground substance is dense, stains deeply with eosin, and
no limiting membrane is present; but by the use of the double
stain Lyons blue and acid carmine, whereby the nucleolus
stains blue and the chromatin red, a deep red line appears to
surround the nucleolus: I cannot determine whether this
line is a nucleolar membrane or a layer of chromatin, or
whether it is merely an appearance caused by the refraction
of the nucleolus.
When the nucleolus first appears it is usually situated at
that pole of the nucleus opposite the chromatin mass and is
not in contact with the nuclear membrane (Fig. 301). In
nuclei of intermediate size, before the nuclear sap commences
to stain with eosin, it is most frequently in contact with the
nuclear membrane (Figs. 302-304); but in the largest ger-
minal vesicles it is never in contact with this membrane,
though often lying excentrically in the nucleus.
As soon as the germinal vesicle has nearly, or quite, attained
its maximum dimensions (quite frequently, however, in those of
still smaller size) two very noticeable changes take place in it:
(1) the chromatin assumes a different form and stains differ-
ently (these chromatin changes shall be delineated later); and
(2) the nuclear sap, which had heretofore remained colorless or
was merely of a light lilac shade (by the double stain haema-
toxylin and eosin), now becomes a yellowish-red color, so that
the nuclei in this stage may be easily distinguished from those
of preceding ones (Figs. 304, 305, 307-310, 316). Simul-
taneously two changes occur in the nucleolus: (1) it stains no
longer a deep red with eosin, as before, but a yellowish red,
and appears more refractive; and (2) the several vacuoles
within it gradually fuse together and so produce a larger one,
which has usually a central position. The fluid, structureless
466 MONTGOMERY. [VoL. XV.
substance of the vacuole stains more faintly than the ground
substance of the nucleolus, and has much the same color shade
as the nuclear sap. In certain germinal vesicles, which appear
to be of a somewhat later stage of development, numerous
small globules (z.D., Figs. 306 and 310) are scattered through
the nuclear sap; they stain with eosin a little more deeply than
the last-named nuclear portion, vary in number and size, and
have no regular distribution. In one case (Fig. 316), which
stood in a stage immediately antecedent to the pole spindle
formation, where there was a centrosome at either end of the
nucleus in the cytoplasm (the nuclear membrane had not yet
disappeared), such globules were present in the nucleus; so
that we may infer that these globules are one of the latest
formations in the germinal vesicle before the pole spindle is
formed. I have not found any stages between the stage just
described and the perfectly formed spindle (Fig. 311). About
fifty or sixty ova were examined in the stage of the first pole
spindle, and in all of them the nucleolus had completely
disappeared, and no trace of it could be found either in the
nucleus or in the cytoplasm.
What has been the manner of this disappearance of the
nucleolus? Its total disappearance must occur within a rela-
tively short time, since otherwise one would expect to find
stages showing this process. The observations which I was
able to make would demonstrate at least the mode of the com-
mencement of the vanishing of the nucleolus. We have seen
that when the germinal vesicle has attained its greatest size
or, in some cases, a little before its maximum size is reached,
its nuclear sap stains red; therefore some substance must be
suspended in the caryolymph at this period which was not con-
tained in it before. Now such a substance must have been
derived either from other elements of the nucleus or from
the cytoplasm. It has probably not been derived from the
cytoplasm, since the nuclear membrane at this stage has its
maximum thickness and hence could not be easily penetrable ;
and also there is no appearance of any similar substance in the
cytoplasm, since no yolk globules or other nutritive elements
seem to be present, but the whole cytoplasm (at least the
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 467
nodules of its meshes) stains a lilac-blue color. And since
it is wholly improbable that it should be derived from the
chromatin we must conclude that it takes its origin from
the nucleolus. In other words, a substance emanates from the
nucleolus and dissolves in the nuclear sap, and this process
must be regarded as the commencement of the dissolution of
the nucleolus. In support of this conclusion is the fact that
in many germinal vesicles the nuclear sap stains most intensely
in the neighborhood of the nucleolus (Fig. 309). Further, the
minute red-staining globules which later occur in the nuclear
sap must also be nucleolar in point of formation, z.e., be either
a substance given off in globular form from the nucleolus, or
be accumulations (perhaps chemically changed by the action of
the nuclear sap) of that nucleolar substance which has already
diffused through the nucleus. Of importance in this connec-
tion is the fact that these globules are often found in contact
with the nucleolus (Figs. 306 and 316). In all preceding stages
the nucleolus is regularly oval or spherical in outline, but in
the largest germinal vesicles not only may the size of its con-
tained vacuole be increased to such an extent that the original
ground substance forms only a thin shell around it (Figs. 308,
312, 314), but also its outline becomes frequently irregular
(Fig. 313); and in one case I found it broken at one pole, so
that its large vacuole communicated with the cavity of the
nucleus (Fig. 315). A morphological change in the shape of
the nucleolus which seems to take place with great regularity
consists in the indentation of the nucleolar wall at that point
where it is thinnest (Figs. 308, 314, 316). It would seem that
the pressure from without, z.e., the pressure of the nuclear sap,
being greater than the pressure of the fluid within the vacuole,
would cause the nucleolar wall to be pressed in at that point
where it is thinnest. The fact remains that the nucleolus
persists in the nucleus until a very short time before the pro-
duction of the pole spindle, and when the latter is formed no
trace of it can longer be found in any part of the nucleus or
cell. And since there is no reason for supposing that it is
extruded from the cell we must assume that it dissolves
within it. The red-stained substance and small globules
468 MONTGOMERY. [Vou. XV.
within the nucleus would show that dissolution commences
in the nucleus; and we must assume that when the nuclear
membrane has disappeared the cytoplasmic substances which
then come into contact with the nucleolus would cause its
rapid and total dissolution. It may be remarked that in the
region of the fully formed spindle (Fig. 311) no trace of the
red-stained nuclear sap is longer to be seen; accordingly this
sap with its contained nucleolar substance must either have
been distributed through the cytoplasm or have been chem-
ically changed by that portion of the latter which immediately
surrounds the spindle.
In the ovary no ova are to be found which have advanced
beyond the production of the first pole body, so that the forma-
tion of the second pole body must occur after the egg has been
discharged from the ovary; I had no material at hand to enable
me to determine the relation of the nucleolar substance in the
female pronucleus.
Of considerable morphological interest are the metamor-
phoses of the chromatin in the various ovarial stages. In those
small ovogonic mitoses (Fig. 300) from which the true egg cells
(first ovocytes) are derived aster and dyaster stages are to be
found ; with the lens used for this study (the homogeneous
immersion ;!; of Zeiss) I could not determine the form of the
chromosomes. As the ovum increases in size the dense chro-
matin mass of the aster gradually loosens, until up to the time
when the nuclear sap commences to stain red (Figs. 301-304)
the chromatin is arranged in the form of rather numerous
granules, which are situated mostly close to the nuclear mem-
brane. Thus far the chromatin has stained intensely blue, with
the double stain haematoxylin and eosin ; but when the nuclear
sap begins to stain with eosin a marked change takes place in
the character and arrangement of the chromatin ; it now stains
a lilac color, often more reddish than bluish, and has no longer
a peripheral position, but becomes arranged in the form of
threads, sometimes in the form of a small number of loops, the
two ends of each loop being joined together (Figs. 304, 305,
307, 309). In some of the larger germinal vesicles absolutely
no trace of chromatin can be found (Fig. 316). In the equator
No. 2.] COMPARATIVE CYTOLOGICAL STUUVIES. 469
of the first pole spindle (Fig. 311) lie twelve small chromosomes,
which stain an intense blue black with haematoxylin and have
an oval or slightly elongate form. It remains for investigators
working with more abundant material and with stronger micro-
scopical lenses, to penetrate more deeply into these phenomena
of the chromatin changes, but it would seem that the chromo-
somes of the first pole spindle have the value of either tetrads
or dyads. The lilac or even reddish stain of the chromatin at
a particular period would seem at first sight to be due to the
assimilation by the chromatin of that nucleolar substance dif-
fused in the nuclear sap ; but even as probably it might be due
to the mere penetration of this substance between the individ-
ual microsomes of each chromatin thread, without any chemical
change of the chromatin substance (Fig. 309). The red-
staining globules in the nuclear sap, which I have assumed
to be of nucleolar derivation, cannot be considered as meta-
morphosed portions of chromatin substance, since they vary
so considerably in size and number ; this point needs to be
emphasized, since in some of the larger germinal vesicles no
trace of chromatin is.to be seen, and it might be thought by
some one that these globules, which occur in such nuclei, repre-
sented the supposedly absent chromatin. (Platner, '89c, had,
in Aulastomum seen only nucleolar fragments and overlooked
the true chromosomes.) Where in the largest germinal vesi-
cles, before the formation of the pole spindle, the chromatin
appears to be absent in the nucleus, we must assume that it
is merely obscured by the large amount of diffused nucleolar
substance.
In the first pole spindle (Fig. 311), after treatment with
Flemming’s fluid or with corrosive sublimate, the mantle fibers
have a remarkable thickness and appear even thicker than in
Fig. 311 ; they stain a reddish-lilac color with the haematoxylin
and eosin stain, not a lilac blue, as do the rays of the asters
and the cytoplasm ; I could not determine whether they extend
quite to the centrosomes. I am also unable to decide whether
each chromosome lies upon a single spindle fiber which extends
from centrosome to centrosome, or whether its ends are con-
nected with separate fibers. The centrosomes are rather large,
470 MONTGOMERY. [VoL. XV.
refractive granules, and stain with eosin ; they were present in
one egg, close to, and opposite, the two poles of the nucleus,
before the nuclear membrane had disappeared (Fig. 316), so that
they may be extranuclear in origin. The radiations of the asters
are very clear, especially after fixation in Flemming’s fluid, and
may be traced nearly to the cell membrane. Immediately
around each centrosome a central portion of the aster is dif-
ferentiated, namely, an attraction sphere (in the terminology of
van Beneden), and this differs from the remaining portion in
staining less intensely, and appears to be quite sharply bounded
from it. In this attraction-sphere the cytoplasmic granules are
smaller and more densely grouped, so that at first sight it
might appear to consist of a homogeneous “archoplasm,” but
careful study shows that in it the cytoplasmic microsomes are
arranged in radial rows around the centrosome, and each of
these rows appears to be continuous with a ray of the outer
aster. Or, to express it differently, the microsomic rays of the
sphere extend to the centrosome, but this terminal part of each
ray differs from the remaining distal portion in that its micro-
somes are smaller and closer together. Thus in P2sczcola the
finer structure of the attraction-sphere seems to have much
resemblance to that of Ascarzs, as described by Kostanecki and
Siedlecki (Arch. mikr. Anat., 48, 1896).
It remains to describe the mode of arrangement of the ova
within each ovarial acinus. The proximal, small end of the latter
is filled with small ovogonia (the youngest stages), and from
mutual contact these are polygonal in form (Fig. 300). As we
proceed towards the distal end of the acinus (Fig. 301) the ova
not only become gradually larger, but have a different arrange-
ment, in such a manner that they become epithelially grouped
along the wall of the acinus, each cell having a pyramidal
shape, with its apical end directed towards the central cavity
of the acinus. A little more distally in the acinus (Figs. 302
and 303), the ova become not only larger, but fewer of them
are to be found on a given transverse section of the acinus ; the
individual ova have more of an oval shape and become sepa-
rated from one another. Now when we proceed still further
towards the distal end of the acinus (Fig. 304) we find a single
No.2.] COMPARATIVE CYTOLOGICAL STUDIES. A7I
ovum commencing to outstrip the others in point of size, 7.¢.,
in rapidity of growth, until we reach a point where this fortu-
nate cell nearly fills the whole cavity of the acinus, driving the
neighboring ova aside. Those cells which come into contact
with such a rapidly growing ovum, as well as those in more
proximal portions of the acinus which did not chance to lie
close to the wall of the acinus, do not develop further, but
disintegrate, and various stages of such disintegration may be
seen in the cavity of the acinus, such as irregular cells with a
nucleus, those which have lost their nuclei, and finally refrac-
tive cytoplasmic masses which stain deeply with eosin (the
cytoplasm of the developing ova stains with haematoxylin).
Perhaps such degenerated masses of cellular substance are des-
tined to be assimilated by their more fortunate brethren. Often
a number of such degenerating ova are to be seen grouped at
one pole of a large ovum, and these cases present a certain
similarity to cleavage stages, the large ovum resembling a
macromere, the others micromeres. It is not difficult to find
an explanation for the disintegration of certain of the ova, for
only those close to the wall of the acinus can procure nourish-
ment in amount sufficient for their growth, since this nour-
ishment must be derived through the wall of the acinus from
the body cavity (there being no yolk in the ova); and the
peripherally situated ova must obtain all the nourishment thus
furnished, so that those in the center of the acinus simply die
for want of food. Further, a particular ovum of those placed
peripherally, if it procures a greater amount of nourishment
than its neighbors do, because, e¢.g., of being in contact with a
greater surface of the wall of the acinus, grows faster than the
others and, pushing them aside, eventually gets full control of
the whole amount of nourishment, so that a slight advantage at
the start would increase in value in a geometrical ratio. Here,
accordingly, we have a beautiful example of that process termed
by Roux ‘‘der Kampf der Theile ums Dasein,”’ that cell becoming
a mature ovum which has succeeded in obtaining the greatest
amount of nourishment. It is also interesting to note the
position of the nucleus within the growing ovum ; in all the
younger stages of the egg it is placed in that part of the cell
472 MONTGOMERY. [VoL. XV.
which is nearest to the wall of the acinus, z.e., nearest to the
source of the food supply ; only then does it come to occupy a
central position within the cell, when the latter has attained its
maximum size and the thickness of the cell membrane shows
that the cell is assimilating little or no nourishment from
without.!
b. Somatic Cells.
12. Ganglion Cells of Doto.
(Plate 21, Figs. 36-49.)
(I have studied those nerve cells which occur in the cerebral,
pleural, and pedal ganglia. Three kinds of these cells may be
readily distinguished and described in succession : (1) colossal
cells, which are found only in the cerebral ganglion; (2) cells of
medium size; and (3) small cells.)
Colossal ganglion cells (Figs. 43-49). — The number of the nu-
cleoli in the nuclei of these cells varies from about six to thirteen;
they are also variable in regard to the position which they
occupy in the nucleus, and though always excentrically placed
they never lie in contact with the nuclear membrane. Some-
times all the nucleoli in a given nucleus are of approximately
equal size, but as frequently one or two are several times larger
than any of the others. Where such larger nucleoli occur along
with a number of smaller ones, the former are usually vacuolar
in structure ; sometimes nearly all the nucleoli contain vacuoles,
in other cases none of them are vacuolar. Quite often the
nucleoli in a nucleus show slight differences in their staining
intensity, and one of them may stain quite differently from the
rest (Figs. 44 and 46). None of the nucleoli have limiting
membranes. No cases of nucleolar division were found, unless
those cases where two nucleoli lie near to one another may
represent the completion of such a division.
Ganglion cells of medium size (Figs. 37-42). —In these the nu-
cleoli varyin number from one to four, two or three being the rule.
Those of the same nucleus frequently show differences in size
and form, as well as slight staining differences. In only one
1 For the observations of other writers on germinal spots in Hirudinea, cf.
O. Hertwig ('76), Leydig ('49), Whitman ('78), and Platner ('89c).
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 473
case (Fig. 41) I found three nucleoli of approximately equal
dimensions and homogeneous ; usually they vary somewhat in
size and contain vacuoles. The shape of the nucleoli is either
spherical or oval, or it may be irregular; certain ones stain
scarcely at all, and appear granular: these might represent
cases of degeneration.
Smallest ganglion cells (Fig. 36). — Here a single nucleolus
is the rule, though two may occasionally be found. They are
spherical or oval, and vary considerably in size. Vacuoles do
not seem to occur in them, though they might well escape
observation from the small dimensions of the nucleoli, which
often renders it difficult to distinguish the nucleoli from the
larger chromatin granules.
In all these ganglion cells the chromatin appears in the form
of small granules, but on a preparation fixed with Hermann’s
fluid and stained with Lyons blue (Fig. 45) it appeared as a
network ; in this preparation the granules seemed to be united
by fine fibers, which stained less intensely than the granules.
But even here the connecting threads might consist rather
of linin than of chromatin, since the solution of Lyons blue
employed by me stained all the nuclear substances except the
nuclear sap (paralinin). Such fibers often appear to radiate
outwards from the surface of the nucleoli, as if the latter were
suspended by them. The nucleoli always stain differently from
the chromatin.
There is, as a rule, a relatively small amount of nucleolar sub-
stance in the cells of the second and third types in comparison
with most of the other nuclei which I have examined ; but the
nuclei of those of the first type, on the contrary, usually contain
a relatively large amount of this substance, for not only may
one or two of the nucleoli in a nucleus be quite large, but also
a considerable number of nucleoli are frequently present.
13. Ganglion Cells of Montagua pilata (Verr.).
(Plate 22, Figs. 90-97.)
(The same types of cells may be roughly distinguished as in
Doto.)
ATA MONTGOMERY. [Vou. XV.
Colossal ganglion cells (Figs. 90-92, 94—97).—In the nuclei of
these there are never more than from one to three nucleoli,
which neither contain vacuoles nor become noticeably irregular
in size, as is the case in Doto. Most frequently only a single
nucleolus is present. It is the rule that they are oval and not
spherical, though in some cases they may appear perfectly
spherical ; perhaps the great majority of them are oval and
seem to be spherical only when they do not chance to be longi-
tudinally sectioned. Their substance is perfectly homogeneous,
without a limiting membrane. When two or three occur in the
same nucleus they are usually of approximately equal dimen-
sions (Figs. 94 and gs). Further, it would seem to be the rule
that when one nucleolus is present ina nucleus it is larger than
any one of the two or three which may be found in other
nuclei; but, nevertheless, the relative amount of nucleolar
substance seems to vary in different nuclei.
Ganglion cells of medium size (Fig. 93). — Here one or two
nucleoli are present in each nucleus, and these are of homo-
geneous appearance.
Smallest ganglion cells.— The nucleoli are similar to those
of the corresponding cells of Doto.
On a preparation preserved in Flemming’s fiuid I find many
of the nucleoli present a different structure from those fixed
with corrosive sublimate or Kleinenberg’s fluid. Thus many
of them do not appear homogeneous, but finely granular and
refractive (Figs. 96 and 97). On the surface of such nucleoli
occur small, refractive, yellowish globules, which appear black or
yellow, according to the focus of the microscope; some of them
are very small. These never occur within the nucleolus, but
only on its periphery. They may easily be distinguished from
the chromatin granules by their rounded form and high degree
of refrangibility, as well as by their deeper yellow color (this
preparation had been stained with haematoxylin and eosin, but
the nuclei had not become stained, probably owing to too long
a fixation in the fluid of Flemming). Numerous other nuclei
on the same sections showed none of these globules, and none
were to be seen on preparations which had been differently
preserved. Accordingly, I consider them to be artefacts,
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 475
caused by (1) the direct action of the fluid of Flemming, or
more probably (2) they might be post-mortem exudations of the
nucleoli, which might well be produced before the slowly pene-
trating fixative had reached to the cells in question. At any
rate, they cannot be regarded as normal structures. Do they
represent the ‘“ Kernkérperchenkreis”” of Eimer ?
The chromatin, as in Doto, occurs in the form of granules,
which are connected by fine fibers. After fixation with Klein-
enberg’s fluid a clear space encloses each nucleolus (Figs. 93
and 94); but this space is not to be found after fixation in
other fluids.
As in Doto, the nuclei of the colossal ganglion cells contain
a relatively greater amount of nucleolar substance than do those
of the secondand third types. But in the former genus there are
in the colossal cells from about six to thirteen nucleoli, and these
vary noticeably in size and structure, while in A/ontagua there
are only from one to three, which are always homogeneous and
usually quite equal in dimensions. Why should there be this
marked difference in the form and number of the nucleoli ??
14. Ganglion Cells of Piscicola rapax (Vert.).
(Plate 23, Figs. 134-136.)
In the ganglia of the brain occur cells of different dimen-
sions. Each nucleus contains most usually a single small
spherical nucleolus ; seldom are there two present, and in these
cases they are unequal in size. None of the nucleoli contain
vacuoles. They are excentric in position, but are never in
contact with the nuclear membrane. These nucleoli are small
in proportion to the size of the nucleus.
15. Muscle Cells of Lineus gesserensis (O. F. M.).
(Plate 21, Figs. 51-56.)
(The nuclei of the circular muscular layer of the body wall
were studied. Those of Cerebvatulus lacteus Verr. are essen-
1 For other observations on nucleoli in ganglion cells of molluscs, cf the
reviews of the papers of Pfliicke ('95), Leydig ('83), and Rohde ('96).
476 MONTGOMERY. [Vov. XV.
tially similar to those of Lzzeus ; in the metanemerteans they
are too small for satisfactory study.)
These nuclei are very variable in shape, all extremes being
found between ovoid or oval and elongate rod-like forms. But
they are rarely angular. I have remarked in a previous con-
tribution that the nuclei of the muscle cells are more variable
in form than those of the cells of any other tissue in the
nemerteans, and now I would offer the following explanation
for this variability : when the muscle fiber (a single, smooth
fiber with attached nucleus constitutes a muscle cell) contracts,
this contraction must produce likewise a contraction (shorten-
ing) of the nucleus ; but when the fiber expands the form
of the nucleus must become more elongate, corresponding to
the elastic extension of the fiber, for the fiber cannot contract
without causing a shortening of its nucleus, since the latter is
closely adherent to it.
One very small nucleolus is usually to be seen in each
nucleus (Figs. 51-54, 56); sometimes it does not appear to be
present (Fig. 55), but whether in these cases it is absent or only
escapes observation by reason of its minute size, it is difficult
to decide; in the greater number of nuclei it may be seen by
careful focussing of the microscope. It most usually lies very
close to the center of the mass of nucleoplasm, so that if the
nucleus be larger at one pole than at the other it is situated in
the larger end, while in elongate nuclei, of nearly equal diam-
eter throughout, it usually lies at an early equal distance from
both ends of the nucleus. The nucleolus may be said, as a
general rule, to occupy the center of the nuclear substance,
and is not often markedly excentric; in none of the other cells
examined in the course of these investigations did the nucleoli
show a similar tendency to occupy the center of the nucleus.
The nucleolus always stains differently from the chromatin.
The relative amount of chromatin varies in different nuclei.
It is always found, after the action of various fixatives, to occur
in the form of small granules, which are connected by delicate
irregular fibers, which stain exactly as the granules do. The
nuclear sap stains faintly with haematoxylin (this has not
been shown in the figures). The nucleolus is either in contact
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 477
with chromatin granules or with fibers of chromatin, which
pass between it and the nuclear membrane; there is never a
clear space around the nucleolus, but it seems to be held in
position by the chromatin.
16. Muscle Cells of Piscicola rapax (Verr.).
(Plate 29, Figs. 325-337-)
(The nuclei of the longitudinal muscle layer of the body wall
were studied. For the examination of the different stages of
these nuclei worms of different sizes must be studied; I exam-
ined the nuclei of leeches of about 6 mm. in length, where the
cells and their nuclei are smailest, as well as of larger and fully
mature individuals, where these cells and their nuclei attain
their maximum dimensions.)
In the smallest nuclei (Fig. 325) a single nucleolus is inva-
riably present and lies centrally ; it is of medium size, more or
less oval in outline, and contains a varying number of small
vacuoles. In larger nuclei it becomes larger and more elongate
in form, lying in the longitudinal axis of the nucleus (Figs. 328
and 331); at the end of this stage its greatest dimensions are
reached. Next commences a process of fragmentation of this
original nucleolus into a number of smaller nucleoli, which are
of different sizes. There appears to be little uniformity in the
mode of this nucleolar division (Figs. 327, 329, 332, 333): the
nucleolus may become dumbbell shaped and then divide into
two larger pieces; or when much elongated it usually breaks
simultaneously into a number of consecutive portions ; or buds
of nucleolar substance may be divided off from its surface. This
segmentation is not strictly dependent upon the size of the
nucleus, nor upon the size or form of the nucleolus. The frag-
mentation continues, the larger daughter-nucleoli also dividing,
until in the largest nuclei (those of the mature worm) as many
as twelve small nucleoli may be present, which are irregularly
distributed through the nucleus (Figs. 335-337). In all these
stages at least some of the nucleoli contain vacuoles, though
they have not been reproduced in all the corresponding figures.
All the nucleoli of the largest nuclei are thus produced by a
478 MONTGOMERY. [Vou. XV.
series of divisions from the single original one. This division
usually commences, then, when the form of the nucleus changes
from the original oval to a more elongate shape. It seems
probable that this elongation of the nucleus may directly cause
the division of the nucleolus, since the long axis of the latter
coincides with that of the nucleus; and were the nucleolus in
any way fixed in position in the nucleus, the nuclear elongation
would draw out the nucleolus and cause it to break into frag-
ments. But the division of the daughter-nucleoli does not
always take place in the direction of the long axis of the
nucleus, so that some other factor might be at work to produce
this division.
The chromatin is arranged in the form of a reticulation (Fig.
326). The nuclei of the younger cells are usually regular in
outline, but those of the larger ones become very irregular ;
this irregularity of the contours of the nuclei is more marked
by fixation with corrosive sublimate than with Flemming’s fluid,
so that it might be regarded as an artefact caused, ¢.g., by the
obstacle offered to the rapid penetration of the preserving
fluid by the dense outer (fibrillar) layer of the cytoplasm in
the largest muscle cells.
17. Blood Corpuscles of Doto.
(Plate 22, Figs. 98-101; Plate 23, Fig. 102.)
(These cells are usually to be found abundantly in the
cavity of the cirratida and of the sheaths of the tentacles,
though their number varies greatly in different cirratida.
They lie in the meshes of the loose network of mesenchym
cells, either singly or grouped together into bundles. I have
been unable to find them in other parts of the body. These
cells appear to be free mesenchym cells, with perhaps the
function of blood corpuscles.)
There is always a single large nucleolus, which is usually
very large in proportion to the size of the nucleus. It varies
in form from a perfect sphere to an elongate oval. The nucleo-
lar substance is usually homogeneous, but in some cases it is
granular (Figs. 99-102) and then it stains faintly as if it were
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 479
undergoing a degeneration. Quite frequently a small spherical
granule lies in the center of the nucleolus and this always
stains more intensely than the surrounding substance (Figs.
99-102). In only about half a dozen cases, out of hundreds of
cells examined, did I find attached to the surface of the nucleo-
lus one or two much smaller bodies, which also stained less
intensely (Figs. 100 and 101). Can it be that in certain cases
the nucleolus becomes differentiated into a “‘ Hauptnucleolus ”
and a “ Nebennucleolus,” in which case these small bodies
would represent the “ Nebennucleoli” ? In certain of the cir-
ratida of a young individual the nucleoli of the greater num-
ber of nuclei were situated at that pole of the nucleus directed
towards the median axis of the cirratidum, 7.e., those in the
nuclei on the right side of the cirratidum were in the left-hand
poles of the nuclei, and those in the nuclei of the left side of
the cirratidum (as seen on sections) were placed at the right-
hand poles of the nuclei. I did not observe this regular posi-
tion of the nucleoli in the cirratida of the other individuals
sectioned and hence would conclude that it was not a normal
phenomenon, but an osmotic consequent of the fixing reagent.1
The size of the nucleolus preserves approximately the same
ratio to that of the nucleus.
The nucleus is either spherical or oval in outline. The
apparent arrangement of the chromatin varies according to the
fixative employed. After picro-nitro-osmic acid (Fig. 102) it
appears granular ; after Hermann’s fluid (Figs. 99-101), in the
form of delicate fibers which radiate from the nucleolus to the
nuclear membrane ; after alcoholic solution of corrosive subli-
mate (Fig. 98) we find a few fibers radiating from the surface
of the nucleolus, but the greater amount of the chromatin
appears in the form of granular masses, which lie mainly near
1 In a previous paper ('96) I figured for the nuclei of those mesenchym cells
which surround the distal end of the ventral nerve cords of Ceredratulus lacteus,
the nuclei with their chromatic masses pressed against that side of the nuclear
membrane which was situated nearest to the central point of the section. At
that time, I regarded this excentric position of the chromatin as a normal but
peculiar phenomenon; but now, on comparison with the cells of Doto, I am
convinced that it is an artefact produced by the osmotic action of the fixing
Teagent.
480 MONTGOMERY. [VoL. XV.
the periphery of the cell, so that the nucleolus is surrounded
by a clear space. These nuclei thus offer a suggestive object
lesson, to teach how careful one must be in the determination
of the form of delicate cellular structures by the study of
preserved material.
Cells which are isolated have a spherical form ; those grouped
together are polygonal, owing to their mutual pressure (Figs.
99 and 101). A cell membrane is present. The cytoplasm is
for the most part finely granular; portions of it, however, are
always more dense and stain more deeply than the former
portion ; there are great individual differences in different cells
(Figs. 100 and 102). Often the cytoplasm is more or less vacuo-
lar or a clear space may partially surround the nucleus and
a similar space be present between the cytoplasm and the cell
membrane, this space being transversed by a few radiating
fibers. Such spaces are best shown after the action of the
fluid of Hermann; they are seldom to be seen after fixation in
picro-nitro-osmic acid ; but whether a coarse alveolar layer of
cytoplasm at the periphery of the cell be normal or be an arte-
fact, there are certainly marked differences in the structure of
the cytoplasm in neighboring cells, and these differences might
be regarded as the morphological changes corresponding to
functional phases in the cells. Cases of degenerating cells are
numerous, and may be recognized by their faint staining
properties and by their granular appearance.
18. Giant Cells of Doto.
(Plate 30.)
(These enormous cells, which are the largest cells in the
body, not excepting the ova, lie at the anterior part of the
body just behind the head region and are closely apposed to
the folds of the nidamental gland. They do not produce a
closed mantle on the outer surface of this gland, but either are
isolated or occur in small groups of from two to four cells
each. In each individual their number appears to be about
thirty or forty. These cells do not seem to have any open
communication with the neighboring tissues, and I cannot
No.2.] COMPARATIVE CYTOLOGICAL STUDIES. 481
conclude from their structure what their function is; per-
haps they have a function similar to that of lymph glands.
Such cells are absent in Montagua.)
The form of these is a more or less polyhedral one, caused
by the pressure of the surrounding organs (Fig. 339). The
nucleus is relatively and absolutely very large and is very vari-
able in form, sometimes irregularly oval, sometimes with obtuse
or pointed processes, or again concavo-convex, that side being
convex which lies near the nuclear membrane (on a transverse
section such a nucleus appears sickle shaped). The chromatin
is arranged in the form of rather coarse granules (Figs. 339
and 342), which after fixation in Hermann’s fluid (Fig. 340)
appear to be the nodal points of a reticulum.
The nuclei (Figs. 338-346) are numerous, vary in number
from about six to about forty, and are irregular in size. Their
shape is usually oval, seldom perfectly spherical, though quite
frequently, as the figures show, they may be more or less
elongate or even very irregular in form. Vacuoles are fre-
quently present in them. The nucleoli stain as do all true
nucleoli, but different degrees of staining density may be
observed in the nucleoli of the same nucleus (Figs. 338, 342,
346). In two cases, one of which is here figured (Fig. 342), a
dense ring of chromatin was found around a nucleolus, but such
cases, judging from their infrequency, must be regarded as very
abnormal, if not attributable to the action of the fixing fluid.
Divisions of the more elongate nucleoli certainly take place.
Thus I have observed dumbbell-shaped nucleoli in three cases
(Figs. 343, 345, 346), and Fig. 340 probably represents a stage
just after a division, where two smaller nucleoli have apparently
been divided off from a larger one, one end of the latter being
drawn out toa point. Thus it might seem that the large num-
ber of nucleoli are produced by divisions of a smaller number
of larger nucleoli. The variability in the size, form, and
number of these nucleoli recalls those of the subcuticular gland
cells of Pzsczcola (cf. infra) ; but in these cells of Doto I have
been unable to make out different morphological phases.
The cytoplasm of these cells is also remarkably differentiated
(Fig. 339). In a given cell certain portions of the cytoplasm
482 MONTGOMERY. [Vou. XV.
may be dense and stain deeply ; other portions are less dense in
structure, with a corresponding less intensity of stain; and still
other portions of the cell substance appear structureless and
do not stain at all. The cytoplasm in at least a portion of the
peripheral area of the cell is always dense and deeply staining ;
rarely is the cytoplasm in the whole cell of this dense structure.
With low powers of magnification (e.g., Zeiss Obj. C, oc. 2 or 4)
there may appear to be either several cavities in the cytoplasm
or asingle large one at one side of the nucleus. These differ-
entiations of the cytoplasm (which fixation in corrosive subli-
mate or in Hermann’s fluid bring out always in the same manner)
probably denote certain metabolic states of the cytoplasm, but
it would be difficult to determine from the structure alone to
what physiological processes these states might correspond.
There is no definite secretion produced by the cytoplasm, z.e.,
no secretion with a definite form. As has been noted, a wholly
or nearly wholly clear space often occurs in the cytoplasm at
one side of the nucleus; such a space usually lies at that
margin of the nucleus situated closest to the center of the cell,
and the nucleus may often surround it to some extent. Where
the nucleus comes into contact with this space its membrane is
thinnest and its outline irregular, and quite frequently this
margin of the nucleus is produced into Jong, irregular, amoe-
boid processes, which extend into the space in question and
pass around it. These appearances would show that the
nucleus stands in a certain functional relation to the metabolic
changes of the cytoplasm, not improbably that it assimilates
certain substances produced in the latter.
To return to the nucleoli, I cannot find any genetic connec-
tion between these structures and the cytoplasm. They are
usually grouped near the center of the nucleus, and though
often quite peripheral in position, never come into contact
with the nuclear membrane, nor are they found in the amoeboid
processes of the nucleus. It will be necessary to study very
young individuals of this mollusc in order to determine the
mode of nucleolar development.
The cell (Fig. 339) is developed by a delicate membrane,
which seems to be interrupted at no point on the surface of the
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 483
cell. The cell has thus no external openings and no ducts or
fibers which penetrate into the enveloping tissues.
19. Subcutical Gland Cells of Piscicola rapax (Verr.).
(Plate 25; Plate 26, Figs. 198-203.)
(These cells lie for the most part in the body cavity, between
the body muscular wall and the intestine. Two modifications
of them may be distinguished : (1) those at the ends of the
body, near the suckers and in the wall of the latter, which are
comparatively small, and the relatively short cell ducts of which
open at all points of the surface of the body at the ends of the
body and on the inner surface of the suckers; these seem to
resemble the second modification in all respects except size ; (2)
the larger type of these gland cells, which I have studied exclu-
sively, are massed together in that portion of the body cavity
which extends from the region a little anterior to the brain,
nearly to the posterior end of the body, the greater number of
them being in contact with the inner surface of the body
wall.)
In order to find all the functional stages of these cells one
must study preparations of worms of various dimensions, since
all the stages cannot be found in a single individual ; I made
consecutive series of sections of seven different individuals, the
smallest being about 6 mm. in length, and the largest being fully
matured. The remarkable cycles of the nuclear and cell stages,
to be described below, were equally well discernible with all
three of the fixatives employed, namely, Flemming’s fluid and
alcoholic and aqueous solutions of corrosive sublimate ; various
double stains were used.
These cells, when they reach their fullest dimensions, are so
enormous that they may be readily seen with the naked eye.
Their single ducts all open on the surface of the body, between
the epithelial cells, a little anterior to the region of the sexual
pore ; their openings are at this point equally numerous on
the dorsal, lateral, and ventral sides of the worm. The most
posterior gland cells of the body send their ducts a distance of
four-fifths the total length of the body before they open on the
484 MONTGOMERY. [Vou. XV.
surface of the latter, these ducts transversing a large number of
body segments (in certain of the enchytraeid O/igochaeta there
have been described subcutical gland cells whose ducts pass
through a number of segments, but I believe that they are not
of the same relative length as those of Pzsczco/a). Each cell has
its own duct, the latter being morphologically merely a process
of the cell (Figs. 178, 181, 202) ; and as these individual ducts
run in bundles parallel to one another, on their way to the sur-
face of the body, they become closely apposed to one another,
but there are apparently no open communications between the
several ducts, nor do they unite to form larger, compound ducts.
The ducts of those gland cells which are situated behind the
sexual pore necessarily have an anterior direction, while those
which are situated near to the head end of the animal send their
ducts posteriorly. The duct departs from the cell more or less
at right angles from its distal end, z.¢., that end which is usually
directed towards the central axis of the worm. Since the greater
number of these cells become filled with secretion only when the
worm is sexually mature, and since they all open on the surface
of the body near the sexual pore, they have probably the same
function as the clitellar glands of the Olzgochaeta, after these
observations had been completed I found that Bourne ('84) had
described such gland cells in Pontobdella as “ clitellar glands,”
but he made no observations on their finer structure.
In studying the cycle of the structural changes of these
cells two main morphological periods may be distinguished :
(1) the prophasis, from the immature cell to the cell filled with
secretion ; and (2) the metaphaszs, from the time when the
cell begins to discharge its secretion until it becomes re-formed
into a functionally immature cell again. I have no means of
determining whether a given cell becomes filled with secretion
only once a year (as, ¢,g., at the period of sexual maturity) or
whether it may secrete several times in succession during the
sexual period. At any rate, all appearances lead me to con-
clude that it secretes periodically, most probably once during
each period of sexual maturity. I have found no evidences that
it secretes only once and then dies to become absorbed by
the other tissues of the body; in other words, there were no
No.2] COMPARATIVE CYTOLOGICAL STUDIES. 485
evidences of cell degeneration or of a formation of new cells, so
that we must conclude that each of these cells continues to
functionate periodically during the whole time of the existence
of the leech.
We may now describe in succession the prophase and the
metaphase of the structural changes.
Prophasis (Figs. 178-196). —In the smallest cells found in
the youngest leech examined no trace of secretion is present
(Figs. 178-180). In these the nucleus is usually central in
position, with a delicate chromatin network, and with a single,
most frequently oval, nucleolus, in which one or a few small vac-
uoles are commonly present. Around the nucleus, and filling
the cell duct, is a somewhat dense cytoplasm, which becomes
more vacuolar at the periphery of the cell. The chief cyto-
plasmic changes from now on are as follows (I have not fig-
ured these changes, since they may be briefly characterized) :
that portion of the cytoplasm close to the nucleus gradually
becomes more dense and begins to stain differently from the
rest, and then becomes quite homogeneous ; most frequently
there is a layer of this homogeneous substance between the
nucleus and the cell duct, only that portion of the cytoplasm at
the proximal end of the cell, as well as a thin layer around
the homogeneous substance, retaining its primitive appearance.
Next, this homogeneous mass gradually breaks up into the
numerous secretion corpuscles (Fig. 181, Secr.), the shape of
the latter being ovoid after fixation in corrosive sublimate, but
more spherical after the action of Flemming’s fluid. These
secretion corpuscles stain at first just like the homogeneous
substance, but gradually commence to stain otherwise, and in
the functionally mature cell stain differently from the primitive
cytoplasm, as well as from the homogeneous substance from
which they were derived. The whole cell thus gradually
becomes filled with these small corpuscles, until finally no
trace of the original cytoplasm is to be seen, except a few
faintly staining fibers. The cytoplasm which fills the duct
undergoes the same morphological changes as that of the cell
body just described, so that the first secretion corpuscles in it
are the derivatives of its own substance ; the cytoplasm of the
486 MONTGOMERY. [Vou. XV.
duct and of the distal portion of the cell are as a rule the first
portions to become differentiated into the secretion. At the
end of the prophase the cell has attained its maximum size,
and the duct its greatest diameter, both containing hundreds
of the mature secretion corpuscles lying in an unstained, struc-
tureless fluid. The duct in all stages is always larger at its proxi-
mal than at the distal end, though it narrows very gradually.
But the most interesting morphological changes are those of
the nucleus. While the secretion is being produced in the
cytoplasm the nucleus increases rapidly in size, and at the
same time becomes very irregular in form, until in the nearly
physiologically mature cell it attains enormous dimensions and
sends out through the substance of the cell long branching
processes, which anastomose with one another and some of
which reach even to the cell membrane (Figs. 178-196). Kor-
schelt has described ('89) branched nuclei in the spinning glands
of certain insect larvae, which are somewhat similar to the
nuclei here delineated. The nucleus attains its greatest dimen-
sions and its most marked degree of ramification when there
is the greatest amount of the homogeneous substance in the
cell, z.e., just before this substance becomes metamorphosed
into the secretion corpuscles. At this stage we find the greater
portion of the nucleus situated at the proximal part of the
cell, and from that point it sends out irregular branches which
envelop the mass of homogeneous substance, and which pene-
trate into it. At this period, further, no two nuclei are alike
in form, so that it would be in vain to attempt to figure all
the shapes which they may assume. The nuclear membrane
becomes very thin, often scarcely perceptible, around the
branched processes. I know of no other nuclei which are more
interesting in point of size and variability of form than these ;
and it would well repay accurate investigation in the endeavor to
decide in what way they may influence or modify the cytoplas-
mic changes which are simultaneously taking place, for they
obviously have a close physiological connection with the forma-
tion of the cellular secretion. Since the nucleus undergoes a
rapid process of growth in these stages, we are obliged to
assume that it is taking up substances from the cell body ; but
No.2.) COMPARATIVE CYTOLOGICAL STUDIES. 487
it probably does not assimilate the mature secretion corpuscles,
since when the latter are produced, as we shall see, the nucleus
commences to retract in size and to withdraw its processes. As
the nucleus increases in size its chromatin reticulum becomes
looser, as if it were elastically stretched by the expansion in
volume of the nucleus; the chromatin is continued into the
ramifying processes of the nucleus.
The nucleolar changes during the prophase are as follows :
in the immature cell there is invariably a single rather large
nucleolus, which occupies a more or less central position in the
nucleus (Figs. 178-181, 184) ; it may be either oval or spindle
shaped, and most frequently contains one or several small vac-
uoles. Its substance appears homogeneous after treatment with
corrosive sublimate, granular after the action of the fluid of
Flemming, and has no limiting membrane ; in all its stages
within the nucleus it stains very intensely, though always dif-
ferently from the chromatin. Now as the nucleus increases
in volume so also does the nucleolus, though at first at a rela-
tively more rapid rate than does the former ; and in growing
larger it gradually becomes more elongated, rod shaped, and at
this stage is most frequently in contact with the nuclear mem-
brane (Fig. 182). When it has taken up this peripheral posi-
tion its period of most rapid growth commences, so that at
this stage there is a proportionately greater amount of nucleo-
lar substance in the nucleus than at any other period in its
history. When it is apposed to the nuclear membrane it has
at first more or less the form of a rod (often of a slightly curved
rod), but as its substance commences to increase in volume this
rod shape gradually becomes changed and the nucleolus becomes
bent inwards (towards the center of the nucleus), frequently in
the form of a V, an S, or a W, though there is marked vari-
ability in regard to the form it may assume, since no two nucle-
oli can be found at this stage which have exactly the same form
(Fig. 189). It is about this time that the nucleolus attains its
greatest staining density. Then this large and irregularly
shaped nucleolus leaves the nuclear membrane and begins to
fragment into pieces, which are very irregular in shape and
variable in number and size ; the nucleolus may show thereby
488 MONTGOMERY. [VouL. XV.
a number of constrictions, or buds of nucleolar substance may
project from its surface ; it may first break into two larger
pieces, and then these may fragment further, or it may at once
break into a number of pieces which are irregular in their
dimensions (Figs. 185-188, 190, 191, 193). These fragments
gradually wander apart from one another, the nucleus now
being larger and already somewhat irregular in shape ; and at
the same time each of the primitive nucleolar fragments divides
into smaller pieces of unequal size, until when the nucleus has
attained its greatest dimensions and most pronounced degree
of ramification it contains a very large number of irregular
nucleoli, which are unequal in their dimensions (Figs. 194-
196). The figures given of this last stage show only sections
of nuclei, and since as many as five or six sections may be made
of one of these colossal nuclei (my sections were between 3 and
5 in thickness), not one of these figures shows more than a por-
tion of the total number of nucleoli in these largest nuclei ; in
some of the latter nuclei I compute the number of the nucleolar
fragments to be at least three hundred. But the total mass of
nucleolar substance in these largest nuclei is certainly consid-
erably greater than the mass of the primitive nucleolus at
the time of its greatest size ; accordingly, though the division
products of the primitive nucleolus might constitute the greater
part of the nucleolar substance in the largest nuclei, they do
not constitute all of it. Therefore there must be a formation
of new nucleolar substance after the primitive nucleolus has
divided, z.e., a production of nucleolar substance not derived
from the primitive nucleolus; I cannot determine the manner
of formation of this new nucleolar substance, but would suggest
that either new nucleoli are formed, or that the fragments of the
primitive nucleolus increase in size by the addition of new nucle-
olar substance to them. The greater number of nucleoli in the
largest nuclei are collected in or near the thicker portion of the
nucleus and few or none lie in the branched processes ; they are
at this time seldom in contact with the nuclear membrane. Only
a few of them contain vacuoles, and those which do may be re-
garded as derivatives of the primitive nucleolus, the vacuoles of
the latter still being preserved in its daughter-nucleoli.
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 489
A nucleolar change now occurs which I have never seen
paralleled, and to my knowledge no similar morphological change
has ever been described. At the time when the homogeneous
substance of the cell is commencing to differentiate into the
secretion corpuscles, the nucleus begins to withdraw its branched
processes and to decrease in size; while so doing it discharges
its nucleoli into the cell body (Figs. 197-199). There can be no
doubt of the genuineness of this process, since I have examined
at least two hundred nuclei at this stage, which showed all
intermediate stages between nuclei which had discharged only
a few nucleoli and those which had discharged all except a
single one of their nucleoli into the cell. The study of these
nuclei gives the impression that successive contractions of the
nucleus take place, whereby at first all the more peripheral nucle-
oli, and later those which are more central in position, become
successively extruded, for in the cell two or three more or less
parallel rows of nucleoli may be found, or more properly speak-
ing, concentric circles of nucleoli (Figs. 197 and 198). In some
cases I have observed nucleoli which were halfway through
the nuclear membrane, but by far the greater number of the
nucleoli are found either within or without the nucleus, and
this would prove that the contractions of the nucleus are sudden
in their action. I think that it is the sudden contractions of
the nucleus which alone cause the expulsion of the nucleoli,
for as the nucleus diminishes in volume its chromatin network
may be seen gradually to become closer and denser, and the
pressure within the nucleus becoming greater than the pressure
without it, the nucleoli, not being fixed in position, are forced
out into the cell body where there is comparatively little pres-
sure, since the secretion corpuscles are not densely grouped,
but lie scattered through a thin and structureless fluid substance.
The nucleoli, when they have arrived in the cell body, are
not found in equal number at all points around the nucleus ;
accordingly they are probably not discharged from all sides of
the nucleolus in equal number, but only there where the nuclear
membrane is thinnest (it is probably thinnest at those points
whither the nuclear processes had withdrawn themselves). But
though the nuclear membrane appears to be thinner at some
490 MONTGOMERY. [VoL. XV.
points than at others, there are no visible pores in it, so that
the nucleolar substance must be squeezed through the nuclear
membrane itself. When one takes a sponge filled with water
and presses it in the hand the water is forced out of it in the
form of jets or columns, which are radial to the surface of the
sponge ; exactly similar seems to be the method of the discharge
of the nucleoli in the case at issue, except that the nucleus is
itself actively contracting. Thus we find the greater number
of the nucleoli which lie in the cell body close to the surface
of the nucleus to be irregularly columnar or rod like in shape
(Fig. 198) and radially grouped around the nucleus. Those
which lie nearer the periphery of the cell, however, and which
had probably been discharged by a previous contraction of the
nucleus, are more irregular in form, and their axes have a less
regular position with regard to that of the nucleus. Further,
those ends of the rod-like nucleoli in the cell which are directed
towards the surface of the nucleus are usually more attenuated
than the opposite ends, z.e., a nucleolus lying in the cell close
to the nucleus has often the form of a pyramid the apex of
which is directed towards the surface of the nucleus, and this
form we would expect to result in the squeezing of a more or
less viscid substance, like that of the nucleoli, through the
nuclear membrane. I give only two figures showing the stage
of the discharge of the nucleoli from the nucleus, simply in
order to save time in the drawing of the numerous nucleoli ;
but my preparations show very clearly all the stages of this
process: one has only to examine sections of the mature leech to
find them in abundance. The extrusion of the nucleoli continues
until only about twenty, then a dozen, then four or five, and
finally only a single nucleolus (Fig. 199) remains in the nucleus ;
corresponding to these successive states of the discharge of the
nucleoli we find cells in which only a few nucleoli, and then
those in which the greater number of the nucleoli, lie in the
cell body. One nucleolus always remains in the nucleus, though
this one appears to differ in no wise from those which are dis-
charged. Those nucleoli which lie in the cell body (Figs. 197—
199) differ from those in the nucleus in their lesser density,
greater size, and different reactions to certain stains (we shall
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 4gI
return to the chemical change later); in other words, the sub-
stance of those nucleoli which have come to be situated in the
cell body undergoes a physical and perhaps a chemical change
in this portion of the cell, and their expansion in volume might
be accounted for on the ground of there being a smaller degree
of pressure in the cell body than there is in the nucleus.
It will be noticed that the prophase and the metaphase of
the cell body and of the nucleus do not exactly coincide in
point of time, the metaphase of the nucleus commencing earlier
than that of the cell body. Thus the nucleus attains its great-
est dimensions and most diverse ramification at the time when
the cell body contains the greatest amount of homogeneous
substance, and the nucleus enters on its metaphase (diminu-
tion in volume, retraction of processes, expulsion of nucleoli)
when the secretion corpuscles are only commencing to arise in
the cell body. At the beginning of the metaphase of the cell
body (when the latter is filled with the secretion corpuscles and
commences to excrete them) the nucleus has already assumed
a nearly spherical or oval form, has greatly decreased in size,
and has discharged most of its nucleoli into the cell, z.¢., the
nucleus has advanced already some distance in the path of
the metaphasis.
The metaphase of the cell body (Figs. 198-203) commences
when the cell is filled with the secretion corpuscles, all traces
of the previous homogeneous substance being absent, and begins
to discharge them through its duct. During this process the
cell gradually decreases in size, and the primitive cytoplasm
again comes into view, at first in the form of delicate fibers.
When the cell has shrunk to about one-third of its former size
(the diameter of the duct does not decrease quite so rapidly,
since it may be still full of secretion corpuscles after they have
all disappeared from the cell body) the nucleus has simultane-
ously decreased in size, but with greater proportionate rapidity
than the cell body, and so at the close of the metaphase (Fig.
202) the nucleus reaches its smallest relative size. The latter
contains at this stage invariably a single nucleolus, of spherical
or oval form, very regular in outline, and exactly similar to the
nucleolus at the commencement of the anaphase except that
492 MONTGOMERY. [VoL. XV.
it does not appear to contain vacuoles. The nucleus itself is
somewhat elongate and irregular in outline, and, owing to its
maximum degree of contraction (a characteristic of the end of
the metaphase), its chromatin builds a dense network within it.
A study of the cell body at this stage allows us to follow
the morphological changes undergone by those nucleoli which
had been discharged by the nucleus (Figs. 198-203). The
cytoplasm gradually assumes a reticulate or a somewhat granu-
lar structure, and finally a most regular vacuolar or alveolar
structure. As the cell body decreases in size the discharged
nucleoli lying in it gradually stain less deeply, they lose their
rod-like form, and no longer remain isolated, but all the nucleo-
lar substance in the cytoplasm gradually becomes confluent,
and becomes arranged in the form of a coarse, irregular network
of substance distributed in the cytoplasm, and readily distin-
guishable from the latter by its different staining properties
(Figs. 201-203). By a hasty inspection this network of nucle-
olar substance might appear to represent branches of the
nucleus, but a careful study shows that at this period of its
growth the nucleus has no branches. As the cell continues to
become smaller the amount of nucleolar substance in the cyto-
plasm gradually becomes less and less, first the network at the
periphery of the cell disappearing, then that in the vicinity of
the nucleus, until at the conclusion of the metaphase no nucle-
olar substance is any longer to be seen in the cytoplasm. I am
unable to determine whether it is finally discharged through
the cell membrane or whether it becomes metamorphosed into
cytoplasm ; it certainly is not excreted through the cell duct,
since no nuclear substance occurs in the latter, and at this
stage the duct is no longer an open tube, but all the secretion
corpuscles having been expelled from it, it is again filled with
cytoplasm. The suggestion may be made that at least a portion
of this nucleolar substance remains in the cytoplasm, so that in
the succeeding prophase the nucleolus within the nucleus might
find the material necessary for its growth in the nucleolar sub-
stance suspended in the cytoplasm ; thus there might be, in the
history of the nucleolar substance, periods of its expedition into
the cytoplasm alternating with those when it is again taken
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 493
into the nucleus. And that in the prophase the single nucle-
olus of the nucleus derives the material necessary for its
further growth from the cell substance, seems highly probable
when we recall the fact that at the time of its most rapid
growth it is usually apposed to the nuclear membrane, which
would denote that it is taking up a substance which penetrates
that membrane from the side of the cell body.
We have alluded to certain chemical changes which occur in
the nucleolar substance when discharged from the nucleus
during the metaphase of the latter. These staining differentia-
tions and the coloration of the cytoplasm as observed on five
different preparations are as follows (the first preparation was
fixed with Flemming’s fluid, the others with corrosive subli-
mate).
First preparation (Ehrlich’s haematoxylin, two hours ; eosin,
ten minutes): cytoplasm pale lilac; nucleoli in the nucleus, and
when first discharged from it, reddish or rusty brown; nucleo-
lar substance at the end of the metaphase lighter in color.
Second preparation (gentian violet in aqueous solution, twenty-
five minutes ; eosin, four and one-half minutes): cytoplasm
very faintly stained ; nucleoli in the nucleus deep violet, those
in the cytoplasm yellowish red.
Third preparation (Ehrlich’s haematoxylin, one hour ; eosin,
five minutes): cytoplasm pale pink ; nucleoli in the nucleus,
and when first discharged from it, purple ; nucleolar substance
in the cytoplasm at the end of the metaphase pure blue.
Fourth preparation (Ehrlich’s haematoxylin, forty minutes ;
eosin, five minutes) : nucleolar substance within and without
the nucleus yellowish red ; cytoplasm of a paler red.
Fifth preparation (Mayer’s acid carmine, twenty minutes ;
Lyons blue, five minutes): cytoplasm unstained ; nucleoli in the
nucleus, and, when first discharged, bluish green; nucleolar
substance at the end of the metaphase reddish purple in the
cytoplasm. These methods of double staining show that the
nucleolar substance, when discharged from the nucleus, under-
goes some chemical change in the cytoplasm; and they
serve to distinguish, further, this substance from the true
cytoplasm.
494 MONTGOMERY. [VoL. XV.
I have no material of Pzsczcola after the breeding season, and
accordingly could not follow the changes of these gland cells
in their metamorphosis from the end of the metaphase to the
commencement of the prophase. But these two end stages do
not differ much from one another, since the cell at the former
stage differs from that of the latter merely in that its nucleus
is smaller and more irregular in shape.
It is not difficult to determine the sequence of the stages
described ; only in the smallest individuals do all the stages of
the prophase occur, and only in the largest those of the
metaphase.
20. Mesenchym Cells of Cerebratulus lacteus (Verr.).
(Plate 29, Figs. 315a, 316a—324.)
(I have described these cells in a previous contribution ('96),
and so shall treat of them in this place mainly with regard to
their nucleoli.)
The smallest nuclei (Figs. 316a and 317) are densely filled
with chromatin, and nucleoli appear to be absent ; the nuclear
sap also stains with haematoxylin, so that these nuclei may
be easily recognized by their deep stain and sometimes nearly
homogeneous appearance. I have made a careful examination
for nucleoli on preparations stained by the Ehrlich-Biondi
method, as well as with haematoxylin and eosin, and am
certain that nucleoli are either wholly absent or, if present,
must be very minute in point of size. Such, then, is the
structure of the smallest nuclei, namely, those found in the body
cavity, and those of the smallest cells of the pseudoepithelia
lining the body cavity.
The non-continuous pseudoepithelia of the body cavity are
layers of differentiated mesenchym cells, which differ from the
primitive cells in their greater dimensions and more oval or
spherical outlines (the undifferentiated cells are bipolar or
multipolar, with long branching processes). In these larger
cells we find for the first time a spherical, deeply staining
nucleolus. Now the size of the latter stands in a pretty con-
stant ratio to that of the nucleus. Further, in the smallest
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 495
nuclei which contain nucleoli, from one to three of the latter
occur, and one or all of these are frequently found in close con-
tact with the nuclear membrane (Figs. 315a, 318, 320), while in
the largest nuclei observed only a single nucleolus is present, and
this one is relatively large and is always at or near the center
of the nucleus, never at its periphery (Figs. 319, 322, 323).
In connection with the problem of the origin of this nucleolus
we recall those small granules contained in the cytoplasm,
which I have (96) termed nutritive particles. These particles
(Nut. Gl.) stain with eosin quite as intensely as the nucleolus,
and in the smallest cells are either wholly absent or present in
only small number ; but in the larger cells they are usually
much more abundant, or when not more numerous they are of
greater size, and are often quite densely grouped around the
nucleus. It would seem probable that the nucleolar sub-
stance is derived from these supposed nutritive particles. Thus
when the nucleoli first appear they are most frequently in con-
tact with the nuclear membrane ; and this shows that they are
formed at the periphery of the nucleus, and only later come to
occupy a central position within it. And since the nutritive
particles are usually very numerous in the immediate vicinity
of the nucleus, we may conclude that the nucleoli are formed
from substance of these nutritive particles, which has been
taken up by the nucleus. In the smallest nuclei alone do more
than one nucleolus appear, so that the nutritive substance
would seem to be taken into the nucleus from several points
on its periphery, and then subsequently these several assimi-
lated portions of nutritive substance may fuse together and so
produce a single large nucleolus. Accordingly, the substance
of the nucleolus would in this way appear to have an extra-
nuclear origin. That these nutritive particles are being succes-
sively absorbed by the nucleus is shown by the fact that the
increase in the size of the nucleus and of the nucleolus go hand
in hand. On the other side, these nutritive bodies in the cyto-
plasm cannot be considered to be of nucleolar origin, since
they usually make their first appearance in the cell body before
a nucleolus arises in the nucleus ; and if they did have a nucle-
olar origin, 2z.e., if they were excreted portions of the nucleolus,
~
496 MONTGOMERY. [VoL. XV.
we should expect to find the largest nucleoli in the smallest
cells and the smallest ones in the largest cells. Further, the
nucleolar substance cannot be regarded as a secretion of the
nucleus itself, since this would leave unexplained the peripheral
position which it at first occupies in the nucleus. Thus the
mode of origin of the nucleolus in these cells would seem to be
similar to that of the nucleoli in the ova of the nemerteans.
A final point may be noted: the nucleolus accepts the same
stains, though more intensely, than do the nutritive particles in
the cytoplasm ; accordingly, the substance of those bodies which
have been absorbed by the nucleus, and then by their fusion in
the nucleus produce the nucleolus, must have undergone either
a slight chemical or physical change within the nucleus.
The largest mesenchym cells of the pseudoepithelia probably
represent the youngest stages of the ova, though in the single
individual of this species at my disposal no gonads were pres-
ent, so that I can bring no proof positive that this is the mode
of origin of the egg cells. In Carinella it is from similar cells
that the genital products are derived, as I have previously
shown (96). Coe (95) described certain of the more mature
egg stages.
In my earlier paper on these cells (/c.) I termed all the
nuclear divisions of these cells ‘“‘amitotic.’’ But renewed study
of these elements shows that only the divisions of those cells
are amitotic (Figs. 316a and 317), from which the free mesen-
chym cells are produced. Whereas, in the nuclear divisions of
the cells of the pseudoepithelia from which the masses of larger
cells are derived I now find evidences of regularity in the
distribution of the chromatin, so that probably these divisions
are mitotic. However, in these small nuclear divisions it is
almost impossible to decide whether we have to do with mitoses
or with amitoses without the use of better lenses than those
which were at my disposal.
21. Ganglion Cells of Nemerteans.
I may here briefly mention the relations of the nucleoli in
these cells, and for other details refer to a previous contribution
of mine (97).
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 497
Lineus gesserensis.— Cells of the first type: one or two small
nucleoli. Cells of the second type: one nucleolus. Cells of
the third type : a single nucleolus, or two of unequal size.
Cerebratulus lacteuws.— Cells of the first type: as in the
preceding species. Cells of the second type: one or two
nucleoli. Cells of the third type : one or two nucleoli, which
in one case stained differently. Cells of the fourth type:
usually one peripheral nucleolus; rarely are two present, and
then they are unequal in dimensions.
In all these cells the nucleolus is comparatively small, homo-
geneous, and no evidences of nucleolar division were seen.
IV. GENERAL COMPARISONS AND CONCLUSIONS.
Here I shall summarize merely the results of my observations
on the nucleolus, and compare them with the conclusions of
other investigators. Numerous other morphological points
have been brought up, however, in the preceding pages, such
as yolk development, differentiation of ova, nuclear divisions,
distribution of the chromatin elements in the germinal vesicle
at different stages in the growth period, changes in the struc-
ture of cytoplasm, etc.
1. Chemistry of the Nucleolus.
I have made no special chemical study of these structures,
except what may be learned from their reactions to stains. In
the gregarines no substance could be demonstrated which chemi-
cally corresponds to the chromatin of the metazoan cell ;! but the
following table represents the mode of staining of true nucleoli
in the somatic and germ cells of the Metazoa:
STAIN. NvucLeo.us. CHROMATIN.
Del. or Ehrl. haematoxylin, eosin . red : A . blue.
Ehrlich-Biondi stain . - : . maroon or red PEpereen.
Acid carmine, nigrosine. “ . blue or greenish . red.
Del. haematoxylin, cochineal 3 . pinkorred . . blue.
Safranin, gentian violet, orange . . yellow . : . blue.
Schwarz (87) distinguishes in plant cells pyrenin, the sub-
stance of the true nucleoli, from the other nuclear substances
1 That is, not to chromatin in the form of pure nucleic acid.
498 MONTGOMERY. [VoL. XV.
and finds that it has a closer chemical affinity to the substance
of the nuclear membrane (amphipyrenin) than to any other
substance. Judging merely from the reactions of these two
substances to stains I would agree in this point with Schwarz.
Zacharias ('82) shows also for plant cells that the nucleolar
substance is sw generis and is allied to plastin. O. Hertwig
(92) terms the nucleolar substance “ Paranuclein’’ and observes:
“ Nuclein und Paranuclein betrachte ich als die wesentlichen
Substanzen des Kerns. ... Beide scheinen mir in irgend
welchen Beziehungen zu einander zu stehen.”’ But it is impor-
tant to note that the true nucleolar substance probably has no
chemical relation to the true chromatin (nuclein). Thus karyo-
somes should not be considered as a particular group of
nucleoli, since they are not nucleoli at all, but nodal points of
the chromatin reticulum. The substance of every true meta-
zoan nucleolus apparently differs chemically from the chromatin,
linin, paralinin, and oedematin (lanthanin) ; and accordingly
“pyrenin”’ is a term preferable to “paranuclein,” though
“pyrenin’”’ may include divers substances.
There are also chemical differences between the nucleoli
proper ( Hauptnucleoli”’) and the paranucleoli (‘« Nebennucle-
oli’), which occur together in many ova and in a few somatic
cells ; the substance of the paranucleoli stains more lightly
than that of the nucleoli proper. List (96) distinguishes three
kinds of true nucleoli, from a chemical standpoint : (1) the
nucleolus of somatic cells ; (2) the nucleolus proper of germinal
vesicles ; and (3) the paranucleolus of germinal vesicles ; and
he considers the substance of the paranucleus of the germ cell
to be closer related chemically to the nucleolus of somatic cells
than either of them is to the nucleolus proper of ova. List
promises a more complete paper on this subject. The so-called
“nucleoli,’”’ which react like chromatin, are of course not true
nucleoli, but either karyosomes (thickened nodal points of the
chromatin reticulum) or chromatin nucleoli (independent lumps
or spheres of chromatin). It is my intention to devote a special
paper to the consideration of the latter structures. Other
papers on the chemistry : Macallum (95), Michel (96), Carnoy
and Lebrun ('97).
No.2.] COMPARATIVE CYTOLOGICAL STUDIES. 499
2. Number of Nucleolz.
As Flemming (82) has stated, the number of nucleoli is
small in most cells, not more than from one to five. But in
certain stages of some cells there may be several hundred (ova
of Reptilia, Amphibia, Selachii, nemerteans, subcuticular gland
cells of Pzsczcola). Even in those cases just mentioned, where the
number of the nucleoli is very large, the immature cell contains
only one or a few nucleoli, so that the large number is attained
only when the nucleus has increased in size, cf the observa-
tions of Auerbach (74a). Among somatic cells a large number
of nucleoliis much more infrequent than among egg cells. At
a given stage of a given cell of any one species of metazoan
the number of nucleoli is pretty constant, and there is less
variability in the number among those cells where the typical
number of nucleoli is a small one than in those where a large
number is present. In cells where the usual number of nucleoli
is one or two, as in those of the nidamental gland of Mon-
tagua, three may quite frequently be found, but no cells are
found in which not a single nucleolus occurs ; in other words,
there is in most cases some degree of variability in the number
of the nucleoli, and the amount of this variability stands in a
more or less direct ratio to the number of the nucleoli, but it is
numerically progressive as a rule, tending to produce more than
the normal number, and in no cases where cells normally con-
tain nucleoli do we find a regressive numerical variation leading
to the total disappearance of nucleoli. In certain few cells no
nucleoli are present, and this is the case in more cells than
Flemming ('g2) was disposed to admit, since not only are spe-
cialized cells like mammalian blood corpuscles without them, but
they are also absent in certain connective-tissue elements of
nemerteans, and in certain other cells of a low degree of vitality.
Auerbach (90) formulated the law that the number of nucle-
oli is more or less constant for all the cells of a given species.
But this conclusion is certainly erroneous, since in Do¢o there
is one nucleolus found in the blood corpuscles and in the ovum,
from one to five in the ganglion cells, from one to three in the
cells of the nidamental gland, and in the giant cells as many as
500 MONTGOMERY. [Vou. XV.
forty ; and in P2sczco/a, usually one in the ovum and the
ganglion cells, about twelve in the mature muscle cells, and
three hundred or four hundred in the subcuticular gland cells.
From the data at hand we accordingly conclude that the
number of nucleoli is not constant for the species. (On the
number of nucleoli at different stages in amphibian ova, cf. Car-
noy and Lebrun, '97Aa).
In order to determine whether the number of nucleoli in egg
cells were fixed for, or in any way determined by, the particular
groups of Wetazoa, I have compiled the following tables (pp. 501—
505) for the larger groups, these tables representing the data
of previous investigators and of my own observations. In them
four classes of germinal vesicles are distinguished according to
differences in the number and kind of the nucleoli; this classifi-
cation is only for convenience’ sake, only arbitrarily chosen, and
is probably not a natural one. On the left hand is given the
name of the genus or group ; the asterisk corresponding to each
form indicates by its position in a particular vertical column the
nucleolar relations of the ovum of the form specified ; and next
to the asterisk is placed the name of the authority. In some
cases two investigators may have reached different conclusions
in regard to the nucleolar relations, so that for these cases two
asterisks were employed.
One must be extremely cautious in any attempt to draw
conclusions from these data, not only because the data are
so meager, but also because where data have been culled
from so many different observers some of the facts may ulti-
mately prove to have been erroneous. Thus many of these
ova may have been examined at only one point in their develop-
ment, and in others paranucleoli may have been entirely over-
looked, or may have been confused with true nucleoli. But
taking this mass of observations as it stands, the following gen-
eral conclusions may be drawn: we find that a large number of
nucleoli is not always characteristic of ova with a considerable
amount of deutoplasmic substances, for a single nucleolus is
typical for the birds and for many of the Arthropoda. Further,
the number of the nucleoli does not seem to be dependent
upon the amount of yolk, nor upon the mode of cleavage,
No. 2.]
Form.
Esperella
Spongilla
Tedamione
Hircinia
fHydrozoa
Aequorea
Hydractinia
Podocoryne
Geryonia
Tubularia
Eucope
Aeginopsis
Nausithoé
{ Pelagia
Physophora
{ Rodalia
Ctenophora
Bothriocephalus
Distomum
Polycladidea
Tricladidea
Rhabdocoele
Prorhynchus
Bothnioplana
Haplodiscus
Carinella
Cerebratulus
Lineus
{ Malacobdella
| Drepanophorus
Tetrastemma
Amphiporus
Stichostemma
Zygonemertes
Proneurotes
Prosadenoporus
Pelagonemertes
COMPARATIVE CYTOLOGICAL STUDIES.
501
| * (H. V. Wilson)
| * (Fiedler)
* |
4 } (H. V. Wilson)
* (Weismann)
* (Hacker)
*
zt (Bunting)
* (Fol)
| * (Doflein)
* (O. Hertwig)
| * (Chun)
Plathelminthes.
* (Schauinsland)
* (Schauinsland) |
* (Lang) |
* (Jijima)
* (Repiachoff) |
# |
i (Vejdovsky)
* (Bohmig)
Nemertini.
* (Biirger)
* (Hubrecht)
* (mihi) |
| * (v. Kennel)
| * (Burger)
A SINGLE More THAN OnE Nv-
NucLeouus. CLEOLUS, ALL ALIKE.
Coelenterata.
NUCLEOLUS AND
PARANUCLEOLUS.
* (O. Hertwig)
* (Brauer)
*
| (O. Hertwig)
*
| * (mihi)
| * (Burger, Coe)
* (Hubrecht)
502 MONTGOMERY. [VoL. XV.
F | A SINGLE More THAN OnE Nvu- NUCLEOLUS AND
ORM.
NucLeo.us. CLEOLUS, ALL ALIKE, PARANUCLEOLUS.
Annelida.
( Nereis * (E. B. Wilson)
Spinther ns _
Ophryotrocha * } (Korschelt)
Sternaspis * (Vejdovsky)
Polydora * (mihi)
Spio * (Giard)
Capitellids * (Eisig)
| Polygordius * (Fraipont)
Oligochaeta * 7 i
+ Rhynchelmis I Welcexsisy)
Lumbricus * (Claparéde)
{ Nephelis * (Leydig)
Branchiobdella as Tade)
Haemopis x ff 2 * (O. Hertwig)
| Piscicola * (mihi)
Clepsine * (Whitman)
l Piscicola * (Leydig)
Arthropoda.
f Homarus * (Herrick)
| Porcellio * (St. George)
Oniscus * (Wielowiejski)
Heterocope EN eer a.
Diaptomus *f (rene
Argulus * (Leydig)
| Astacus * (Wielowiejski)
Cyclops *
Sida - =
Canthocamptus mee)
Moina oe
L Euchaeta * (vom Rath)
{ Epeira * (Korschelt)
| Dolomedes * (Korschelt)
Phalangium * (Korschelt,
Leydig) * (Henking)
J Lycosa * (Leydig)
Theridium * (v. Wittich) * (Leydig)
Tetragnatha * (Leydig)
Araneina ws : oecerhe
Vey : \ (Wielowiejski)
| Zilla * (Van Bambeke)
No. 2.]
COMPARATIVE CYTOLOGICAL STUDIES.
593
Form.
A SINGLE
NucLeotus.
Julus
Geophilus
| Glomeris
\ Lithobius
Peripatus
{ Blatta
Nepa
Notonecta
Carabus nemoralis
Gryllotalpa
Pieris
Anabolia
Bombus
Anomalia
Ophion
Ephialtes
Pemphigus
Musca
Necrophorus
Geotrupes
Banchus
Pimpla
Stenobothrus
Meloe
Libella
Melolontha
Lina
Lycus
Sphinx
Ambyteles
rm
Psammechinus
Echinocardium
Echinus
Toxopneustes
Asteracanthion
Sphaerechinus
Amphidetus
Solaster
f Chaetoderma
| Proneomenia
* (Stuhlmann)
* (Stuhlmann)
* (Brandt)
‘ \ (Will)
|
#
*
.
*
» 7 (Stuhlmann)
*
*
e
* (Leydig)
* (Wielowiejski)
| More THAN One Nv- |
CLEOLUS, ALL ALIKE.
NUCLEOLUS AND
PARANUCLEOLUS.
* (Balbiani)
|
*
i | (Stuhlmann)
*
«J
% } (Leydig)
Echinodermata.
A »
i (Bergh)
*) :
ii (Ludwig)
Mollusca.
* (Wirén)
* (Leydig)
* (Stuhlmann)
* (Leydig)
* (Stuhlmann)
* (St. George)
* (Wagner)
*
(Stuhlmann)
| BS no
5 (Hacker)
*
at (O. Hertwig)
* (Hubrecht)
504
MONTGOMERY.
[VoL. XV.
Form.
A SINGLE
NvucLEoLus.
More THAN OnE Nvu-
|
CLEOLUS, ALL ALIKE.
NUCLEOLUS AND
PARANUCLEOLUS.
{ Neritina
Doto
Montagua
Tellina
_ Helix
~ Limax
Arion
Doris
Aeolidia
Amphorina
Paludina
( Anodonta
Unio
4 Mytilus
Pholas
( Cyclas
Distaplia
Phallusia
Botryllus
Clavelina
Ciona
Styelopsis
Ascidia
Amphioxus
Petromyzon
( Scyllium
4 Torpedo
\ Pristiurus
Scorpaena
Conger
Gadus
< Trigla
Gasterosteus
Anguilla
Cyprinus
Rana
- Amblystoma
\ Triton
Emys
~ Lacerta
eeomurtle +?
|
* (Blochmann)
* (mihi)
* (Platner)
Tunicata.
| * (Davidoff)
* (Bergh)
* (Pizon)
* (Seeliger)
|
|
Vertebrata.
| * (Van d. Stricht)
* (Bohm)
*)
|« | (Riickert)
| * J
* (Van Bambeke)
*
| * \ (Scharff)
A)
* (Ransom)
* (Brock)
* (Eimer)
* (Born)
* (Fick)
* (Leydig)
| * (mihi)
* (Eimer)
* (Agassiz)
* (mihi)
*
zs (O. Hertwig)
* (Mark)
* (Platner)
a (Lonnberg)
* (Trinchese)
(Leydig)
* (Flemming)
* (Hessling)
* (Lonnberg)
* (List)
| * (Stepanoff)
*
| | (Floderus)
* (O. Hertwig)
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 505
Forax A SINGLE More THAN OnE Nv- NucLEoLus AND
NUCLEOLUs. CLEOLUS, ALL ALIKE. PARANUCLEOLUS.
Gallus * (Holl) |
Fringilla * (v. Wittich) |
Columba * (mihi)
Felis * (St. George)
Cavia * (Rein)
Mus * (Holl) |
Vespertilio | * (v. Beneden)
Sus *
Myoxus | * \ (Leydig)
| Talpa a)
Ovis |
Lepus | * (St. George)
Homo | * (Nagel) * (Flemming)
nor yet upon the mode of deposition of the egg (z.e., whether
it is pelagic, hatched in a cocoon, or nourished in an uterus).
These facts hardly warrant an attempt to explain the factors
limiting the number of nucleoli, and perhaps such explanations
should rather be expected from experimental workers than from
purely structural observers. On examining the metazoan groups
in detail we find in certain of them a degree of uniformity in
regard to the number of nucleoli. Thus the only vertebrate
ova with two kinds of nucleoli are those of Lepus and Ovis. A
single nucleolus is the rule for Amphioxus, Petromyzon, the
birds, and most of the mammals; the Refzclia, Amphibia,
Teleostiz, and the Selachiz have numerous nucleoli. In the
Tunicata there is either a single nucleolus or a nucleolus and
paranucleoli; this is also the rule for the Echinodermata,
Mollusca, and Annelida. In the Arthropoda there is consider-
able diversity in regard to the number and differentiation of the
nucleoli. In the nemerteans we find most usually either a
single nucleolus or a large number of small ones. In the
Plathelminthes one or two is the rule; this is also most
frequently the case for the coelenterates, but in some of the
latter paranucleoli have been described.
506 MONTGOMERY. [Vou. XV.
3. Position of the Nucleolus in the Nucleus.
Where a single nucleolus is present it almost always lies
excentrically, though not against the nuclear membrane. Those
cases where it regularly occupies the center of the nucleus must
be regarded as exceptional ; thus I am unable to agree with
Macfarlane that the nucleolus is either the morphological or
the tropic center of the cell. At the time of its origin, and
often at the time of mitosis, the nucleolus may be in contact
with the nuclear membrane. Where a number of nucleoli are
present they may be scattered irregularly through the nucleus,
or grouped at one point in it, or be concentrically arranged ;
their position is often dependent upon the stage of the develop-
ment of the nucleus. Thus in the metanemerteans examined
by me they lie at the periphery in the smallest germinal
vesicles, then wander towards its center, and finally migrate to
the periphery again.
The nucleoli lie in the nuclear sap, as a rule not in any close
connection with the chromatin reticulum. But in those cases
where the nucleolus may be unusually large it appears to be
suspended by the fibers of this reticulum, but not in such a way
that the fibers penetrate into its substance, but become simply
wound around its surface; thus it appears that when the
nucleolus increases in size it forces apart the fibers of the
nuclear network in such a way that the latter gradually pro-
duce a latticework on its surface. In this way the nucleoli
may be more or less held in position in the nucleus, but
Herrick’s observations on the gravitation of the nucleolus
show that it is not firmly held by the chromatin fibers. The
nucleolus is, as it were, a ball lodged in the branches of a
tree, its movements hindered by the ‘intervening branches, but
nevertheless not immovable. Various views on the mode of
suspension of the nucleolus : Pfliicke (95), Heidenhain (92),
Rosen (95), Jensen (83), Zimmermann ('96). Note also its
peculiar position in Syxapta (Leydig, '52).
1 For the opinions of other authors, cf the reviews of the papers of Pfliicke
('95), Heidenhain (92), Rosen ('95), Jensen ('83), Leydig ('52), Zimmermann
(96), Schneider ('91).
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 507
4. General Morphological Structure of the Nucleolus.
The ground substance of the nucleolus is more or less dense,
but not brittle, and either homogeneous or finely granular,
rarely coarsely granular. It may be either fluid or viscid in
consistency.
In the greater number of cases it has no limiting membrane.
Such a membrane was found by me only in the germinal spot
of Polydora, and here it appeared to be merely a denser portion
of the ground substance. When any small nucleolus is viewed
in its totality a membrane appears to surround it, but this
phenomenon is due to the refraction of light from its convex
surface, and many observers have been misled by this appear-
ance into supposing that a membrane is present. Others in
describing those states of nucleoli in which a large vacuole is
present have erroneously described the peripheral layer of true
ground substance as a nucleolar membrane ; it is necessary to
distinguish between such a peripheral layer, which consists of
true ground substance, and a nucleolar membrane proper, which
is a differentiation of the ground substance. Some authors,
é.g., Lavdowsky ('94), have described a membrane of chromatin
enveloping the nucleolus, and I have found that those of the
giant cells of Doto may sometimes be surrounded by a mass of
chromatin. But this apposition of a mass of chromatin in Dozo
is certainly an artefact, though it would seem probable that
the nucleolus in some cases has an envelope of chromatin
forming a distinct capsule separated from the chromatin net-
work of the nucleus. I am able, however, to corroborate the
observations of Macfarlane ('81) and Pennington (97), that the
nucleolus in Spzvogyra has a true membrane.!
A very unusual structure of the nucleolus is that afforded by
the salivary gland cells of Chzvonomus as described by Balbiani
(81), Leydig (83), Korschelt (84), and Macallum (95). C.
Schneider ('91) supposes the nucleoli, as well as the rest of the
nuclear substance, to consist of ‘ Geriist”’ (linin?) and chro-
1 The following writers have described nucleolar membranes: Macallum (’95),
Carnoy and Lebrun ('97a), Will ('85), Holl ('93), Roule (’83), Biirger ('90),
Ogata ('83), Vejdovsky ('82), Meunier ('86), Carnoy ('86), Mann ('92).
508 MONTGOMERY. [VoL. XV
matin, and considers the nucleoli to be only isolated masses of
chromatin surrounded by linin sheaths ; these observations
have not been corroborated by any other writers and would seem
to be due to faulty methods of fixation.
In opposition to Meunier (86), and in agreement with most
investigators, I must conclude that vacuoles are normal struc-
tures in nucleoli, since they may be seen after the most diverse
methods of fixation, and their size and number are not only to
some extent limited for the particular cell, but are also different
at different periods in the metamorphoses of the nucleus. It is
the rule that the youngest nucleoli are homogeneous, and that
vacuoles first arise when they have increased in size. Their
size and number vary at different phases in the development
of the nucleolus. Very frequently a number of smaller ones
appear, and then these subsequently fuse together and produce
a larger one. The nucleoli of egg cells are characterized as a
rule by more numerous or larger vacuoles than those of somatic
cells, and in many somatic cells these vacuoles appear to be
wholly absent. The vacuolar substance appears in some cases
not to be a derivative of the ground substance of the nucleolus,
but to be derived from without the nucleolus (ova of Doo and
Montagua). Perhaps this vacuolar substance always has an
extranuclear origin, since in many cases a germinal spot grows
larger merely by an increase in its volume, while the ground
substance seems neither to increase nor diminish.
The alveolar structure of nuclei as described by Purcell (94),
Schaudinn (94), Korschelt (95), and Lauterborn ('95b) is prob-
ably referable to the regular distribution of equal-sized vacuoles
in the nucleolus.
A “Kernkorperchenkreis,” a shell of minute granules
arranged concentrically around the nucleolus, has been de-
scribed by Eimer (71,°72), Auerbach (74a, who considered it
to be the result of opposing repulsive forces of the nucleolus
and nuclear membrane), Brass (’g9), Pfliicke (95), Platner ('89a),
Smirnow (90), Engelmann (’g0), Carnoy and Lebrun (97a). A
more or less similar phenomenon has been described by me for
ganglion cells of Doto. Such a nucleolar circlet must be con-
sidered, in most cases at least, an artefact. But in this cate-
No.2.] COMPARATIVE CYTOLOGICAL STUDIES. 509
gory should not be classed small masses of nucleolar substance
grouped around a larger one, these being normal phenomena
during the growth period of nucleoli.
Reticulations within the nucleolar substance have been de-
scribed by some few authors. Thus Carnoy ('85, '97a), Meunier
(86), and Moll (93) described nucleoli containing a skein of
chromatin, but Zacharias and Strasburger (ss) did not find
anything resembling these supposed skeins described for
Spirogyra. Leydig (88) states that the germinal spot of Lycosa
“‘pbietet das Bild eines Knauels dar.’’ Fromann ('g4) described
the nucleolar substance as consisting of granules connected by
fibers, Biitschli (80) found the nucleoli of Dznoffagellata to
contain a fine reticulum, and Davidoff (89) states that the
germinal spot of Dzstaplia takes up portions of the nuclear
reticulum into itself (but cf Bancroft, '98, and Shafer, '80). The
only structure which was found by me to resemble a skein was
present in the later stages of the germinal spot of Polydora ;
but in this object, owing to the gradual confluence of the vacu-
oles, which thus produce anastomosing channels of vacuolar
substance in the ground substance of the nucleolus, it is the
true ground substance which represents a skein-like appear-
ance. It is very probable that Carnoy and his followers have
mistaken the vacuolar substance for the ground substance, and
have considered the true ground substance to be chromatin ;
I am forced to conclude that in all probability there are no
skeins of chromatin lying in any metazoan nucleolus, since I
have never found any evidence of chromatin in it in any meta-
zoan cell. But it is not improbable that in the nuclei of
gregarines chromatin may be massed in some or all of the
nucleoli.
Nucleolini, granules within the nucleolus, have frequently
been observed. A single nucleolinus to a nucleolus has been
described by Vejdovsky ('95b), Morgan (96), Agassiz ('57),
Kleinenberg (72), Leydig (88), Macfarlane (85), Lavdowsky
(98), A. Brandt (78), Van Bambeke ('g6), Kosinski ('87, '93) ;
several nucleolini to a nucleolus, by Biirger (90), Rhumbler
(93), Holl (93), Wolters (91), Schron (65), Scharff (88), See-
liger (82), Gjurasin (93), Haeckel (174), Mann (92), Van Bam-
510 MONTGOMERY. [Vou. XV.
beke ('97b), Mark (77), Bancroft (98). Compare also the follow-
ing: Huie (97), Van Bambeke ('97), Kosinski (87, '93), Mark
(77), Zimmermann ('96), Hodge (94). I have found these bodies
occurring in varying number, though most frequently absent,
in the nucleoli of various cells, and they appeared to be merely
loosened portions of the ground substance which had come to
lie within a vacuole. Macfarlane and his pupil Mann have
described nucleolini under the names “ endonucleolus”’ and
“nucleolo-nucleus”’ as occurring singly and with great con-
stancy in certain plant cells, though Zacharias ('g5) studied
Macfarlane’s object (Chara) and makes no mention of any of
these structures. Macfarlane ascribes the utmost importance
to his “endonucleolus,” regarding it as the tropic center of the
cell and as an important mechanical agent during nuclear
division. Mann has not only described a most complex struc-
ture of the nucleolus, such as no other observer has yet seen,
but also has found fine fibrils radiating out from it, which he
supposes to penetrate through the nuclear cavity. From my
own observations, and in agreement with the majority of ob-
servers, I can attach no particular morphological significance to
the nucleolinus; it appears to be only a detached portion of
the nucleolar ground substance, to be in most cases absent,
and when present to vary greatly in regard to size, position, and
number. It is undoubtedly the case that many structures
which have been described as nucleolini are in reality minute
vacuoles, which from their refrangibility appear to be granules ;
such is the case with the minute vacuoles of Polydora and
Montagua when studied after the action of certain stains, and
has been shown for other objects by Zimmermann and Huie,
Lavdowsky found in the nucleolus a central vacuole, and in
the latter a small granule, which he supposed to be ‘das noch
in Entwicklung begriffene Centrosoma,” destined to finally
pass out of the nucleolus; he was unable to determine how
it does wander out of the nucleolus and become the centrosome,
so that his suggestion has merely the value of a hypothesis.
Van Bambeke describes the nucleolinus of the germinal spot
of Amaurobius as “doué d'un mouvement trés vif” ; this
interesting phenomenon certainly deserves investigation, though
No. 2.]} COMPARATIVE CYTOLOGICAL STUDIES. 511
it is not impossible that the supposed nucleolinus was in reality
a microorganism inclosed in the vacuole of the nucleolus.
(&. also Flemming’s observation on the egg of Asczdia, '97.)
Supposed nerve fibrils in the nucleolus have been described by
Eimer ('73, '90).
5. Polarity of the Nucleolus.
In the gregarine (Goxospora?) from the intestine of Lzxeus
gessevensis it is the rule that the vacuoles make their first
appearance at that pole of the nucleolus which is nearest to the
nuclear membrane. In the germinal spot of Montagua the
opposite position of the large excentric vacuole is the rule,
though the percentage of cases in which the vacuole has a
particular position with regard to the nuclear membrane is
less than in the gregarine. On the contrary, in the germinal
spots of Pzsczcola and Rodalia there is no regularity in regard
to the position of the vacuoles, and in that of Polydora the
vacuoles are, at the time of their first appearance, usually
central in position. In the germinal spots of many other
Metazoa, where a single large vacuole is present it more
usually lies excentrically than centrally, though its position
appears to be independent of the proximity of the nuclear
membrane ; so that in these cases we can speak of a certain
polarity in regard to the position of the vacuole within the
nucleolus, and not of a polarity of the axis of the nucleolus
in regard to the position of the nuclear membrane. But
in the two gregarines examined by me the substance of the
nucleolus, or of some of the nucleoli, is differentiated at
two poles of the nucleolus, so that the portion of the ground
substance at one end stains differently from that of the other
end of the nucleolus ; this state apparently does not occur in
the nucleoli of metazoan cells. It remains to be solved whether
in the gregarines the chromatin or its physiological equivalent
is localized at some particular point or pole of the nucleolus,
z.e.,. whether or not such nucleoli should be compared to the
nucleoli of the Mezazoa.
512 MONTGOMERY. [Von. XV.
6. Amoeboid Movements, Divisions, and Fusions of Nucleolt.
Amoeboid movements have been seen in life in metazoan
cells by the following observers (germinal vesicles): A. Brandt
(74, Blatta), Eimer ('75, Szlurus), O. Hertwig (76, Rana,
Pterotrachea), La Valette St. George ('66, Lzbella; '83, /sopoda),
Bergh (79, Gonothryaea), Van Beneden ('69,'76, Polystomum,
Rana), Balbiani (64, several genera of spiders), Leydig (ga,
Libella), A. Brandt (78, numerous /zsecta, Distomum), Van
Bambeke (86, atta), Knappe (86, Bufo), Auerbach ('74a,
Teleostii). In somatic cells: Schwalbe (76, sympathetic gan-
glion cells of Rana), Kidd (75, epithelial cells from the mouth
of Rana), Hodge (94, nerve cells of Rana), Auerbach ('74b,
salivary gland cells of Musca). In Protozoa: Van Beneden
(69, '76, Gregarina, Monocystis). In plants, Zacharias ('85) has
observed amoeboid movements in the nucleoli of Chara (an
observation overlooked by Zimmermann, who states that such
movements have not been seen in plants).
These observations would show that amoeboid movements
are probably natural phenomena of certain nucleoli, but one
should not be too positive of the naturalness of these phe-
nomena, since some of the observations were made upon the
heated stage, and in all of them the object was probably more
or less compressed and placed in artificial conditions. But they
are in all probability frequently normal phenomena, since, as we
shall see, divisions and fusions of nucleoli are certainly normal
and of wide occurrence, and the latter can only be classed as
forms of amoeboid motion. The question arises, Are these
movements wholly passive, caused by movements in the other
parts of the nucleus, or should they be considered an inherent
function of the nucleolus? The latter alternative would seem
the more probable, since no movements of the other nuclear
elements are known in the resting cell. Van Beneden ('69) has
described rhythmic expansion and contraction of the volume of
nucleoli in gregarines. But all these movements of nucleoli
should not be regarded as automatic motions of the nucleolus
in the sense that an Amoeba forms and retracts processes ; but
rather with Rhumbler (93) they should be regarded as “ Auf-
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 513
lésungsvorgange,” due to chemical changes in its substance.
Cf. the movements described by Flemming (97) for the ovum
of Ascidia.
The nucleolus has in some cases a viscid consistency (as
described by me for Stzchostemma) and then may be irregular
in form ; in other cases it is more fluid, and this is probably
the case when it has regularly a spherical shape, 7.¢., the globu-
lar form characteristic of drops of a thin liquid. Its more or
less fluid consistency allows changes of form, division into
particles, and fusions of neighboring nucleoli.
The division of a nucleolus into two or more parts is a
normal and regular phenomenon in many cells, though all
nucleoli do not show this property. Two kinds of nucleolar
division may be distinguished : (1) that mode by which the
nucleolus becomes elongated and then breaks into two or more
parts, whereby the daughter-nucleoli are usually capable of
further division; and (2) that mode by which the nucleolus
fragments nearly simultaneously into a number of small gran-
ules. From my own observations the former mode is evinced
by the nucleoli of the muscle and giant gland cells of Pzsczcola,
the giant cells of Doto, and the germinal spots at certain
stages in the ovogenesis of the metanemerteans. This mode of
division cannot be regarded as a phenomenon of nucleolar
degeneration, since the nucleolus and its products may often
continue to increase in size during the process of division. But
the second mode, that by which the nucleolus breaks into a
large number of granules, since it is particularly characteristic
of the nucleolus in nuclear division, may be regarded as a
process of degeneration ; the case of divisions during nuclear
division shall be considered later. A strange mode of nucleolar
division has been described by A. Schneider (83). According
to his observations on K/ossza, the smaller nucleoli are portions
of the inner substance of the larger nucleoli and wander out of
each larger one by passing through the pore (“canal micropy-
laire”’) of the cortical substance of the latter; this intranucleolar
origin of the smaller nucleoli is still open to question, since it
was not observed in life, and since the canal micropylaire was
observed in only one nucleolus. Marshall (92) has described
514 MONTGOMERY. [VoL. XV.
a somewhat similar method of formation of the smaller nucleoli
of Gregarina blattarum. Now I found in the nucleus of the
gregarine from ZLzzews numerous nucleoli of different dimen-
sions, and often irregular in their outlines ; and this irregularity
in form would point not only to amoeboid movements of the
nucleoli, but also to nucleolar divisions, since in the largest
nuclei we find a large number of small nucleoli. All appear-
ances showed that these smaller nucleoli are division products
of the larger ones ; but it seems that they simply bud off from
the surface of the latter, and are not preformed in their interior.
In other words, Schneider and Marshall are probably correct
in concluding that the smaller nucleoli are disassociated portions
of the larger ones; but they may perhaps be mistaken in
assuming that they are preformed in the interior of the latter,
since these investigators may have mistaken vacuoles for intra-
nucleolar nucleoli. (Other observations on nucleolar divisions
in resting cells : Hermann, '89 ; Vejdovsky, ‘95a ; Biitschli, '8o;
R. Hertwig, '76 ; Kultschitzky,'ss8 ; Bergh, 79; Bannwarth, '92;
Stuhlmann, ‘86; A. Brandt, 78; Scharff, ‘88; Eisig, '87 ;
Cunningham, '95 ; Kosinski, '87, '93 ; Carnoy and Lebrun, '97a ;
Steinhaus, '88 ; Cuénot, ‘91 ; Metzner, '94.)
Fusions of nucleoli are not as widely known as divisions, but
there are some facts which would show that the former processes
are by no means unusual in their occurrence. Such fusions
have been described for cells of plants by Zacharias (85), Mann
(92), and Wager (93) ; for animal cells by Rhumbler ('98, ’95),
Brauer (91), Leydig ('50), Pfitzner (83), and Riickert ('92). I
have found fusions of the nucleoli to be characteristic phenomena
of certain stages in the maturation of the germinal vesicles of
nemerteans, an extreme case being furnished by Stichostemma,
where sometimes all the nucleoli may fuse together at the
center of the nucleus, and so produce a single large one. The
nucleolus at the time of its origin may be said to be undergoing
a process of fusion, since it is produced by the coalescence of
numerous smaller portions of nucleolar substance. There is
nothing problematical in regard to the fusion of nucleoli, since
it is a physical property for bodies of like nature (when fluid)
to fuse together when they come into contact, though this
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 515
process is to some extent dependent upon the nature of the
medium in which they are suspended (cf Rhumbler, '93). (Cf.
also Hermann, ’89b ; Bouin, '97; Mertens, '93 ; Debski, '97 ;
Carnoy and Lebrun '97a; Koernicke, '96.)
7. Paranucleolt and Pseudonucleoli, Double Nucleolt, etc.
The term paranucleolus is here adopted as equivalent to
Flemming’s ‘“ Nebennucleolus,” and I shall use simply the
name “nucleolus,” or “nucleolus proper,” instead of “ Haupt-
nucleolus.” E. B. Wilson’s terms, “principal nucleolus ’”’ and
“accessory nucleolus,’ are somewhat inconvenient on account
of their length, and may be misleading, since the “ principal
nucleolus” is often smaller than the ‘accessory nucleolus.”
“ Paranucleolus,” as used here, is not employed in the same
sense as by Stuhlmann ('86), since he expresses by this term
portions of the nuclear reticulum ; in my paper the term
“nucleolus” has not been used for any part of the chromatin
elements of the nucleus.
In many egg cells, especially those of the Mollusca, Annelida,
Tunicata, and Echinodermata, two kinds of nucleoli occur accord-
ing to the writers on these objects, which differ from one
another chemically and in some cases also structurally ; these
are the nucleolus proper and the paranucleolus. Of these it
is the nucleolus proper which seems to be morphologically
comparable to the nucleoli of somatic cells, however the two
may differ chemically. The paranucleolus may be either larger
or smaller than the nucleolus, and appears usually to be distin-
guishable from the latter by staining less deeply with the
specific nucleolar stains. In the spermatoblast of the mouse
these two kinds of nucleoli have been found by Hermann (gg) ;
and in somatic cells by Lénnberg (92, liver cells of Doris, Poly-
cera, Acolidia, and Astacus) ; perhaps the smaller of the two
nucleoli found by me in the blood corpuscles of Doto might
represent a paranucleolus. In plant cells apparently only one
kind of nucleolus is present, this being comparable morphologi-
cally to the nucleolus proper of the germ cells and to the nucle-
oliof the somatic cells of Metazoa. Thus paranucleoli are quite
51 6 MONTGOMERY. [VoL. XV.
frequent in many egg cells, infrequent in somatic cells of the
Metazoa, and apparently never present in plant cells. In each
such egg cell there may be either one nucleolus proper and
from one to several paranucleoli (this being the most usual case),
or there may be a single paranucleolus and a few nucleoli
proper. In the ova of three forms examined by me there were
two kinds of nucleoli present, namely, in MWontagua, Polydora,
and Rodalia. In my descriptions I have employed the term
“‘pseudonucleolus ”’ for these secondary nucleoli, since in this
form they have a different structure from that of the nucleolus
proper, but nevertheless stain in the same way, so it is difficult
in this case to decide whether they correspond to paranucleoli,
and hence I have used the indifferent name “ pseudonucleoli”
for them. In Polydora we found from one to three paranucleoli
in the larger germinal vesicles, and these are always apposed to
the nucleolus. Then the smaller, deeply staining bodies in the
maturer stages of the ovum of Rodalza may be comparable to
paranucleoli. Whether the remarkable structures of the germi-
nal vesicles of Tetrastemma catenulatum are paranucleoli, I am
wholly unable to decide. This problem of different types of
true nucleoli in the same nucleus is one of the most difficult
in the study of nucleolar structures, so that it is necessary to
discuss it more in detail.
A. Schneider ('83), Brauer ('91), and Floderus (96) consider
the paranucleoli to be derivatives of the nucleolus proper, more
especially to be buds from its surface. Hicker ('93a) considers
them to be secretions of the chromatin. Flemming (82) doubts
whether “ die Unterscheidung von Haupt- und Nebennucleolen
eine durchgehende Geltung beanspruchen kann ”’; he finds that
in Anodonta the two are at first in contact, but that later they
become separated. Giard ('81) finds in the ovum of a Spionid
one nucleolus, and later there appears in the nucleus a much
smaller body, which fuses with the former. Lonnberg (92)
thinks that the paranucleoli may serve for the acquisition of
nourishment, or may contain reserve nourishment. List ('96)
considers that the paranucleoli and the nucleoli of the somatic
cells are more closely allied to one another than to the nucle-
olus proper of the ova, and that the former two “ mindestens
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. S17
verschiedene Modificationsstufen des Paranucleins .. . darstel-
len.” Hessling (54) found that in the ovum of Unzo the smaller
paranucleolus is divided off from the larger nucleolus proper.
Hacker, in his last paper on the subject ('95), considers that the
paranucleoli are of later formation than the nucleolus proper.
Now in many of those cases where a paranucleolus and a
nucleolus have been described lying in contact with one another
it is very probable that the vacuolar portion of the vacuole has
been described as a paranucleolus. I have no doubt that many
of the earlier observers, who studied the nucleolus mainly in the
living egg, have been thus misled, since only sections of nucleoli
can show the true nature of the nucleolus. Thus Loénnberg, in
speaking of “helle Kugeln” in the germinal spot of AZytzlus,
says : “Es ist schwer zu entscheiden, ob es sich hier nur um
Vacuolen handelt”’; and any one studying the unsectioned nucle-
olus of A/ontagua would be misled into supposing that here two
nucleoli of different consistency are apposed to one another.
Accordingly, we must be very careful in treating as facts some
of the observations of the earlier workers, which were made
upon unstained and unsectioned material.
But there are undoubtedly many cases in which two kinds of
nucleoli do occur,! and this is especially so in germinal vesicles.
The nucleolus and the paranucleolus may be in contact with
one another, may be always separated, may at first be in con-
tact and later become separated, or finally may be at first
separated and later come into mutual contact. Are these para-
nucleoli derived from the nucleolus proper, or have they a
distinct origin? In the ovum of Polydora the paranucleoli
appear towards the close of the maturation period, and then are
always in contact with the outer surface of the nucleolus proper.
1 Cf. the reviews of the following papers: Floderus ('96), Hermann ('89a, b,
97), Vejdovsky ('95a), Flemming ('74, '82), Hacker ('93a), Kultschitzky ('88),
Lukjanow ('87b), Brauer ('91, '92), Nussbaum (’87), Rein ('83), Henking (’87),
Van Beneden (’80), Leydig ('55a, '50), Stauffacher ('93), Stepanoff (’65), Giard
(‘81), Mark (77, '81), Lonnberg (92), Stuhlmann (’86), List ('96), Van Bemme-
lin ('83), Platner ('86), Claparéde (’69), Hessling ('54), Riickert ('94), Bouin ('97),
Vom Rath ('95b), Moore ('95), Weismann and Ishikawa ('89), Fol ('89), Lacaze-
Duthiers ('57), Fauvel ('97), Held ('95), Michel ('96), Steinhaus ('88), Metzner
(94), Braem ('97), Siebold ('39), Reinhard ('82), Kraepelin ('92), Davenport
(91).
5 18 MONTGOMERY. [VoL. XV.
In the ova of MWontagua and Rodala they are never in contact
with the nucleolus. In none of these three cases observed by
me does there seem to be any genetic connection between the
paranucleoli and the nucleoli proper. And in other cases,
where the two are separated (this separation is the most usual
state), no genetic connection between the two has been de-
scribed ; and even in that smaller number of cases where
they are in contact with each other at some period of their
development, no positive proof of their genetic relation has
been offered. Therefore we might conclude, though with re-
serve, that in the greater number, if not all, cases the para-
nucleoli are not derivatives of the nucleolus, but are products
sut generis. It is the rule that the nucleolus proper appears
in the nucleus before the paranucleoli arise, the latter usually
arising first towards the close of the growth stages. Accord-
ingly, though I cannot corroborate Hacker's (95) conclusions
as to the origin of the nucleolar substance, I am inclined to
agree with him that portions of nucleolar substance are succes-
sively deposited in the nucleus, and that those portions which
are deposited last, after the nucleus has undergone important
physiological and chemical changes, would differ from the
portion first produced (that of the nucleolus proper), and so
would represent the paranucleolus. And there are certain facts
from my own observations which would support this view. In
the earlier stages of the maturation of the ovum of Tetrastemma
and Zygonemertes there are a large number of nucleoli produced
successively at the periphery of the nucleus; these then wander
successively to the center of the nucleus, and then from that
point again to the periphery. Now in this last stage, when the
nuclear filaments are commencing to arise, we find, usually in
contact with the latter, much smaller, more deeply stained
nucleoli, and these I have termed ‘nucleoli of the second
generation.” We have found, accordingly, that after the nu-
cleus has passed through very marked physiological changes
(increase in size, redistribution of chromatin), another kind of
nucleoli appears, which may or may not be morphologically com-
pared to the paranucleoli of other ova. These nucleoli of the
second generation have neither a genetic nor a physiological
No.2.] COMPARATIVE CYTOLOGICAL STUDIES. 519
relation to those of the first generation ; and their difference
from the latter is probably due to the fact that they have been
produced at a time when very different physiological conditions
exist in the nucleus.
It is not my intention in this contribution to deal in any
detail with those cases where double nucleoli occur in a cell, or
those where two chemically and morphologically different kinds
of “nucleoli”? occur in the same nucleus ; to these cases it is
my intention to devote a special study. But preliminarily, from
those observations which I have made on this subject, the
following conclusions are in order. In a nucleus there some-
times occurs a double nucleolus, the component parts of which
may each represent a true nucleolus ; or such a double nucle-
olus may consist of a true nucleolus apposed to a chromatin-
nucleolus (according to my unpublished observations on the
spermatocytes of the beetle Havpfa/us). Further, and this is fre-
quently the case in resting spermatocytes of the first order, the
nucleus may contain a true nucleolus separated from a chromatin-
nucleolus ; and in Pexzatoma, the account of the spermatogenesis
of which will be shortly published by me, the unique process
occurs of the chromatin-nucleolus being a metamorphosed
chromosome (one of the fourteen chromosomes of the last
spermatogonic division becoming the chromatin-nucleolus of
the first spermatocyte) !_ This peculiar structure of Pentatoma
divides with the true chromosomes in the first reduction divi-
sion. In another case where I have been able to follow all
the developmental stages of a chromatin-nucleolus, namely, in
cells of the hypodermis of the larva of Carpocapsa, I found it
to originate from one of the granules of the nuclear reticulum,
—a particular one of these granules (karyosomes) gradually
increasing in size until it attains large dimensions ; during its
growth period it is usually attached to one of the true nucleoli
of the cell. What is of importance in these two cases (Penta-
toma and Carpocapsa) is the distinction emphasized between the
true nucleolus and a karyosome or chromatin-nucleolus : the
latter always standing in genetic connection with the true
chromatin, while the former, so far as my observations go, is
never derived from this substance. These observations are not
520 MONTGOMERY. [VoL. XV.
wholly out of place in the present paper on the true nucleolus,
since they are necessary to prove that the true nucleolus is in
all cases never derived from the chromatin ; where “ nucleoli ’”’
have been described as arising from the chromatin elements of
the nucleus, such structures cannot correctly be included under
the term “nucleolus,” when the latter is used in the proper
sense.
8. Relation between Nucleoli and Centrosomes.
The greater number of cytologists agree that there is no
genetic relations between these two structures ; and my obser-
vations on the egg of Pzsczco/a as well as more recent studies on
other objects corroborate this view. But some few have been
led to contrary conclusions by observing the fact that in mitosis
the nucleolus often disappears about the time that the centro-
some becomes apparent. Thus Karsten ('93) assumes that the
nucleoli wander out of the nucleus into the cytoplasm, and
there become the centrosomes of the spindle ; this observation
has been refuted by Humphrey (94). Also Wasielevsky ('93)
believes that the centrosomes of the egg of Ascaris stand in
some connection to the nucleoli, but this stands in direct oppo-
sition to the conclusions of all other workers on this object,
except those of Carnoy and Lebrun ('97b), and the supposition
of Sala (95). Then Lavdowsky ('94) concludes that the nucleo-
linus is the centrosome in the process of formation, but he failed
to observe the steps by which this body develops into a centro-
some. Further, Julin ('93b) is said by Delage (95) to have
assumed a genetic relation between the centrosome and the
nucleolus. Other’supporters of the nucleolar origin of the cen-
trosome : Balbiani (95), Wilcox ('95), Bremer ('95b), F. Toyama
(94). I believe that these are the only investigators who have
assumed this genetic relation. We may conclude, from the
greater number of observations at hand, that there is probably
no connection between these structures in the metazoan cell.
But it is difficult to decide the homologies of the body found by
Keuten (95) in the nucleus of Cevatiwm, and termed by him
nucleolo-centrosoma ; he considers it as equivalent to the central
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 521
spindle and centrosome of Ascaris, but might it not be com-
pared to the nucleolus alone, or to the nucleolus plus centro-
somes of the metazoan cell? However, the significance of
most protozoan “nucleoli” is very problematical. (Cf the later
observations of Lauterborn, '95a.)
9. Ontogenetic Origin of the Nucleolus.
Very few observations have been made to determine the
mode of origin of the nucleolus, though there are numerous
hypotheses intended to explain it. We may leave aside, for the
time being, its mode of reappearance in the daughter-nuclei
after nuclear division, since a special section will be devoted to
that subject.
In order to determine the mode of origin of the nucleolus in
resting stages of nuclei, I have studied those cells in which at
first no nucleolus is present, but which after a certain period of
growth acquire one. Objects well adapted for such investiga-
tion are the ova of the nemerteans and the mesenchym cells
of Cerebratulus. For details of these processes the reader is
referred to the observations.
In the ova of the nemerteans the nucleoli at the time of their
first appearance are always in close contact with the nuclear
membrane ; this is also the case for the mesenchym cells of
Cerebratulus, and probably for the paranucleoli of the ova of
Rodalia. In all these cells the nucleoli only then leave the
periphery of the nucleus and wander towards its center, after
the nucleus has increased more or less in size. There is only
one explanation for the peripheral position of the nucleoli at the
time of their first appearance, namely, that their substance is
extranuclear in origin. This process of formation has already
been discussed in detail for the several cells, and it is not
necessary to repeat here all the detailed observations on which
the main deduction is based. If the nucleolar substance were
a secretion of the nucleus, as Hacker ('95) assumes, how would
this assumption explain the strictly peripheral position of the
nucleoli when they first arise? For on Hiacker’s hypothesis
we should expect the supposed nucleolar secretions to be de-
522 MONTGOMERY. [VoL. XV.
posited evenly throughout the nucleus, and not only at the
periphery. And his deductions are based in great part, as those
of most other investigators, on the study of maturation mitoses,
and he had not observed their first mode of origin, namely, their
origin in nuclei which are not in the prophases of mitosis, but
are only gradually becoming differentiated from somatic cells.
I have found no evidences in any cell that the nucleoli stand
in any genetic relation to the chromatin elements of the
nucleus ; and while the chromatin may derive substances from
the nucleoli, I am unacquainted with any observations which
show that the nucleoli derive any part of their substance from
the chromatin. In all the cases observed by me, the nu-
cleus appears to assimilate a substance or substances from the
cytoplasm, and after this substance has entered the nucleus it
apparently undergoes there a chemical change, and becomes
deposited on the inner surface of the nuclear membrane in the
form of masses of varying dimensions, which may be either
globular or irregular in shape, according as they are fluid or
viscid in consistency. In the case of the ova of the nemerteans
the substance taken up into the nucleus, and which there
becomes deposited in the form of nucleoli, is sometimes exactly
similar to the substance of the yolk-balls which lie in the cyto-
plasm; in other cases it is probably similar to those metabolically
changed portions or inclusions of the cytoplasm, out of which
the yolk-balls are later differentiated. In Lznzeus, indeed, the
yolk-balls may often be found halfway through the nuclear mem-
brane, and their appearance is exactly similar to that of the
nucleoli. Inthe mesenchym cells of Ceredvatulus the substance
of the nucleoli appears to be identical with that of the numer-
ous nutritive granules which are dispersed in the cytoplasm ;
the latter globules arise in the cytoplasm before the nucleolus
appears in the nucleus, and as soon as they become numerous
in the neighborhood of the nucleus, peripheral nucleoli begin to
appear in the latter. In the subcutical gland cells of Pzsczcola
the nucleolus, at the time of its most rapid growth, is apposed
to the nuclear membrane; but when this period of volume-
increase has ceased, it is never found in this position. Further,
the paranucleoli of Rodalia appear first in contact with the
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 523
nuclear membrane. Schwalbe (76) found in the nuclei of
various vertebrate embryos that when the nucleoli first arise
they appear as thickenings of the inner surface of the nuclear
membrane.
From these observations I conclude, accordingly, that the
nucleolar substance, in many if not all cells, has an extranu-
clear origin ; and that, though it may undergo a chemical change
after entering the nucleus, it can be regarded neither as a secre-
tion nor as an excretion of the latter. In making this con-
clusion I can corroborate the views of only one investigator,
namely, Korschelt (89), though he changed this opinion in a
later paper (97). He concluded that the nucleolar substance
stands in some connection with the nutritive processes of
the cell, and that the nucleus probably derives it from the
cytoplasm.
Other views on the origin of the nucleolus (those of Hacker
have already been mentioned): Auerbach ('74a, '76) first supposed
the nucleolus to be cytoplasmic in origin ; more recently (90)
he appears to champion its nuclear origin. Rhumbler (’93)
assumes that the “ Binnenkérper” of Protozoa are products of
the nucleus, but he does not attempt to decide whether those
of the Metazoa have a similar origin. Strasburger ('82b) also
postulates a nuclear origin for the nucleolus, and assumes that
its substance is allied to chromatin. Jordan (93) holds that
the nucleoli probably arise from the chromatin threads. Flem-
ming (82) considers them to be “specifische Produkte des
Kernstoffwechsels.” Schwalbe (76) supposes the nucleolar
substance to be at first identical with that of the nuclear mem-
brane, since he found it to arise as thickenings of the latter.
C. Schneider ('91) supposes it to be a metamorphosed portion
of the chromatin. Leydig (83) concludes that the nucleoli are
portions of the chromatin reticulum. Guignard ('85) assumes
that they are derivatives of the chromatin filaments. Watasé
(94) considers them to be metabolic products of the cell, but
he gives no detailed observations in regard to their mode of
formation. Mertens (93) and Retzius ('81) consider them to
arise by concentration of the chromatin reticulum.
524 MONTGOMERY. [Vou. XV.
10. Discharge of Nucleolar Substance from Resting Nuclet.
Will (84) holds that the larger nucleoli of the amphibian ger-
minal vesicle pass out into the cytoplasm, and there become the
yolk-nuclei ; and Scharff ('88) corroborates this view for the ova
of Trzgla, though it is opposed by Cunningham ('95). Macallum
(91) concludes that in amphibian ova the peripheral nucleoli
generate a substance which diffuses first in the nucleus and from
there into the cytoplasm, and that this substance combines with
the cytoplasm to form the yolk substance ; Jordan (93) expresses
a somewhat similar view in regard to the yolk formation of the
newt. Henneguy (93) assumes that the corpuscle of Balbiani
in the ova of Vertebrata “est trés probablement une partie de
la tache germinative, ou une tache germinative entiere, qui sort
de la vésicule [germinative] pour penetrer dans le vitellus,” and
Mertens (93) holds a similar view. And for egg cells of
Tunicata, Floderus (96) confirms Roule’s (84) observations,
that the “ intravitelline Korper”’ are paranucleoli which have
wandered into the cell body. Cf also Bremer ('95a, b).
Leydig (88) finds that in ova of Geophilus, Stenobothrus,
Rana, and Tyiton particles of nucleolar substance penetrate
into the cytoplasm. Lukjanow (88) concludes that in the case
of the cells of the stomach mucosa of Sa/amandra, the nucleo-
lus discharges a portion of its substance from the nucleus.
Humphrey (94), from observations on plant cells, maintains
that in some cases portions of nucleolar substance may pass
into the cytoplasm.
Fol (83a, b) concludes that the follicle cells of the ascidian
egg arise as buds from the surface of the germinal vesicle,
and that each of these buds contains a particle of nucleolar
substance ; these conclusions are affirmed by Roule (83).
Scharff ('88) supposes that the follicle cells of the ovum of
Gadus are derived from nucleoli which have left the germinal
vesicle, such nucleoli becoming the nuclei of the new cells.
(Ogata '83) studied human pancreas cells and finds that a
nucleolus wanders out of the nucleus, becomes a ‘‘ Nebenkern,”
and the latter finally changes into the nucleus of a new cell, a
conclusion which is opposed by Platner ('g9b).
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 525
I have found a wandering of nucleolar substance out of rest-
ing nuclei in one very beautiful and unique case, namely, in the
subcuticular gland cells of Pzsczco/a ; at one stage in its cycle
of development the nucleus commences to contract in volume,
and in so doing discharges all except a single one of its nucleoli
into the cytoplasm. This and certain of the observations cited
from other investigators show that a discharge of nucleolar
substance from the resting nucleus takes place in some cells.
But the more recent observations of Morgan, Floderus, and
others on Tunicate development render it very probable that
Fol and Roule were mistaken in assuming that the nucleoli
which pass out of the germinal vesicle become the constituents
of follicle cells. There is still some question, also, as to whether
the nucleolar substance in the cytoplasm takes any part in the
formation of the yolk substance. Other pertinent observations :
Mertens (93), Bremer ('95a, b), Kosinski ('87, '93), Galeotti (95),
Melissinos and Nicolaides (90), Auerbach ('74), Ver Ecke
(93), Steinhaus ('88), Rohde ('96).
11. Behavior of Nucleoli during Nuclear Division.
It is in cases of nuclear division that the nucleolus has
received the most attention from morphologists. The behav-
ior of the nucleolus in mitosis and amitosis may be treated
separately.
1. Amitosis. — In this mode of nuclear division it is frequently
the case for the nucleolus to divide first, so that each of the
daughter-nuclei receives a half, or approximately a half (for the
division of the nucleolus is not always into two equal parts),
of the parent-nucleolus. In support of this deduction the fol-
lowing observations may be mentioned : Schaudinn ('94, Amoeba
erystalligera); F. E. Shulze ('75, A. polypodia); Will ('85, ova of
Nepa, Notonecta) ; Doflein ('96, degenerating ova of Tubularia);
Carnoy (85, ova of Gryllotalpa, Lithobius, Geotrupes) ; Korschelt
(95, intestinal cells of Ophryotrocha); my observations on the
peritoneal cells of Polydora; Hoyer ('90, intestinal epithe-
lium of Rhabdonema); Frenzel ('93b, hepatopancreas cells of
Astacus) ; Platner ('89a, Malpighian tubes of Dytzscus ; Wheeler
526 MONTGOMERY. [Vou. XV.
(89, follicle cells of Blatta); de Bruyne ('97, follicle cells of
Nepa, Periplaneta, Meconema, Aeschna). E. B. Wilson (96),
in speaking of amitosis, states: ‘““In many cases, however,
no preliminary fission of the nucleolus occurs ; and Remak’s
scheme must therefore be regarded as one of the rarest forms
of cell division.” But the list of cases which I have given
shows that such cases of nucleolar division are frequent in
amitosis, so that I conclude that a fission of the nucleolus, if
not exactly typical for this mode of nuclear division, is never-
theless well represented and occurs here much more frequently
than in mitosis. Dr. E. G. Conklin has demonstrated to me
preparations of nucleolar division in follicle cells of Gry/lus,
which he has kindly allowed me to mention here,
2. Mitosis.—In karyokinesis the nucleolus may either not
disappear, or, and this is the most usual case, it disappears
before the spindle is formed. These two modes may be con-
sidered in turn.
(a) The nucleolus does not disappear.—In some few cases
the nucleolus wanders out into the cytoplasm after the disap-
pearance of the nuclear membrane and may remain there for
some time without undergoing any change. Such cases have
been described by Hacker (92a, egg of Aegworea), Wheeler ('95,
that of Myzostoma), H. V. Wilson (94, ova of Zedamione and
Hircinia), Tang] (82, flower buds of Hemerocallis), Gjurasin
(93, Pesiza), and Karsten (93, sporangia of Psz/otum). In all
these cases the nucleolus ultimately disappears in the cytoplasm,
though in Aeguorea it may be observed still in the cell body of
one of the blastomeres at the thirty-two cell stage, and the
daughter-nuclei produce their own nucleoli. (Similar are the
observations of Mead, '95; Hacker, ’96, '97; Rosen, '95; Zimmer-
mann, '96; Metzner, '94; Foot '94; Poirault and Raciborski, '96.)
In the other cases where the nucleolus does not disappear it
remains within the nucleus. In some of these cases it appears
to divide into two or more parts; in other cases it may be that
one of the daughter-nuclei receives the whole parent-nucleolus,
while in the other one a new nucleolus is produced. There are
a few observations which show that it sometimes divides ; thus
Strasburger (’82b, embryo sac of Galanthus) and Rosen ('92b,
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 527
Synchytiium) ; Reinke studied the mitosis of the spleen cells
of the mouse, and found that the single parent-nucleolus divides
into three or four pieces, while at the end of the mitosis each
daughter-nucleus contains a single nucleolus. In the mitoses
of the ovogonia of Lzxeus and Polydora my own observations
show that the nucleolus persists in the nucleus, and each
daughter-nucleus contains one nucleolus, so that it is very
probable that in these cases the parent-nucleolus divides into
two, and each daughter-nucleus thereby receives a half of it ;
but these mitoses were so small that I was unable to decide
this point definitely. Rosen (95) finds nucleolar division in
root cells of Phaseolus ; J. Wagner ('96a) describes a similar
division of a ‘nucleolus’ in spermatocytes of Arachnids, though
this case, like that described by Henking (90), probably repre-
sents a chromatin nucleolus. This persistence of the nucleolus
in the nucleus during mitosis must be considered atypical.
(6) The nucleolus disappears during mitosis. — This is the
most usual mode of behavior of the nucleolus during mitosis.
The nucleolus either gradually diminishes in size, and so finally
vanishes, or else it first fragments into a number of smaller
pieces, and then these disappear. The only cell which I had
for the study of this phenomenon was the ovum of Prsczcola
during the formation of the first pole spindle. When this
spindle is complete no trace of nucleolar substance is to be
seen anywhere in the cell. In stages immediately antecedent
to that of the spindle, numerous minute granules, as well as a
smaller number of larger globules, are dispersed through the
nuclear sap; all these stain with eosin, and I regard them as
particles of nucleolar substance which had become separated
from the nucleolus. Thus a dissolution of the nucleolar sub-
stance commences before the nuclear membrane has disap-
peared, and after this membrane has vanished it is probable
that all the nucleolar substance must be dissolved by the action
of the cytoplasm, or at least become dispersed through the
latter, so that no remnant of it is to be found in the region of
the spindle or of the chromosomes. During the process of
dissolution of the nucleolar substance in the nuclear sap the
chromatin elements stain red (with eosin), and this fact may be
528 MONTGOMERY. [VoL. XV.
explained either by the assumption that the nucleolar substance
unites chemically with the chromatin, or that it simply pene-
trates into the meshes of the latter; since no nucleolar
substance appears to be united with any of the twelve chromo-
somes we may conclude that it does not unite chemically with
the chromatin, and therefore the chromosomes probably do not
serve to carry it over into the daughter-nuclei. We may now
briefly review the results of other observers on the mode of
disappearance of the nucleolus during mitosis.
It is not necessary to discuss the earlier view of O. Hertwig,
which he has since discarded, that “der Eikern der aus dem
Keimblaschen frei gewordene oder ausgewanderte Keimfleck
ist,” nor yet the view of Kolliker. Kleinenberg ('72) believes
that the germinal spot of Hydra dissolves during mitosis ;
Brauer (91) finds that it breaks into fragments, of which a part
seems to be dissolved in the cytoplasm, ‘ein Theil tritt unver-
andert nach dem Schwinden der Membran in das Eiprotoplasma
iiber.” Fick (93, germinal spot of Amdlystoma) finds that the
nucleoli disappear at the time of the longitudinal splitting of the
chromosomes ; and Bohm ('gs) reaches the same conclusion for
Petromyzon. Davidoff (89, ovum of Distaplia) concludes “ dass
aus dem Nucleolus ein Kern mit Kernnetz, mit einem Nucleolus
und Nucleolinus hervorgegangen ist’’; and Vejdovsky ('88,
Rhynchelmis), Blochmann ('82, Veritina), and Marshall ('92, Gre-
garina) conclude that the nucleoli become chromosomes. In the
egg of Ascaris the nucleoli gradually disappear, according to most
observers. Strasburger ('82b) first contended that the nucleolar
substance is taken up into the nuclear filaments ; later ('88) he
writes: ‘“ Auf Grund meiner neueren Erfahrungen erscheint es
mir iiberhaupt unwahrscheinlich, dass die Nucleolarsubstanz,
auch nach ihrer Auflosung im Kernsafte, den Kernfaden als
Nahrung dienen sollte,” and he considers that after it is
dissolved in the nuclear sap a portion of it forms the cell
membranes of the daughter-cells (cf also his paper of '93).
Rein (83, ova of Zepus and Cavia) finds that the nucleolus
breaks into small fragments, which finally disappear in the
substance of the nucleus. Pfitzner (83, ectoderm cells of
Hydra) terms the nucleolar substance “ prochromatin,” since
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 529
he finds that in mitosis it changes into chromatin. Rabl (’85,
larval cells of amphibians) and O. Schultze ('87, ova of Rana
and 7yiton) contend that the nucleolar substance takes some
part in the formation of the nuclear filaments ; but Born (94)
subsequently found that these filaments stand in no connection
with the nucleolar substance. Holl ('93, ovum of Jus) finds
that the central granules of the nucleoli wander out of them
and so become the chromosomes. Van Beneden (75, ovum of
Lepus) originally supposed that the nucleolus becomes the first
pole body. Kastschenko ('90, ova of Se/achzz) finds that all the
nucleoli disappear in the spirem stage, while Riickert (92)
finds that a few of them pass into the cytoplasm. Stuhlmann
(86, ova of /xsecta) finds that the nucleoli gradually disappear
during the maturation of the egg; and similar conclusions
were reached by Stauffacher (93, Cyc/as), Rhumbler ('95,
Cyphoderia), Sheldon ('90, Peripatus), Heathcote (86, ulus),
Van der Stricht (95, Amphzoxus), Brauer ('92, Branchipus), and
Vejdovsky (82, Sternaspis). Auerbach ('96, spermatogonium
of Paludina) holds that the nucleolar substance becomes incor-
porated with the chromatin elements. Meunier ('86) and Moll
(93) for Spzvogyra, and Carnoy (’85) for other cells also, hold
that the chromosomes are derivatives of the chromatin skein of
the nucleolus. Heuser ('84, mitoses of various plant cells) con-
tends that the nucleoli become gradually apposed to the nuclear
filaments, and that their substance unites with these elements,
though in some cases a superfluous portion of the nucleolar sub-
stance may be discharged from the nucleus. Korschelt ('95,
ovum of Ophryotrocha) finds that the nucleolus gradually dis-
appears by dissolving in the nuclear sap, and believes that a part
of this substance may be introduced into the nuclear filaments.
Zacharias ('85) somewhat prematurely concludes that the nucleoli
always disappear in mitosis. Tang] (82) finds that in Hemerocal-
/ts, in uninucleolar nuclei, the nucleolus dissolves in the nucleus,
but in those which are multinucleolar one may pass out into the
cytoplasm ; in Hesperus and Cisium they gradually disappear.
Humphrey ('94, plant cells) holds that “die Nucleolen in einigen
Fallen aus der Kernhohle, bevor sie von den karyokinetischen
Kraften angegriffen werden, austreten kénnen.”’
530 MONTGOMERY. [VoL. XV.
Wager (93, Agaricus) describes the nucleoli as becoming
dissolved in the caryolymph, and then, this dissolved substance
penetrating the chromatin elements, the latter serve to carry
it over into the daughter-nuclei. Went ('87, plant cells) holds
“dass in vielen Fallen wenigstens der Nucleolus beim Anfang
der Kerntheilung im Kernfaden aufgenommen wird,” and that
“er sich nach der Theilung auch wieder daraus_bildet.”
Riickert (94, egg of Cyclops) finds that the nucleoli gradually
break into fragments and the latter disappear. But there is
not space here to mention all the views of students of mitosis.
There are only a few observations which would show that in
mitosis the chromosomes are derived from the nucleoli (David-
off, Vejdovsky, Blochmann, Marshall, Sobotta, 95, Macallum,
'95, Carnoy, '97a, R. Hertwig, '96, not corroborated by Brauer,
'94), and these cases stand in such marked contradiction to the
observations of other morphologists that a reinvestigation of
them is very necessary.!. Then we have the observations of
Carnoy, Meunier, and Moll, which would show that the chro-
mosomes are derived from a part of the nucleolus ; but the
existence of a ‘nucléole-noyau,” z.c., of a nucleus within a
nucleus, as assumed by Carnoy and his followers, in any meta-
zoan cell, seems to be very problematical. On the other hand,
most observers agree that the nucleoli disappear more or less
gradually during mitosis, and that the chromosomes are not
derived from them. Now we have reached the crucial ques-
tion: What is the mode of transference of the nucleolar
substance to the daughter-nuclei? In answer to this, some
observers hold that this substance may be distributed in the
cytoplasm and taken up therefrom into the daughter-nuclei;
others, that it combines with the chromatin elements and is
transferred with these; still others maintain a position inter-
mediate between these two.2 But when we find so much vari-
ance in the conclusions of competent investigators only one
deduction is allowable, namely, that the mode of transportation
1 On the relation of nucleoli to chromosomes, cf also Cunningham ('97),
Sobotta (95), Macallum (’95), Platner ('89c), Carnoy ('97a), R. Hertwig ('96),
Van Beneden (’83), Zimmermann ('96), Lauterborn ('96), Boveri ('88), Wheeler
(97).
2 Cf. also Belajeff ('94), Mottier ('97), and Rosen ('95).
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 531
of the nucleolar substance is probably different in different
objects.
We have found above that in the simplest though secondary
nuclear divisions, the amitotic, the nucleolar substance of the
parent-cell is transported into the daughter-nuclei by the me-
chanically simplest process, namely, by a direct division of the
parent-nucleolus ; this is very frequently the case in amitosis,
though it does not always occur. But in most mitotic divisions
the nucleolus first disappears, 7z.¢., there would seem to be an
indirect mode of transference of its substance corresponding to
the indirect mode of transference of the chromatin and linin ele-
ments. Now all mitotic divisions do not proceed on exactly the
same plan, for we find differences in regard to the presence of a
central spindle, in regard to the number of the chromosomes, etc.
Accordingly, one would expect also different modes of transfer-
ence of the nucleolar substances. Thus in some cases, as
Wager ('93) suggests, the chromosomes may serve as mechan-
ical vehicles for the transportation of this substance. In many
other cases it is very probable that this substance, after the
disappearance of the nuclear membrane, becomes dispersed in
the cytoplasm ; and then each of the daughter-nuclei may
either take up this substance from the cytoplasm again, or may
produce its own nucleolus from a new substance, owing to the
primitive nucleolar substance having been assimilated by, or
even discharged from, the cytoplasm. There are observations
in support of each of these three modes of re-formation of
nucleoli in the daughter-nuclei. But since when the nuclear
membrane disappears the cytoplasm probably comes into con-
tact with the substance of the nucleoli, it is most probable that
it would produce either a physical or a chemical change in the
latter, and hence the second and third modes would appear
the more probable. Accordingly, I agree with Humphrey (94)
that there is no substantial basis for Zimmermann’s ('93) con-
clusion ‘‘omnis nucleolus e nucleolo,” or more strictly speaking,
that the nucleolus in most cases is not derived from a previously
existing one. But the third mode of diffusion of the nucleolar
substance is in reality not a transference of this substance at
all, since it probably becomes lost in the cytoplasm ; and hence,
532 MONTGOMERY. [VoL. XV.
though the mode of disappearance of this substance may be
more or less dependent upon the mode of mitosis, the substance
of the parent-nucleolus may be in many cases not transferred
to the daughter-nuclei, but the latter (perhaps as a rule) may
produce their own nucleoli de xovo.
Strasburger ('93, '97) assumes that the small granules found
by Kostanecki (92) in the equatorial plate of the central spindle
may be nucleolar particles, and accordingly that the nucleolar
substance may be in this way very evenly distributed to the
daughter-nuclei; but it is not as yet clearly shown that these
granules are derivatives of the nucleolus (cf also Debski, '97,
Sala, ’95, Pfitzner, ’séb, and Rosen, ’95).
Zacharias ('85), Carnoy (’85), and Platner ('86) have concluded
that in some cases the achromatic spindle fibers are derived
from the nucleolus ; similar views are held by Strasburger ('95,
'97), Harper ('97), and Fairchild (97), but most facts would show
this view untenable.
Rhumbler (93) assumes that a greater amount of nucleolar
substance is accumulated in the nucleus before mitosis than is
necessary for its growth, and this superfluous amount would
serve for the formation of the nucleoli in the daughter-nuclei.
12. The Function of the Nucleolus.
The attempt to deduce the physiological economy of a struc-
ture from a mere study of its morphological relations is always
difficult, and this is especially the case with regard to the
nucleoli.
Balbiani (64) found contractile and discharging vacuoles in
the germinal spot of Pal/angiwm, and notes that they differ
from the contractile vacuoles of the Rzzopoda in that they
are not formed again at the same point. Hacker ('93c) regarded
the nucleolus of the ovum of Echzmus as an excretory organ,
since he found its large vacuole to be contractile ; he compared
it directly to the contractile vacuole of /zfusoria. Balbianj
('65b) also observed contractile vacuoles in the germinal spots
of Helix, Vortex, and Prostomum, and in these the vacuole dis-
charges through a small orifice in the cortical substance of the
No. 2.]} ° COMPARATIVE CYTOLOGICAL STUDIES. 533
nucleolus. Bohm described (88) the vacuole of the germinal
spot of Petromyzon as connected by a fine duct with the sur-
face of the nucleolus. Lukjanow (ss) found in the stomach
cells of the salamander that the nucleolus is apposed to the
nuclear membrane, through which it discharges an excretion.
Compare also Van Bambeke (97a) and Michel (96). These
observations would show that the nucleolus in some cases con-
tains a contractile vacuole, and that the fluid substance of the
latter is periodically discharged from it (cf. Hodge '94, Van
Bambeke, ’97, and Michel, '96).
Flemming (82) considers the nucleoli to be nuclear organs,
and regards them either as containers or reserve supplies of
chromatin, or as “eine chemische Modification, Vorstufe oder
Doppelverbindung”’ of the latter substance; this view is also
held by Van Bambeke (85). Zacharias ('85) also thinks that
they are organs, but does not agree with Flemming that they
are reserve masses of chromatin; Gjurasin ('93) corroborates
the views of Zacharias. Strasburger originally contended ('84)
that they represent reserve material, a view shared by many
later observers ; more recently (88) he shows that the nucleolar
substance may play some part in the formation of the cell
membrane, but considers that they may also have some other,
as yet unknown, function. Korschelt ('89) concludes that they
are formed as depositions of nutritive substances, and that their
substance “in und vielleicht ausserhalb des Kernes zur Ver-
wendung gebracht werden sollte.’ Rhumbler (93) assumes
that the nucleoli (‘‘ Binnenkérper”’) of the Protozoa represent
“Reservestoffe’”’ deposited in the nucleus and consumed in
the growth of the latter, standing in some connection with the
chromatin ; they are not organs, but secretions of the nucleus.
Hacker (95) concludes that they are not nuclear organs, but
secretions of the nucleus formed in or from the chromatin
elements and destined to be discharged from the nucleus dur-
ing mitosis; he observes that the nucleolar substance “ein
Stoffwechselsprodukt darstellt, dessen Erzeugung in einem
gewissen Abhangigkeitsverhaltniss zur Intensitat der vegeta-
tiven Leistungen von Kern und Zelle steht,” and that its amount
stands in a direct ratio “zur Intensitat der Wechselbeziehungen
534 MONTGOMERY. [VoL. XV.
’
zwischen Kern und Zelle’’; he opposes the view “dass die
Kernkorper aus dem Zellplasma in den Kern hineingelangen
und hier in die Bildung des Chromatins eingehen.” Leydig
(85) holds that certain of the nucleoli are differentiations of
the chromatin reticulum, others of the “ Kernplasma.”’ Watasé
(94) considers that they may be metabolic products of the cell.
Auerbach (90) holds them to be the fundamental constituents
of the nucleus, which is a retrogression to the earlier views of
O.and R. Hertwig. Born ('94) states: “Die Nucleolen stehen
in Beziehung zum individuellen Zellleben, nicht zur Fortpflan-
zung.” Lavdowsky (94) considers them to be reserve masses
of chromatin. Macfarlane ('81,'85) regards them as the tropic
centers of the cell, and as the most important mechanical agents
in cell division. Julin ('96b) believes they conduct the vegetal
processes of the cell. Mottier (97) considers the nucleolus
“ein Kraftvorrath, welcher der Zelle nach Bedarf zur Verfii-
gung steht” ; and Swingle (97), as a reserve fund of nourish-
ment for the kinoplasm in mitosis. Metzner ('94) considers
them to be of importance in the processes of mitosis (compare
his observations). Henneguy ('93) regards the nucleolus and
Balbianian corpuscles as corresponding with the macronucleus
of the /zfusorta (cf. Julin, '93b). These, then, are the most
important views on the nature of the nucleolus.!
From my own observations the nucleolar substance would
seem to be extranuclear in origin, and not a secretion or excre-
tion of the nucleus. To be sure it may, and probably does,
undergo chemical changes within the nucleus, but it is derived
in the first place from the cytoplasm. I regard the nucleoli as
1 The following list includes, I believe, all who have written on the function of
the nucleolus: Korschelt ('89), Hacker ('93a, '95, '97b), O. Hertwig ('77a, '92),
Rhumbler ('93), R. Hertwig ('76, '96), Fick ('93), Lukjanow (’88), Brauer (91),
Nussbaum ('82), Strasburger ('82b, '84, '88, '95, '97), Jordan ('93), Flemming
(80, '82), Van Beneden ('75), Wasielevsky ('93), A. Schneider ('83), Henneguy
(93), Riickert ('92, '94), C. Schneider ('91), Born ('94), R. Wagner ('36, '37),
Auerbach ('74a), Kolliker ('43), Lonnberg ('92), Klein ('78), Macallum (91,
95), Stuhlmann (’86), O. Brandt (’78), Schwarz ('87), Giugnard (’85), Macfar-
lane ('81, '85, '92), Zacharias ('85), Watasé (94), Humphrey ('94), Gjurasin
(93), Mann (92), Julin ('93b), E. B. Wilson ('96), Van Bambeke (’85), Mottier
(97), Swingle ('97), Rosen ('95), Metzner ('94), Wheeler ('97), Carnoy (’84,
‘86, '97a, '97b).
No. 2.) COMPARATIVE CYTOLOGICAL STUDIES. 535
consisting of a substance, or different substances, taken into
the nucleus from the cell body. It seems probable, further,
that these substances stand in some relation to the nutritive
processes of the nucleus, and in a relation to the growth of the
latter. Thus those nuclei which are characterized by an espe-
cially large amount of nucleolar substance are growing nuclei,
z.e., those of egg cells in the maturation period, those of the
subcuticular gland cells of Pzsczcofa, the mesenchym cells of
Cerebratulus. In the gland cells of Pzsczcola the volume of the
nucleolar substance rapidly increases in amount during the
phase of growth of the nucleus, but diminishes when the latter
decreases in volume. Somatic cells, on the contrary, at least
those which are undergoing no dimensional changes, contain
a relatively small amount of this substance. It is doubtful
whether Hacker (95) is quite correct in assuming that the
amount of the nucleolar substance stands in a direct proportion
to the intensity of the functional changes which take place
between the nucleus and the cytoplasm; at least there are but
few criteria to enable one to compute the degree of such an
intensity. Thus one would suppose that in nerve cells there
was a close and intimate correlation between nucleus and cell
body, but the nucleoli of the ganglion cells of the nemerteans
and Piscicola are very small. Hacker’s deduction might be
modified as follows: where there is a close physiological vap-
port, in regard to processes of nutrition, between the nucleus
and the cell body a relatively large amount of nucleolar sub-
stance occurs in the former.
Accordingly, we find a relatively large amount of nucleolar
substance in growing nuclei, and hence conclude that this sub-
stance stands in some connection with the processes of nutrition,
is itself either nutritive in function or represents that portion of
substances assimilated by the nucleus from which all nourish-
ment has been extracted, and in this case it would be a waste
product. A third possibility is that the nucleoli may represent
accumulations of nutritive substance retained in the nucleus as
a reserve supply ; but this does not seem to be very probable,
for by this assumption it would be difficult to explain the uni-
formity in the size of the nucleoli in a given species of cell.
5 36 MONTGOMERY. [Vou. XV.
It would be premature to attempt to decide the exact manner
in which the nucleolar substance is concerned in the metabolism
of the cell. But the facts at least show that it has an extranu-
clear origin, and is especially abundant in growing nuclei, which
shows that it stands in intimate connection with the phenomena
of nutrition of the nucleus.
Vacuoles are characteristic for certain stages in the develop-
ment of many nucleoli, especially those of germinal vesicles.
For the nucleoli of the ova of MZontagua and Doto, I showed
that the vacuolar substance is at first present in the form of
small globules in the nuclear sap, that these become applied
against the surface of the nucleolus, and, finally penetrating into
the latter, represent within it the vacuoles. I was unable to
decide the mode of derivation of the vacuoles for the other
nucleoli studied. So in some cases this vacuolar substance
would appear not to be a derivative of the ground substance of
the nucleolus, but to be derived from without the latter. Thus
such nucleoli may be considered as diosmosing structures. The
manner of growth of nucleoli is apparently by a process of
apposition of smaller particles of nucleolar substance to their
surfaces, and the addition of vacuolar substance to them differs
from this only in that the vacuolar substance is intussuscepted.
This vacuolar substance may be also a product of the nutritive
processes of the nucleus.
It is a difficult question to determine whether the nucleolus
at some stage of its development should not be considered a
nuclear organ. In most nuclei it has a regular shape, in
others it may be oval; in many cases the nucleolus has no
regular shape, and in the salivary gland cells of Chzronomus
(according to Balbiani) it is convoluted. From the facts at
hand we may conclude that the shape of the nucleolus is
pretty constant for the particular species of cell. Now, tak-
ing constancy in form as a criterion of an organ, one might
conclude that the nucleoli are organs. But, on the other hand,
the most frequent form of the nucleolus, namely, the spherical,
might simply be due to its thin fluid consistency, and when it
is more viscid in consistency its shape would be more irregular.
Thus Rhumbler (93) concludes that the irregular nucleoli of
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 537
Foraminifera “durch Zusammenfliessen anfanglich leicht fliis-
siger, dann zahfliissiger und schliesslich erstarrender Massen
entstanden sind.” It may be asked: Why does the nucleolus
persist through the whole resting state of the nucleus if it be
not an organ? It may be simply stored in the nucleus until at
the time of mitosis, when the nuclear membrane disappears,
it has an opportunity to leave the nucleus. The only observa-
tions which would prove that the nucleolar substance may
functionate as an independent organ are those according to
which the nucleolus contains a contractile vacuole, and thus
rhythmically contract and expand ; in these cases the nucleolus
‘might be regarded as a pulsating excretory organ of the nucleus.
The hypothesis might be suggested that though the nucleolus
probably consists of substances which stand in some relation to
the nutritive processes of the nucleus, and so at the time of its
first formation may be a functionless, inert mass of substance,
yet it may at later periods in the history of the resting nucleus
acquire some active function and thus gradually come to
acquire the value of a nuclear organ; this hypothesis is put
forward merely as atentative one. According to this view the
nucleolus might be considered as an organ which serves to
accumulate in itself the waste products of the nucleus, thus
serving as a reservoir for such substances ; or it might be con-
sidered as an organ of excretion, to discharge waste products
out of the nucleus : in either case the nucleolus would seem to
stand in direct connection with the nutritive substances and
forces of the nucleus.
’
13. Comparison of the Nucleoli in Plants, Protozoa, and
Metazoa. :
I have made no morphological studies on the nucleoli of
plant cells, but would judge from the results of botanical inves-
tigators that they are probably strictly comparable to the
nucleoli of the metazoan cells.
Rhumbler (93) doubts whether the nucleoli of the Metazoa
and the “Binnenkorper” of the Pvotozoa are homologous
structures ; and, indeed, there are certain nucleolar structures
5 38 MONTGOMERY. [VoL. XV.
in Protozoa which are unique, such as the nucleolo-centrosome
of Keuten (95). Henneguy considers that the corpuscle of
Balbiani, together with the nucleolar elements of the metazoan
cell, corresponds to the macronucleus of the /zfusoria ; in con-
nection with this view may be mentioned the observations of
Biitschli (80), according to which only the macronuclei of the
Ciliata contain nucleoli. Henneguy’s hypothesis is very ingen-
ious, and opens an interesting field for investigation, but it is
difficult to determine whether it corresponds to the facts at
hand, or whether it does not.! Some of the nucleoli of Protozoa
are comparable to those of Metazoa, but it is doubtful whether
all of them are.* Thus it may be the case in some of the
gregarines that the chromatin (or its physiological equivalent)
is localized in some or all of the nucleoli, and such structures
could not be compared with the nucleoli of the metazoan cell.
As to the metazoan nucleoli, there is the question whether
the nucleoli of egg cells and of somatic cells should be consid-
ered homologous. In my opinion this may be answered in the
affirmative, since the nucleoli of both kinds of cells appear to
be depositions of substances which are concerned in the nutri-
tive processes of the nucleus. In making this conclusion I
limit myself to the true nucleoli and do not consider those
structures which have been erroneously termed nucleoli, but
which in reality are portions of the chromatin reticulum of the
nucleus. Numerous writers have considered the thickened
nodal points of the nuclear network to be nucleoli, and here
may be mentioned Leydig, Klein, Waldeyer, and others. The
“«cyanophilic ”’ nucleoli of Auerbach ('90), the «‘ pseudonucleoli’”’
of Rosen ('92a), the “nucléoles nucléiniens”” of Carnoy ('85),
and the “Karyosomata”’ of Ogata (83), Lukjanow ('87b), and
Macallum ('91) are undoubtedly not nucleoli but portions of
the nuclear reticulum. While the “erythrophilic” nucleoli
of Auerbach, the “Eunucleoli’’ of Rosen, the “nucléoles
1 On the genetic relation of nucleoli to Balbianian corpuscles (true yolk-nuclei),
a relation which seems to me very doubtful, cf Mertens (’93), Galeotti ('95),
Melissinos and Nicolaides ('90), Weismann and Ishikawa ('89), Ver Ecke (’93),
Steinhaus (88), Henneguy ('93), Julin (’93b).
2 For the central masses of chromatin found in many protozoan nuclei, Doflein
('98) proposes the term “ chromatosphere.”
No.2.] COMPARATIVE CYTOLOGICAL STUDIES. 539
plasmatiques’”’ of Carnoy, and the ‘ Plasmosomata’’ of the
other observers correspond to true nucleoli in the sense in
which this term should be used. The existence of Carnoy’s
“ nucléoles mixtes ’’ and “ nucléoles-noyaux”’ in cells of Metazoa
appears to be doubtful. List ('96) considers that the paranu-
cleoli of the egg cells and the nucleoli of the somatic cells
are homologous, but that the nucleolus proper of the ova
is different from both; but the chemical differences which he
finds between these kinds of nucleoli do not prove that they
are morphologically distinct structures.
APPENDIX TO THE LITERATURE REVIEWS.
Siebold ('39) noticed “in den Eiern von Plumatella campanu-
lata Lam. . . . ein deutliches Keimblaschen mit gedoppeltem
Keimflecke.”’
Koelliker (43) concludes: “Es bestande ... das Ei aus
einer primitiven Zelle, dem Keimblaschen, die sich um einen
Kern, den Keimfleck, gebildet, und um die sich nachher Korner
und eine secundare Zelle, die Dotterhaut, gelegt hatte.”’
Auerbach ('74a) was the first to emphasize and prove clearly
that the number of nucleoli is usually quite large, and that they
are frequently irregular in form (before this time it was generally
assumed that the usual number of nucleoli was one or two).
The nucleus is filled with “‘Grundsubstanz”’ (the “ Zellsaft” of
Kolliker) and ‘“ Zwischenkérnchen”’; the latter are distin-
guishable from the nucleoli by their smaller size and different
refraction. He explains the clear zone around the nucleolus
and the “ Kernkorperchenkreis”’ of Eimer by the action of a
repulsive force on the part of the nucleolus and of the nuclear
membrane. He distinguishes several successive stages of the
nucleus with regard to the number of the nucleoli : exucleolar
nuclei, at an early embryonal stage ; paucinucleolar nuclei,
with one or two nucleoli ; p/urznucleolar nuclei, with two to four ;
and mzltinucleolar, with more than four. ‘‘ Die Zahl der Kern-
kérperchen in einem Kerne betragt 1-16, und in extremen
Fallen selbst noch viel mehr, bis tiber 190. Und zwar ist nur
eine kleine Minderheit aller Kerne durch den Gehalt von nur
540 MONTGOMERY. [VoL. XV.
einem oder zwei Nucleoli ausgezeichnet.’”’ He gives a large
series of data on the number and size of nucleoli in embryonal
and adult cells of vertebrates and Musca. The enucleolar con-
dition is characteristic for embryonal cells; later a nucleolus
makes its appearance in the center of the nucleus, though its
substance is probably derived from the cytoplasm ; new nucleoli
are formed by successive divisions of the first one. In Zeleostez
the nuclei have fewer nucleoli than those of Amphzbza, and those
of Reptilia fewer than those of Mammalia ; from which is con-
cluded that the number increases in advancing phylogeny as in
the ontogeny. ‘Je schneller und absolut bedeutender das
Wachsthum der Zellen ist, desto mehr scheint auch die Ten-
denz zur Vervielfaltigung der Kernkérperchen obzuwalten.”
The nuclei of the stomach mucosa of Rava are multinucleolar
in summer and autumn, while after hibernation they contain
only one to four nucleoli, which may be due to a process of
fusion. The substance of nucleoli is similar to that of the
cytoplasm in structure, capability of movements and of produc-
ing vacuoles ; just as the nucleus is first formed as a vacuole in
the cytoplasm, so in the substance of a nucleolus (which is
cytoplasmic in origin) a vacuole is formed which has the same
relation to the nucleolus as the nucleus has to the cell; ‘“ bei
dieser Betrachtungsweise erscheint demnach der Zellkern als
ein hohler Brutraum, bestimmt, eine junge Zellenbrut in sich zu
entwickeln, die Nucleoli aber als wahrhaft endogen entstandene
Tochterzellen.” In higher animals all nucleoli do not become
daughter-cells, but fulfill some new function ; ‘und so werden
wir auch die urspriingliche Bedeutung der Nucleoli als Fort-
pflanzungszellen nicht fiir ganz unmoglich halten diirfen, wenn
wir auch auf der anderen Seite nicht zweifeln kénnen, dass sie
in den meisten Kernen der héheren Organismen ganz andere
Aufgaben zu erfiillen haben miissen.”’
Auerbach ('74b) studied in life the fecundation and cleavage
of Stvongylus and Ascaris. A short time after the appearance
of the two copulation nuclei in the ovum, arise in each from
one to five nucleoli ; ‘wenn eine Mehrzahl sich einfindet, so
kommen sie nicht alle gleichzeitig, sondern eines nach dem
anderen, in Intervallen von einer halben bis zu einigen Minuten
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 541
zum Vorschein, und zwar in unregelmassigen, oft betrachtlichen
Entfernungen von einander.” When the nuclei wander towards
one another the nucleoli move about, ‘“indem sie innerhalb
des Kernraums allerlei gerade, zickzackformige, bogenformige,
Bahnen durchlaufen, mit einer vergleichsweise erheblichen
Geschwindigkeit, so dass zuweilen in weniger als einer Minute
Strecken von der Lange des Kern-Durchmessers zuriickgelegt
werden”; during these movements the nucleoli remain perfectly
spherical. When the copulation nuclei are apposed the nucleoli
in them suddenly disappear, and the mode of this disappearance
was determined in one case, though it is exceedingly rapid ; “das
Kiigelchen wurde allmahlich blasser und etwas grésser und fuhr
dann plotzlich auseinander, ein Wolkchen bildend, welches einen
Augenblick darauf nicht mehr zu sehen war.” The nucleoli
reappear in the resting nuclei, and in the successive genera-
tions up to the eight-cell stage have the same cycle of changes,
except that in each generation they are somewhat larger than
in the preceding. These nucleoli are formed independently of
one another. By the re-formation of the nuclear vacuole a
number of cytoplasmic granules pass into the cavity of the
nucleus, and there fuse to form the nucleoli.
Reinhard (82, cited by Braem, '97) describes in the egg of
Plumatella different stages of the nucleoli, which may be single,
double, or even trilobular.
WISTAR INSTITUTE OF ANATOMY AND BIOLOGY,
PHILADELPHIA, February 3, 1897.
542 MONTGOMERY. [VoL. XV.
LITERATURE LIST.
(An asterisk marks those papers which I have not seen.)
‘57 AGAssiIz, L. Contributions to the Natural History of the United
States of America. First Monograph. Part III. Embryology of
the Turtle. Boston.
‘74a AUERBACH, L. Organologische Studien. Zur Charakteristik und
Lebensgeschichte der Zellkerne. Heft 1. Breslau.
'74b AUERBACH, L. Organologische Studien. Zur Charakteristik und
Lebensgeschichte der Zellkerne. Heft 2. Breslau.
'76 AUERBACH, L. Zelle und Zellkern. Cohn’s Beitr. z. Biol. der Pflan-
Zen. 2.
‘90 AUERBACH, L. Zur Kenntniss der tierischen Zellen. I. Sztzungsber.
a. preuss. Akad. a. Wiss. Berlin.
‘96 AUERBACH, L. Untersuchungen tiber die Spermatogenese von Palu-
dina vivipara. Jena. Zett. f. Naturw. 30.
84 Ayers, H. On the Development of Oecanthus niveus, etc. Jem.
Boston Soc. Nat. Hist. 3.
‘64 BALBIANI, E.G. Sur les mouvements qui se manifestent dans la tache
germinative chez quelques animaux. Compt. Rend. de la Soc. Biol.
Paris Serr 4e ale
*'65a BALBIANI, E.G. Sur les mouvements qui se manifestent dans la
tache germinative chez quelques animaux. Gazette médic. de Paris.
’65b BALBIANI, E.G. Observations sur le rdle du noyau dans les cellules
animales. Compt. Rend. Acad. Paris. 61.
'76 BALBIANI, E.G. Sur les phénoménes de la division du noyau cellu-
laire. Compt. Rend. Acad. Paris. 83.
‘81 BALBIANI, E.G. Sur la structure du noyau des cellules salivaires chez
les larves de Chironomus. Zool. Anz. 4.
83 BALBIANI, E.G. Sur Vorigine des cellules du follicule et du noyau
vitellin de l’ceuf chez les Géophiles. Zool. Anz. 6.
*'95 BALBIANI, E.G. Sur la structure et la division du noyauchez la Spiro-
chona gemmipara. Ann. de Microgr. 7.
85 BAMBEKE, C. VAN. Etat actuel de nos connaissances sur la structure
du noyau cellulaire a l’état de repos. Ann. Soc. de Méd., Gand.
’86 BAMBEKE, C. VAN. Contribution pour servir a l’histoire de la vésicule
germinative. Bull. Acad. roy. Belg. 11.
93 BAMBEKE, C. VAN. Elimination d’éléments nucleaires dans l’ceuf
ovarien de Scorpaena scrofa L. Bzdl. Acad. roy. Belg. 25.
'97a BAMBEKE, C. VAN. L’oocyte de Pholcus phalangioides Fuessl. Com-
munication préliminaires. Bruxelles.
'97b BAMBEKE, C. VAN. L’oocyte de Pholcus phalangioides Fuessl.
Verh. d. anat. Gesell. Jena.
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 543
‘98 Bancrort, F. W. Ovogenesis in Distaplia occidentalis Ritter
(MSS.), with Remarks on Other Forms. Sczence. Vol. viii, N. s.,
No. 189.
'92 BANNWARTH. Untersuchungen tiber die Milz. I. Die Milz der Katze.
Arch. f. mikr. Anat. 38.
'94 BELAJEFF, W. Zur Kenntnis der Karyokinese bei den Pflanzen.
Flora, Erganzungsband.
'83 BEMMELEN, J. F. vAN. Untersuchungen itiber den anatomischen und
histologischen Bau der Brachiopoda Testicardines. /ena. Zeit.
J. Naturw. 16.
'69 BENEDEN, E. VAN. Sur une nouvelle espéce de Grégarine, etc. Bz//.
Acad. roy. Belg. 28.
'75 BENEDEN, E. vAN. La maturation de l’ceuf, la fécondation et les
premiéres phases du développement embryonnaire des mammifeéres,
etc. Bull. Acad. roy. Belg. 40.
'76 BENEDEN, E. vAN. Contributions 4 l’histoire de la vésicule germina-
tive et du premier noyau embryonnaire. Sxl. Acad.roy. Belg. 41.
'80 BENEDEN, E. vAN. Contributions A la connaissance de l’ovaire des
mammiféres. L’ovaire du Vespertilio murinus et du Rhinolophus
ferrum-equineum. Arch. de Biol. Belg. 1.
’83 BENEDEN, E. vAN. Recherches sur la maturation de l’ceuf et la fécon-
dation. Arch. de Biol. 4.
'85 BENEDEN, E. VAN and JuLIN, C. Recherches sur la morphologie des
Tuniciers. Arch. de Biol. 6.
'79 BERGH, R. S. Studien tiber die erste Entwicklung des Eies von Gono-
thryaea Loveni. Morph. Jahrb. 5.
89 BEeRGH, R.S. Recherches sur les noyaux de l’Urostyla grandis et de
l’Urostyla intermedia n. sp. Arch. de Biol. 9.
*'94 BERGH, R. S. Vorlesungen iiber die Zelle und die einfachen Gewebe
des thierischen Kérpers. Wiesbaden.
‘42 BiscHorr, T. Entwicklungsgeschichte des Kaninchen-Eies. Braun-
schweig.
*'45 BiscHoFFr. Entwicklungsgeschichte des Hunde-Eies. A/iil/er’s Archiv.
'82 BLOCHMANN, F. Ueber die Entwicklung der Neritina fluviatilis.
Zeit. f. wiss. Zool. 36.
'94 BLOCHMANN, F. Ueber die Kerntheilung bei Euglena. B7o/. Cen-
tralol. 14.
’'88 Boum, A.A. Ueber Reifung und Befruchtung des Eies von Petromy-
zon Planeri. Arch. f. mikr. Anat. 32.
’93 BOumic, L. Zur feineren Anatomie von Rhodope Veranii Kolliker.
Arb. d. zool. Inst. Graz. 5.
'95 BOumMiG, L. Die Turbellaria acoela der Plankton-Expedition. Z7ged.
ad. Plankton-Exped., herausgeg. von V. Hensen. 2.
'92 Born, G. Die Reifung des Amphibieneies und die Befruchtung
unreifer Eier bei Triton taeniatus. Amat. Anz. 7.
344 MONTGOMERY. [Vo. XV.
794 Born, G. Die Struktur des Keimblaschens im Ovarialei von Triton
taeniatus. Arch. f. mikr. Anat. 43.
97 Bourn, P. Etudes sur l’évolution normale et l’involution du tube
séminifére. Avch. de Anat. Micr. 1.
°84 BourNE, A. G. Contributions to the Anatomy of the Hirudinea,
COLI BAU Sy NOR 2
87 Boveri, T. Zellen-Studien, 1. Jena.
’88 Boveri, T. Zellen-Studien, 2. Jena.
197 BRAEM, F. Die geschlechtliche Entwickelung von Plumatella fungosa.
Zoologica, herausgegeb. von Leuckart und Chun. Heft 23.
74 BrRanptT, A. Ueber active Formveranderungen des Kernkorperchens.
Arch. f. mikr. Anat. 10.
"78 BRANDT, A. Ueberdas Ei und seine Befruchtungsstatte, etc. Leipzig.
’82 BRANDT, K. Kerntheilungsvorgange bei einigen Protozoen. Szol.
Centralbl. 3.
’89 Brass, A. Die Zelle das Element der organischen Welt. Leipzig.
791 BRAUER, A. Ueber die Entwicklung von Hydra. Zezt. f.wiss. Zool. 52.
92 Brauer, A. Ueber das Ei von Branchipus Grubii v. Dyb. von der
Bildung bis zur Ablage. Aédh. ad. Akad. d. Wiss. Berlin.
93 BRAUER, A. Zur Kenntnis der Spermatogenese von Ascaris megalo-
cephala. Arch. f. mikr. Anat. 42.
’94 BRAUER, A. Ueber die Encystirung von Actinosphaerium Eichhornii.
Zeit. f. wiss. Zool. 58.
95a BREMER, L. Ueber das Paranuclearkérperchen der gekernten Erythro-
cyten, etc. Arch. f. mikr. Anat. 45.
’95b BREMER, L. Die Identitat des Paranuclearkérperchens der gekern-
ten Erythrocyten mit dem Centrosom. Arch. f. mikr. Anat. 46.
‘78 Brock, J. Beitrage zur Anatomie und Histologie der Geschlechts-
organe der Knochenfische. J/orph. Jahrb. 4.
93 Brooks, W. K. The Genus Salpa. Mem. Biol. Lab. Johns Hopkins
Univ. 2.
97 BRUYNE, C. DE. Les “Cellules doubles.” Verh. d. anat. Gesell. f. 1897.
Jena.
94 BuntiING, M. The Origin of the Sex-Cells in Hydractinia and Podo-
coryne, etc. Journ. of Morph. 9.
90 BURGER, O. Untersuchungen iiber die Anatomie und Histologie der
Nemertinen, etc. Zezt. f. wiss. Zool. 50.
95 BURGER, O. Die Nemertinen des Golfes von Neapel. /auna und
Flora des Golfes von Neapel. XXII. Monographie.
'76 BuTscuLt, O. Studien iiber die ersten Entwicklungsvorgange der Ei-
zelle, die Zelltheilung und die Conjugation der Infusorien. Adz. a.
Senckenberg. naturforsch. Ges. 10.
’80 BUTSCHLI, O. Protozoa. Bronn’s Klassen und Ordnungen. 1.
’85 BUTSCHLI, O. Einige Bemerkungen iiber gewisse Organisationsver-
haltnisse der Cilioflagellaten und der Noctiluca. Morph./Jahré. to.
No. 2.] COMPARATIVE CYTOLOGICAL STUUVIES. 545
84 CaRNoy, J. B. La biologie cellulaire. Etude comparée de la cellule
dans les deux régnes. Lzerre.
'85 CarRnoy, J. B. La cytodiérése chez les arthropodes. Za Cellule. 1.
86 CaRnoy, J. B. La cytodiérése de l’ceuf, etc. La vésicule germinative
et les globules polaire chez quelques Nématodes. Za Cellule. 3.
97a CarRnoy, J. B. and LEBRUN, H. La vésicule germinative et les glo-
bules polaires chez les Batrachiens. La Cellule. 12.
'97b CaRNOY, J. B. and LeBrun, H. La fécondation chez l’Ascaris
megalocephala. Verh. d. anat. Gesell. f. 1897. Jena.
’80 CHUN, C. Die Ctenophoren des Golfes von Neapel. Fauna und
Flora des Golfes von Neapel. 1.
’69 CLAPAREDE, E. Histologische Untersuchungen tiber den Regenwurm
(Lumbricus terrestris Linné). Zezt. f. wiss. Zool. 19.
195 Cor, W. R. On the Anatomy of a Species of Nemertean (Cerebratu-
lus lacteus Verr.), etc. Zvans. Connect. Acad. 9.
791 CONKLIN, E. G. and Brooks, W. K. On the Structure and Develop-
ment of the Gonophores of a certain Siphonophore belonging to the
Order Auronectae. Johns Hopkins Univ. Circulars. 10.
*'84 COURCHET, L. Dunoyau. Paris(?).
*'95 CreETy, C. Contributo alla conoscenza dell’ ovo ovarico. Richerche
Lab. Anat. Roma. 4.
91 CuéNoT, L. Etudes morphologiques sur les Echinodermes. Aych. de
Biol. i.
795 CUNNINGHAM, J. T. Experiments and Observations made at the
Plymouth Laboratory. 2. The Development of the Egg in Flat
Fishes and Pipe Fishes. /Journ. Mar. Biol. Assoc. London. 3.
797 CUNNINGHAM, J.T. On the Histology of the Ovary and of the Ovarian
Ova in Certain Marine Fishes. Q. /. M@. S. N.S., 40.
91 Davenport, C. B. Observations on Budding in Paludicella and Some
Other Bryozoa. Bull. Mus. Comp. Zool. Warvard. 22.
*89 DaviporF, M. v. Untersuchungen zur Entwicklungsgeschichte der
Distaplia magnilarva Della Valle, etc. I. MMztthecl. d. zool. Stat. v.
Neapel. 9.
‘97 DeEBsKI, B. Beobachtungen iiber Kerntheilung bei Chara fragilis.
Jahrb. wiss. Botan. 30.
795 DELAGE, Y. La structure du protoplasma et les théories sur l’heré-
dité, etc. Paris.
93 Demoor, J. Contribution a l'étude de la physiologie de la cellule.
Arch. de Biol. 13.
96 DoFLeEIN, F.J.T. Die Eibildung bei Tubularia. Zezt. f. wiss. Zool.
62.
’98 DoFLEIN, F. J. T. Studien zur Naturgeschichte der Protozoen. III.
Ueber Myxosporidien. Sfengel’s Zool. Jahrb. Anat. Abtheil rr.
‘71 Eimer, T. Die Schnauze des Maulwurfs als Tastwerkzeug. Arch.
Jj: mikr. Anat. 7.
540 MONTGOMERY. [VoL. XV.
‘72 Ermer, T. Untersuchungen tiber die Eier der Reptilien. Arch. f-
mikr. Anat. 8.
‘73 Eimer, T. Zoologische Studien auf Capri. I. Ueber Beroé ovatus.
Leipzig.
'75 Eimer, T. Ueber amédboide Bewegungen des Kernkorperchens.
Arch. f. mikr. Anat. W1.
'78 Ermer, T. Die Medusen physiologisch und morphologisch auf ihr
Nervensystem untersucht. Tubingen.
*'90 Ermer, T. Organic Evolution. (English translation.)
'87 E1stc, H. Monographie der Capitelliden des Golfes von Neapel.
Fauna und Flora des Golfes von Neapel. 16.
*'86 ELLENBERGER. Beitrag zur Lehre von den Kernk6rperchen. ‘Arch.
f. wiss. u. prakt. Tierheilkunde. 12.
'80 ENGELMANN, T. W. Zur Anatomie und Physiologie der Flimmerzel-
len. Arch. f. ges. Physiol. 23.
'97 ERLANGER, R. von. Ueber die Morphologie der Zelle und den
Mechanismus der Zellteilung. Zool. Centralbl. 4.
'97 FAIRCHILD, D.G. Ueber Kerntheilung und Befruchtung bei Basidio-
bolus ranarum Eidam. /ahrd. f. wiss. Botan. 30.
'97 FAUVEL, P. Recherches surles Ampharetiens. Avx/l. soc. de la France
et Belgique. Paris. 30.
‘93 Fick, R. Ueber die Reifung und Befruchtung des Axolotleies. Ze7¢.
f. wiss. Zool. 56.
'88 FiepLer, K. Ueber Ei- und Samenbildung bei Spongilla fluviatilis.
Zett. f. wiss. Zool. 47.
‘74 FLEMMING, W. Ueber die ersten Entwicklungserscheinungen am Ei
der Teichmuschel. Arch. f. mtkr. Anat. 10.
*'75 FLEMMING, W. Studien in der Entwicklungsgeschichte der Najaden.
Sitzber. ad. Akad. d. Wiss. Berlin. 71.
’80 FLEMMING, W. Beitrage zur Kenntniss der Zelle und ihrer Lebens-
erscheinungen. Arch. f. mikr. Anat.
'82 FLEMMING, W. Zellsubstanz, Kern und Zelltheilung. Leipzig.
94 FLEMMING, W. Referat “ Die Zelle.” Merkel und Bonnet’s Ergebn.
d. Anat. u. Entw. Wiesbaden. 3.
'97 FLEMMING, W. Zelle. Merkel und Bonnets Ergebn. d. Anat. u.
Entw. 5.
'96 FLopERus, M. Ueber die Bildung der Follikelhiillen bei den Ascidien.
Zeit. f. wiss. Zool. O61.
'73 FoL, H. Die erste Entwicklung des Geryonideneies. Jena. Zett. f.
Naturw. 7.
'83a For, H. Sur l’ceuf et ses enveloppes chez les Tuniciers. ec. zool.
suisse. 1.
'83b For, H. Sur Vorigine des cellules du follicule et de l’ovule chez les
Ascidies et chez d’autres animaux. Comp. Rend. Acad. Sci. Paris.
96.
No.2.] COMPARATIVE CYTOLOGICAL STUDIES. 547
89 For, H. Sur l’anatomie microscopique du Dentale. Arch. zool. ex-
per. et gén. 7.
*'81 FONTANA. Traité sur le venin de la vipére. Florence.
'94 Foot, K. Preliminary Note on the Maturation and Fertilization of
the Egg of Allolobophora foetida. Journ. of Morph. 9.
87 FRAIPONT, J. Le genre Polygordius. fauna und Flora des Golfes
von Neapel.
85 FRENZEL, J. Einiges uber den Mitteldarm der Insecten, sowie tber
Epithelregeneration. Arch. f. mikr. Anat. 26.
’92 FRENZEL,J. Dienucleolare Kernhalbirung. Arch. f. mikr. Anat. 39.
'93a FRENZEL, J. Ueber einige Argentinische Gregarinen, etc. ena.
Zett. f. Naturw. 27.
'93b FRENZEL, J. Mitteldarmdriise des Flusskrebses und die amitotische
Zelltheilung. Arch. f. mikr. Anat. 41.
*'67 FROMMANN, C. Untersuchungen iiber die normale und pathologische
Anatomie des Riickenmarks, Jena.
'84 FROMMANN, C. Untersuchungen tiber Struktur, Lebenserscheinungen
und Reaktionen thierischer und pflanzlicher Zellen. Jena. Zeit. f.
Naturw. 17.
*’'53 GABRIEL. De Cucullani elegantis evolutione. Berolini.
’95 GALEOTTI, G. Ueber die Granulationen in den Zellen. Jzternat.
Monatsch. f. Anat. u. Physiol. 12.
'96 GEROULD, J. H. The Anatomy and Histology of Caudina arenata
Gould. Bull. Mus. Comp. Zool. Harvard Coll. 29.
81 GraRD, A. Sur un curieux phénoméne de préfécondation, observé
chez une Spionide. Compt. Rend. Acad. Sci. Paris. 93.
‘93 GyuURASIN, S. Ueber die Kerntheilung von Peziza vesiculosa Bullard.
Ber. deutsch. botan. Gesell. 11.
82 GRAFF, L. von. Monographie der Turbellarien. I. Rhabdocoelida.
Leipzig.
’88 GrarFF, L. von. Die Annelidengattung Spinther. Zezt. f. wiss. Zool.
46.
’96 GREENWOOD, M. On Structural Change in the Resting Nuclei of
Protozoa. Part I. The Macronucleus of Carchesium polypinum.
Journ. Physiol. London. 20.
’83 GruBER, A. Ueber Kerntheilungsvorgange bei einigen Protozoen.
Journ. Physiol. London. 38.
785 GUIGNARD, L. Nouvelles recherches sur le noyau cellulaire et les
phénoménes de la division communs aux végétaux et aux animaux,
Ann. Sct. Nat. Paris, Botanie. Sér.6. 20.
*'94 GUIGNARD, L. L’origine dessphéres directrices. Journ. de Botan. 8.
92a HAckeER, V. Die Furchung des Eies von Aequorea Forskalea.
Arch. f. mikr. Anat. 40.
'92b Hacker, V. Die Eibildung bei Cyclops und Canthocamptus. Sfen-
gel’s zool. Jahrb. 5.
548 MONTGOMERY. [VOL. XV.
'93a HACKER, V. Das Keimblaschen, seine Elemente und Lageverande-
rungen. I. Arch. f. mikr. Anat. 4I.
'93b HACKER, V. -Ueber die Bedeutung des Hauptnucleolus. Vorlaufige
Mittheilung. Ber. d. naturf. Gesell. Freiburg i. B. 7.
'93c Hacker, V. Das Keimblaschen, seine Elemente und Lageverande-
rungen. II. Arch. f. mikr. Anat. 42.
’°95 HACKER, V., Die Vorstadien der Eireifung. Arch. f. mikr. Anat. 45.
°96 HACKER, V. Ueber eine neue Form der Geschlechtszellen-Sonderung.
Ber. d. naturf. Gesell, Freiburg i. B. to.
'97a HACKER, V. Die Keimbahn von Cyclops. Neue Beitrage zur
Kenntniss der Geschlechtszellen-Sonderung. Arch. f. mikr. Anat.
49.
’97b HACKER, V. Ueber weitere Uebereinstimmungen zwischen den Fort-
pflanzungsvorgangen der Tiere und Pflanzen. Bzol. Centralbl. 17.
'74 HAECKEL, E, Anthropogenie oder Entwicklungsgeschichte des Men-
schen. Leipzig.
97 Harper, R. A. Kerntheilung und freie Zellbildung im Ascus. /ahrd.
fj. wiss. Botan. 30.
'86 HeatuHcoTE, F. G. The Early Development of Julus terrestris.
OW, Ue SS. 20:
92 HEIDENHAIN, M. Ueber Kern und Protoplasma. Festschr. f. Kéllt-
ker. Leipzig.
95 HeLp, H. Beitrage zur Struktur der Nervenzellen und ihrer Fort-
satze. Arch. f. Anat. u. Physiol.
87 HENKING, H. Untersuchungen iiber die Entwicklung der Phalangiden.
I. Zeit. f. wiss. Zool. 45.
°90 HENKING, H. Untersuchungen iiber die ersten Entwicklungsvorgange
in den Eiern der Insekten. II. Ueber Spermatogenese und deren
Beziehung zur Entwicklung bei Pyrrhocoris apterus L. Zezt. f. wiss.
Zool. Si.
93 HenNneEGUY, L. F. Le corps vitellin de Balbiani dans l’ceuf des Ver-
tébrés. Journ. de l’ Anat. et de la Physiol. Paris. 29.
96 HENNEGUY, L. F. Legons sur la cellule. Paris.
’89a HERMANN, F. Beitrage zur Histologie des Hodens. Arch. f. mtkr.
Anat. 34.
’89b HERMANN, F. Die postfotale Histiogenese des Hodens der Maus
bis zur Pubertat. Arch. f. mikr. Anat. 34.
°97 HERMANN, F. Beitraige zur Kenntniss der Spermatogenese. Arch. /.
mtikr. Anat. 50.
795 Herrick, F. H. Movements of the Nucleolus through the Action of
Gravity. Anat. Anz. to.
'76 HeRtTWIG, O. Beitrage zur Kenntniss der Bildung, Befruchtung und
Theilung des thierischen Eies. I. Morph. Jahrb. 1.
77a HERTWIG, O. Beitrage zur Kenntniss der Bildung, Befruchtung und
Theilung des thierischen Eies. II. Morph. Jahrd. 3.
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 549
‘77> HERTWIG, O. Weitere Beitrage zur Kenntniss der Bildung, Befruch-
tung und Theilung des thierischen Eies. Morph. Jahrb. 3.
‘78a HERTWIG, O. Beitrage zur Kenntniss der Bildung, Befruchtung und
Theilung des thierischen Eies. III. Abtheil. 1. Morph. Jahrb. 4.
'78b HERTWIG, O. Beitrage zur Kenntniss der Bildung, Befruchtung und
Theilung des thierischen Eies. III. Abtheil. 2. Morph. Jahrb. 4.
’80 HERTWIG, O. Die Chaetognathen. Jena. Zeit. f. Naturw. 14.
'90 HERTWIG, O. Vergleich der Ei- und Samenbildung bei Nematoden.
Arch. f. mikr. Anat.
'92 HERTWIG, O. Die Zelle und die Gewebe. Jena.
‘76 HeERTWIG, R. Beitrage zur einheitlichen Auffassung der verschiedenen
Kernformen. Morph. Jahrb. 2.
‘79 HeRTWIG, R. Der Organismus der Radiolarien. Jena.
'84 HeERTWIG, R. Ueber die Kerntheilung bei Actinosphaerium Eichhornii.
Jena. Zeit. f. Naturw. 17. N. F., 10.
88 HertTwiG, R. Ueber Kernstruktur und ihre Bedeutung fiir Zelltheilung
und Befruchtung. S7ztzber. d. Gesell. f. Morph. u. Physiol. Miin-
chen. 4.
’96 HeRTWIG, R. Ueber die Entwickelung des unbefruchteten Seeigel-
eies. Festschr. f. Gegenbaur. 2. Leipzig.
'54 HeEsSLING, T. v. Einige Bemerkungen zu des Herrn Dr. Kebers
Abhandlung “ Ueber den Eintritt der Samenzellen in das Ei,” etc.
Zeit. f. wiss. Zool. 5.
*'59 HESSLING, T. v. Die Perlmuschel und ihre Perlen. Leipzig.
‘93 HEUSCHER, J. Zur Anatomie und Histologie der Proneomenia Slui-
teri Hubrecht. Jena. Zeit. f. Naturw. 27.
’84 HEUSER. Beobachtungen iiber Zellkerntheilung. Botan. Centralbl.
7s
'94 Hopce, C. F. A Microscopical Study of the Nerve Cell during Elec-
trical Stimulation. Journ. of Morph. 9.
"90 Hott, M. Ueber die Reifung der Eizelledes Huhns. Sitzb.d. Ahad.
ad. Wiss. v. Wien. 99.
'93a Hoii, M. Ueber die Reifung der Eizelle bei den Sdugethieren.
Verh. d. anat. Gesell., 7te Versammlung, in Gottingen. Jena.
*93b Horii, M. Ueber die Reifung der Eizelle bei den Saugethieren.
Sttzb. d. Akad. ad. Wiss. v. Wien, math.-naturw. Cl. 102.
'81 HuBRECHT, A. A. W. Proneomenia Sluiteri gen. et sp.n. Vieder-
lind. Arch. f. Zool. 1. Supplement.
’'87 HuBrecuT, A. A. W. Report on the Nemertea Collected by H. M.S.
“Challenger.” Challenger Reports. 19.
‘97 Hute, L. Changes in the Cell-organs of Drosera rotundifolia, pro-
duced by Feeding with Egg-albumen. @Q./. M.S. N.S., 39.
'94 HuMPHREY, J. E. Nucleolen und Centrosomen. Ber. deutsch. botan.
Gesell. 12.
83 JENSEN, O.S. Recherches sur la spermatogénése. Arch. de Biol. 4.
550 MONTGOMERY. [VoL. XV.
'84 Jijima, J. Untersuchungen tiber den Bau und die Entwicklungsge-
schichte der Stisswasserdendrocoelen (Tricladen). Zezt. fi wiss.
Zool. 40.
'°35 Jones, T. W. On the Ova of Women and Mammiferous Animals, as
they Exist in the Ovaries before Impregnation. London and Edin-
burgh Philos. Mag. 7.
'37 Jones, T. W. On the First Changes in the Ova of the Mammifera in
Consequence of Impregnation, etc. Phz/. Trans. 37.
93 JorDAN, E.O. The Habits and Development of the Newt (Diemyc-
tylus viridescens). Journ. of Morph. 8.
*'93a JULIN, C. Structure et développement des glandes sexuelles ; ovo-
génése, spermatogénése et fécondation chez Styelopsis grossularia.
Bull. soc. de la France et Belgigue. Paris. 25.
*'93b JULIN, C. Le corps vitellin de Balbiani et les éléments de la cellule
des Métazoaires qui correspondent au macronucleus des Infusoires
ciliés. Bull. soc. de la France et Belgigue. Paris.
93 Kaiser, J. E. Die Acanthocephalen und ihre Entwicklung. Chun x.
Leuckart’s Bibliotheca zoologica. 7.
’93 KARSTEN, G. Die Beziehungen der Nucleolen zu den Centrosomen
bei Psilotum triquetrum. Ber. d. deutsch. botan. Gesell. 11.
’90 KASTSCHENKO, N. Ueber den Reifungsprocess des Selachiereies.
Zeit. f. wiss. Zool. 50.
‘77 KENNEL, J. v. Beitrage zur Kenntnis der Nemertinen. <Avréezt. zool.
Inst. Wiirzburg. 4.
"95 KEUTEN, J. Die Kerntheilung von Euglena viridis Ehrenberg. Zev¢.
fj. wiss. Zool. 60.
’75 Kipp, P. Observations on Spontaneous Movement of Nucleoli.
ONTe MS.) NaS) 5.
'78 KLEIN, E. Observations on the Structure of Cells and Nuclei. I.
Drip te SS, We
'79a KLEIN, E. Observations on the Structure of Cells and Nuclei. II.
OT. aS. 10:
’79b KLEIN, E. Ein Beitrag zur Kenntniss der Struktur des Zellkerns.
Centralbl. f. d. med. Wiss. Berlin. 17.
"72 KLEINENBERG. Hydra, eine anatomisch-entwicklungsgeschichtliche
Untersuchung. Leipzig.
’86 KnapprE, E. Das Bidder’sche Organ, ete. Morph. Jahrb. 11.
*96 KOERNICKE, M. Untersuchungen iber die Entstehung und Entwick-
lung der Sexualorgane von Triticum, etc. Verh. d. naturf. Ver. d.
preuss. Rheinlande. 53.
'43 KOLLIKER, A. v. Beitrage zur Entwicklungsgeschichte wirbelloser
Thiere. I. Ueber die ersten Vorgange im befruchteten Ei.
Miiller’s Arch. f. Anat. und Physiol.
*49 KOLLIKER, A. v. Beitrage zur Kenntniss niederer Thiere. Zezt. f.
wiss. Zool. 1,
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 551
'84 KorSCHELT, E. Ueber die eigenthtmlichen Bildungen in den Zell-
kernen der Speicheldriisen von Chironomus plumosus. Zool. Anz. 7.
’89 KoRSCHELT, E. Beitrage zur Morphologie und Physiologie des Zell-
kernes. Sfengel’s Zool. Jahrb. 4.
°95 KoRSCHELT, E. Ueber Kerntheilung, Eireifung und Befruchtung bei
Ophryotrocha puerilis. Zez¢t. f. wiss. Zool. 60.
’96 KoRSCHELT, E. Ueber die Struktur der Kerne in den Spinndriisen
der Raupen. Arch. f. mikr. Anat. 47.
'97 KORSCHELT, E. Ueber den Bau der Kerne in den Spinndriisen der
Raupen. Arch. f. mikr. Anat. 49.
*'87 KostnskI. Zur Lehre von verschiedenen Typen der Kernkérperchen
beim Menschen. (In Russian.) /eschenedjelnaja klin. Gazeta.
24.
*'93 KosinskI. Einige Bemerkungen iiber die safraninophilen Kernk6r-
perchen in den Krebszellen. (In Russian.) Arch. d. Lad. f. allg.
Pathol. a. Univ. v. Warschau.
'92a KOSTANECKI, K. v. Ueber Centralspindel-Kérperchen bei karyo-
kinetischer Zelltheilung. Vorlaufige Mittheilung. JJerkel und
Bonnet’s Anat. Hefte.
'92b KosTANECKI, K. v. Ueber die Schicksale der Centralspindel bei
karyokinetischer Zelltheilung. Merkel und Bonnet’s Anat. Hefte.
‘98 KosTANECKI, K. v. Die Befruchtung des Eies von Myzostoma gla-
brum. Arch. f. mikr. Anat. 51.
*'92 KRAEPELIN, K. Die deutschen Siisswasserbryozoen. II. Abh. naturw.
Ver. Hamburg. 10.
*88 KuLtscHitzky, N. Ueber die Eireifung und die Befruchtungsvor-
gange bei Ascaris marginata. Arch. f. mikr. Anat. 32.
’54 LAcCAzE-DUTHIERS. Recherches sur les organes génitaux des Acé-
phales lamellibranches. Az. scz. nat. Sér. 4. 2.
°57 LacaAze-DuTHIERS. Organisation du dentale. Ann. scz. nat. Sér. 4.
he
84 Lanc, A. Die Polycladen (Seeplanarien) des Golfes von Neapel.
Fauna und Flora des Golfes von Neapel. 11.
‘93 LAUTERBORN, R. Ueber Bau und Kerntheilung der Diatomeen.
Vorlaufige Mittheilung. Verh. d. nat.-med. Ver. v. Heidelberg. 5.
’95a LAUTERBORN, R. Protozoenstudien. I. Kern- und Zelltheilung von
Ceratium hirundinella O. F. M. Zezt. f. wiss. Zool. 59.
'95b LAUTERBORN, R. Ueber eine Siisswasserart der Gattung Multicilia
Cienkowski (M. lacustris n. sp.), etc. Zezt. f, wiss. Zool. 60.
‘96 LAUTERBORN, R. Untersuchungen tiber Bau, Kerntheilung und Bewe-
gung der Diatomeen. Leipzig.
66 La VALETTE ST. GEORGE, Vv. Ueber den Keimfleck und die Deutung
der Eitheile. Arch. f. mikr. Anat. 2.
*'83 LA VALETTE ST. GEORGE, Vv. Commentatio de Isopodibus. (Fest-
schrift.)
552 MONTGOMERY. [VoL. XV.
‘94 LAvpowsky. Von der Entstehung der chromatischen und achromati-
schen Substanzen in den tierischen und pflanzlichen Zellen. Merkel
und Bonnet’s Anat. Hefte.
‘53 LeuckartT. Artikel “Zeugung” in Wagner's Handwoérterbuch der
Physiologie, Bad. iv.
‘49 Leypic, F. Zur Anatomie von Piscicola geometrica, mit theilweiser Ver-
gleichung anderer einheimischer Hirudineen. Zeit. fi wiss. Zool. 1.
‘50 LrypiG, F. Ueber Paludina vivipara, etc. Zezt. f. wiss. Zool. 2.
‘52 LreypiGc, F. Anatomische Notizen tiber Synapta digitata. Arch. f.
Anat. u. Physiol.
‘55a Leypic, F. Ueber Cyclas cornea Lam. Miiller’s Arch. f. Anat. u.
Physiol,
‘55b Leypic, F. Ueber den Bau und die systematische Stellung der
Raderthiere. Zezt. f. wzss. Zool. 6.
'83 LrypiGc, F. Untersuchungen zur Anatomie und Histologie der Thiere.
Bonn.
’85 LerypiG, F. Zelle und Gewebe. Bonn.
’88 LreypiGc, F. Beitrage zur Kenntniss des thierischen Eies im unbe-
fruchteten Zustande. Sfengel’s Zool. Jahrb. 3.
'97 LipForss, B. Zur Physiologie des pflanzlichen Zellkernes. Acta
Univ. Lundensis. 33.
96 List, T. Beitrage zur Chemie der Zelle und Gewebe. I. Ueber die
Farbung thierischer Gewebe mit Berlinerblau. M7ztthez/. d. zool.
Stat. v. Neapel. 12.
'92 LONNBERG, E. Kernstudien. ferh. a. biol. Ver. v. Stockholm. 4.
'49 LovEN, S. L. Ueber die Entwickelung der Mollusca acephala. (Von
dem Schwedischen von Creplin tibersetzt.) Arch. f. Naturg. 15.
'74 Lupwic, H. Ueber die Eibildung im Thierreich. Avrédezt. zool.-zoot.
Inst. Wirzburg. 1.
87a LuKJANOW, S.M. Beitrage zur Morphologie der Zelle. I. Arch. f.
Anat. u. Physiol., physiol. Abtheil., Supplement.
'87b LuKJANOW, S. M. Beitrage zur Morphologie der Zelle. II. Ueber
die Kerne der glatten Muskelzellen bei Salamandra macul. <A7ch.
Sf. mikr. Anat. 30.
88 LukKJANOow, S. M. Ueber eine eigenthtimliche Form des Kernkérper-
chens. Arch. f. mikr. Anat. 32.
°89 LuKJANOw, S. M. Einige Bemerkungen iiber sexuelle Elemente beim
Spulwurme des Hundes. Arch. f. mikr. Anat. 34.
*'93 LusTIG UND GALEOTTI. Cytodologische Studien tiber pathologische
menschliche Gewebe. Zéegler’s Beitr. 2. pathol. Anat. 14.
91 Maca.ium, A. B. Contributions to the Morphology and Physiology
of the Cell. Zyvans. Canadian Inst. 1.
‘95 Macatium, A. B. On the Distribution of Assimilated Iron Com-
pounds, other than Haemoglobin and Haematins, in Animal and
Vegetable Cells. Q./. 4. S. N.S., 38.
No.2.] COMPARATIVE CYTOLOGICAL STUDIES. 553
’81 MACFARLANE, J. M. The Structure and Division of the Vegetable
Cell. Trans. of the Botan. Soc. of Edinburgh. 14.
'85 MACFARLANE, J. M. Observations on Vegetable and Animal Cells ;
Their Structure, Division, and History. I. The Vegetable Cell.
Trans. of the Roy. Soc. of Edinburgh. 30.
'92 MACFARLANE, J. M. A Comparison of the Minute Structure of Plant
Hybrids with that of their Parents, and its Bearing on Biological
Problems. Tvans. of the Roy. Soc. of Edinburgh. 37.
'91 Mann, G. Methods of Differential Nucleolar Staining. TZvrans. and
Proc. of the Botan. Soc. of Edinburgh. 19.
'92 Mann, G. The Embryo-sac of Myosurus minimus L.: a Cell Study.
Trans. and Proc. of the Botan. Soc. of Edinburgh. 19.
‘77 Mark, E. L. Beitrage zur Anatomie und Histologie der Pflanzen-
lduse, insbesondere der Cocciden. Arch. f. mikr. Anat. 13.
’81 Mark, E. L. Maturation, Fecundation, and Segmentation of Limax
campestris Binney. Bul. Mus. comp. Zool. Harvard Coll. 6.
792 MARSHALL, W. S. Beitrage zur Kenntniss der Gregarinen. A7ch. f.
Naturg. 59.
‘90 Mastius, J. Contribution a l'étude des Rotateurs. Avch. d. Biol. 10.
'95 Meap, A. D. Some Observations on Maturation and Fecundation in
Chaetopterus pergamentaceus Cuvier. Journ. of Morph. 10.
'89 MELISSINOS AND NICOLAIDES. Untersuchungen tiber einige intra-
und extranucleare Gebilde im Pankreas der Saugethiere, etc. Arch.
f. Anat. u. Physiol., physiol. Abtheil.
'93 MERTENS, H. Recherches sur la signification des corps vitellins de
Balbiani dans Vovule des mammiféres et les oiseaux. Arch. de
Biol. 13.
*'67 METSCHNIKOFF, E. Zur Entwicklungsgeschichte der rothen Blut-
kérperchen. Virchow’s Arch. f. pathol. Anat. 41.
*'86 METSCHNIKOFF, E. Embryologische Studien an Medusen. Wien.
'94 Metzner, R. Beitrage zur Granulalehre. Arch. f. Anat.u. Physiol,
physiol. Abtheil.
°86 Meunier, A. Le nucléole des Spirogyra. La Cellule. 3.
‘96 MEvEs, F. Ueber die Entwicklung der mannlichen Geschlechtszellen
von Salamandra maculosa. Arch. f. mikr. Anat. 48.
’97 Meves, F. Zur Struktur der Kerne in den Spinndriisen der Raupen.
Arch. f. mikr. Anat. 48.
'96 MicHEL, A. Des nucléoles composés, notamment dans l’ceuf des anné-
lides. Comp. Rend. Acad. Sci. de Paris. 123.
*'97 MicueL, A. Sur la composition des nucléoles. Comp. Rend. de la
Soc. Biol. de Paris. 4.
'93 MincHIN, E. A. Observations on the Gregarines of Holothurians.
OR RMS NS 3346
'93 MoLL, J. W. Observations on Karyokinesis in Spirogyra. Verh. &.
Akad. Wetensch. Amsterdam. 1. :
554 MONTGOMERY. [VoL. XV.
'95 MontGom_ry, T. H., JR. Stichostemma eilhardi nov. gen. nov. spec.
Ein Beitrag zur Kenntnis der Nemertinen. Zezt. f. wiss. Zool. 59.
‘96 Montcomery, T. H., Jk. On the Connective Tissues and Body Cavi-
ties of the Nemerteans, etc. Sfengel’s Zool. Jahrb. to.
'97a MONTGOMERY, T. H., JR. Studies on the Elements of the Central
Nervous System of the Heteronemertini. /Journ. of Morph. 13.
*97b Montcomery, T. H., Jr. Preliminary Note on the Chromatin
Reduction in the Spermatogenesis of Pentatoma. Zool. Anz. 546.
’95 Moors, J. E.S. On the Structural Changes in the Reproductive Cells
during the Spermatogenesis of Elasmobranchs. Q./.M.S. N.S.,
38.
96 MorGan, T. H. The Production of Artificial Astrosphaeres. Arch.
Sj. Entwicklungsmech. d. Organismen. 3.
’97 MottieER, D. M. Beitrage zur Kenntniss der Kerntheilung in den
Pollenmutterzellen einiger Dikotylen und Monokotylen. /ahré. f.
wiss. Botan. 30.
‘94 Mursacnu, L. Beitrage zur Kenntnis der Anatomie und Entwickelung
der Nesselorgane der Hydroiden. Arch. f. Naturg. 60.
’88 NAGEL, W. Das menschliche Ei. Arch. f. mikr. Anat. 31.
'85 NANSEN, F. Bidrag til Myzostomernes Anatomi og Histologi. Bergens
Mus. Aasberetn.
’82 Nussbaum, M. Ueber den Bau und die Thatigkeit der Driisen.
Arch. f. mtikr. Anat. 21.
’87 NusspAaum, M. Ueber die Theilbarkeit der lebendigen Materie. II.
Arch. f. mikr. Anat. 29.
'83 OGaTta, M. Die Veranderung der Pankreaszellen bei der Sekretion.
Arch. f. Anat. u. Physiol., physiol. Abtheil.
'97 PENNINGTON, M. E. A Chemico-Physiological Study of Spirogyra
nitida. Pzblic. Botan. Lab. Univ. of Pennsylvania. 1.
90 PFEFFER, W. Zur Kenntniss der Plasmahaut und der Vacuolen, nebst
Bemerkungen tiber den Aggregatzustand des Protoplasmas und tiber
osmotische Vorgainge. Adh. da. konigl. sachs. Akad. ad. Wiss.,
math.-physik. Cl. 16.
’°81 PFITZNER, W. Ueber den feineren Bau der bei der Zelltheilung auf-
tretenden Differenzirungen des Zellkerns. Morph. Jahrb. 7.
'83 PritzNeR, W. Beitrage zur Lehre vom Bau des Zellkerns und seinen
Theilungserscheinungen. Arch. f. mikr. Anat. 22.
'86a PFITZNER, W. Zur morphologischen Bedeutung des Zellkerns.
Morph. Jahrb. it.
'86b PFITZNER, W. Zur Kenntnis der Kerntheilung bei den Protozoen.
Morph. Jahrb. 1.
*'86c PFITZNER, W. Zur morphologischen Bedeutung des Zellkerns,
Virchow’s Arch. f. pathol. Anat. 103.
’95 PrLiicke, M. Zur Kenntnis des feineren Baues der Nervenzellen bei
Wirbellosen. Zezt. f. wiss. Zool. 60.
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 555
'63 PFLUGER, E. F. W. Ueber die Eierstécke der Saugethiere und des
Menschen. Leipzig.
'93 Pizon, A. Histoire de la blastogénése chez les Botryllidés. Avn.
sect. mat. Paris. 14.
’86 PLATNER,G. Zur Bildung der Geschlechtsprodukte bei den Pulmona-
ten. Arch. f. mikr. Anat. 26.
89a PLATNER, G. Beitrage zur Kenntnis der Zelle und ihrer Theilungs-
erscheinungen. III. Die direkte Kerntheilung in den Malpighi’schen
Gefassen der Insekten. Arch. f. mikr. Anat. 33.
'89b PLATNER, G. Beitrage zur Kenntnis der Zelle und ihrer Theilungs-
erscheinungen. IV. Die Entstehung und Bedeutung der Neben-
kerne im Pankreas, etc. Arch. f. mikr. Anat. 33.
'89c PLATNER, G. Beitrage zur Kenntnis der Zelle und ihrer Theilungs-
erscheinungen. VI. Die Bildung der ersten Richtungsspindel im
Ei von Aulastomum gulo. Arch. f. mikr. Anat. 33.
96 PorRAULT, G. and RacrgorskI, M. Ueber konjugate Kerne und kon-
jugate Kerntheilung. Bzol. Centralbl. 16.
94 PURCELL, F. Ueber den Bau der Phalangidenaugen. Zezt. f. wiss.
Zool. 58.
94 QUATREFAGE, A. DE. Etudes embryogéniques. Mémoire sur l’em-
bryogénie des tarets. Ann. scz. nat. Sér. 3, II.
'85 Rapi, K. Ueber Zelltheilung. I. Morph. Jahrb. 10.
*'93 Racisporski, M. Zur Morphologie des Zellkerns der keimenden
Samen. Azz. d. Akad. d. Wiss. v. Krakau.
*’'95 RacovitzA, E.G. Notes de biologie. III. Mceurs et reproduction
de la Rossia macrosoma (D. Ch.). Arch. de zool. expér. et gén.
Sér. 3, 2.
67 Ransom, W. H. Observations on the Ovum of Osseous Fishes.
Phil. Trans. London. 157.
95a Ratu, O. vom. Neue Beitrage zur Frage der Chromatinreduktion
in der Samen- und Eireife. Arch. f. mikr. Anat. 46.
'95b Ratu, O. vom. Ueber den feineren Bau der Driisenzellen des Kopfes
von Anilocra mediterranea Leach, etc. Zezt. f. wiss. Zool. 60.
'82 Rauber. Neue Grundlegungen zur Kenntniss der Zelle. Morph.
Jahrb. 8.
*'41 REICHERT. Miiller’s Arch. f. Anat. und Physiol.
'83 Rein, G. Beitrage zur Kenntniss der Reifungserscheinungen und
Befruchtungsvorgange am Saugethierei. Morph. Jahrb. 22.
*'82 REINHARD, W. Skizze des Baues und der Entwickelung der Siiss-
wasserbryozoen. Charkow. (Russian.)
94 REINKE, F. Zellstudien. I. Morph. Jahrb. 43.
58 Remak, R. Ueber die Theilung der Blutzellen beim Embryo. Mz/-
ler’s Arch. f. Anat. u. Physiol.
'93 REPIACHOFF, W. Zur Spermatologie der Turbellarien. Ze7zt. f. wiss.
Zool. 56.
550 MONTGOMERY. [VOL. XV.
*'81 Retzius, G. Zur Kenntniss vom Bau des Zellkerns. Bzolog. Un-
tersuch.
'93 RHUMBLER, L. Ueber Entstehung und Bedeutung der in den Kernen
vieler Protozoen und in Keimblaschen von Metazoen vorkommenden
Binnenkorper (Nucleolen), ete. Zezt. fi wiss. Zool. 56.
‘95 RuHUMBLER, L. Beitrage zur Kenntnis der Rhizopoden. IV. Cypho-
deria margaritacea Schlumb. Zezt. f. wiss. Zool. 61.
’96 RoHDE, E. Ganglienzellen und Neuroglia. Ein Kapitel iiber Vermeh-
rung und Wachsthum von Ganglienzellen. Arch. f. mikr. Anat. 47.
'92a ROSEN, F. Beitrage zur Kenntniss der Pflanzenzellen. I. Ueber
tinktionelle Differenz verschiedener Kernbestandtheile und der Sex-
ualkerne. Cohn’s Beitr. 2. Biol. d. Pflanzen. 5. Breslau.
’92b ROSEN, F. Beitrage zur Kenntniss der Pflanzenzellen. II. Studien
iiber die Kerne und die Membranbildung bei Myxomycetes und Pil-
zen. Cohn’s Beitr. z. Biol. d. Pflanzen. 5. Breslau.
'95 ROSEN, F. Beitrage zur Kenntniss der Pflanzenzellen. III. Kerne
und Kernkorperchen in meristematischen und sporogenen Geweben.
Cohn’s Beitr. z. Biol. ad. Pfhlanzen. 7. Breslau.
*'95 Rossi, U. Contributo allo studio della struttura, della maturazione
e della distruzione delle uova degli Anfibi (Salamandrina perspicil-
lata e Geotriton fuscus). Pwbdl. Istit. Stud. Sup. Firenze, Sez.
Med. Chir.
’83 RouLe, L. La structure de l’ovaire et la formation des ceufs chez les
Phallusiadées. Compt. Rend. de l’Acad. de Paris. 96.
'92 RUCKERT, J. Zur Entwicklungsgeschichte des Ovarialeies bei Sela-
chiern. Anat. Anz. 7.
'94 Rickert, J. Zur Eireifung bei Copepoden. Merkel und Bonnet's
Anat. Hefte. 12.
’95 SACHAROFF, N. Ueber die Entstehung der eosinophilen Granulationen
des Blutes. Arch. f. mikr. Anat. 45.
95 SaLA, L. Experimentelle Untersuchungen tiber die Reifung und
Befruchtung der Eier bei Ascaris megalocephala. Arch. f. mtkr.
Anat. 44.
‘88 SANFELICE. Spermatogénése des vertébrés. Arch. ztal. de Biol. 10.
'88 SCHARFF, R. On the Intra-ovarian Egg of some Osseous Fishes.
OJ-MaS. NYSaiZ8e
'94 SCHAUDINN, F. Ueber die Kerntheilung mit nachfolgender K6rper-
theilung bei Amoeba crystalligera Gruber. Sitzber. d. k.-k. Akad.
ad. Wiss. uv. Berlin. 38.
'83 SCHAUINSLAND, H. Beitrag zur Kenntniss der Embryonalentwicklung
der Trematoden. /ena. Zeit. f. Naturw. 16.
'86 SCHAUINSLAND, H. Die embryonale Entwicklung der Bothriocepha-
len. Jena. Zett. f. Naturw. 19.
'88 SCHEWIAKOFF, W. Ueber die karyokinetische Kerntheilung der Eu-
glypha alveolata. Morph. Jahrb. 13.
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 557
°78 SCHINDLER, E. Beitrage zur Kenntniss der Malpighi’schen Gefasse
der Insecten. Zezt. f. wiss. Zool. 30.
°38 SCHLEIDEN, M. J. Beitrage zur Phytogenesis. Muller's Arch. f.
Anat., Physiol. u. wiss. Med.
95 ScHLOTER, G. Zur Morphologie der Zelle. Arch. f. mikr. Anat. 44.
*'80 ScHMITz. Untersuchungen iiber die Struktur des Protoplasmas und
der Zellkerne der Pflanzenzellen, Sitzber. d. Niederrhein. Gesell. f.
Naturk. Bonn.
* SCHNEIDER, A. Grégarines nouvelles ou peu connues. Tad/ettes
soologiques. 1.
’75 SCHNEIDER, A. Contributions 4 V/histoire des Grégarines des inverté-
brés de Paris et de Roscoff. Arch. de zool. expér. et gén. 4.
’°83 SCHNEIDER, A. Nouvelles observations sur la sporulation du Klossia
octopiana. Arch. de zool. expér. et gén. Sér. 2. 1.
'91 SCHNEIDER, C. Untersuchungen tuber die Zelle. d7d. zool. Inst.
Wien. 9.
*92 SCHOTTLANDER, P. Beitrage zur Kenntniss des Zellkerns und der
Sexualzellen bei Kryptogamen. Cohn’s Bettr.z. Biol.d. Pflanzen. 6.
°65 SCHRON, O. Ueber das Korn im Keimfleck und in dem KernkGérper-
chen der Ganglienzellen bei Saugethieren. Ustersuch. z. Natur-
lehre ad. Thiere, herausgeg. von J. Moleschott. 9.
*'82 ScHUTZz, J. Ueber den Dotterkern, dessen Entstehung, Vorkommen
und Bedeutung. Dissertation. Bonn.
’87 SCHULTZE, O. Untersuchungen tiber die Reifung und Befruchtung
des Amphibieneies. Zezt. fi wiss. Zool. 45.
°75 SCHULZE, F. E. Rhizopodenstudien. V. Arch. f. mikr. Anat. 11.
’76 SCHWALBE. Bemerkungen tiber die Kerne der Ganglienzellen. Jena.
Zeit. f. Naturw. 10.
*'39 SCHWANN, T. Mikroskopische Untersuchungen tber die Ueberein-
stimmung in Struktur und Wachsthum der Thiere und Pflanzen. Berlin.
’87 SCHWARZ, F. Die morphologische und chemische Zusammensetzung
des Protoplasmas. Cohn’s Bettr. z. Biol. d. Pflanzen. 5.
'82 SEELIGER, O. Eibildung und Knospung von Clavelina lepadiformis.
Sitzber. a. k.-k. Akad. ad. Wiss. v. Wien. 85.
*'80 SHAFER, E. A. On the Structure of the Immature Ovarian Ovum
in the Common Fowl and in the Rabbit. Proc. of the Roy. Soc. of
London. 30.
'90 SHELDON, L. The Maturation of the Ovum in the Cape and New
Zealand Species of Peripatus. Q. /. M.S. N.S., 30.
*'39 SIEBOLD, C. T. von. Beitrage zur Naturgeschichte der wirbellosen
Thiere. Meneste Schr. d. naturf. Ges. Danzig. 3.
90 SmirNow, A. Die Struktur der Nervenzellen im Sympathicus der
Amphibien. Arch. f. mikr. Anat. 35.
95 SopoTTa, J. Die Befruchtung und Furchung des Eies der Maus.
Arch. f. mikr. Anat. 45.
558 MONTGOMERY. [Vou. XV.
93 STAUFFACHER, H. Eibildung und Furchung von Cyclas cornea L.
Jena. Zeit. f. Naturw. N.F., 28.
97 STAUFFACHER, H. Die Urniere bei Cyclas cornea (Lam.). Zeitt. f.
wiss. Zool. 63.
'88 STEINHAUS, J. Les metamorphoses et la gemmation indirecte des
noyaux dans l|’épithélium intestinal de la Salamandra maculosa.
Arch. physiol., norm. et pathol. Sér. 4, 2.
'65 STEPANOFF, P. Ueber die Geschlechtsorgane und die Entwicklung
von Cyclas. Arch. f. Naturg. 31.
’87 STOLNIKOW. Vorgange in den Leberzellen, insbesondere bei der
Phosphorvergiftung. Arch. f. Anat. u. Physiol., physiol. Abtheil.,
Supplement.
*’80 STRASBURGER, E. Zellbildung und Zelltheilung. 3te Auflage. Jena.
'82a STRASBURGER, E. Ueber den Zellkern. Botan. Zeitung. 40.
'82b STRASBURGER, E, Ueber den Theilungsvorgang der Zellkerne und
das Verhaltniss der Kerntheilung zur Zelltheilung. Arch. f. mtkr.
Anat. 21.
84 STRASBURGER, E. Die Controversen der indirekten Kerntheilung.
Arch. f. mikr. Anat. 23.
88 STRASBURGER, E. Ueber Kern- und Zelltheilung im Pflanzenreiche,
nebst einem Anhang iiber Befruchtung. Jena.
‘93 STRASBURGER, E. Zu dem jetzigen Stande der Kern- und Zellthei-
lungsfragen. Anat. Anz. 8.
'95 STRASBURGER, E. Karyokinetische Probleme. /ahré.f. wiss. Botan.
28.
'97 STRASBURGER, E. Ueber Cytoplasmastrukturen, Kern- und Zellthei-
lung. Jahrb. f. wiss. Botan. 30.
’86 STUHLMANN, F. Die Reifung des Arthropodeneies. Nach Beobach-
tungen an Insekten, Spinnen, Myriapoden und Peripatus. Ber. d.
naturforsch. Gesell. v. Freiburg i. B. 1.
*'69 SVIERCZEWSKI. Zur Physiologie des Kerns und Kernkérperchens
der Nervenzellen des Sympathicus. Centralbl. f. med. Wiss. Berlin.
'97 SwINGLE, W. T. Zur Kenntniss der Kern- und Zelltheilung bei den
Sphacelariaceen. /ahré. f. wiss. Botan. 30.
'82 TANGL, E. Die Kern- und Zelltheilungen bei der Bildung des Pollens
von Hemerocallis fulva L. Denkschr. ad. Wien. Akad. d. Wiss.,
math.-naturw. Cl. 45.
*’94 ToyAMA. On the Spermatogenesis of the Silkworm. Sxl. Coll.
Agricult. Imp. Univ. Tokio. 2.
80 TRINCHESE, S._ I primi momenti dell’ evoluzione nei Mollusci. 4 ¢#z
R. Accad. Lincet. Ser. 3, 7.
*’81 TRINCHESE,S. Per la fauna marittima italiana. Aeolididae e familie
affni. Arti R. Accad. Lincet. 11.
*'36 VALENTIN. KRepertorium. T. 1.
*'39 VALENTIN. Repertorium. T,. 4.
No. 2.] COMPARATIVE CYTOLOGICAL STUDIES. 559
'95 VAN DER STRICHT, O.° La maturation et la fécondation de l’eeuf
d’Amphioxus lanceolatus. Bz. Acad. Roy. Belg. Sér.3. 30.
82 VEJDOvSKY, F. Untersuchungen tiber Anatomie, Physiologie und
Entwicklung von Sternaspis. Denkschr. d. Wien. Akad. d. Wiss.,
naturw.-math. Cl. 43.
'84 VEyDOVSKY, F. System und Morphologie der Oligochaeten. Prag.
'88 VEJDOVSKY, F. Entwicklungsgeschichtliche Untersuchungen. I.
Reifung, Befruchtung und Furchung des Rhynchelmis-Eies. Prag.
‘95a VEyDOvSKY, F. Zur vergleichenden Anatomie der Turbellarien. 1.
Zett. f. wiss. Zool. 60.
'95b VEJDOVSKY, F. Zur vergleichenden Anatomie der Turbellarien. IJ.
Zeit. f. wiss. Zool. 60.
93 VER EEcKE. Modifications de la cellule pancréatique pendant I’ac-
tivité sécrétoire. Arch. de Biol. 13.
'86 VIGELIUS, W. J. Zur Ontogenie der marinen Bryozoen. J/i¢thezl. d.
zool. Stat. v. Neapel. 6.
'42 Voct, C. Embryologie des Salmones. Neuchatel.
93 WaGeER, H. On Nuclear Division in the Hymenomycetes. Ann. of
Botany. London. 7.
'96a WAGNER, J. Einige Beobachtungen tiber die Spermatogenese bei
den Spinnen. Zool. Anz. 19.
*'96b WAGNER, J. Beitrage zur Kenntnis der Spermatogenese bei den
Spinnen. Ard. nat. Gesell. St. Petersburg. 26.
’35 WAGNER, R. Einige Bemerkungen und Fragen tiber das Keimblaschen
(vesicula germinativa). Miller's Arch. f. Anat., Physiol. u. wiss. Med.
*36 WAGNER, R. Prodromus historiae generationis hominis atque mam-
malium. Leipzig.
'37 WAGNER, R. Beitrage zur Geschichte der Zeugung und Entwickelung.
Abn. da. k. bay. Akad. d. Wiss. Miinchen. 2.
*'39 WaGner, R. Artikel “Ei” in Eysch und Gruber’s Encyclopadie.
1. Abtheil.
*'70 WALDEYER, W. Eierstock und Ei. Ein Beitrag zur Anatomie und
Entwicklungsgeschichte der Sexualorgane. Leipzig.
88 WALDEYER, W. Ueber Karyokinese und ihre Beziehungen zu den
Befruchtungsvorgangen. <Ayrch. f. mikr. Anat. 32.
*'95 WALDEYER, W. Die neueren Ansichten tiber den Bau und das
Wesen der Zelle. Deutsch. med. Wochenschrift.
‘93 WASIELEVSKy. Die Keimzone in den Genitalschlauchen von Ascaris
megalocephala. Deutsche med. Wochenschrift. 41.
"94 WarTaské, S. On the Nature of Cell-Organization. Biol. Lectures at
Woods Holl for 1893.
*'82 WEISMANN, A. Beitrage zur Kenntniss der ersten Entwicklungsvor-
gange im Insektenei. Festgabe fiir Henle. Bonn.
'83 WEISMANN, A. Die Entstehung der Sexualzellen bei den Hydrome-
dusen. Jena.
560 MONTGOMERY. [VOL. XV.
'89 WEISMANN UND ISCHIKAWA. Ueber die Paracopulationszelle im
Daphnidenei. Sfengel’s Zool. Jahrb., Abtheil. f. Anat. 4.
'87 WENT, F. A. F. C. Beobachtungen tiber Kern- und Zelltheilung.
Ber. a. deutsch. botan. Gesell. 5.
89 WHEELER, W. M. The Embryology of Blatta Germanica and Dory-
phora decemlineata. Journ. of Morph. 3.
’95 WHEELER, W. M. The Behavior of the Centrosomes in the Fertilized
Egg of Myzostoma glabrum. Journ. of Morph. to.
"96 WHEELER, W. M. The Sexual Phases of Myzostoma. M?ttheil. d. ,
zool. Stat. v. Neapel. 12.
‘97 WHEELER, W. M. The Maturation, Fecundation, and Early Cleav-
age of Myzostoma glabrum Leuckart. Arch. de Biol. 15.
‘78 WHITMAN, C. O. The Embryology of Clepsine. Q./. M.S. 18.
'84 WIELOWIEJSKI, H. v. Vorlaufige Bemerkungen tuber die Eizelle.
Biol. Centralol. 12.
95 WiLcox, E. V. Spermatogenesis of Caloptenus femur-rubrum and
Cicada tibicen. Bull. Mus. Comp. Zool. Harvard Univ. 27.
84 WiLL, L. Ueber die Entstehung des Dotters und der Epithelzellen
bei den Amphibien und Insekten. Zool. Anz. 7.
85 WILL, L. Bildungsgeschichte und morphologischer Werth des Eies
von Nepa cinerea L. und Notonecta glauca L. Zezt. fi wiss. Zool. 41.
86 WILL, L. Oogenetische Studien. I. Die Entstehung des Eies von
Colymbetes fuscus L. Zezt. f. wiss. Zool. 43.
’96 WILSON, E.B. The Cell in Development and Inheritance. New York.
94 Witson, H. V. Observations on the Gemmule and Egg Development
of Marine Sponges. /ourn. of Morph. 9. F
'92 WiREN, A. Studien tiber die Solenogastres. I. Monographie des
Chaetoderma nitidulum Lovén. Kongl. Svenska Vet. Akad. Hand-
ling. Stockholm. 24.
*'45 WITTICH, W. v. Observationes quaedam de aranearum ex ovo evo-
lutione. Halle.
‘49 WiTTICH, W. v. Die Entstehung des Arachnideneies im Eierstock,
etc. Arch. f. Anat. u. Physiol.
‘91 WoLTERS, M. Die Conjugation und Sporenbildung bei Gregarinen.
Arch. f. mikr. Anat. 37.
82 ZACHARIAS, E. Ueber den Zellkern. Botan. Zeitung. 40.
85 ZACHARIAS, E. Ueber den Nucleolus. Botan. Zeitung. 43.
’93 ZACHARIAS, E. Ueber Chromatophilie. Ber. ad. deutsch. botan.
Gesell. 11.
"94 ZAcCHARIAS, E. Ueber Beziehungen des Zellenwachsthums zur Be-
schaffenheit des Zellkerns. Ber. d. deutsch. botan. Gesell. 12.
*'93 ZIMMERMANN, A. Beitrage zur Morphologie und Physiologie der
“ Pflanzenzellen. Bd. 2. Tuiibingen.
'96 ZIMMERMANN, A. Die Morphologie und Physiologie des pflanzlichen
Zellkernes : eine kritische Litteraturstudie. Jena.
No. 2.]
COMPARATIVE CYTOLOGICAL STUDIES.
501
EXPLANATION OF PLATES XXI-XXx.
All the figures have been drawn with the aid of the camera lucida, and rep-
resent sections of the structures delineated. Those parts of them which are
colored represent as accurately as possible the stained preparations from which
they were copied; in most of the figures only certain portions are colored, the
other details being filled in with the pencil.
In order to show the correct pro-
portionate size of the various cells and nuclei the greater number of the figures
have been made at a magnification afforded by the homogeneous immersion lens
jy of Zeiss, with the ocular 4, and unless otherwise specified this may be under-
stood to have been the magnification employed. The following abbreviations have
been used in the figures:
G.
(Go 7ah
Cen.
Chr.
Chr. F.
Chrom.
Cc. Mb.
C. Sp.
(EB INE
(EE AY
Cut.
Cy. Pl.
an;
cell.
cell duct.
centrosome.
chromatin.
chromatin filament.
chromosome.
cell membrane.
centrosphere.
nucleus of connective tissue.
connective tissue sheath of
the ovarial acinus.
cuticula.
cytoplasm.
degenerated cells (or cell
substance).
endoplasm.
gonadal membrane.
intravitelline membrane.
nucleus.
problematical nuclear body.
nuclear fibers.
nuclear granules.
nuclear membrane.
LVR.
LV. Sap.
Nut. Gl.
n.
n. 2.
n. D.
n. Gr.
n. Mb.
mn.
n. Sub.
n. Vac.
Nx.
Ps.n.
Secr.
SP.
Sp. F.
Vac.
Va. Bi.
Yk. Gi.
metamorphosed portion of
nucleus.
nuclear sap.
nutritive globule.
nucleolus.
nucleolus of the second gen-
eration.
derivatives of the nucleolar
substance.
granules of degenerated nu-
cleoli.
nucleolar membrane.
nucleolinus.
nucleolar ground substance.
nucleolar vacuole.
nucleolar body of unknown
origin.
pseudonucleolus.
secretion corpuscles.
spores.
spindle fibers.
vacuole.
yolk ball.
yolk globule.
re
Diss
COMPARATIVE CYTOLOGICAL STUDIES. 563
EXPLANATION OF PLATE XXI.
Figs. 1-19: Gregarines from Lineus gesserensis.
Fic. 1. Smallest individual found (hom. immers., oc. 2. Hermann’s fluid;
Del. haematoxylin, eosin).
Fic. 2. Outline of the largest individual. Obj. C., oc. 2.
Fic. 3. Nucleus (corros. sublimate ; Del. haematoxylin, eosin).
Fic. 4. Portion of a longitudinal section, though an individual in which
spores were present (as in 3).
Fic. 5. The smaller of the two nuclei of Fig. 1.
Fic. 6. The same gregarine drawn in Fig. 1, but with obj. C., oc. 2 to show
its relative size to the one of Fig. 2.
Fics. 7-9. Nuclei (alcohol. sublimate ; Ehrlich-Biondi stain, 3% hrs.).
Fic. 10. Nucleus (Flemming’s fluid; Del. haematoxylin, eosin).
Fic. 11. Idem (Flemming’s fluid ; Ehrlich-Biondi stain, 23% hrs.).
Fics. 12-16. Nuclei (Flemming’s fluid; Del. haematoxylin, eosin).
Fics. 17-19. Idem (sublimate with 2% acetic acid; aq. sol. methylen blue,
30 min.; aq. sol. brasilin, 214 hrs.).
Figs. 20-35: Gregarines from Carinella annulata (fixation with alcohol. sol.
sublimate).
Fics. 20 and 21. Outlines of two individuals. Obj. C., oc. 2.
Fics. 22-25. Nuclei (Del. haematoxylin, 15 min., alum carmine, 6 hrs.).
Fic. 26. Nucleus (Ehrlich-Biondi stain, 3 hrs.).
Fics. 27 and 28. Nuclei (Del. haematoxylin, eosin).
Fic. 29. Nucleus, only the outlines of the nucleoli drawn.
Fics. 30-35. Nuclei (as in 26).
Figs. 36-49: Nucler of ganglion cells from the brain of Doto (Fig. 36, of the
smallest type of cell; Figs. 37-42, of medium-sized cells; Figs. 43-49, of
the colossal cells).
Fic. 36 (Hermann’s fluid, 114 hrs. ; Lyons blue, 15 min.).
Fics. 37 and 38 (Hermann’s fluid, 1% hrs.; Ehrl. haematoxylin, 1% hrs.,
eosin, 7 min.).
Fics. 39 and 40 (alcohol. sol. sublimate ; Ehrlich-Biondi stain, 314 hrs.).
Fics. 41 and 42 (as in 37).
FIG. 43 (as in 39).
Fic. 44 (Hermann’s fluid, 114 hrs.; safranin, 92 hrs., gentian violet, 114
hrs., orange G., 2 min.).
Fic. 45 (as in 36).
Fics. 46 and 47. Two sections of one nucleus (as in 37).
Fic. 48 (as in 37).
FIG. 49 (as in 39).
Fic. 50. Immature germinal vesicle of Hmys (picric acid; Del. haematoxylin).
564 MONTGOMERY.
Figs. 51-56: Nuclei from the muscle cells of the circular musculature of
Lineus gesserensts.
Fic. 51 (alcohol. sol. sublimate ; Ehrlich-Biondi stain, 31% hrs.).
Fic. 52 (aq. sol. sublimate ; cochineal, 1 hr., Del. haematoxylin, 20 min.).
Fics. 53 and 54 (aq. sol. sublimate with 2% acetic acid; Ehrl. haematoxylin,
eosin).
Fic. 55 (Hermann’s fluid, 30 min. ; Ehrl. haematoxylin, 3 hrs.; eosin, 5 min.).
Fic. 56 (as in 54).
Journal of Morphology. Vol.xv.
566 MONTGOMERY.
EXPLANATION OF PLATE XXII.
Figs. 57-63, 65-87: Germinal vesicles of Montagua pilata; Figs. 64, 88, 89:
germinal vesicles of Doto.
Fics. 57~59 (alcohol. sol. sublimate ; Ehrlich-Biondi stain, 3 hrs.).
Fic. 60 (alcohol. sol. sublimate; Ehrl. haematoxylin, 1 hr. ; eosin, 5 min.).
Fics. 61-63 (aq. sol. sublimate; Del. haematoxylin, 25 min. ; eosin, 5 min.).
Fic. 64 (alcohol. sol. sublimate; Ehrlich-Biondi stain, 31% hrs.).
Fics. 65-69 (as in 60).
FIG. 70 (as in 61).
FIG. 71 (as in 57).
FIGs. 72-75 (as in 61).
Fic. 76. Outlines of pseudonucleoli from various ova of one individual (aq.
sol. sublimate).
Fic. 77 (as in 61).
Fics. 78-80 (alcohol. sol. sublimate ; Mayer’s acid carmine, 15 min.; nigrosine,
10 min.). 5
Fics. 81-87 (Flemming’s fluid ; Del. haematoxylin, eosin).
Fics. 88 and 89 (as in 64).
Figs. 90-97: Nuclet of ganglion cells from the. brain of Montagua pilata (Fig.
93, froma cell of medium size ; the others from the colossal cells).
Fics. go and 91 (alcohol. sol. sublimate; Ehrlich-Biondi stain, 3 hrs.).
Fics. 92-94 (picrosulphuric acid; Del. haematoxylin, 25 min.; eosin, 5 min.).
FIG. 95 (as in 90).
Fics. 96 and 97 (Flemming’s fluid; Del. haematoxylin, eosin).
Figs. 98-101: Blood corpuscles of Doto.
Fic. 98 (alcohol. sol. sublimate; Ehrlich-Biondi stain, 3% hrs.).
Fics. 99-101 (Hermann’s fluid, 114 hrs.; safranin, 92 hrs.; gentian violet, 1%
hrs.; orange G., 2 min.).
a
7
Journal of Morphology. Vol. xv.
PUXNXM.
568 MONTGOMERY.
EXPLANATION OF PLATE XXIII.
Fic. 102. Blood corpuscle of Doo (picro-nitro-osmic acid, 35 min.; Del.
haematoxylin, 30 min.; eosin, 5 min.).
Figs. 103-133: Egg development of Tetrastemma catenulatum.
FIGs. 103-106. Germinal vesicles (aq. sol. sublimate ; Ranvier’s picrocarmine ;
Del. haematoxylin).
Fic. 107. Portion of a young gonad (as in 103).
Fic. 108. Immature ovum (as in 103).
Fic. 109. Germinal vesicle (aq. sol. sublimate; Del. haematoxylin, 25 min. ;
eosin, 5 min.).
Fics. 110 and 111. Germinal vesicles (aq. sol. sublimate ; Del. haematoxylin,
15 min.; eosin, 5 min.).
Fic. 112. Germinal vesicle with portion of the surrounding cytoplasm (as in
II).
Fic. 113. Germinal vesicle (as in 110).
Fic. 114. Idem, with portion of the surrounding cytoplasm (as in 110).
Fics. 115 and 116. Outlines of young ova (as in I10).
Fics. 117-119. Germinal vesicles (as in 110).
Fic. 120. Outline of germinal vesicle, the natural color of the nucleoli shown
(aq. sol. sublimate).
Fic. 121. Tangential section of the inner surface of the nuclear membrane,
the dotted line representing the greatest diameter of the nucleus (as in 109).
FIGs. 122-133. Germinal vesicles (as in 109).
Figs. 134-136: Outlines of the nuclei of ganglion cells of Piscicola.
Fics. 134 and 135 (alcohol. sol. sublimate).
Fic. 136 (Flemming’s fluid, 1 hr.).
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EXPLANATION OF PLATE XXIV.
FIGs. 137-139. Germinal vesicles of Tetrastemma catenulatum, the nucleoli
omitted in Fig. 139 (aq. sol. sublimate ; Del. haematoxylin, 25 min.; eosin, 5
min.).
Figs. 140-158; Egg development of Amphiporus glutinosus.
Fics. 140-143. Germinal vesicles (aq. sol. sublimate ; Del. haematoxylin, 20
min.; eosin, 5 min.). :
Fics. 144-146. Germinal vesicles with surrounding cytoplasm (as in 143).
FIGs. 147-150. Germinal vesicles (as in 143).
Fic. 151. An abnormally large yolk ball (aq. sol. sublimate ; aq. sol. dahlia,
15 min.; eosin, 5 min.).
Fics. 152-154. Germinal vesicles (aq. sol. sublimate; haematoxylin, 45
min. ; ferro-ammonio-sulphate, 45 min.; haematoxylin, 45 min.).
Fic. 155. Ovum and a part of the gonadal cavity in which yolk balls lie, only
a portion of the cytoplasm drawn (as in 152).
Fics. 156-158. Germinal vesicles (as in 154).
Figs. 159-177: Egg development of Lineus gesserensis.
Fic. 159. Nuclei from which the germinal vesicles are derived, from the cyto-
plasm of the gonad (Hermann’s fluid ; safranin, 70 hrs.; gentian violet, 1 hr.;
orange G., 2 min.).
Fic. 160. Ovum (as in 159).
Fics. 161 and 162. Germinal vesicles (as in 159).
Fic. 163. Group of neighboring nuclei from a gonad, showing mitotic stages
(aq. sol. sublimate ; Del. haematoxylin, 20 min. ; eosin, 5 min.).
Fics. 164-172. Nuclei from gonads (as in 163).
Fics. 173-176. Germinal vesicles (as in 163).
Fic. 177. The largest ovum found, only a part of the cytoplasm drawn (as in
163).
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Journal of Morphology Vol.Xv.
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572 MONTGOMERY.
EXPLANATION OF PLATE XXV.
All the figures refer to the large subcuticular gland cells of Piscicola rapax
Figs. 178-196 show stages of the prophase, and Fig. 167 the commencement
of the metaphase of the nucleus.
Fic. 178. Outline of an immature cell, only a portion of its duct drawn (aq.
sol. sublimate).
Fics. 179-181. Immature cells (aq. sol. sublimate; Mayer’s acid carmine, 20
min. ; nigrosine, 25 min.).
Fic. 182. Immature nucleus (alcohol. sol. sublimate).
Fic. 183. Idem (alcohol. sol. sublimate ; Ehrlich-Biondi stain, 3 hrs.).
Fics. 184-189. Nuclei (alcohol. sol. sublimate).
Fics. 190-194. Stages of the ramification of the nucleus (Flemming’s fluid).
Fics. 195and 196. Nuclei at the end of the prophase (alcohol. sol. sublimate).
Fic. 197. Nucleus at the commencement of the metaphase, discharging its
nucleoli (Flemming’s fluid).
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574 MONTGOMERY.
EXPLANATION OF PLATE XXVI.
Figs. 198-203: Large subcuticular gland cells of Piscicola, in stages of the
metaphase.
Fics. 198 and 199. Nuclei discharging their nucleoli, only outlines drawn
(Flemming’s fluid).
Fics. 200-203. Subsequent stages of the metaphase (as in 198).
Figs. 204-212: Egg development of a siphonophore (Rodalia(?); all fixed in
alcohol and stained with Del. haematoxylin).
Fic. 204. Ovum from gonophore, chromatin unstained. Obj. A, oc. 4.
Fic. 205. Ovum from egg pouch. Obj. C, oc. 4.
Fic. 206. Germinal vesicle from egg pouch. Obj. C, oc. 4.
Fics. 207-209. Germinal vesicles from gonophores. Obj. C, oc. 4.
Fic. 210. Ovum from egg pouch. Obj. C, oc. 4.
Fic. 211. A large and a small ovum from an egg pouch. Obj. C, oc. 4.
Fic. 212. Nucleolus from a large ovum of a gonophore.
~ Jpurnal of Morphology Val. XV.
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576 MONTGOMERY.
EXPLANATION OF PLATE XXVII.
Figs. 213-235: Egg development of Stichostemma eilhardi.
FIG. 213. Portion of the cell syncytium of an immature gonad (aq. sol. sub-
limate ; aq. sol methylen blue, 5 min.; brasilin, 20 min.).
Fic. 214. Germinal vesicle (as in 213).
Fic. 215. Yolk balls (as in 213).
Fic. 216. Germinal vesicle (as in 213).
Fics. 217 and 218. Portions of cell syncytia of gonads (as in 213).
Fic. 219. Germinal vesicle (aq. sol. sublimate ; Del. haematoxylin, 15 min.;
borax carmine, 20 hrs.).
Fic. 220. Portion of the cell syncytium of a gonad (aq. sol. sublimate ;
Ehrlich-Biondi stain, 3 hrs.).
Fics. 221-223. Germinal vesicles (as in 220).
Fics. 224-227. Idem (Flemming’s fluid; alum carmine, 24 hrs.).
Fic. 228. Portion of a gonadal syncytium (aq. sol. sublimate ; Del. haema-
toxylin, 15 min.; alum carmine, 22 hrs.).
Fics. 229 and 230. Germinal vesicles (as in 228).
Fic. 231. Germinal vesicle (aq. sol. sublimate; Del. haematoxylin, 15 min. ;
alum carmine, 45 hrs.).
Fic. 232. Idem (aq. sol. sublimate ; picrocarmine, 22 hrs.).
Fic. 233. Ovum, only a portion of the cytoplasm drawn (Lang’s fluid; alum
carmine; Del. haematoxylin, 15 min.).
Fic. 234. Germinal vesicle (aq. sol. sublimate ; Del. haematoxylin, 15 min. ;
alum carmine, 16 hrs.).
Fic. 235. Germinal vesicle and portion of the cytoplasm (aq. sol. sublimate ;
Del. haematoxylin, 15 min. ; alum carmine, 24 hrs.).
Figs. 236-248: Egg development of Zygonemertes virescens.
Fics. 236-241. Germinal vesicles (aq. sol. sublimate ; Del haematoxylin, 20
min.; eosin, 5 min.).
Fics. 242 and 243. Idem (aq sol. sublimate; Ehrlich-Biondi stain, 3 hrs.).
Fics. 244 and 245. Idem (alcohol. sol. sublimate ; picrocarmine; Del. hae-
matoxylin, 20 min.; eosin, 5 min.).
Fic. 246. Portion of an ovum (as in 242).
Fics. 247 and 248. Germinal vesicles (as in 242).
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578 | MONTGOMERY.
EXPLANATION OF PLATE XXVIII.
Figs. 249-281: Egg development of Polydora.
Fic. 249. Nuclear division in a peritoneal cell (alcohol. sol. sublimate; Ehrl.
haematoxylin, 1 hr.; eosin, 5 min.).
Fics. 250-254. Free cells of the body cavity (as in 249).
Fic. 255. Nuclear mitosis (as in 249).
Fics. 256-259. Mitoses of genital cells (as in 249).
Fic. 260. Nuclear mitosis (as in 249).
Fics. 261-266. Immature ova (as in 249).
Fics. 267 and 268. Germinal vesicles (as in 249).
Fic. 269. Ovum, only a portion of the cytoplasm drawn (as in 240).
Fics. 270and 271. Ova (aq. sol. sublimate with 5% acetic acid; Ehrlich-Biondi
stain, 3 hrs.). :
Fics. 272-275. Germinal vesicles (as in 270).
Fics. 276 and 277. Idem (Perenyi’s fluid, 1 hr. ; Ehrlich-Biondi stain, 2% hrs.).
Fic. 278. Ovum, only a portion of the cytoplasm drawn (Flemming’s fluid ;
safranin, 70 hrs.; gentian violet, 21% hrs.; orange G, 2 min.).
Fics. 279-281. Germinal vesicles (as in 278).
Figs. 282-299: Egg development of Tetrastemma elegans.
Fics. 282-291. Germinal vesicles (alcohol. sol. sublimate; Del. haematoxy-
lin, 25 min. ; eosin, 5 min.).
Fic. 292. Ova (Hermann’s fluid; Del. haematoxylin, 45 min. ; eosin, 5 min.).
Fics. 293-299. Germinal vesicles (as in 292).
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580 MONTGOMERY.
EXPLANATION OF PLATE XXIX.
Figs. 300-316: Egg development of Piscicola rapax.
Fics. 300-304. Transverse sections of ovarial acini (alcohol. sol. sublimate ;
Ehrl. haematoxylin, 1 hr.; eosin, 5 min.).
Fics. 305 and 306. Germinal vesicles (as in 300).
Fic. 307. Ovum (as in 300).
Fic. 308. Germinal vesicle (as in 300).
Fics. 309 and 310. Ova (as in 300).
Fic. 311. First pole spindle in the ovum; only one attraction sphere is drawn,
and that only partially, the dotted line showing how far its rays extend into the
cytoplasm (as in 300).
Fics. 312 and 313. Germinal vesicles (alcohol. sol. sublimate ; Mayer’s acid
carmine, 20 min.; Lyons blue, 5 min.).
Fic. 314. Germinal vesicle; the dotted line shows the extension of the
indented surface of the nucleolus, the unstained small oval space being the exter-
nal opening into it (alcohol. sol. sublimate ; fuchsine, Io min.).
Fic. 315. Germinal vesicle (Flemming’s fluid; Ehrl. haematoxylin, 2 hrs. ;
eosin, 10 min.).
Fic. 316. Ovum with attraction spheres at opposite ends of the nucleus; the
rays of only one attraction sphere drawn (alcohol. sol. sublimate; Ehrl. haema-
toxylin, 40 min. ; eosin, 5% min.).
Figs. 3150-324: Mesenchym cells of Cerebratulus lacteus (fixation with alcohol.
sol. sublimate).
Fic. 315a. Nucleus (Ehrlich-Biondi stain, 2 hrs.).
Fics. 316a and 317. Nuclear division in free cells (Ehrl. haematoxylin, 2
hrs.; eosin, 5 min.).
Fics. 318 and 319. Nuclei (as in 317).
Fics. 320-324. Whole cells (as in 317).
Figs. 325-337: Nuclei of the muscle cells of the longitudinal musculature of
Piscicola rapax.
FIG. 325 (aq. sol. sublimate).
Fic. 326 (alcohol. sol. sublimate; Ehrl. haematoxylin, 1 hr.; eosin, 5 min.).
Fic. 327 (Flemming’s fluid, 1 hr.).
Fic. 328 (aq. sol sublimate).
FIG. 329 (as in 327).
Fics. 330 and 331 (as in 328).
FIG. 332 (as in 327).
FIG. 333 (Flemming’s fluid, 1 hr.).
Fics. 334 and 335 (as in 326).
Fics. 336 and 337 (as in 333).
.
— Journal of Morphology Vo
LXV.
—Chrk
520,
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OP
582 MONTGOMERY.
EXPLANATION OF PLATE XXX.
All figures refer to the giant cells of Doto.
Fic. 338. Nucleus (alcohol. sol. sublimate; Ehrlich-Biondi stain, 314 hrs.).
Fic. 339. Cell (as in 338; hom. immers., oc. 2).
Fics. 340 and 341. Nuclei (Hermann’s fluid, 114 hrs.; safranin, 92 hrs. ; gen-
tian violet, 1% hrs. ; orange G., 2 min.).
Fics. 342 and 343. Two sections of a single nucleus (as in 338).
Fic. 344. Nucleus (Hermann’s fluid; Ehrl. haematoxylin, 11% hrs.; eosin,
7 min.).
Fic. 345. Dividing nucleolus (as in 344).
Fic. 346. Nucleus (as in 344).
PUXXX. -
=
= s'
JOURNAL
MORPHOLOGY.
EDITED BY
Cc. O. WHITMAN,
Head Professor of Biology, Chicago University.
WITH THE CO-OPERATION OF
EDWARD PHELPS ALLIS.
Milwaukee.
FEBRUARY, 1899.
BOSTON, U.S.A.:
GINN & COMPANY.
AGENT FOR GREAT BRITAIN: AGENTS FOR GERMANY: AGENT FOR FRANCE:
EDWARD ARNOLD, R, FRIEDLANDER & SOHN, JULES PEELMAN,
37, Bedford Street, Strand, Berlin, N.W., 2 rue Antoine-Dubois,
London, W.C. Caristrasse 11. Paris, France.
CONTENTS OF No. 3, FEBRUARY, 1899.
I. Studies on the Maturation, Fertilization, and tat
Cleavage of Thalassema and Zirphaea. . . 583-634
BRADNEY B, GRIFFIN.
Il. Ox the Blood-Plates of the Human Blood, with
Notes on the Erythrocytes of Amphiuma and — :
NERUIUS 25 os aht Plo BEL tee, Fos. ela aay, pe O95 OOO
Gustav EISEN, PH.D.
Ill. Yhe Phosphorescent Organs in. the Toadfish, : a
Porichthys Notatus Givard . . . . . . 667-696 z en
re _ CHARLES WILSON GREENE.
IV. On the Species Clinostomum Heterostomum . . 697-710
W. G. MacCaLium. ; ’ ;
V. Mitosis in Noctiluca Miliaris and its Bearing
on the Nuclear Relations of the Protozoa and ie
Metadod a. Sk SIAR AS Cah hes Ror oe
Gary N. CALKINS. :
Che Atheneum press.
GINN & COMPANY, BOSTON, U.S.A.
Volume XV. February, 1899. Number 3.
JOURNAL
OF
MORPHOLOGY.
STUDIES ON THE MATURATION, FERTILIZA-
TION, AND CLEAVAGE OF THALASSEMA
AND ZIRPHAEA.!
BRADNEY B. GRIFFIN.
CONTENTS.
MONA DV CULO once concene cm asuspnscenig ee len cen engeoten Ct nacs snestevensed suseoedeseavedesschessvocusivsnevasssssadsuvs
PPAR TSO, UELAUASS EMGAS oso ccs oocspscev dusts. Stascaetensscsuadacaduassestactetcanciovanivedusetvceadessaseccsese
Te Phe Ovarian vb p oyand sNUCle ONS eos ccecsc recs etennect censtocescacwerchicrantsenestseeucenas
DP PATCHromatic: Structures: s.ccsc0.ctecocecacevecs-ceaccesS-sucses evs scecsassnscscstcctovecer sentatstnas
é. The Second Polar Division
c. The Sperm-Amphiaster........
d. The Cleavage-Amphiaster .
Lee ne Chromatin! ec. so de crcescesscct soteeeasc res ccesesgessseevescessse:cuescuesutendece:
MERPIERT NEA CARY A ELON = cee feet 2 oct scoys oe asak vada atenecat ceca) & eaccce vaca cesszcacheced
Prstniehiertiliza tion and) CLEAVAGE ac. oy secssc-ceceemaencasenstveace see secscesesncssnteces
Wy hes Polar Bodiesiic.-s.cce-.-sc-2000:. occu snuctsesbasoce utissedeusuel secsscuseutyocccreedcostteceds
ParT II. ZIRPHAEA......
V. Development of Ova...
VTL DOR GOmtTGS Om cermin eee te sass sacs see ces col sect ceas Seats cee daedeon daca ree eee eee
VII. The Chromatin
1 The death of Mr. Griffin, in April, 1898, occurred before the final revision of
the manuscript of the following paper, which had been placed in my hands as a
584 GRIFFIN. [Vou. XV.
INTRODUCTION.
In the autumn of 1895, Prof. E. B. Wilson placed in my
hands a very complete series of maturation and fertilization
stages of the echiuroid 7kalassema mellita Conn, collected by
him at Beaufort, N. C., for a careful study of the achromatic
structures with especial reference to the structure and function
of the centrosome in maturation and fertilization. The research
was at first exclusively confined to 7halassema, which proved
an extremely favorable object. During the following summer,
however, it was the writer’s good fortune to be able to collect
a series of stages of the large piddock, or “boring clam,” Z7z7-
phaea crispata from the Pacific shores of the United States.!
As the maturation and fertilization phenomena in the Lamelli-
branch also were at that time wholly unknown, the investiga-
tion of Zirphaea was immediately undertaken in connection
with further study of 7Zalassema.
The work upon the achromatic elements produced results so
clear and convincing that the writer was encouraged to take up
a study of the chromatin with reference to the reduction ques-
tion, which was then, as now, in fully as unsettled a condition
as that respecting the centrosome. It was in this portion of
the research that Zz7phaea yielded the most important evidence,
furnishing in fact the key to the understanding of the process
in Thalassema.
dissertation for the degree of Doctor of Philosophy just before his last illness.
Besides slight verbal alterations, which have not modified in any essential way
either the substance or the form of his conclusions, the only changes that have
been made are omissions of detail and the insertion of references to the figures.
No literature-list accompanied the manuscript, and it has seemed best not to
attempt the preparation of one. The text-figures have been prepared from
sketches made as memoranda on the margin of the manuscript; but the author’s
methods of work were so conscientious that their accuracy may safely be assumed.
An earlier and briefer paper on 7%a/assema, containing some details not included
in the present work, was published by Mr. Griffin in the 7yamnsactions NV. Y. Acad.
Sci., vol. xv, June, 1896, under the title “ The History of the Achromatic Struc-
tures in the Maturation and Fertilization of Zhalassema.” I am indebted to
Mr. Henry E. Crampton, Jr., for revision of the proofs of the present paper. —
EDMUND B. WILSON.
1 Cf. Harrington and Griffin on Distribution and Habits of Some Puget
Sound Invertebrates,” Zyansactions of N. Y. Acad. Sci., March, 1897.
No. 3.] THALASSEMA AND ZIRPHAEA. 585
In accordance with the above, the problem to be first attacked
was that of the centrosome, —its morphology, its physiological
significance, and its behavior towards fixing agents; on all of
which points the accounts of previous investigators are, in
a greater or less degree, conflicting. It was especially hoped
that some positive evidence might be obtained respecting the
continuity of the centrosome, a question which has of late
seriously engaged the attention of cytologists. In opposition
to the earlier view of van Beneden and Boveri, a considerable
number of later authors (e.g., Biirger, '92 ; Watasé, '94 and '97;
Farmer, 96; Foot, '97; Carnoy, 97; and numerous botanical
investigators) regarded the centrosome as a more or less tran-
sient structure, arising by the modification of preéxisting cyto-
plasmic elements (e.g., microsomes, Watasé, ’97), persisting for
a shorter or longer period, and being dissolved and reformed
according to the varying conditions of mitotic activity —the
expression rather than the cause of the aster-formation (Foot,
'97). On this point my results are, I think, clear and convin-
cing as far as they go, though they certainly do not bring the
question to a conclusion. In both Thalassema and Zirphaea
the centrosome (sperm-centrosome in TZkalassema) can be
traced uninterruptedly from the first appearance of the aster
throughout a@// intermediate stages into the cleavages, and
at no time disappears from view. My results here agree
entirely with those of van Beneden and Boveri (Ascavis), Mead
(Chaetopterus), Wheeler (Myzostoma), Kostanecki and Wier-
zejski (Physa), and others. Neither in 7alassema nor in
Zirphaea could the centrosomes be demonstrated during the
growth period of the ovum, but it must be borne in mind
that in the absence of rays or characteristic envelopes, a
granule so minute as the centrosome would be indistinguish-
able from numerous cytoplasmic and yolk granules which fill
the egg.
A third and no less important question touches the deriva-
tion of the cleavage-centrosomes, on which point my observa-
tions in the case of Zhalassema have, I think, no room for
doubt, while in the case of Zivphaea the facts are too equiv-
ocal to justify a positive conclusion.
586 GRIFFIN. [Vou. XV.
In the fourth place, by reason of the varying accounts of
different authors respecting the tetrads or quadruple groups,
the nature of the so-called maturation divisions is still involved
in doubt. Boveri('87) (ovocyte of Ascarzs) and Brauer ('93) (sper-
matocyte of Ascaris), ('92) (ovocyte of Branchipus) agree that
the tetrad arises by a double longitudinal splitting of the spi-
reme thread, thus giving no reduction in the Weissmannian
sense. Other investigators, however (notably vom Rath, '92 and
'94, in insects, and Rickert and Hacker in copepods), describe
the process as consisting of one longitudinal and one transverse
division of the primary chromatin rod, thus giving the theo-
retically required reduction. Here the numerical reduction
(pseudo-reduction of Riickert) results from a suppression (or,
more properly speaking, a postponement) of one-half of the
transverse divisions of the spireme thread. According to some
authors, however (e.g., Wilcox, '95; Korschelt, '95; and Calkins,
'95), the spireme segments into the normal number of chromo-
somes, and the numerical reduction is accomplished by a sub-
sequent conjugation or association in pairs of the chromosomes.
In some types, again (notably many plants, Elasmobranchs, etc.),
no tetrads occur, and the chromosomes persist in their original
rod-shaped or ring form. A number of more recent authors,
working upon these forms, fail to find any evidence of reduc-
tion, and believe the maturation divisions to involve simply two
successive longitudinal divisions of the chromosomes (e.,¢.,
Farmer, '95, in plants; Meves, '96, in the spermatocytes of the
salamander; and quite lately, Miss Sargant, '97, and Strasburger,
'97, in Lilium).
The reduction question has been so often reviewed of late,
that I may dispense with detailed references to the work of
others at this point. My own observations, I believe, leave no
reasonable doubt that in both forms studied a true reducing
division occurs, and in this respect the Echiuroids (7alassema)
and Lamellibranchs (Zzrphaea) fall into line with the insects
and copepods.
I wish to take this opportunity to express to Prof. E. B.
Wilson my sincere appreciation of his interest, advice, and
continued encouragement.
No. 3.] THALASSEMA AND ZIRPHAEA. 587
MeEtuHops. In both forms the material included stages fixed with picro-
acetic (1%-2% acetic acid), sublimate acetic (20%, 10%, and 3% acetic acid),
and pure sublimate. In general, the picro-acetic series gave the best results,
while in Zz7fhaea the pure sublimate was a complete failure. The eggs
were imbedded in paraffine cut into sections 2-1ou thick, mounted and
stained on the slide by various stains. For the achromatic structures, by
far the best results were obtained with iron haematoxylin, either alone or
followed by acid fuchsin, Congo red, orange, or eosin. For study of the
chromatin, the sections, after remaining in the haematoxylin for thirty-six
to forty-eight hours, were extracted until the cytoplasm became colorless,
and then restained with one of the plasma stains just mentioned. By this
method the chromosomes stand out conspicuously as intensely black bodies
upon a pale red or orange background. Their structure and changes can
then be traced with but little trouble. Good results were also obtained by
use of Flemming’s triple stain and Auerbach’s ('96) methyl-green acid-
fuchsin mixture.
PART I. —THALASSEMA
I. THE Ovarian EGG Aanp NUCLEOLUS.
The sexual products of Thalassema are developed, as Conn
(86) has described, from the peritoneal covering of one of the
muscular bands about 5 mm. cephalad of the anus, and extend-
ing from the alimentary canal to the body wall. The ova and
sperm-cells very early become detached in masses from the
sexual glands, and undergo their subsequent development float-
ing freely in the body cavity. My studies upon the early ova
were made in large part from such masses imbedded and
sectioned by chance along with the earlier maturation stages.
These were not, however, in all cases to be relied upon, since
degeneration had, in many instances, already set in. More
satisfactory material was obtained by fixing the coelomic fluid,
whereby many such egg masses were obtained along with
innumerable coelomic corpuscles.
The earliest clusters obtained consist entirely of minute cells
3 in diameter, all possessing extremely little cytoplasm and
with nuclei in the spireme stage. After the segmentation
of the spireme into the reduced number of chromosomes
(viz., 12), the ovum commences to grow and all parts rapidly
increase in size, although the cytoplasmic growth is by far the
588 GRIFFIN. [VoL. XV.
most rapid. In all these stages, up to eggs 47m in diameter,
the cytoplasm shows an extremely dense reticulum with no
yolk-spheres. As growth continues, these appear in increasing
number in the meshes. It would, therefore, seem that growth
results as a distention of the reticulum by deposition of deuto-
plasm-spheres in the meshes, as in Chaetopterus (Mead, '97).
In mode of growth, the ova of 7halassema present some
interesting resemblances to those of the allied genus Boneliia,
though differing in several important particulars. In Bonedllia,
as Vejdovsky ('78) has most clearly shown, the eggs arise from
a peritoneal fold, and breaking away in clusters, undergo their
subsequent development floating freely within the body cavity.
But ove (the central) cell of each cluster develops into an ovum,
the remainder functioning as nutritive and follicle cells. Vej-
dovsky shows that the egg develops at the expense of the
nutritive cells, where the follicle cells become flattened and
give rise to an outer egg membrane. In 7%alassema only the
peripheral cells develop into ova, though the precise fate of the
more central ones is difficult to determine. Differential stain-
ing alone yields no evidence that they contribute in any way
to the growth of the eggs. With Auerbach’s acid-fuchsin
methyl-green stain (96), the reactions of the nuclei and cyto-
plasm are characteristic and opposite, the chromosomes and
nucleoli taking a pure green, and the cytoplasm and nuclear
reticulum a pure red. This yields equally little evidence that
any of the chromatin is expelled from the nucleus to give ri8e to
yolk material, as described by numerous observers (Blochmann,
v. Bambeke, Balbiani, Calkins, and others). The cell outlines,
especially those of the growing egg, are sharp, and the single
hyaline membrane is distinct. There is here no follicular or
cellular membrane, as in Bonellia.
Nucleus. — It is important to note that as the egg increases
in size, the nucleus takes up an eccentric position on the side
of the free surface of the egg (Pl. XXXI, Figs. 1-3). Thus
the early, if not the ultimate polarity of the egg is to be
referred to its position in the cluster, the attached side becom-
ing the vegetative, and the free end the animal pole, as in Cyclas
(Stauffacher, '93) and other forms. The bearing of this will
No. 3.] THALASSEMA AND ZIRPHAEA. 589
be seen later, when discussing the rotation of the first polar
amphiaster.
The nucleolus was first noticed upon the breaking up of the
spireme. Its intense blackness and rough and irregular outline
give the impression of an irregular remnant of the spireme, and
with Auerbach’s fluid it can often be made to take a purer green
than the chromosomes. The persistent nucleolus undergoes
no change until the early prophase of the first polar mitosis. If
it then happen to be near either of the asters, but not other-
wise, it assumes an elongate ovoid outline, with the narrow end
directed toward the center of the aster (Pl. XXXI, Fig. 11).
Two portions are now distinguishable, a larger and more darkly
staining area involving the pointed end, and a smaller cap-like
portion at the blunt extremity. The two portions are not,
however, to’ be compared to the Haupt and Nebentheil of the
lamellibranch nucleoli, since they stain similarly and are not
constant features. The nucleolus subsequently undergoes reab-
sorption along with the remnants of the nuclear skein (Pl. XX XI,
Fig. 10).
II. GENERAL HIsTorRY OF THE ACHROMATIC STRUCTURES.
The egg of Yhalassema is in many respects exceptionally
favorable for cytological study. It is large (7o—80p diameter)
and transparent, and the yolk is distributed in the form of large
deutoplasm-spheres in the meshes of an extremely coarse cyto-
plasmic reticulum (Pl. XXXI, Figs. 10-12). The yolk distri-
bution is, furthermore, such as to leave a relatively unobstructed
field for the play of the mitotic phenomena, and in consequence
the achromatic structures reach a powerful development, and
comparatively little difficulty is experienced in following the
history of the centrosome.
a. The First Polar Division.
Origin of the Amphiaster. — The earliest stage that yields
unequivocal traces of centrosomes is found among preparations
of eggs fixed three minutes after fertilization. Their presence
is then revealed by two excessively minute asters situated close
590 GRIFFIN. [VoL. XV.
to the wall of the germinal vesicle, and about 45-90 degrees
from each other (Pl. XXXI, Fig. 7). The rays of each aster
converge directly to a minute dark-staining granule, the centro-
some, which may at times be double even in this early stage.
I find no certain indication of a rupture of the nuclear membrane
behind the centrosomes, such as Mathews (95) finds in Aszerzas,
indicating their possible nuclear origin. The slight flattening
or folding of the membrane seems to be rather the effect of
inwardly growing rays, since it becomes steadily more marked
as the asters develop. Not infrequently in these early stages
one aster seems a trifle more advanced than the other, since
the membrane behind it is considerably folded, while behind
the other it is only flattened or but slightly folded (Pl. XX XI,
Fig. 7). This inequality soon disappears.
In later stages the asters remain the same distance apart and
undergo no further divergence. This is quite at variance with
what occurs in most other forms. Platner (89) (Aw/astomum),
Korschelt (95) (Ophryotrocha), Wheeler (95) (Myzostomum),
Garnault (88) (He/ix), Mathews ('95) (Asterias), and others, all
describe diverging daughter-asters or centrosomes immediately
derived by division from a single one. It apparently agrees
with Zhysanozoon (Van der Stricht, '94) and Chaetopterus (Mead,
'97). The definitive spindle, in consequence, arises in all cases
secondarily by the meeting of rays from the opposite and inde-
pendent asters, as in M/yzostoma (Wheeler), Salamander (Driiner,
94), Selachians (Moore, '95), Opisthobranchs (MacFarland, '97),
and some other forms.
Centrosomes.—In stages earlier than two to three minutes
after fertilization, a most careful search has failed to reveal any
structure that can, with certainty, be identified as a centrosome
or definitive aster. The unfertilized, and some of the early
fertilized eggs, however, exhibit an interesting phenomenon,
which, from its possible bearing upon certain facts recently
brought to light by experimental and other investigators (e.¢.,
Reinke, '94; Hertwig,'96; Mead, 97; Morgan, '96; Osterhout,’97;
Mottier, '97) well merits description. In preparations of both
full-grown and sometimes younger ova the strands of the cyto-
reticulum show a tendency to arrange themselves in radiating
No. 3.] THALASSEMA AND ZIRPHAEA. 591
lines, giving rise at various nodal points to aster-like appear-
ances (Pl. XXXI, Fig. 6). Where the strands converge to a
point between two microsomes, there is no focal granule, but
when they focus upon a granule (microsome) the latter is often
larger and more deeply stained than the surrounding micro-
somes, and a centrosome is thus simulated. This tendency to
form centers of radiation seemed especially marked in a slide
of eggs fixed by sublimate acetic (20%) while still within the
sexual pouches. It may be interesting to note that where the
eggs taken from the pouches have been allowed to remain in
sea water for some time without being fertilized, this appearance
becomes far less noticeable. Several preparations, one minute
after fertilization, show numerous minute centers of radiation
scattered irregularly throughout the cytoplasm, although more
abundant or more powerful about the animal pole. At least
ten were counted in a single section, some of which are closely
approximated, while others are some distance apart. The rays
are few (5 or 7), straight, and often short, though sometimes
equal to or longer than the egg radius. These can sometimes
be traced continuously without curving from one center to
another. At the center a granule is generally present, which
is distinguished from the neighboring microsomes by larger
size and a deeper staining power. Similar granules, however,
not the centers of radial systems, are to be met with through-
out the cytoplasm. These ‘“asters’’ are to be distinguished
from the somewhat similar appearances in the unfertilized ege
by their greater distinctness and straighter rays. In prepara-
tions made two minutes after fertilization the radiations are
much more obscure, while in three-minute preparations and
later, they are scarcely, if at all, traceable, their place being now
taken by the definitive asters.
It is evident that we have here a phenomenon at bottom
identical with that described by Mead (97) in Chaetopterus. In
Thalassema, however, the “secondary asters’’ are smaller and
less distinct, and do not push in the nuclear membrane as they
often do in Chaetopterus (cf. Mead). Moreover, I find no evi-
dence of a fusion of the “asters” or of any genetic relation
between them and the definitive polar asters, though it is not
592 GRIFFIN. [Vot. XV.
improbable that ¢wo of the centers of radiation at the animal
pole are caused by and belong to the true centrosomes.
In most cases the definitive asters appear on the side of the
nucleus nearest the egg surface, z.e., at the animal pole (PI.
XXXI, Figs. 7, 8); but sometimes they appear laterally (PI.
XXXI, Fig. 9), as in Myzostoma, and they may occupy any
intermediate position. In such early stages I have never seen
both at the vegetative pole, though occasionally one may occupy
this position. From this it appears that the line joining the
centers of the asters (the future spindle-axis) may be inclined
to the egg-axis! at any angle from o—go degrees.
Considerable variation also exists in the distance separating
the asters at their first appearance; for among eggs fixed at the
same period after fertilization and in which the asters have
attained the same degree of development, the angular distance
was found to measure from 45-90 degrees, or more. This pro-
duces a variation, not only in the length of the completed
spindle, but also in its position relative to the germinal vesicle.
In extreme cases, when the angle is very small, the main rays
may meet without penetrating the membrane, and thus results
a short spindle wholly without the nucleus and quite near the
surface of the egg. In this case it is the more lateral rays that
enter the nucleus and come into relation with the chromosomes.
The larger the angle, the longer and more centrally situated is
the spindle. When, as in most cases, the angle is great enough
to render the line joining the centrosomes secant to the nucleus,
then the median rays, which are destined by meeting equatori-
ally to form the spindle, push in the membrane and enter the
germinal vesicle. In this case the completed spindle is more
or less completely surrounded by the discarded chromatin-skein
(Pl. XX XI, Fig. 10). This variation is of interest as bridging,
to some extent, the gap between such cases as the salamander
spermatocytes, where the amphiaster lies wholly without the
nucleus, and forms like Ophryotrocha (ovum), Ascaris (spermato-
cytes), etc., where the nucleus comes to lie midway between the
asters (cf. Driiner, ’95, and Braus, '95), and would seem to indi-
1 By “egg-axis” is here meant the line joining the centers of the egg and of the
germinal vesicle.
No. 3.] THALASSEMA AND ZIRPHAEA. 593
cate that the difference between these extreme cases may be
primarily only that of position of the asters.
Still another fact has to be considered as influencing the
length of the spindle. By average of actual micrometer meas-
urements (full account being taken of the above-mentioned
variation in the distance between the asters) it was found
that the completed spindle is approximately one-fourth shorter
than the distance between the centrosomes previous to meeting
of the rays —2.e., the asters approach at completion of the spindle.
This often causes the asters to become more or less imbedded
in the nuclear skein. A shortening of both maturation-spin-
dles occurs in Ascaria (Boveri, '87), Artemia (Weismann und
Ischikawa ('88), Ophryotrocha (Korschelt, 7. c.), and Branchipus
(Brauer, ’82), and is explained by the first and last named authors
as a result of contraction of the spindle-fibers. In 7halassema,
however, the shortening takes place at a much earlier stage,
probably while the chromosomes are still severally connected
with but one of the asters. It more nearly resembles Mac-
Farland’s (97) account of the approach of the cleavage-asters
to form the cleavage amphiaster in Plewrophyllidia. Erlanger
(98) has recently described a shortening of the cleavage-spindle
just previous to metaphase in the sea-urchins.
From its completion (Pl. XXXI, Fig. 12) until the polar
body is separated, the first maturation-spindle maintains very
nearly a constant length, measuring about } the egg-diameter.
A few measurements made during the expulsion of the polar
body show that also during this process the spindle retains a
fairly constant length, and hence must rise bodily with the
elevation of the protoplasm that gives rise to the polar body.
This has been also observed in Physa (Kostanecki and Wier-
zejski, '96). At this stage the egg, as a whole, elongates in
the direction of the spindle-axis only ;'; of the mean diameter.
The development of the asters, the progressive increase in the
number, length, and strength of the rays which at first throw
the nuclear membrane into folds and then rupture it and enter
the nucleus (Pl. XXXI, Figs. 7—12), have all been described
in a previous paper (Griffin, '96). During the early stages of
the asters, while they are commencing to break through the
594 GRIFFIN. [Von. XV.
nuclear membrane, their rays are approximately similar in struc-
ture. As soon, however, as the chromosomes enter, in any
considerable extent, into the system, certain rays commence to
stand out more prominently. They are thicker, more deeply
staining, than the remaining rays, and more homogeneous in
that they are not microsomal in structure. As they can be
traced to the chromosomes, they are evidently the developing
traction-fibers (“ Zugfasern”’) and are to be regarded as devel-
oping out of rays at first identical with the astral rays (of
Driiner, '94). In both maturation-spindles these fibers stand
out prominently, and in structure convey the impression as of
threads spun of numerous strands. But a single traction-fiber
is attached to each daughter-chromosome (Pl. XX XI, Fig. 12),
as has been described by so many other observers in other
forms.
Besides those attached to the chromosomes, the spindle con-
tains numerous other fine and light staining fibers, which in
some cases can be traced continuously from pole to pole. As
there is here no distinction between central spindle and mantle
jibers, these central spindlefibers occur scattered throughout
the entire spindle. In equatorial section they can be seen as
numerous dots among which the chromosomes of the solid equa-
torial plate are interrupted.
Movements of the Polar Amphiaster. — In the stages previous
to completion of the spindle, the line joining the centrosomes is
very seldom situated in an egg radius. The completed spindle
is, however, radial in position, with its aster still quite distant
from the surface of the egg. It must, therefore, have under-
gone some rotation or change of position, however slight.
Owing to absence of all means of orienting the spindle with
reference to a fixed point of the egg, it was impossible to
determine the precise extent of this shifting, though the occur-
rence of sections in which the spindle is oblique to the egg
surface proves conclusively that it rotates to some extent.
Sections through either aster of the just completed spindle
show how completely the discarded nuclear skein has become
involved in the astral system. The rays then appear to be but
rearranged strands, the granules serving as microsomata (PI.
No. 3.] THALASSEMA AND ZIRPHAEA. 595
XXXI, Fig. 11). Owing to this intimate connection, the dis-
carded nuclear skein is, I believe, shifted along with the spindle,
and there is, therefore, no means of orientation such as was
found by Hertwig ('78) in Astevacanthion, where the spindle is
figured as leaving behind the remains of the nucleus as it rises
to the surface.
These considerations involve an important problem touching
the polarity of the egg in relation to the cleavage-planes. We
have seen (p. 588) that the eccentricity of the germinal vesicle,
and hence that of the primary egg-axis, are to be referred to
the position of the young egg in the cluster, the nucleus shift-
ing toward the free side. If the rotation of the spindle is such
as to bring it to always lie in the egg-axis, then the position of
the polar bodies and the first cleavage-plane will, of course,
depend upon the position of the young egg in the cluster. But
another possibility is open. The outer aster remaining station-
ary, the spindle might rotate about it as a pivot just sufficient
to bring the spindle-axis in an egg radius, in which case the
position of the spindle, and hence that of the polar bodies and
cleavage-plane, would be independent of the early egg-axis, and
would vary according as the asters appeared in one or another
of numerous possible positions about the germinal vesicle.
A rotation of the first maturation-spindle — generally through
90 degrees of arc—has been described in numerous forms.
Thus Sobotta ('95) observed it in the mouse, Boveri ('87) in
Ascaris, Weismann and Ischikawa ('88) in Artemia and Eupa-
gurus, Brauer (92) in Branchipus, etc. It appears, however,
from the accounts of these authors, that it takes place in these
forms much later than in Zhalassema. Other observers find
no evidence of rotation and believe the spindle to reach the
surface by a motion of translation only, in the direction of its
axis (cf Korschelt, '95; Wheeler, '95 ; Kostanecki and Wier-
zejski, '96).
As the spindle rises, the outer aster soon encounters the
membrane. The more outwardly directed rays in consequence
steadily shorten, while the more lateral ones are deflected and
curved backward —a phenomenon also well shown in Physa
(Kostanecki and Wierzejski, '96). In the definitive position
596 GRIFFIN. [Vo XV.
(Pl. XXXI, Fig. 12) the outer aster is closely applied to the
inner wall of the membrane and often flattened against it. The
funnel-like depression of the egg surface next the outer centro-
sphere, not infrequently seen in other forms (notably Physa
and the mouse), is here quite noticeable. It is probably to be
referred to the influence of the aster, which causes the bound-
ing surface of the egg to conform to the radial arrangement.
During the divergence of the daughter-chromosomes, the
respective halves are united by fibers considerably finer than
the traction-fibers, and which present a wavy and granular
appearance (Pl. XXXI, Fig. 15). These interzonal fibers (Ver-
bindungsfasern) are still present at telophase and can often be
traced from the chromosomes in the extruding polar body to
the corresponding ones within the egg. They soon disinte-
grate and break up into separate granules in the equatorial
region, though a cell-plate (zwischenkorper) is formed.
b. Zhe Second Polar Division.
In mode of formation the second maturation amphiaster pre-
sents a marked contrast to the first. It arises shortly after the
chromosomes have reached the poles of the first polar spindle,
and apparently as a new formation between the already diverged
daughter-centrosomes in the previously clear and homogeneous
centrosphere (cf Boveri, '90; Mead, 95; Korschelt, 95; Mac-
Farland, '97; and others). The chromosomes remain for some
time on the outer side of the spindle, stretching in a curved line
from pole to pole (Pl. XX XI, Figs. 15-18). From now on, the
spindle steadily elongates until its definitive length (1 to } the
egg-diameter) is attained, and at no time shortens. A rotation
through go degrees of arc brings it radial with its outer aster
at the point on the egg surface at which the first polar body
was expelled. At division the extremely coarse, straight, and
homogeneous interzonal fibers are gathered together as the polar
body is constructed off, but the resulting cell plate disappears
shortly after separation of the body (Pl. XXXII, Figs. 19-26).
The inner centrosome has meanwhile divided exactly as
though preparing for a third mitosis (Pl. XX XII, Figs. 19-26).
No. 3.] THALASSEMA AND ZIRPHAEA. 597
Such a mitosis, however, never occurs; for shortly after the
chromosomes remaining within the egg have passed into ves-
icles, the centrosomes, with all trace of rays, disappear com-
pletely. The reconstructed egg-nucleus, which results from
the close aggregation, if not complete fusion, of the vesicles
(Pl. XXXII, Fig. 28), now advances, unaccompanied by any
sort of aster or rays, to meet the sperm.
ce. The Sperm-Amphiaster.
The sperm-aster first appears about the time of second polar
telophase, at the edge of the clear area surrounding the sperm-
head, and, roughly speaking, on the side nearer the center of
the egg. It contains a single focal centrosome to which the
rays converge directly (Pl. XXXII, Fig. 19). In many cases a
process staining the same as the sperm-head extends from the
latter through the clear area to the center of the aster (cf.
Michaelis, ‘96, and Kostanecki, '96, both of whom have described
a similar phenomenon). When no such process can be demon-
strated, the aster appears independent of the sperm-head. The
early division and divergence of the centrosomes give rise to
a minute amphiaster already some distance in advance of the
sperm-head (Pl. XXXII, Figs. 26, 27). This distance varies
considerably (cf MacFarland, '97), as do also the relative time
and amount of the divergence, which may occur before or after
the sperm-head has become vesicular. The daughter-centro-
somes occupy the apices of an elliptical clear area outside
of which the rays are grouped, those at the apices being
focused at the respective centrosomes, while the intermediate
rays still converge to the previous center, a phenomenon not
uncommon in other forms. In some preparations a cloudy, rod-
like structure, which may be called the centrodesmus (primary
Centrodesmus of Heidenhain, '94), was seen extending between
the daughter-centrosomes (Pl. XXXII, Fig. 27), but this bears
no relation to the definitive spindle, since it soon disappears
and leaves the asters independent. The further development is
marked by steady increase in the number, length, and strength
of the rays of these asters and the approach of the now hemi-
598 GRIFFIN. [Vou. XV.
spherical sperm-nucleus so as to lie closely applied to the amphi-
aster, with an aster at each pole (Pl. XXXII, Fig. 32). The
application of the fused or more often incompletely fused egg
vesicles to the base of the sperm-nucleus and their subsequent
fusion give rise to a segmentation-nucleus with the asters at
opposite poles (Pl. XXXII, Figs. 33-37).
When, as often happens, the sperm enters from the side, the
axis of the amphiaster is more or less nearly parallel to the egg-
axis, as marked by the polar bodies. With the progress of the
sperm-nucleus along its curved copulation path, the amphiaster
is gradually rotated so as to bring its axis (the axis of the future
cleavage-spindle) perpendicular to that of the egg. The relative
position of the germ-nuclei at contact depends upon the extent
to which the rotation has already progressed. A tardy rotation
may cause the egg-vesicles to be received first upon one of the
asters (Pl. XXXII, Fig. 32. Cf. Brauer, '92, for same phe-
nomenon in Branchipus). Eventually, however, the rotation is
effected so as to bring the egg-nucleus detween the asters in
the normal copulation position.
In some forms (¢.g., Ascaris, Mouse, Unio, Prostheceraeus,
Pleuroyphyllidea, and others) the astral systems completely fail
to appear, or disappear entirely about the time of copulation of
the germ-nuclei; and in others (notably the sea-urchins) the
rays become far less distinct, and a resting-stage results. On
account of the extreme difficulty of detecting the centrosomes
in these stages, such facts as the above have been considered by
some observers as strong evidence against the persistence of
the centrosome. In 7halassema, the “ pause” is of short dura-
tion, and while the asters are a trifle less distinct, they never-
theless show clearly throughout, and the persistence of their
focal centrosome is easily demonstrated. With the approach
of the germ-nuclei the egg-vesicles seem to exert a disturb-
ing influence upon the amphiaster, or render it more easily
affected by reagents ; for the astral centers are often somewhat
distorted, the centrosomes pushed aside (carrying the asters
with them) against the membrane of one of the nuclei or fur-
ther out into the cytoplasm. In most instances the presence of
the centrosomes can be made out with comparatively little diffi-
No. 3.] THALASSEMA AND ZIRPHAEA. 599
culty. With the commencing fusion of the nuclei, the centro-
somes take up a polar position, and immediately become the
centers of renewed activity, for many additional rays commence
to start up about then.
From the above it is quite evident that the centrosomes persist
entire throughout the whole of the critical stage where, in so
many forms, they have been lost sight of (cf. Figs. 32-37).
d. The Cleavage-Amphiaster.
After taking up a nearly central position, with its axis perpen-
dicular to the egg-axis, the cleavage-nucleus rapidly elongates,
sometimes even before the pronuclei have completely fused.
Meanwhile a minute centrosphere becomes differentiated about
each centrosome (Pl. XX XIII, Figs. 37-39), which has persisted
unchanged up to this period, with the rays converging directly
to it. The centrosomes remain single until shortly before
metaphase, when each divides (Pl. XX XIII, Fig. 39); but the
daughter halves do not diverge to any extent. The inner rays
soon commence to push in the membrane at the poles, and
later throw it into folds (Pl. XX XIII, Fig. 38) in the manner
described by Platner (Aulastomum), Watasé (Squzd), Braus
(Salamander), and others. These in-growing spindle-fibers are
essentially similar in behavior to those of the first maturation-
asters. Like the latter, they are first similar to the remaining
astral rays, but among them traction-fibers are soon distinguish-
able by their greater thickness and homogeneity (Pl. XX XIII,
Figs. 39-41). For some time after the nucleus has become
fusiform, with the chromatin-spireme crowded together equato-
rially, the membrane still persists laterally and conforms strictly
to the astral system. Later its two halves merge into and
become indistinguishable from its rays.
Equatorial sections during metaphase (Pl. XX XIII, Fig. 40)
show the chromosomes arranged in a solid plate set in the center
of a circular area of granules (cross-sections of spindle-fibers
and those astral rays that pierce the equatorial plane), in all
respects similar to the first maturation-spindle, save the double
number (24) of chromosomes. Immediately upon divergence
600 GRIFFIN. [Vou. XV.
the daughter-chromosomes are seen to be united by wavy and
very granular interzonal fibers (Pl. XX XIII, Fig. 42), most
of which later disintegrate (cf Wilson, '95), though after the
cleavage-furrow is complete, a few of these are still present
and converge to several black granules in the furrow or to a
single small black cell-plate (Pl. XX XIII, Fig. 46).
No sooner is the equatorial plate fairly established than the
cloudy area, with its contained centrosomes, commences to
migrate toward the outer periphery of the centrosphere where
the cloudy area, having grown much less distinct, finally fades
out entirely (Pl. XXXIII, Fig. 41). The centrosomes now
diverge, and by mid-anaphase set up a minute amphiaster super-
imposed upon the crown of astral rays (Pl. XXXIII, Fig. 43).
Aside from a slight elongation, it persists in this condition
during the rest of the anaphase, the formation and fusion of
the vesicles, and throughout the persistence of the reconsti-
tuted daughter-nucleus (Pl. XXXIII, Figs. 43-46). The cen-
trosomes, by continued divergence, finally reach opposite poles
of this nucleus, and the prophase of the second cleavage is thus
initiated. Every step in this whole process can easily be fol-
lowed out in detail, and there can be no question that the
centrosomes persist throughout.
The initiation of a new and independent astral system under
the apparent stimulus of the centrosome, while the persisting
rays of the old still converge to a point previously occupied by
the centrosome, is a fact noted by other investigators, although
it has not, I believe, received the attention it deserves. Besides
instances figured by Boveri ('90), MacFarland ('97), and others,
the most striking case is perhaps that of the trout, as figured by
Henneguy (91). Here, within the old centrosphere, a minute
aster is developed about each of the diverging daughter-centro-
somes. Outside the long astral rays of the old system still
converge toward the center of the former centrosphere. While
he does not emphasize the point in the text, a comparison of
his figures clearly brings out the migration of the daughter-
centrosomes to the outer periphery, exactly as in Thalassema.
Such facts as the above, I believe, lend the strongest possible
support to the theory of the centrosome as an active stimulat-
No. 3.] THALASSEMA AND ZIRPHAEA. 601
ing agent in mitosis rather than the mere “expression of cell
activity.”
As this is an important point, it will be well to treat it a
little more in detail. So long as the center of a new astral
system coincides with that of a previous one, the mere ferszst-
ence of the focal granules accords equally well with either of the
theories representing the centrosome ; for all are agreed that
such a structure, once formed, might well persist for some
time. A crucial test would seem to be supplied by those cases
in which a new system is formed about a point more or less
removed from the old center. If the centrosome is but an
expression of the forces that give rise to the aster, we should
expect to see it deserted zz sztw at the old center and a new
one formed at the focus of the new aster. We have shown
that quite the contrary is the case, the new aster starting up
about the old centrosome. In this, 7/a/assema furnishes the
strongest evidence, since the purposeful migration of the cen-
trosomes takes place defore the formation of the newaster. In
thus accepting the physzological theory of the centrosome as an
active originating agent in mitosis, we must, however, clearly
bear in mind that it by no means necessarily implies the 07
phological theory that it is a permanent cell organ, — though
the balance of evidence seems at present to favor the latter
view as well as the former. If subsequent research should
render the morphological view untenable, this would not, I think,
invalidate the physiological theory ; though if, on the other
hand, the physiological theory be proved untenable, the main
support would thereby be withdrawn from the morphological
theory. In other words, if it ever be proved unequivocally
that a centrosome can arise de zovo, it would not thereby
follow that the granule thus formed is solely an expression or
by-product of the aster formation and not an active agent.
In all stages previous to the establishment of the equatorial
plate, the astral rays can, in favorable preparations, be traced
directly to the centrosome, the centrosphere, when present,
being constituted by rays that stain more lightly and lack the
grossly microsomal structure. From metaphase onward, how-
ever, a fine light-staining reticulum becomes developed in the
602 GRIFFIN. [Vou. XV,
centrosphere between the rays and gradually replaces the latter.
By the early anaphase, during the peripheral wandering of the
centrosomes, the centrosphere has assumed a strictly reticular
appearance, as described by Wilson and others in the Echino-
derms (Pl. XX XIII, Figs. 41, 42).
This progressive development of the reticulum pazz passu,
with decrease of rays within the centrosphere, and following
close upon metaphase, constitutes strong evidence that the
reticulum is developed out of the substance of the rays ; and
this evidence is all the stronger from the fact that both rays
and reticulum stain exactly the same. We have here a radial
structure breaking up into a reticular one. But in cells of this
type the radial structure is indicative of mitotic activity, the
reticular of repose (‘‘resting”’ condition). These considera-
tions lead us to the view that the reticular centrosphere is a
degeneration phenomena consequent upon the withdrawal (or
cessation of activity) of the centrosome to initiate a new system,
and heralds the breakdown of the astral system. From now
on, the old system with its rays actually does disintegrate.
Evidence drawn from comparison of other forms lends addi-
tional support to this view, for in all cases, as far as I know,
the reticular centrosphere is secondary and preceded by stages
in which the rays focus directly to the centrosome or its more
zmmediate envelopes.
The enormous number of rays in the dense astral crown
during anaphase renders it impossible to regard them as entirely
consisting of rearranged cyto-strands, as Wilson and others have
so interpreted them. As there is obviously insufficient material
in the preéxisting cyto-reticulum for this purpose, we are forced
to look upon them as in part a newformation. The additional
material is probably derived as a product of some specific form
of metabolic activity set up by the centrosome, while their
radial arrangement is most likely due to a radial disposition of
the forces thereby disengaged, more or less analogous, though of
course not strictly comparable, to the magnetic “lines of force.”
1 Cf. Sea-urchins (Wilson, Kostanecki, and others) ; Mereis (Wilson) ; Chae-
topterus (Mead) ; Triton (v.d. Stricht,’92) ; Trout (Henneguy, ’91) ; RAynchelmis ?
(Vejdovsky), etc.
No. 3.] THALASSEMA AND ZIRPHAEA. 603
III. THe CuHromatin.
a. In Maturation.
Among the early egg-clusters are some in which the chro-
matin of every cell is arranged in a thick, rough, dark-staining
spireme, in which occasional indications were observed of the
longitudinal splitting so frequently observed in other forms at
this period. It is impossible to say with certainty whether this
spireme consists of a single thread or several —a difficulty
that has frequently confronted other observers. It is coiled in
several turns about the nuclear membrane, against the inner
wall of which it seems to be principally situated (Pl. XXXI,
Figs. 1, 2). Lack of earlier stages has prevented the work-
ing out of the genesis of this spireme. In later egg-clusters
are cells containing a spireme in all respects similar to the
above-described, except the commencing transverse division
and marked indications of the longitudinal fission (Pl. XX XI,
Figs. 1, 2). In other cells of the same cluster this process
has advanced one step further, the spireme being now com-
pletely divided into segments connected by numerous linin
strands (Pl. XXXI, Figs. 3-5) and partially cleft longitudi-
nally to form stout and much-flattened rings. The transverse
division appears to be followed by a shortening and concentration
of the resulting segments, obliterating their granular appear-
ance (see Hacker, '95, Canthocamptus, for similar phenomenon).
Although the exact number of these segments is not easy to
determine, they are readily seen to be nearer twelve than
twenty-four, which clearly indicates that the chromosomes first
appear in the reduced number as in so many other forms.
Meanwhile a fine nuclear reticulum is gradually developed,
which readily takes the plasma stains, and could not be made
to take the pure chromatin stains. Throughout the entire
growth period it increases in bulk and distinctness until, in the
full-grown ovum, it fills the entire germinal vesicle (Pl. XX XI,
Figs. 3-6). During prophase of the first maturation divi-
sion, after exhibiting a marked increase in its affinity for iron
haematoxylin, it is rejected bodily by the spindle and degen-
604 GRIFFIN. [Von. XV.
erates in the cytoplasm (Pl. XXXI, Figs. 10, 11). The source
and mode of growth of this reticulum are impossible to deter-
mine. It evidently does not arise through an anastomosing of
chromosome processes as in the germinal vesicle of Pristiurus
(Riickert, '92) and epithelial cells of Salamander (Rabl, '85) ;
for all the facts suggest that its development is independent of
the chromosomes which are passive during its growth. I would
conclude, therefore, that this nuclear reticulum is a secondary
and special structure, developed to preside over the metabolic
functions of the egg during the growth period, when the true
chromatin (idioplasm), in the form of chromosomes, is passively
awaiting the formation of the spindle. This view may be
elucidated by a comparison with the Infusorian nuclei. It is
here generally conceded that the macronucleus is the more
especially concerned with the purely vegetative functions of
the animal, while the micronucleus represents a reserve idio-
plasm especially concerned with reproduction. Exactly such a
difference in function I conceive to exist between the nuclear
reticulum and the chromosomes in ova of the 7alassema type.
The following rough parallel may be drawn :
INFUSORIAN. Eco-CELL.
1. Vegetative period, when the cell is
dominated by the Macronucleus.
Growth period, presided over by the
nuclear reticulum.
2. Macronucleus breaks up, disap-
pears, to be followed by
3. Division and persistence of Micro-
nucleus.
Up to this stage my results
with those of vom Rath in Gryllotalpa.
Nuclear reticulum breaks up, disap
pears, to be followed by
Division and persistence of the Chro
mosomes (polar mitosis).
on the tetrad-formation agree
Instead, however, of
immediately concentrating into tetrads, as in Gryl/otalpa, the
rings now begin to elongate, and their granular composition
becomes very apparent. In full-grown ova within the sexual
pouches they appear as small, much-coiled, or zigzag, granular
rods (Pl. XXXI, Fig. 6), generally near the membrane, though
sometimes a few are more nearly central. This coiled or zig-
1 In the Selachians it would appear (Riickert,’92) that its function is assumed
by the true chromatin, a similar reticulum arising by the chromosomes putting
forth anastomosing processes, and losing their affinity for chromatin stains.
No. 3.] THALASSEMA AND ZIRPHAEA. 605
zag condition greatly obscures their true form, but in favorable
cases they appear double. In other preparations of unfertilized
eggs removed from the pouches, the chromosomes stand out
very plainly as black and often clearly double rods.!_ In thus
demonstrating the presence of the chromosomes as double rods
throughout the entire growth period, my results agree with
those of Hacker ('92), Riickert ('92), vom Rath ('92), and others.
The chromosomes persist in the above-mentioned shape until
the asters commence to break through the wall of the germinal
vesicle, when they seem to undergo a concentration and approach
the asters. In entering the spindle, the chromosomes some-
times arrange themselves about the distal ends of the inner
rays, simulating the daughter-chromatin plates of an anaphase
(Pl. XXXI, Fig. 10). By growth of the rays these two
groups become pushed together, meeting equatorially to form
the equatorial plate. Bolles Lee ('97) has described a similar
phenomenon in Hedzx.
The nuclear reticulum now shows a marked increase in its
affinity for haematoxylin, which increase continues until after
completion of the spindle; the stain is retained with a tenacity
not surpassed by even the chromosomes or the centrosomes.
After complete extraction of the haematoxylin from the cyto-
plasm, and replacement by Congo red, the discarded reticulum
appears as a dense, thick-stranded, dark blue or black skein upon
a red background (Pl. XXXI, Fig. 11). After Flemming’s
Triple, however, it is indistinguishable from the surrounding
cytoplasm, though after Auerbach’s fluid, it takes the fuchsin
rather more deeply than the cytoplasm. The skein gradually
undergoes resorption, decreases in bulk and distinctness, and
finally fades out entirely, though traces of it are sometimes met
with as late as first polar metaphase with peripherally situated
spindle.
While entering the spindle in the prophases, the chromo-
somes exhibit a great variety of forms, which in most cases are
1 It was these irregular black chromosomes that I described (Griffin, '96) as
“here and there . . . light thickenings or bunching together of strands of the
chromatic reticulum, which is doubtless a prelude to chromosome formation.”
More extended study of later and earlier stages now shows them to be much-
twisted ring or rod chromosomes.
606 GRIFFIN. [Vou. XV.
easily reduced to the type of a double rod. In its simplest
form this rod consists of slender halves terminating at each end
in a common knob or swelling (Fig. I, a). In drawing apart
in the middle, the halves may give rise to a knobbed ring
(Fig. I, 4), and by a continuation of this process a perfect ring
arises, with or without one or two bead-like swellings (Fig. I, c, 2).
These rings may be open and
rs —- simple, or variously coiled or
twisted into the shape of a
b <> Ft figure 8. Occasional slender
rods, of extreme thinness
sc and variously coiled or bent,
are most readily interpreted
Y/
Oe
as segments of rings cut or
, eg broken by the knife. In most
cases the granular nature of
both these latter rings and
7, — rods is quite apparent.
aa? Similar figures have been de-
k W aaa scribed by Kastschenko ('90)
and Riickert (92) in the Se-
lachians, Fick (93) in the
_ Axolotl, Hacker in Copepods,
n Farmer and Moore (’96) in
Fic. I.— Formation of chromosome groups (tetrads) Lilium, and others.
in the prophases of the first maturation-division Shorter, much thicker, and
in Diotima a~drngfomation; 7-200" often apparently homogene-
ous rods also occur, which
may be very short (Fig. I, e) or longer and generally with a central
swelling (Fig. I, f). These may be fairly straight or variously
coiled, or even bent into more or less of a V shape (Fig. I,
g, f, 7). In certain favorable cases, clear evidence of a double
character appear, as a distal forking or breaking apart of the
halves in the center (Fig. I, 7). From this fact, as well as
from study of later and earlier stages, and analogy with
Zirphaea, 1 do not hesitate to consider all these varied forms
double rods in which the halves are so closely appressed
that the line of separation is obliterated. The central knob
No. 3.] THALASSEMA AND ZIRPHAEA. 607
is probably the beginning of a process that will be treated
in detail after the description of the morphology of the
chromosomes in metaphase. From the complete failure to find
forms indicating a direct transformation of the open rings into
the metaphase figures, it seems not improbable. that the thick
rods may represent a later and more concentrated and com-
pressed stage of the rings. This also appears to be indicated
by measurements which show the rings to be of greater length
than the rods. The open ring, however, is not to be consid-
ered a necessary stage, for it is quite evident that many of the
rods arise directly from the similar structures present in the
germinal vesicle.
More difficult to determine are the occasional two short rods
lying side by side (4, 4, m), as well as crosses or ophiurid forms
(z), the arms of which appear perfectly solid without the slight-
est indication of a division into halves. A few may possibly
be explained by supposing an accidental adherence of two
distinct chromosomal rods. Others may have resulted from a
premature division, either longitudinal or transverse, of a single
rod, and the subsequent rotation of the halves; or the cross
might be considered a ring compressed along two perpendicular
diameters, whereby the four included quadrants were converted
into loops. The close adhering of the halves of these loops
might well obscure the line of separation and cause the arms
to appear solid. Analogy with Zz7phaea would strongly sug-
gest the last-mentioned view.
Despite the varied forms presented during prophase, the
chromosomes of the equatorial plate exhibit considerable uni-
formity. Hence the various prophase forms must in some man-
ner be convertible into a uniform type of metaphase figure.
The commonest and, as we shall consider it, the typical form
assumed by the chromosomes in the latter stage is that of a
cross, with a pair of broad arms in the equatorial plane, and
the narrower perpendicular arms directed toward the poles
of the division figure (Pl. XXXI, Fig. 12, Text-fig. II, 9, p).
The latter pair or polar arms are of varying length, and invari-
ably end distally in a solid knob, to which a single traction-
fiber is attached. The more proximal portion is often seen
608 GRIFFIN. [VoL. XV.
with greater or less clearness to be divided into halves by a
pale longitudinal streak extending along the middle. Viewed
laterally, these arms are quite thin, and invariably single (PI.
XXXI, Fig. II, 7). The broad lateral arms, also of varying
length, appear in general quite solid, although occasionally
what seemed like a similar streak was observed to divide them
in halves (f). Rarely straight, and in the same plane as the
polar arms, they are more often curved or bent toward each
t
tit [Eq
Fic. I1.—The chromosome groups (tetrads) of the first maturation-division in Thalassema.
The narrow lines represent the traction-fibers; 0, early, 7, late typical cross; g, curved
cross; 7, curved cross with fused lateral arms in lateral view; s, the same seen from
above ; ¢, %, origin of the cross from rod-form; v, early stage of curved cross; w-z, un-
usual and asymmetrical forms.
other (g, v). By increased bending, the arms may come into
contact, whereby a chromosome, 7-shaped, in lateral view
results (ry, s). Similar 7-shaped chromosomes, but without the
distal knobs, are figured by Fick (93) in the corresponding divi-
sion in the Axolotl, Moore ('95) also obtains not dissimilar
figures in the Z/asmobranchs, and interprets them in an analo-
gous manner.!
1 The possibility is not completely excluded that these 7-figures may be due to
a second longitudinal division of the ring, the two double halves immediately
diverging. In view, however, of the prevalency of the crosses and other shapes,
in which a second longitudinal division is excluded, it seems more probable that
the above interpretation is the correct one, unless, indeed, we accept the possi-
No. 3.] THALASSEMA AND ZIRPHAEA. 609
The crosses are to be derived from the double rods by a
swelling or looping up of the halves in the middle. The begin-
ning of the process is represented by the previously described
thick rod with central bead-like swelling (Fig. II, f). Ina
slightly later stage, a central hollow appears which, separating the
swelling into two knobs, shows the rod to be double (0, ~). In
the form figured at the rod is greatly curved, but it may some-
times be straight, when a dagger-shaped figure results (0, x),
often with a clear central split. In this respect also it differs
from Klinckowstrom’s figure of a dagger-like chromosome in
Prostheceraeus. The metaphase crosses, with curved lateral
arms, may have resulted from rods curved like z or v. A further
increase in size of the lateral swellings produces a cross (gq)
quite similar to those of the equatorial plate.!
The chromosomes now being taken into the spindle (PI.
XXXI, Figs. 12, 13), it becomes of importance to determine
whether the arms formed by the mid-swelling of the halves of
the rod become the polar or the lateral arms. In the former
case the axis of the double rod (which is that of the original
spireme segment), being coincident with the equatorial plane,
the first division would be an equation division. If, on the
other hand, they become the lateral arms, the reverse would
hold, and the resulting division would be a reducing division.
The former I believe to be the correct alternative. For, in
the prophases, the lateral swelling seems considerably shorter
than the two other arms of the cross, while in early metaphase,
the eguatorial arms are often quite long, and the polar ones
bility suggested by Wilcox (’95), that in one and the same spindle some chromo-
somes undergo a reducing, and others an equation division. The occurrence of
varied prophase chromosome figures, all of which subsequently develop into a
uniform type of metaphase figure, recalls Calkins’s ('97) results in the fern. In
Thalassema, however, the end result is not a four-sphered tetrad (as in the fern),
but a ring variously looped and, as we have seen, a careful comparison of all
intermediate stages shows that the interpretation given by Calkins of the crosses
in the fern does not hold in 7ha/assema.
1 Considerable variation exists in the degree of approximation of the halves of
these figures, and hence in the clearness with which the central split appears.
Rarely it appears with great clearness, but more often it can be satisfactorily
detected only by the very best optical means (e,g., Zeiss, Apoch. 1.5 mm. oculars
6-12, with most powerful light), while sometimes even these fail to bring it out,
and the arms then appear perfectly solid.
610 GRIFFIN. [VoL. XV.
very short. From this it would seem that the longer axis of
the prophase cross corresponds to that of the equatorial arms
of the metaphase figure and hence the following division is
an equation or longitudinal division.
As previously mentioned, the arms of the metaphase crosses
vary extraordinarily in length, and may be of any size from a
short knob to a long process. The variation, however, con-
forms to an obvious law, since the longer the lateral arms, the
shorter the polar, and vice versa, as in Liliwm (Farmer and
Moore, '95). Moreover, those spindles containing the greatest
number of crosses, with short lateral arms and short-bodied 7’’s,
are the more advanced. The meaning of all this is very obvi-
ous; the lateral loops are gradually unfolding, and their sub-
stance passing into the polar arms. Throughout this process
the central furrow, separating the halves of the polar arms, is
sometimes clearly apparent, and sometimes not. The process
continuing, the lateral arms steadily decrease in size, and with
their disappearance the chromosomes round out to more or less
of a ring, in which the central furrow is sometimes most clearly
shown (Pl. XXXI, Figs. 13, 14). The chromosomes now divide
in the equatorial plane.
Upon separation, the halves or daughter-V’s may become
immediately contracted, and their bases squared off, or for
some time may remain elongate and more or less drawn out.
The split separating their limbs is sometimes very clear
(Pl. XXXI, Fig. 15). During divergence they progressively
shorten and contract into sausage-shaped rods, and at or even
before telophase they break apart at the angle, and the limbs
becoming entirely free, give rise to double chromosome of two
short sausage-shaped rods lying side by side (Pl. XXXI, Fig.
16). However varied the prophase, or even the metaphase
figures may be, the daughter-halves behave precisely alike in
all cases examined, invariably dividing transversely at the apex
of the V.
We are now ina position to understand certain, at first sight,
aberrant chromosomes occasionally met with in the equatorial
plate. Observed but once or twice in metaphase, is the doubly
curved or elongate S-shaped rod (II, w), with a slight central
No. 3.-] THALASSEMA AND ZIRPHAEA. 611
swelling, and the traction-fibers attached some distance from
the distal extremities. Another form (II, x) is similar to one
figured by Flemming in the salamander, and considered as a
ring split at but one point. Here, however, it evidently has
a different interpretation, being probably similar to the above,
but under greater tension, and with the free distal ends bent
further. Both show the faintest possible indication of a central
split. These may be rings just previous to division and after
the disappearance of the lateral loops, or those in which lateral
loops have failed to develop. In the latter case they would
divide transversely, while the remaining chromosomes split
longitudinally, and an equation and a reducing division would
occur simultaneously in the same spindle —a possibility urged
by Wilcox (95). A third deviation, observed but once, is the
thick 7-shaped form (II, y, z), in which the rounded extremities
and faint streak in the center of each arm show it to be a ring
with but one lateral loop instead of two. Occasionally the ring,
failing to develop lateral loops, becomes attached to the traction-
fibers, near one extremity (II, z), as Farmer and Moore ('96)
figure in the pollen mother-cells of Zz/zwm, except that it does
not break apart at the attached end. The just-mentioned thick
T-form may represent a more contracted condition of this ring,
or may have been derived from one of the open rings.
After completion of the first division the dyads are taken
immediately into the second polar spindle and arranged at right
angles to the spindle-axis, with the halves directed toward either
pole (Pl. XXXI, Fig. 18; Pl. XXXII, Figs. 19, 20). A single
traction-fiber is attached to one end of each of the rods. In
those on the periphery of the spindle, this is the zuner
extremity.1
The division of the daughter-”’s in anaphase or telophase
recalls Ophryotrocha, except that in Thalassema the halves
remain associated in pairs, and it is certain that one member
of each pair, and not a whole pair, passes into the polar body.
1 In cases where the halves are closely appressed in the middle, and swollen at
the extremities, an appearance suggesting tetrads sometimes results. This, I
believe, is all the significance there is in “tetrads” described by some authors
(e.g, Klinckowstrém, '97) in the second polar spindle.
612 GRIFFIN. [Vov. XV.
At anaphase the ends attached to the traction-fibers are the
first to diverge, while the free extremities cleave together, pro-
ducing a more or less open V (Pl. XXXII, Fig. 25). This
opens further, the two limbs become straight and then sepa-
rate, producing the appearance as though a single rod had just
divided transversely, precisely as Wilson has described in the
cleavage-spindle of Zoxropneustes. The diverging halves maintain
a pretty constant length, and are as long as the daughter-V’’s
in the first polar telophase. At telophase the chromosomes
remaining within the egg pass into minute vesicles which, by
fusion or close aggregation, constitute the egg-nucleus.
SUMMARY.
Z. By longitudinal fission and transverse segmentation of the
spiveme-thread, there arise 12 (reduced number) ellipse-shaped
chromatin masses.
2. These persist throughout the growth period of the egg.
3. During prophase they concentrate into crosses, the arms of
which are tight loops.
4. In the first polar division, these are drawn out again into
ellipses which divide to form daughter-V’s (equation division).
5. The V's break apart at the angle in the second polar divi-
sion (reducing division).
b. Jn Fertilization and Cleavage.
In general, the sperm enters nearer the vegetative pole, though
it occasionally penetrates above the equator.! After entrance
it increases enormously in size, but remains not far from the
surface as a large black sphere within a clear area (Pl. XXXI,
Fig. 12; Pl. XXXII, Fig. 19). By second polar telophase,
roughly speaking, it becomes vesicular (Pl. XXXII, Fig. 26)
and ultimately gives rise to a fine reticulum like that of the egg-
1 In one or two cases a sperm (probably a supernumerary one) had entered at
the animal pole, near the outer aster of the first polar figure. A similar fact has
been reported by Foot ('95) in A//olobophora, and Kostanecki and Wierzejski in
Physa.
No. 3.] THALASSEMA AND ZIRPHAEA. 613
nucleus (Pl. XXXII, Figs. 32, 33). The more or less complete
fusion of the germ-nuclei extends to the chromatin network of
each, which even before the fading of the separating membrane
shows dark-staining thickenings (Pl. XX XIII, Figs. 35, 36).
The ingrowing astral fibers now crowd towards the equator,
the chromatin (Pl. XX XIII, Figs. 37, 38), which, by this time,
forms a more or less perfect spireme. Intervening strands of
a finer and more granular nature (linin?) are, however, still
traceable. With the complete disappearance of the nuclear
membrane, the spireme becomes closely compacted in the equa-
torial plane (Pl. XXXIII, Fig. 39). Bead-like swellings occur
at regular intervals along the entire length of the thread;
these mark out the chromosomes. By transverse division at
these swellings, there result 24 pin-shaped chromosomes, at-
tached by their heads to the traction-fibers (one fiber to each
chromosome). The whole spireme thus going over into the
chromosomes, there is here no bodily casting out of chromatin.
The above-mentioned finer strands probably represent portions
of the original chromatic reticulum that break down into linin,
as claimed by Wilson (95).
The subsequent history, the divergence and conversion into
vesicles of the pin-shaped daughter-chromosomes, the recon-
stitution of the nuclei and genesis of the second cleavage
amphiaster (Pl. XX XIII, Figs. 41-46), does not appear to
contain anything worthy of especial remark. At no time was
any chromatin rejection observed such as Boveri (92) has
described in Ascaris.
IV. Tue Povar BopIies.
The formation of the polar bodies in Thalassema and the
almost invariable division of the first have already been minutely
described by Conn (86). As his researches were limited almost
entirely to the living egg, he was unable to follow out the
details of the mitotic phenomena. The latter are easily studied
in sections. The first polar body divides by a complete and
typical mitosis, although in minor details it shows evident signs
of degeneration.
614 GRIFFIN. [Vou. XV.
The constriction of the cytoplasm which separates the polar
bodies does not commence until telophase, when the chromo-
somes are at the poles of the division figure (Pl. XX XI, Fig. 16 ;
Pl. XXXII, Fig. 19). At this stage there commences to appear
a small projection or knob of clear granular cytoplasm, entirely
devoid of yolk-spheres and containing the outer group of chro-
mosomes and the outer centrosome (or centrosomes). The knob
increases in height and with a deepening of the constriction
becomes balloon-shaped, connected with the egg by a narrow
cytoplasmic bridge. Often this constriction advances more
rapidly on one side, giving a slight obliquity to the polar body,
as in L2max (Mark, ’81) and in the corresponding spermatocyte
division in Amphibians (Flemming, '87). With the severing of
the bridge the body rounds out, leaving the surface of the egg
rough and uneven, as described in Ophryotrocha (Korschelt, '95).
The second polar body arises in an essentially similar manner.
Within the rising polar body the centrosomes and chromo-
somes take up an extreme distal position (Pl. XX XI, Figs. 15-18;
Pl. XXXII, Figs. 19, 20). Inappearance and behavior the cen-
trosomes are in no way to be distinguished from those remaining
within the egg. Early divergence, which may take place while
the polar body is as yet represented by only a slight hummock
(Pl. XX XI, Fig. 15), followed by the appearance of rays, gives
rise to a minute amphiaster. Further divergence of the centro-
somes is accompanied by an’elongation of the polar body, which,
in most cases, takes place tangentially, although sometimes,
doubtless owing to a mechanical rotation of the polar body, the
spindle lies with its axis in an egg-radius (Pl. XXXII, Figs.
21-25).
The history of the chromosomes is, in all essential points,
but a repetition of that of those remaining within the egg. Soon
after the polar body becomes separated, or even while it is con-
nected with the egg (Pl. XX XI, Fig. 16), the line separating the
limbs of the daughter-V’s becomes very clear. Ai little later
the limbs break apart at the angle and shorten up into sausage-
shaped bodies associated in pairs (Pl. XXXII, Fig. 22). An
equatorial grouping of these dyads follows upon sufficient diver-
gence of the centrosomes, but as the individual dyads tend to
No. 3.] THALASSEMA AND ZIRPHAEA. 615
scatter some distance on each side of the equatorial plane, the
result is by no means a compact and sharply defined “ plate,”’
as in the second polar spindle. The telophase (Pl. XXXII,
Fig. 30) shows the chromosomes aggregated in a mass at each
pole, and the commencing cytoplasmic division. The latter,
however, may be retarded (Pl. XXXII, Fig. 31), or even fail
to occur, in which case the two chromatin bunches remain at
the poles (Pl. XXXII, Fig. 29). The centrosomes were only
occasionally to be made out at telophase. Not infrequently in
these stages a number of dark-staining bodies were observed in
an equatorial position (Pl. XXXII, Figs. 30, 31) in all respects
similar to those figured by Korschelt in Ophryotrocha ('95), and
considered by him to be chromatin particles left behind in the
anaphase.
After this division the three polar bodies remain clustered
together, and may attempt the formation of resting-nuclei. In
the two-celled stage one or more of them is to be found between
the blastomeres (Pl. XX XIII, Fig. 46), evidently there under-
going degeneration and absorption. One or two centrosomes
pass into the second polar body; but I have not observed the
latter to divide or commence any of the initial stages.
A mitotic division of the first polar body is a very common occurrence,
and has been repeatedly observed by previous investigators, though few
attempts have been made to follow out the process in full detail. This is
no doubt due to the minuteness of the elements and the often abortive
nature of the mitosis. The earliest reference to a d7véston of the first polar
body appears to be attributed to KGlliker in 1852 (Fick, ’93). Hertwig
('77) describes the phenomenon in the leech Mefhelis, though unable to
make out the behavior of the nucleus. From the mention of vacuoles that
fuse into one, we are led to infer that a resting-nucleus is formed or attempted.
No spindle nor centrosomes are figured. Trinchese ('80) observed the
mitosis of the first and also of the second, as did also Blochmann (’g9) in
the honey bee, and Nussbaum (’89) in Pod/icipes. Blochmann ('82) gives
a very complete account in Meretina. He figures a spindle in metakinesis
lying paratangentially with its axis in the direction of the greatest diameter
of the body. No distinct centrosome is demonstrable. The chromosomes
are described as uniting into “ ein solider Kern.” The anaphase with Ver-
bindungsfaden is figured and also the cytoplasmic division. Platner (86)
finds the division of the first a constant occurrence in Arzon, giving three
bodies into which spermatozoa sometimes enter. He observed a spindle in
616 GRIFFIN. [Vou. XV.
metakinesis and various stages in the division. Garnault ('88) studied both
Arion and Helix and describes the first polar body as dividing mitotically
with or without the formation of a resting-nucleus. In numerous insects
(Blochmann, '87,'89, and Hertwig, '90) each of the two groups of daughter-
chromosomes of the first maturation-spindle divide at telophase while still
within the egg, giving rise to four groups of chromosomes. These pass into
four resting-nuclei (the female pronucleus and three polar nuclei). Vejdovsky
(88) has described the mitosis of the first polar body in Lumobricus rubellus
and Allolobophora foetida. A completed spindle is figured of the latter, with
one well-developed aster. The spindle lies in the greatest diameter of the
polar body, which is here radial and not paratangential.
A mitotic division, or at least some of the initial stages, has been also
noted in numerous insects (Blochmann, ’87), in Amphibians (Schultze, ’87),
Myzostomum (Wheeler, '95), the Mouse (Sobotta, '95), Phallusia (Hill,
95), and other forms. Quite recently Korschelt (95) has described, in
some detail, the nuclear changes in the polar bodies of Ophryotrocha, in
which considerable variation appears to exist.
From the above it appears that the phenomenon in question is a pretty
general one ; that in some cases it results in a complete division typically
mitotic; but that more often the attempt is abortive and the process ceases
with the attainment of some intermediate stage. In a few cases a resting-
nucleus may intervene previous to the division.
PART II.— ZIRPHAEA.
V. DEVELOPMENT OF OVA.
The earliest ova that can be easily recognized as such have
more or less oval nuclei, measuring about 6 by 9p in diameter.
They lie imbedded in the stroma of the ovarian tubules,
with a minute quantity of cytoplasm heaped on either side.
A cell membrane could not be demonstrated with certainty.
The large, dark-staining nucleolus is single and eccentric.
The nuclear reticulum is as yet represented by a few strands
only. Peripherally situated chromosomes are occasionally to
be observed. The ovum has clearly passed out of the spireme
stage and has entered the growth period. During the growth
period numerous nuclei that stain a brilliant green with Auer-
bach’s fluid are present in considerable number, some scattered
irregularly throughout the stroma, others closely appressed
against the growing egg. They are evidently nutritive nuclei.
In the earliest stages the cytoplasm of the growing egg takes,
No. 3.] THALASSEMA AND ZIRPHAEA. 617
with Auerbach’s fluid, a uniform bluish green, sometimes almost
as brilliant as that of the nuclei. As the egg increases in size
and protrudes into the lumen of the tubule, the cytoplasm takes
the fuchsin in increasing quantity and assumes more and more
of a reddish tint. This ultimately predominates over the green,
and by the time the egg has broken away from the wall of
the tubule the green is scarcely noticeable. Iron haematoxy-
lin, followed by orange or other plasma stain, gives a similar
result ; the cytoplasm, which in early stages takes the haema-
toxylin intensely, shows with growth a steady increase in its
affinity for orange. These changes can hardly be due to expul-
sion of nuclear elements into the cytoplasm, for all elements
within the nucleus show a steady growth and differentiation.
The chromosomes and nucleolus increase in size, and the latter
in complexity, while pari passu the formerly sparse and scat-
tered nuclear reticulum becomes close and compact, filling the
entire nucleus. Moreover, in some preparations, the base of
the growing egg showed a more marked affinity for the nuclear
stains than did the cytoplasm near the nucleus. These points,
taken in connection with the behavior of the free nuclei, point
to the latter as being the active agent in elaborating cyto-
plasmic material.
When full grown, the ovum is but half the diameter (vzz.,
40) of that of Zhalassema. Its close and dense cytoplas-
mic reticulum, in whose meshes the fine granular yolk is dis-
tributed, renders the egg excessively opaque. The asters and
spindles do not, in consequence, show as brilliantly as in 7ha/-
assema, while the centrosome is often lost to view among the
innumerable granules that fill the egg. The germinal vesicle
is quite eccentric and filled by a close reticulum. The complex
double nucleolus (Pl. XXXIV, Fig. 47), characteristic of Lame!
libranchs and some vertebrates (Flemming, '82), some anne-
lids (Wilson, '96) and invertebrate liver cells (Lomberg, '92),
here shows to advantage. The smaller portion (“ Haupttheil’
of Flemming) stains black with haematoxylin and green with
Auerbach’s fluid. Generally hemispherical in outline, it sits
like a cap upon the larger vesicular “‘ Nebentheil” (Flemming).
It is, however, sometimes apparently spherical, or even bi- or
618 GRIFFIN. [VoL. XV.
tri-lobed, as described by Stauffacher (93), in Cyclas. The
Nebentheil varies in appearance, according to the fixing-fluid
employed. After picro-acetic it is large and clear, about three
or four times the diameter of the Haupttheil, and contains a
bunch of granules staining black with haematoxylin and red
with Auerbach’s fluid. After sublimate acetic, and occasionally
also after picro-acetic, it is larger, bordered by a denser and
more deeply staining zone of the nuclear reticulum, and is filled
with a finer and sparser reticulum, which shows even greater
affinity for plasma stains than does the nuclear reticulum.
From the accounts of numerous authors, it would appear that
this latter appearance represents more nearly the normal con-
dition, and that the bunch of granules is an artifact.
In the living egg the two portions of the nucleolus appear as
two spherical bodies, but slightly different in texture. Com-
parison with sections plainly corroborates Flemming’s obser-
vation that the Nebentheil is more or less swollen by reagents.
VI. THE CENTROSOME.
In the earliest condition observed, the polar asters, with their
minute dark-staining focal granule or centrosome, have already
attained their maximum divergence, and their ingrowing rays
commence to break through the wall of the germinal vesicle
(Pl. XXXIV, Fig. 48).
As in Thalassema, a large part of the nuclear skein is thrown
out into the cytoplasm, where it undergoes a change in staining
power similar to that in 7zalassema ; but instead of becoming
diffused throughout the egg, as in the latter form, it here
generally sinks into an irregular black mass on the edge of a
vacuole (Pl. XXXIV, Fig. 50).
During metaphase or later, the centrosomes divide (PI.
XXXIV, Fig. 52) and, rapidly diverging, leave in their path
a grayish rod-like streak (centrodesmus) which may possibly
be the remnant of the cloudy area seen in earlier stages (PI.
XXXIV, Fig. 50). The outer two soon degenerate, while the
inner pair, gathering the rays about them in two asters, give
rise to the second polar spindle. This, receiving the inner
No. 3.] THALASSEMA AND ZIRPHAEA. 619
group of dyads (Pl. XXXIV, Fig. 54), rotates from its paratan-
gential into a radial position, and rises to the surface extremely
near the point at which the first polar spindle underwent division.
The divergence of the daughter-chromosomes now follows,
during which the inner centrosome does not seem to undergo
any noticeable change until telophase, when it divides and
appears as two black granules surrounded by a common grayish
envelope (Pl. XXXIV, Fig. 53). The rays, while diminishing
somewhat in distinctness, still persist with sufficient clearness
to enable the aster to be easily recognized as such. During
the formation and fusion of the vesicles, the rays still persist
and focus to one or two or more granules. In many prepara-
tions it is extremely difficult to make sure which of these are
the true centrosomes, as the astral center is often somewhat
disturbed, whereby other and undoubled cytoplasmic granules
become thrust into it. In one very favorable preparation, dur-
ing the fusing of the vesicles, there are seen at the focus of the
persisting egg-aster two black granules, each invested by its
own grayish envelope, and exactly similar to the centrosomes
of the second polar telophase (Pl. XXXIV, Fig. 56). Little
doubt can exist that these are the centrosomes. Remains of
the rays are still to be seen after completion of the egg-nucleus,
and sometimes the centrosomes show as well (Pl. XXXIV,
Fig. 57).
During second polar anaphase and constitution of the egg-
nucleus, the sperm-head becomes vesicular and develops an
aster (Pl. XXXIV, Fig. 53) focused about a distinct centro-
some. An amphiaster, such as Lillie ('97) finds in Unzo, never
arises, and a later division of the centrosome could not be deter-
mined with certainty by reason of the numerous cytoplasmic
granules surrounding it.
Approach of the germ-nuclei increases the difficulty of mak-
ing out the behavior of the astral systems, although the indica-
tions are that one (either sperm or egg) degenerates, while the
other gives rise to the cleavage-amphiaster. This difficulty
arises from the fact that renewed activity sets in only after the
nuclei have so near approached as to make it impossible to
decide to which the centrosomes belong. In some cases the
620 GRIFFIN. [VoL. XV.
persistent pair of centrosomes remain beside each other until
the germ-nuclei are nearly in contact (Pl. XXXIV, Fig. 58), so
that it is here impossible to say which nucleus furnished the
centrosomes. In other instances the centrosomes have already
considerably diverged when the nuclei approach (Pl. XXXIV,
Figs. 59 and 60); but here again the evidence is not satisfactory.
Sometimes the centrosomes appear nearer the sperm-, some-
times closer to the egg-nucleus, or equidistant between the two.
The close proximity of the centrosomes to the egg-nucleus
cannot be taken as a certain indication of their egg origin; for
we know that in other forms the sperm-amphiaster may early
leave the sperm and wander in toward the egg-nucleus.
From now on the process is clear. With further divergence
of the centrosomes, the nuclei meet and incompletely fuse about
the developing amphiaster, while pavz passu the membrane at
the poles become more and more pushed into folds by the
ingrowing spindle-fibers. The completed spindle lies between
the two nuclei, so that the chromosomes come to lie in two
distinct groups (Pl. XXXIV, Fig. 61).
VII. THE CHROMATIN.
a. In Maturation.
The demonstration of the chromosomes in the young egg
still attached to the wall of the ovary, is not always an easy
task. They were observed often enough, however, to dispel all
reasonable doubt as to their normal presence and _ staining
capacity in these early stages. They occur in the form of a
straight or curled rod,a minute ring, or even a cross. In _prep-
arations of unfertilized eggs (sometimes still within the ovary)
and early maturation stages, the chromosomes are seen to have
undergone considerable growth, and they stand out with great
prominence, staining intensely black with haematoxylin. They
are situated mostly peripherally along the inner wall of the
membrane (Pl. XXXIV, Fig. 47), and have the form of the
large double rods, the halves of which may be closely apposed,
spread out to form a ring, or disposed in some intermediate
No. 3.] THALASSEMA AND ZIRPHAEA. 621
manner, and may be further complicated by becoming curled,
coiled, twisted, or bent in a zigzag manner. Their rough and
granular composition is quite apparent. Analogy with other
forms possessed of ring chromosomes leaves little doubt that
these are the product of several transverse and one partial
longitudinal division of a spireme thread.
By the concentration of the chromatin, observed so fre-
quently at this period in other forms (e.g., Copepods, Hicker,
95; Insects, vom Rath, ‘92 ; Wilcox, '95; Ophryotrocha, Kor-
schelt, '95 ; Selachians, Riickert, '93, etc.), each chromosome
entirely loses its granular appearance and becomes converted
into a compact tetrad, which consists of four closely apposed
loops or spheres (Pl. XXXIV, Figs. 49 and 50).
Along with the rings and rods in the germinal vesicle, cross-
shaped chromosomes are also to be met with, and the arms of
these may be straight or more or less bent or curved so as to
recall the “ ophiurid-shaped”” chromosomes of Hertwig ('90) (PI.
XXXIV, Figs. 47 and 49). The arms seem never to exceed
four in number. In favorable preparations these crosses are
seen to be hollow in the center, with indications of a split or
seam extending up into two or more of the arms. These chromo-
somes may in consequence be regarded as rings compressed
along two mutually perpendicular diameters, with the four
included quadrants thereby converted into loops. Additional
evidence is here furnished of the correctness of the interpreta-
tion given to the crosses in Thalassema (p. 607).
From the open rings it would seem that the tetrad may
arise directly by concentration of material at four different
points on the circumference. While this may possibly take
place sometimes by a looping, such pictures as Pl. XXXIV,
Fig. 47, seem to show that the tetrad arises by a curling or
tangling up of the ring into a knot at the four points. The
result, however, is essentially the same in all cases —a further
concentration or contracting of the loops gives rise to a fairly
compact tetrad. Upon reaching this stage, or sometimes while
still “ophurids,” the tetrads commence to enter the forming
spindle, and often become thereby temporarily elongated again
or variously distorted (Pl. XXXIV, Fig. 49).
622 GRIFFIN. [Vor. XV.
When once within the equatorial plate they again assume
this compacted condition (Pl. XXXIV, Fig. 50). Often they
are not dissimilar to the ring chromosomes in the first sperma-
tocyte division in Elasmobranchs, according to Moore’s figures
(95), and if we imagine the latter still further compressed lat-
erally so as to bring the halves into contact, the resemblance
would be perfect. Their arrangement within the plate agrees
essentially with that of Z/a/assema and is subject to but little,
if any, variation (Pl. XXXIV, Fig. 50). The loops are each
attached to a single traction-fiber, leaving the lateral ones quite
free — an arrangement that occurs as an exception in the first
spermatocyte division of Caloptenus (Wilcox, '90). Rarely one
of the tetrads has adjacent spheres attached to the traction-
fibers, as is the rule in Ca/optenus.
In Caloptenus and all forms with similar tetrads the spheres
retain their individuality and diverge in pairs. In Zirphaea,
however, the “spheres,”’ being but contracted loops, the whole
chromosome becomes pulled out into a homogeneous ring
(Pl. XXXIV, Fig. 51). The quadruple appearance is thus
obliterated and is never regained ; so that henceforth we have
to deal with rings and daughter-V’s. The details of the
process are easily followed; the elongation of the polar loops
(Pl. XXXIV, Fig. 51) and the gradual shortening and disap-
pearance of the lateral loops, followed by division of the ring
into daughter-V’s (Pl. XXXIV, Fig. 52), as well as the subse-
quent behavior of the latter, all take place exactly as in Zka-
/assema, but with greater clearness.
It is highly probable that the rings and double rods present
throughout the growth period arise by a transverse segmenta-
tion and partial longitudinal splitting of an original spireme
thread, as shown in 7halassema and other forms characterized
by similar chromatic elements. This being granted, it follows
that the polar mitoses represent one “ equation ”’ (longitudinal)
and one ‘reducing ” (transverse) division. Whether the reduc-
tion takes place in the first or the second polar mitosis, is, how-
ever, impossible to decide in this case, because of the close
similarity in appearance of the four loops.
No. 3.] THALASSEMA AND ZIRPHAEA. 623
b. Jn Cleavage.
In mode of entrance, in appearance and general behavior,
the sperm-head of Zzrvphaea perfectly resembles that of Zha-
lassema. By second polar telophase the vesicular condition is
assumed and the volume enormously increased (Pl. XXXIV,
Fig. 53).. The intermediate stages in this transformation were
not observed. The vesicular sperm-nucleus is completely filled
by a rather close chromatic reticulum feebly staining with iron
haematoxylin, but deeply with Congo red. It is somewhat
pointed in outline, with the sharp end directed toward the
centrosome. The egg-nucleus, which soon becomes consti-
tuted by the fusion of the vesicles left within the egg after the
second polar division, is quite similar in all respects to the
sperm-nucleus.
With continued approach, the germ-nuclei increase in size
and become perfectly smooth and spherical in outline. Their
size is, to all appearance, equal. Meanwhile the chromatin
shows a marked increase in its staining power, and just previ-
ous to copulation it stains as deeply as the chromosomes (cf.
Klinckowstrom, '97, for similar behavior of chromatin in Pvos-
theceraeus). The chromatin now exhibits a disposition to arrange
in dark-staining strands, the initial stage in spireme-formation.
The nuclei have now approached and partially fused to form a
bi-lobed segmentation-nucleus. The ingrowing spindle-fibers
indicate the commencement of spindle-formation.
In the next stage obtained, the spindle is completed and con-
tains a dense equatorial plate of rod-like chromosomes. Both
equatorial (Pl. XXXIV, Fig. 61) and longitudinal sections show
these to be arranged in two separate groups that are evidently
maternal and paternal, respectively. This agrees with the
facts observed by Boveri ('90) in Pterotrachea; Hacker (92) ;
Riickert (93) in Cyclops; Herla (93) in Ascaris; Sobotta
(95) in the mouse; and others that have described a similar
independence of the egg- and sperm-chromosomes. By careful
orientation of the spindle with reference to the polar bodies, it
is seen that the line joining the centers of these chromosomal
624 GRIFFIN. [VoL. XV.
groups, which roughly corresponds to that connecting the cen-
ters of the copulating germ-nuclei, may be parallel or more or
less inclined to the egg-axis.
PART III.— SUMMARY AND CONCLUSION.
Achromatic Structures.
In respect to gross morphology, the astral systems of Tha-
lassema and Zirphaea agree closely with Boveri’s description of
them in Ascaris. The minute focal granule may be compared
to his ‘centriole,’ the cloudy area to his “centrosome,’’ and
the centrosphere to the “heller Hof,’’ which with the dense
crown of astral rays is equivalent to his “astrosphere.”’
A detailed study of all stages in their successive order
leaves, however, no possible doubt that in the forms I have
studied the minute black focal granule (‘centriole’’) is func-
tionally here the true centrosome, as understood by Boveri —
the ‘single permanent cell-organ which forms the dynamic
center of the cell and multiplies by division to form the centers
of the daughter-cells.” It alone of all elements of the astral
system persists throughout all stages, divides, and apparently
initiates mitotic activity. The rays, centrosphere, and cloudy
area, which are in turn differentiated about the centrosome, are
formed only during the prophases and metaphases. No sooner
is the anaphase fairly under way than these begin to break
down, while the centrosome, its mission accomplished, migrates
to the periphery of the sphere and there sets up a new system
often superimposed upon remnants of the old.
By a careful study of these processes, the impression is most
strongly conveyed that throughout all these stages the centro-
some ts the cause vather than the mere expression or bye-product
of the aster-formation. This is especially clear in late anaphase,
where the centrosome deserts the old system and, moving toa
different locality, furnishes there the stimulus to the formation
of a new one. It is hardly less obvious at the close of the
“pause’’ (during copulation of the germ-nuclei), where after
almost complete dying down of the asters the centrosome again
furnishes the stimulus to renewed activity.
No. 3.] THALASSEMA AND ZIRPHAEA. 625
Chromatin.
In comparing the foregoing description of the chromatin in
Thalassema and Zirphaea, we observe that, while a close par-
allelism exists between these forms, each complements and
throws light upon the other. During the growth period of
Thalassema, the ring segments of the spireme greatly elongate
and, in the full-grown egg, become so distorted that their true
form is recognized with difficulty. Sometimes, probably by
reason of a temporary loss of staining power, even their pres-
ence is hard to demonstrate. Here Zirphaca yields important
evidence, for the chromosomes are very conspicuous as thick
rings in various stages of condensation. During prophase
the chromosomes in Ykalassema again show clearly as open
rings or double rods, while in Zz7phaea the already concen-
trated tetrads become partially drawn out into rings. In both
forms the division takes place by drawing out of the chromo-
somes into narrow ellipses followed by one cross division
whereby V’s result. At telophase these break at the angle
and do not split longitudinally, as Meves (95) finds in the Sala-
mander. The process shows most clearly in Z7rphaea, where
the facts demonstrate that the V’s cannot arise by bending of
a single rod, as Miss Sargant describes in the case of Lilium.
The ring or double-rod chromosome is a very common type
in the maturation of animal germ-cells, and the rings in most
cases undergo a subsequent transformation into quadruple
groups or tetrads. The ring is probably the more primitive
type, and the four-sphered tetrad probably arises secondarily to
facilitate the transference of the chromatin masses during the
ensuing divisions. But in all forms heretofore studied, the quad-
ripartite form, when once assumed, is retained and the four
quarters distributed among the four daughter-cells. In all forms
where a reducing division occurs the elements of the tetrad are
so arranged that each sphere comprises exactly one-half of one
of the daughter-segments produced by longitudinal splitting of
the spireme segments. In Zivphaea we find a structure closely
simulating an ordinary tetrad, yet the spheres are in this case
not homologous with those of a quadruple group. This is
626 GRIFFIN. (Vou. XV.
shown by both their origin and their fate; for each sphere is
really a loop representing the approximated halves of two adja-
cent quarters, as shown in the accompanying diagram (III).
In both figures the horizontal and vertical lines represent the
division planes, and the included portions (similarly lettered in
the figures) homologous parts. The horizontal lines in both
coincide with the long axis of the spireme and the transverse
axis of the spindle (equation division), while the vertical line
A B
Fic. III.— A, tetrad of the Copepod type; B, spurious tetrad (cross-form) of
Thalassema or Zirphaea.
is transverse to the spireme-axis or parallel to the spindle-axis
(reducing division). In A whole spheres are thus separated, in
B each sphere (loop) is halved.
Should the tetrad shown at B be shifted 45 degrees so as to
make the division planes separate entire spheres, as is the case
b
in A, the formula of the tetrad would be not Ae as in the first
b
alia ae,
Sai ole ie
case, but ale 7 There seems to be no theoretical
reason why such a mode of division may not occur in nature,
although up to the present time none has been definitely ob-
served. If the “ object” of reducing divisions be, as Weismann
supposes, to provide a source of variation, there would be
obviously an advantage in the above, since the number of
possible combinations is greater in the second case than in
the first. The Elasmobranch rings figured by Moore ('95)
show four thickenings, which, however, are halved like the
loops in Zrphaea, but unfortunately, as nothing is said about
No. 3.] THALASSEMA AND ZIRPHAEA. 627
the origin of these thickenings, we do not know whether they
are homologous with the loops of Zzvphaea or the spheres of
an ordinary tetrad. Bolles Lee ('97) figures and describes a
type in Hefx in which tetrads arise from rings, but later
become compacted into solid chromatin masses. His figures
suggest the possibility of a rotation whereby the spheres are
halved.
It seems not impossible that the mode of division in 7a/as-
sema and other forms characterized by persistent ring chromo-
somes may vary in some degree. We have seen that in cases
where the ring fails to loop, it may become attached to the
fibers in varying ways. If we suppose the attachment to be
fixed and to fall anywhere except in the normal division planes,
the result will be a division in planes more or less inclined to
the normal ones.! Weismann (91) has especially urged this
possibility in ring-form chromosomes.
I have found evidence of a type more or less similar to that
of Thalassema or Zirphaea in Teredo, Pholadidea, and Nerets.
ZOOLOGICAL LABORATORY, COLUMBIA UNIVERSITY,
March, 1898.
1 Persistent ring chromosomes have been described or figured in Plants
(Farmer and Moore), Platodes (v. Klinckowstrom), Elasmobranch (Moore), Mouse
(Sobotta).
628 GRIFFIN.
All of the figures from camera outlines.
EXPLANATION OF PLATE XXXI.
(Thalassema mellita.)
Fic. 1. Cluster of minute ova in spireme stage; occasional indications seen of
the longitudinal splitting of the chromatin-thread.
Fic. 2. Cluster further advanced ; spireme transversely segmented into flat-
tened ellipses ; growth period already entered upon.
Fic. 3. Cluster much further advanced; persistence of chromosomes as flat-
tened ellipses within germinal vesicle.
Fic. 4. Cluster of nearly full-grown eggs; persistence of chromosomes ; forma-
tion of alveoli in meshes of cytoplasm.
Fic. 5. Full-grown unfertilized egg, still in brood-pouch ; persistence of chro-
mosomes, rectangular disposition of cytoplasmic strands.
Fic. 6. Egg one minute after fertilization, showing persistent chromosomes
and multiple “asters.”
Fic. 7. Three minutes after fertilization, showing definitive asters.
Fic. 8. Later stage, asters pushing in wall of germinal vesicle.
Fic. 9. Same stage, showing variation in relation of asters to germinal vesicle.
Fic. 10. Spindle nearly completed, chromosomes in two groups.
Fic. 11. Spindle completed, still central; chromosomes, discarded nuclear
reticulum, nucleolus.
Fic. 12. First polar spindle, radially situated, early stage of chromosomes.
Sperm-head at @.
Fic. 13. First polar spindle, showing variations in form of chromosomes.
Fic. 14. First polar spindle, showing later stage of chromosomes. Achro-
matic structures in this and preceding figure entirely schematic.
Fic. 15. Anaphase of first polar division ; chromosomes diverging as daughter-
V’s or double rods.
Fics. 16 and 17. First polar telophase, showing double chromosomes.
Fic. 18. Second polar amphiaster undergoing rotation.
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630 GRIFFIN.
EXPLANATION OF PLATE XXXII.
(Thalassema mellita.)
Fic. 19. First polar telophase, showing inner centrosomes already diverged,
in preparation for formation of second polar amphiaster. Sperm-head and aster
atrar.
Fic. 20. Second polar spindle radially situated. Prophase of mitosis of first
polar body.
Fic. 21. Same, mitosis of first polar body further advanced.
Fics. 22-24. Stages in mitosis of first polar body.
Fic. 25. Beginning of second polar anaphase; spindle in polar body situated
vertically.
Fic. 26. Second polar telophase; extruding polar body, “cell-plate,” double
inner centrosome. Sperm-nucleus and amphiaster at ¢.
Fic. 27.. Sperm-head with amphiaster.
Fic. 28. Anaphase of mitosis of first polar body. Egg-chromosomes formed
into vesicles.
Fic. 29. Polar bodies; abortive attempt at division of the first has resulted in
formation of two masses of chromatin, one at each pole.
Fics. 30 and 31. Telophase of mitosis of first polar body.
Fic. 32. Germ-nuclei approaching; persistence of sperm-centrosomes and
asters, disappearance of the egg-center.
Fic. 33. Copulation of germ-nuclei, persistence of centrosomes.
PUXNXXII.
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632 GRIFFIN.
EXPLANATION OF PLATE XXXIII.
(Thalassema mellita.)
FIGs. 34-36. Various stages in copulation of germ-nuclei, showing persistence
of centrosomes throughout entire period.
Fic. 37. Segmentation nucleus; renewed activity on part of centers.
Fic. 38. Later stage, chromatin of nucleus in spireme stage.
Fic. 39. Mitotic figure almost completed ; spireme segmenting into the chro-
mosomes ; centrosomes divided.
Fic. 40. Transverse section across equatorial plate.
Fic. 41. Beginning of anaphase; centrosomes migrating to periphery of
centrosphere.
Fic. 42. Anaphase ; the centrosomes at the periphery and widely separated.
Fic. 43. Later anaphase; the new amphiaster on the periphery of the former
centrosphere.
Fic. 44. Telophase; chromosomal vesicles formed and beginning to fuse; a
distinct amphiaster.
Fic. 45. Later stage; nucleus reconstituted.
Fic. 46. Two-cell stage, preparing for the second cleavage ; “cell-plate”” and
engulfed polar bodies between the blastomeres.
Journal of Morphology Vol. xv:
fas, ss _ > PS a a SS PLXXNU.
,
634 GRIFFIN.
EXPLANATION OF PLATE XXXIV.
(Zirphaea crispata.)
Fic. 47. Unfertilized egg, showing ring-like chromosomes and double nucle-
olus.
Fic. 48. Asters pushing in the wall of the germinal vesicle; chromosomes
persistent.
Fic. 49. Later stage, with the chromosomes entering the spindle.
Fic. 50. Fully formed first polar amphiaster; the discarded mass of nuclear
chromatin at w.
Fic. 51. Later stage, the chromosomes pulling out into rings.
Fic. 52. Anaphase; chromosomes diverging as daughter-V’s.
Fic. 53. Second polar amphiaster, showing double chromosomes. 4s ?#. 4.,
first polar body.
Fic. 54. Second polar telophase. ¢, sperm-nucleus and aster. s J. 6., first
polar body; 2%. 4., second polar body.
Fic. 55. Slightly later stage ; chromosomes becoming vesicles.
Fic. 56. Approach of sperm-nucleus to the fusing egg-vesicles ; persistent egg-
centrosomes.
Fic. 57. Egg-nucleus and centrosome.
Fics. 58-60. Approach of germ-nuclei and divergence of centrosomes.
Fic. 61. Transverse section through equatorial plate of first cleavage-figure,
showing separate paternal and maternal chromosome groups.
Fic. 62. Two-cell stage.
Fic. 63. Same, preparing for division.
Fic. 64. Blastula.
a e
sa
ON THE BLOOD-PLATES OF THE HUMAN BLOOD,
WITH NOTES ON THE ERYTHROCYTES
OF AMPHIUMA AND NECTURUS.
GUSTAV EISEN, Pu.D.
CONTENTS.
A. THE BLoop-PLATES OF THE HUMAN BLOOD. >hGE
Tp CXOC UGE OL ypeeseeee enters arse cnne cee seer toc enensec cnceenean ent aestaner ae nettereeeerersupeaneataeres 635
Ie Methods of Investiga ton ic cccccceceseccesnarestecesesccnsccuconcnecseues Gsecersdntenccvstncdtvas 636
III. Historical Notes on the Nature of the Blood-Plates......0........sese es 637
IV. Blood-Plates, Plasmocytes, and Fusiform Corpuscles ............0.000000+5 641
V. Life-History of a Plasmocyte
VI. True and False Blood-Plates
VII. General Description of the Blood-Plates ..0.0........essssccecesseee seeeeeeeeeeseees 646
VIII. Detailed Description of the Blood-Plates. a, 8647)
IX. Blood-Plates and Plasmocytes ........---.c:.-:--00+ ... 649
X. Functions of the Blood-Plates or Plasmocytes ................:12ceceeeeeeeeees 650
B. THE BLoop-CorRPuscLEs OF AMPHIUMA AND NECTURUS.
xe General Remarksion the Prythrocytesy.ccrsccecs.tecsceeeesonscnsweoncesesecensnesesen= 652
XII. Two Kinds of Erythrocytes and Two Kinds of Fusiform Corpuscles 652
MIT. (Structure of the Oblong Erythrocytes .cccececcesseccecesces-cnecoescsesnacvencecsececor 653
PRAT AVieme EIN CLIO MTOts hen GOD ULES ee eceternrets rect tnces arent seesancneveerrnctayartereeeevensctecreroseensn
RSV fede LALIRINI NAY fecens anes eeeestsesversnsussecsscsecnens recercerercese stare rrerse7s
Bibliography
Explanation of the Figures -
IS Cel Ts psepreeerece renee ceeeenestece cone eet te enneee trey ancl st aeanssssouasevencectrererceteerececennse
A. THE BLOOD-PLATES OF THE HUMAN BLOOD.
I. INTRODUCTORY.
In a paper on the plasmocytes of Batrachoseps, I stated that
I had also found plasmocytes in the human blood. At the
time of publication of that paper, I had not yet had oppor-
tunity to study these structures in the human blood, and could
only affirm their presence and suggest that they probably had
636 EISEN. [VoL. XV.
been confounded with blood-plates. I believe that I am now
able to show that there exist various kinds of blood-plates, and
that the true blood-plates in the human blood must be consid-
ered as plasmocytes with a complicated internal structure.
This is the principal object of this paper. Some observations
on the erythrocytes of Amphiuma and Necturus are appended.
For the material of Vectwrus, I am indebted to Prof. William M.
Wheeler, of Chicago University, while the Amphiumas were
procured for me by the California Academy of Sciences, through
Messrs. Brimley, of Raleigh, N. C.
II. MertuHops oF INVESTIGATION.
In the preparation of slides, I have found only the dry method
to be of any value. The blood is spread in the usual way on
cover-glasses, by pulling the latter quickly apart. The glasses
must be chemically clean, otherwise the fine filaments of the
blood-plates will not be extended. In wiping and polishing the
surface of the cover-glasses, a double thickness of soft linen
cloth should always be used, as with a single thickness per-
spiration from the hand will affect the surface. Air-drying for
twelve hours, and subsequent fixing with absolute alcohol, are
the next two steps in the process, requiring no special descrip-
tion. Fixation by osmic acid and corrosive sublimate-alcohol,
as well as with numerous other fixatives, was tried, but found
to be injurious. None would give as fine differentiation as the
absolute alcohol.
For staining, I found only a few stains of any value. The
covers may be most advantageously stained with a weak solu-
tion of toluidine in water, for from several minutes to many
hours. The best differentiation was had after floating the
covers for twenty-four hours in a watch-glass containing a I
per cent. solution of toluidine. After subsequent washing for
a few seconds with distilled water, the covers are quickly dried
by the aid of a vaporizer and then mounted in thus-xylol. By
this method only blood-plates and leucocytes are stained, and
the plates are differentiated very much in the same way as are
the plasmocytes in the blood of Batrachoseps.
No. 3.] BLOOD-PLATES OF THE HUMAN BLOOD. 637
The second method consists in a double staining of eosin
and hemalum. Stain with eosin for five minutes, then wash
with water for several minutes, or until no diffuse red stain is
visible in the serum-film outside of the red blood-cells. The
latter must stand out sharply. Counter stain with a strong
undiluted hemalum for from ten minutes to one-half hour.
Wash with distilled water, and mount in thus-xylol. The
advantage of this method is that the filaments of the blood-
plates are intensely stained red, and can be followed with great
facility. The toluidine method stains the filaments less dis-
tinctly. The disadvantage of the double staining consists in
less differentiation of the inner spheres, as well as in a more
or less intense staining of degenerating and broken red cells
and other fragments and débris. I have found hemalum much
superior to any of the other hematoxylin compounds, in fact
the only one which will stain the inner sphere and centro-
somes.
The preparations were all studied with a Zeiss Apochromat
3 mm., Aperture 1:40, Ocs. 8 and 12; and with an Abbe
achromatic oil immersion substage condenser. The latter is
indispensable in order to get a view of the finer details of the
blood-plates. In addition I consider it indispensable to use the
achromatic light filter, as without it the light will be neither
sufficiently pure nor steady enough to differentiate the inner
structures of the blood-plates.}
III. Historicat Norres ON THE NATURE OF THE BLoop-—
PLATES.
A complete history of the discovery of the blood-plates, and
of the various and contradictory views held by investigators in
regard to the origin and nature of these bodies, lies outside the
scope of this paper, and a few remarks must suffice.
As is well known, the blood-plates in the human blood were
first discovered, or at least first described, by Max Schultze,
in 1865. He described the outward appearance of the blood-
1See Zettschrift f. wiss. Mikroskopie u. f. mikroskopische Technik, Bd. xiv,
1897, PP: 444-447.
638 EISEN. [VouL. XV.
plates as they appear under a narrow angle lens and without
staining. His figure shows the plates, or, as he calls them,
“Plaques” and “ Kornchenbildungen,” to be simply minute
globules of various size, with smooth outlines, although in the
text of the paper he mentions that they frequently show fine
filaments projecting in all directions, which he contends are
nothing but fibrin threads. As to the nature of the “ Plaques,”
Schultze hints at two possible views. Either they may be
elementary parts capable of development, or they are simply
“ Detritusbildungen ” or débris. In order not to prejudice any
one in favor of the one or the other of these views, he selects
for these bodies the name ‘“ Kérnchenbildungen’”’ as least com-
promising. Schultze refers also to other granulations in the
blood, previously described by Kolliker, H. Miiller, Zimmerman,
Hensen, Virchow, and others; but I think it of minor impor-
tance to decide whether these investigators observed the true
blood-plates or simply had before them artifacts or débris.
Bizzozero has, undoubtedly, next after Schultze, contributed
most to our knowledge of the blood-plates. In a paper pub-
lished almost twenty years after Schultze, he describes the
blood-plates as a new and independent element of the blood,
and one to which he assigns a most important function, — that
of causing coagulation of the blood. Since the first researches
of Bizzozero several investigators have taken up the study of
these minute bodies, especially as regards their origin and
functions. The structure and inner organization of the plates
have, however, hardly been touched upon, and the discussions
have mainly turned on the point of whether the plates are
preformed in the blood, or whether they consist simply of
chemico-mechanical precipitates or degenerations. To these
respective views I can here only briefly refer.
Bizzozero contends that the blood-plates are preformed in
the blood, and that they supply material for the fibrin. Hayem,
who has studied this subject minutely, not only supposes that
the blood-plates, to which he gives the name “ hzmatoblasts,”
are preformed in the blood, but considers them to be erythro-
cytes, or, in other words, he believes that in time they develop
into perfect red blood-corpuscles. His theory has, however,
No.3.] BLOOD-PLATES OF THE HUMAN BLOOD. 639
not found many adherents, as it is readily demonstrated that
we do not find in the blood any transition forms between
blood-plates and erythrocytes.
On the other hand, a large number of investigators hold that
the blood-plates originate from the breaking up of the leuco-
cytes, or that they are in some way altered leucocytes. This
view is taken by such biologists as A. Mosso, Ries, Halla,
Griesebach, Weigert, Botkin, Lilienfeld, Hlava, and others.
Among these, A. Mosso considers the blood-plates to be altered
leucocytes, while Lilienfeld and Hlava claim that they consist
mainly of nuclein derived from broken-up nuclei, on which
account these investigators suggest the name “ nuclein-plates.”’
Hayem, already referred to, must be counted in the above
group, as he supposes that the blood-plates originate in the
protoplasma of the leucocytes and are projected out from the
latter before they enter into the circulation of the blood.
Casimiro Mondino and Luigi Sala compare the blood-plates
to the fusiform corpuscles of batrachian, reptilian, and bird
blood ; views adopted by nearly all succeeding investigators.
We now come to a large group of investigators, who are of the
opinion that the blood-plates are in some way derived from the
red corpuscles (the erythrocytes) of the blood. Among these
observers we count Klebs, Ponfick, Welte, Bremer, Wlassow,
and Prof J. Arnold of Heidelberg, than whom none stand
higher, and whose investigations are worthy of the highest
consideration. Arnold’s earliest investigations were made on
blood treated with iodide of potassium, or with normal salt
solutions (0.6 per cent.). He found that erythrocytes thus
treated disintegrate in various ways, and that the fragments
greatly resemble blood-plates. But as through this method
only a probability in the result was reached, new investiga-
tions were made on living blood in the mesenterium of the
mouse, also with the addition of normal salt solution. Arnold
thus found that the erythrocytes show minute wart-like or
globular elevations or buds, which latter separate themselves
from the mother erythrocytes; or some erythrocytes assume
a mulberry form, the minute globules of which either separate
singly or in mass. The separated globules differ greatly in
640 EISEN. [Vo.. XV,
regard to the quantity of haemoglobin contained in them. In
other words, Arnold does not hesitate to consider it as defi-
nitely settled that the blood-plates are derived directly from the
erythrocytes through ‘“ Abschniirung”’ and “ Zerschniirung.”
Arnold also points out that he has found nuclear fragments
in the erythrocytes, which fact would explain their supposed
presence in the blood-plates. Objections to this theory of
the origin of the blood-plates are made by Léwit, Lavdowsky,
Scherer, and Wooldridge, who point to the great difference in
chemical composition between the erythrocytes and the blood-
plates, — differences in hemoglobin, as well as in staining
qualities.
We now mention those according to whose theories the blood-
plates are derived from chemico-mechanical precipitations in
the blood.
Wooldridge is of the opinion that precipitations of fibrinogen
are not to be distinguished from blood-plates ; thus assigning
to the latter a chemico-mechanical origin.
Lowit, who has entered into this discussion with much skill
and energy, tries to demonstrate that the blood-plates are not
preformed in the blood, but that they are partly precipitated
from the blood plasma and are partly the débris of leucocytes.
From the above short review it will be seen how different
are the views held by the various investigators, hardly any two
exactly agreeing as to the origin and nature of the blood-plates.
As to the functions of the blood-plates, I believe that most
investigators agree that the plates stand in some relationship to
the coagulation of the blood. While Bizzozero holds that they
furnish the fibrin for coagulation, others, like Ebert and Schim-
melbush, contend that they do not form any fibrin but simply
conglutinations.
Though many investigators have studied the blood-plates,
few have finally agreed upon any points pertaining to their
origin, while their structure has hardly been the object of
serious research. The summary of our knowledge of the func-
tion and origin of the blood-plates is, I think, most clearly
contained in the words of Friedenthal (Bzo/. Centralblatt, Bd.
xvii, No, 19 (October, 1897), p. 713): It is with certainty
No.3.] BLOOD-PLATES OF THE HUMAN BLOOD. 641
established through observations under the microscope, that
fibrin threads always radiate from heaps of blood-plates; but
whether the latter originate from the red or from the white
corpuscles of the blood cannot be decided.
In conclusion, I will only state that, according to my own
views, the true blood-plates are neither derived from budding
erythrocytes, nor from leucocytes, but that they constitute the
archosomes — centrosomes with spheres —of erythroblasts or
of other nucleated cells, which archosomes have separated
themselves from their attachment to the nuclei in the same
manner as the plasmocytes of the batrachian blood separate
themselves from the fusiform corpuscles.
IV. BLoop—PLATEs, PLASMOCYTES, AND FuSsIFORM
CoRPUSCLES.
As has been mentioned, Bizzozero, Casimiro Mondino, Luigi
Sala, and others compare the blood-plates in the human blood
to the fusiform corpuscles of some of the cold-blooded verte-
brates. Between these respective elements there is, however, a
very great difference, both as regards size and structure. The
blood-plates in the human blood are many times smaller than the
red corpuscles, while the fusiform corpuscles are always of very
nearly the same size as the red corpuscles of the same blood.
As regards structure, again, we find that the fusiform corpuscles
are each characterized by a more or less perfect nucleus, while
it can be shown that in the blood-plates no such nucleus exists.
The only similarity between the blood-plates and the fusiform
corpuscles is that the nature of each causes them to adhere
together and to other objects through the aid of peculiar
fringed filaments ; thus causing them to form masses of lesser
or greater extent.
In the great majority of cold-blooded animals no blood ele-
ments are found which can be compared to the blood-plates in
the human blood. In the small batrachian, Batvachoseps atte-
nuatus, I have, however, recently described minute bodies,
exteriorly, which somewhat resemble fusiform corpuscles, but
are of smaller size and of a different structure, there being no
642 EISEN. [Vou. XV.
nucleus or chromatin present. As will be shown in this paper,
these plasmocytes have almost the identical structure of the
human blood-plates and must be considered to be of the same
nature. I have also shown that these plasmocytes bud out
and separate from the fusiform corpuscles, after which the
latter decay and disintegrate. It is but a logical conclusion
to suppose that the blood-plates of the human blood must
have a similar origin ; that they are derived from nucleated
corpuscles or erythroblasts, and possess a complicated, distinct,
and invariable structure. The blood-plates cannot possibly be
considered as precipitations of globulin, fibrinogen, or of other
substances in the blood. On the contrary, we must recognize
in them a distinct physiological and morphological element of
the blood—an element with a phylogenetic life-history and
with important physiological functions.
V. Lire—History oF a PLASMOCYTE.
As my original paper on the plasmocytes of the batrachian
blood may not be accessible to every one interested in this
subject, the following short summary of my former researches
may be acceptable. In the blood of batrachians, reptiles,
and birds there exist corpuscles void of cell membrane, but
furnished with a nucleus. These “fusiform corpuscles,” as
they are generally termed, are nothing but disintegrating nucle-
ated red corpuscles, which have lost their hemoglobin as well
as their cell membrane. At each opposite pole of this corpuscle
is seen at first a very small cytoplasmic projection or bud. In
this bud or “plasmocytoblast’’ we can distinguish the centro-
somes of the original red blood-cell, surrounded by several
differentiated layers of cytoplasm and archoplasm. This bud
grows rapidly, the centrosomes separate from each other, sur-
round themselves with cytoplasmic envelops and show otherwise
great activity. At last the bud or buds separate themselves
entirely from the nucleus of the fusiform corpuscles and become
free and independent elements in the blood. These independ-
ent elements I have named “plasmocytes.’”’ At first they are
very small, but they soon grow and reach even the size of small,
No.3.] BLOOD-PLATES OF THE HUMAN BLOOD. 643
red blood-corpuscles. The cytoplasmic envelops or spheres
close up around the centrosomes thus giving the plasmocyte
aregular form. Plasmocytes, then, are corpuscles consisting
of the original centrosomes surrounded by several cytoplasmic
spheres, but not possessing a nucleus. They are the surviv-
ing elements of a nucleated erythrocyte, the nucleus and some
other parts having disintegrated. The life-history of a plas-
mocyte can be followed without any difficulty in the blood of
Batrachoseps, where they are of large size. The blood-plates
of the human blood, though very much smaller than the plas-
mocytes of Batrachoseps, possess the same general structure as
the latter, and must on this account also possess a similar origin.
Within the last few months this plasmocyte theory has been criti-
cised by Dr. E. Giglio-Tos, who contends that the plasmocytes
described by me are nothing but altered fusiform corpuscles,
and that the granospheres are nothing but disintegrating nuclei.
I have no intention of entering into any controversy with
Dr. Giglio-Tos upon the merits of his theory, as his criticism
is based exclustvely upon theoretical knowledge and not upon
the study of the blood of Batrachoseps. An examination of
properly prepared material would soon dispel any doubts as to
the origin of the plasmocytes, as whole and unbroken series
between plasmocytoblasts and plasmocytes are seen on almost
every slide, whereas absolutely no connecting links or inter-
mediate stages are found between fusiform corpuscles and
plasmocytes ; neither are any to be seen between disintegrat-
ing nuclei and plasmocytes ; nor can any possible process of
disintegration account for the constant and complicated struc-
ture of the plasmocytes. Dr. Giglio-Tos offers his criticism as
an “omaggio alla verita scientifica.’ Would not science have
been more honored if the critic, before publishing his conclu-
sions, had actually viewed some plasmocytic blood ; I should
certainly have offered him every facility to do so.1
1 Since the above was written, in November, 1897, I have had the pleasure of
communicating with Dr. E. Giglio-Tos. After having studied slides of the blood
of Batrachoseps, as well as the blood of living specimens sent him by me, Dr.
Giglio-Tos has changed his views as regards the identity of the plasmocytes with
the fusiform corpuscles, or, as he most appropriately calls them, the ¢romdocytes.
In private letters to me Dr. Giglio-Tos has acknowledged his error in this respect,
644 EISEN. [Vor. XV.
VI. TruE anp FatsE BLoop—PLaATEs.
The reason why so much difference of opinion exists in
regard to the nature of the plasmocytes or blood-plates must, I
think, be sought for in the fact that different structures have
been confounded under the common name of blood-plates. In
human as well as in batrachian blood, it is quite apparent that
the name blood-plate has been given, not only to the human
blood-plates described by Bizzozero, which are highly organized
bodies, but also to fragments, — disintegrated parts of erythro-
cytes and leucocytes, — as well as to purely chemical or mechan-
but has not yet expressed any view as to the origin of the plasmocytes. The only
published account of his changed views is found on pages 195 and 1096 of 7 7rom-
bociti degli Itiopsidi e dei Sauropsidi, Memoria del Dott. Ermanno Giglio-Tos
(Accademia Reale delle Scienze di Torino, Anno 1897-98). This is, of course,
satisfactory so far as it goes, though I can but think that the proper place for a
correction of this kind would have been in the publication where the criticism
originally appeared; that is, in the Anatomischer Anzeiger. Upon the appear-
ance of the article by Dr. Giglio-Tos, and before I had had the pleasure of
corresponding with the Doctor personally, I wrote to the publisher of the Axa-
tomischer Anzeiger, asking him to request some one entirely disinterested to study
and report upon the microscopic preparations of Batrachoseps blood which were
sent at the same time. Owing to the very sharp criticism, I thought that this
courtesy was due me. The publisher of the journal did not, however, answer my
letter.
In connection with this I will refer to the view held by Dr. Giglio-Tos in regard
to the origin of the trombocytes. He entirely disagrees with my opinion that the
trombocytes are derived from nucleated erythrocytes, and contends that they con-
stitute perfect and independent elements of the blood. Without entering into a
full discussion of the subject, I will only call attention to the fact that the nucleus
of the trombocyte is invariably found to be ina state of disintegration. Compare
the nucleus of the trombocyte with the nucleus of the erythrocyte and with that
of the leucocyte and we see at once that it is a degenerating nucleus and not one
in a perfect state of preservation. None of the organs or structures of the trom-
bocyte nucleus can be distinguished, no matter what methods are used for fixing
or staining. If the trombocyte is a perfect element it must have a perfect struc-
ture, and we should at some period find some of these trombocytes possessing a
perfectly organized and preserved nucleus. The very beautiful figures accompany-
ing the highly interesting memoir of Dr. Giglio-Tos show the justness of these
remarks. Any one who has carefully studied these cells will see at a glance that
the nuclei of a// the trombocytes figured by Dr. Giglio-Tos are in a state of dis-
integration or degeneration, as far as their morphological structure is concerned.
This is the more evident as the author figures, side by side with them, the perfect
nuclei of the erythrocytes. (See Figs. 34 to 36, etc.) [Note made at the reading
of proof, Oct. 28, 1898.]
No.3.] BLOOD-PLATES OF THE HUMAN BLOOD. 645
ical precipitations of globulin and fibrin. In the blood of the
lower vertebrates the fusiform corpuscles have been confounded
with plasmocytes or true blood-plates, while the latter have been
overlooked. As true blood-plates or plasmocytes, we must con-
sider only those structures possessing a definite, finer, inner
organization ; those in which may be distinguished outer
spheres forming a cytosome, and inner spheres containing cen-
trosomes, as well as highly refractive secreted or food granules.
Such blood-plates are true plasmocytes, partaking of the same
origin as the plasmocytes of Batrachoseps, having budded off
from fusiform corpuscles or from nucleated red blood-cells.
Among the false blood-plates we must distinguish between
those which are caused by morphological degeneration and those
caused by chemical precipitation. Among the former we must
place the fusiform corpuscles which give birth to the plas-
mocytes ; in the latter class must be included the disintegrating
parts of leucocytes, erythrocytes, and nuclei, which are found,
respectively, in the blood of the lower as well as in that of the
higher vertebrates, including man. Another class of false
blood-plates are those caused by purely mechanical and chem-
ical decomposition — precipitations of fibrin and globulin, bodies
amorphous as regards shape and without morphological struc-
ture. This multiple nature of what has been described as
blood-plates has been fully recognized by Friedenthal and
Arnold, who both agree that the blood-plates have no unity of
origin, but originate by budding and disintegration of red and
white corpuscles, as well as from precipitations of fibrin. This
assumption is due to a misunderstanding as to the structure of
the blood-plates, and cannot be accepted as final. On the con-
trary, it shows that there must be a distinction made between
true and false blood-plates, between blood-plates and débris,
between organized and unorganized blood-plates. A diagram-
matic view of this would be as follows:
646 EISEN. — [Vou. XV.
TABLE OF VARIOUS KINDS OF BLOOD-PLATES.
A. Bodies with Organic Structure.
1. True blood-plates or plasmocytes, consisting of various spheres with
centrosomes and refractive granules. These bodies possess an organization,
and the power of growth, movement, secretion, and assimilation, so far
found only in mammals and batrachians.
2. Fusiform corpuscles, possessing a nucleus and cytoplasmic spheres.
In one species, at least, they give origin to plasmocytes. The nucleus
rapidly disintegrates. Found in birds, reptiles, fishes, and batrachians.
3. Disintegrating erythrocytes, leucocytes, and other cells with no special
function. Probably found in the blood of all animals.
B. Bodies without Organic Structure.
4. Precipitates, chemical or mechanical, of globulin and fibrin. They
possess no structure, and must be considered as waste or accidental
products.
VII. GENERAL DESCRIPTION OF THE BLOOD—PLATES.
As I have suggested above, the only elements in the human
blood which should be considered as true blood-plates are those
small disc-like bodies which possess distinct marginal filaments
and show a decided interior organization.
The habit and location of the blood-plates have so frequently
been described that detailed reference to them is not necessary.
Suffice it to say that they occur singly or in groups, —from a
dozen or less up to several hundred, though generally number-
ing from few to twenty. When in groups, the individual blood-
plates may be packed more or less closely together, apparently
adhering to each other with their outer fringed edges.
Figs. 1 and 2 illustrate two such groups, stained with
eosin-hemalum. As will be seen, the plates vary considerably
both in shape and size. The shape, however, is generally round,
or slightly oval, or even irregular, with numerous cytoplasmic
filaments. In Fig. 1 some of the blood-plates are seen to be
surrounded by a red-staining, homogeneous protoplasm of doubt-
ful nature, while in Fig. 2 very little of such protoplasm is
visible, most of the blood-plates being entirely free or isolated.
No. 3.] BLOOD-PLATES OF THE HUMAN BLOOD. 647
Many groups of blood-plates are not surrounded by such a
plasma, and it can be seen that they adhere to each other
exclusively through their cytoplasmic projections. As regards
size, the blood-plates differ greatly from each other. Compared
with the red corpuscles of the blood, they are of course very
much smaller. In a general way it can be said that, taking the
groups of blood-plates as they are found, it takes from seven
to twenty plates to equal in size a red corpuscle, the varying
distance of the plates from each other being taken into account.
Some of the blood-plates are four or five diameters larger than
others. I have frequently found blood-plates the long diameter
of which equaled two-thirds the diameter of a red corpuscle.
Generally, however, they are much smaller, and I think it safe
to say that it takes about ten of the average size blood-plates to
equal a red corpuscle, the surface of the latter alone being
considered. Owing to the exceeding minuteness of the blood-
plates, their uncertain boundaries, and filamentous, cytoplasmic
projections, as well as to their variability in size, direct measure-
ments would be difficult to make and unreliable.
VIII. DertaILeD DESCRIPTION OF THE BLOOD-—PLATES.
Even a superficial examination of a blood-plate shows that it
consists of three distinct envelops or zones, one interior to the
other, and more or less concentrically arranged.
A. In or near the center we find usually one, sometimes
two (seldom more), very bright, highly refractive globules, of
roundish, compact, irregular or regular form, and of a white or
yellowish color. They appear like a diamond in a dark setting.
These highly refractive globules do not stain.
B. Surrounding these highly refractive bodies is seen a
darker area containing one or more dark granules, the latter
generally situated close together and connected by a dark-
staining film or irregular zone.
C. Exterior to this is seen an outer, lighter-staining zone,
with many filamentous, cytoplasmic projections, by which the
blood-plates are made to adhere to each other and to other
objects in the blood.
648 EISEN. [VoL. XV.
If we now examine these respective zones more in detail, we
find that they show characteristics of great constancy. The
inner, highly refractive bodies are nearly always well defined,
with avery sharp outline. In many are seen concentric layers,
though in the majority none such can be recognized, probably
on account of the minuteness of the objects. The refractive
power is always very great and very striking. These granules
vary greatly in size, some being barely perceptible (Pl. XX XV,
Figs. 4, 13); others are so large as to occupy a large part of the
blood-plate (Pl. XXXV, Figs. 5, 12,17). The inner, dark-
staining mass surrounding these refractive bodies is differenti-
ated into several parts, all of which cannot, however, always be
shown to be present at the same time. In the center of the
mass, generally in close proximity to the refractive body, is
seen a darker zone with from one to three or even more gran-
ules of a yet darker color. This darker zone may be either
small or large, sometimes occupying the larger part of the
blood-plate (Pl. XXXV, Fig. 17); at other times it is confined
to the vicinity of the refractive body (Pl. XX XV, Fig. 18).
In some blood-plates (Pl. XXXV, Figs. 6, 12, 13, 16, etc.)
the central dark zone contains one or more light areas sur-
rounding the darker granules. The minuteness of the blood-
plates is such that these, the smallest of the interior structures,
lie near the limit of vision, and only the most delicate manip-
ulation of the light will show them with any degree of satisfac-
tion. There is also a great difference between the individual
blood-plates, some being successfully stained, others not. But
even in those less differentiated enough can be seen to show
that there exists a very remarkable and constant differentiation
as regards structure.
We now come to the outer envelop or zone of the blood-
plate. In the toluidine preparations this zone is very faintly
stained and barely visible, and the fine filaments can be followed
only in isolated instances. With the eosin-hemalum_prepara-
tions the case is different; here the filaments are distinctly
stained, sometimes even intensely brought into view, but there
is less differentiation of detail. In the blood-plate stained with
toluidine we can often see a slight differentiation of this outer
No. 3.] BLOOD-PLATES OF THE HUMAN BLOOD. 649
zone into an inner and outer layer, the inner one being much
lighter in color. The outer layer sends out a number of cyto-
plasmic projections and filaments, which frequently are longer
than the diameter of the main body of the blood-plate.
Recapitulating, we find that the blood-plate contains the
following zones or spheres, more or less concentrically arranged.
Counting the innermost first we have:
1. A highly refractive, unstainable granule (food granule or ferment).
z. A darker-staining zone, generally containing darker granules (somo-
sphere with centrosomes).
3. A lighter, irregular zone (centrosphere).
4. A darker, granulated zone (granosphere).
5. A lighter, unstainable zone, not always distinguishable (hyalosphere).
6. An outer, fringed zone (plasmosphere), stainable with eosin.
IX. Broop—PLaTEs AND PLASMOCYTES.
From the above description it becomes apparent that the
blood-plates of the human blood show a very great similarity
to the plasmocytes of the blood of Batrachoseps. In many
instances, indeed, the structure is identical even to the minutest
details. A comparison of the various figures given in this paper
with Pl. II, Figs. 57, 60, 80, 81, etc., of my former paper ('97),
shows this similarity to be so great as to leave no doubt as to
the indentical nature of the blood-plates and the plasmocytes.
The prominent, inner, refractive body or bodies in the blood-
plates are also seen in many of the plasmocytes. I have sug-
gested that these refractive bodies may constitute food granules
or secreted matter, and I think they are entirely distinct from
the centrosphere (/.c., p. 10). Among the dark-staining struc-
tures of the blood-plates we meet with zones corresponding to
the centrosomes, somosphere, centrosphere, and granosphere of
the plasmocytes; but the minuteness of the blood-plates is
such as to make a segregation of these zones most difficult,
even under favorable circumstances, and in most instances it
is impossible. There is, however, no doubt but that the inner
dark-staining granules, which frequently occur in groups of three,
must be identified as centrosomes, having the same nature,
appearance, and staining qualities as the centrosomes in the
650 EISEN. [Vox. XV.
plasmocytes. The somosphere and centrosphere in the blood-
plates are less apparent, though in many instances (Pl. XXXV,
Figs. 13, 16) I have satisfied myself of their identity. The
large, dark-staining, granulated area, which is always present
in the blood-plates, I do not hesitate to identify with the grano-
sphere of the plasmocytes of Batrachoseps. It is large enough
to be readily seen and recognized. The outer filiferous zone
of the blood-plates so greatly resembles the two outer zones of
the plasmocytes that their identity must be apparent even after
the most superficial examination. The filaments of the blood-
plates are longer than those of the plasmosphere of the plas-
mocytes, but their nature is otherwise the same. Through the
aid of these cytoplasmic projections, the plasmocytes as well as
the blood-plates adhere both to each other and to other objects
in the blood. On account of this great similarity in structure,
as well as in outward appearance and behavior, I do not hesitate
to say that the true blood-plates in the human blood are
plasmocytes, and, as such, must have the same origin as the
plasmocytes in the blood of Batrachoseps ; that they are really
composed of the centrosomes, with several cytoplasmic spheres
originally budded off from some nucleated red cells in the
human body, probably the erythroblasts in the bone marrow.
The process of separation has not been studied, but it is safe
to presume that it must, at least in a general way, be similar to
that observed in the blood of Batrachoseps (l. c., p. 18, etc.).
X. Functions OF THE BLOOD—PLATES OR PLASMOCYTES.
According to Bizzozero and others, the blood-plates in the
human blood, as well as the fusiform corpuscles in the blood of
the lower vertebrates, must be considered as being the direct
cause of coagulation of the blood. They are great, or perhaps
even exclusive, producers of fibrin. That the blood-plates act as
repairers of injuries to blood-vessels is now hardly ever denied by
investigators. The principal difference of opinion arises in the
question as to whether this function of the blood-plates is not also
shared by leucocytes and perhaps even by red blood-corpuscles.
To this controversy I cannot add any opinion based on ex-
No. 3.] BLOOD-PLATES OF THE HUMAN BLOOD. 651
tended investigations, but can only point out a few observations
which I believe have not been recorded by others. In my
former paper on the plasmocytes, attention has been called to
the fact that these plasmocytes actually engulf and digest food,
such as bacteria and débris of erythrocytes and other cells, and
that they frequently possess in their interior one or more
highly refractive granules, which it is suggested may be food
granules, stored for future use. The plasmocytes of the human
blood are too minute to allow of a detailed investigation of any
such engulfed food particles, but still they are sufficiently large
to enable us to clearly distinguish the presence of highly refract-
ive granules of the same nature as those in the plasmocytes
of Batrachoseps, and similarly located as regards the various
spheres.}
In coagulated blood and in thrombosis the finer structure of
the blood-plates appears to be greatly confused, and so far I have
not been able to devise any method by which this structure can
be satisfactorily studied under these conditions. I have found
in coagulated blood many highly refractive granules scattered in
the serum, but am not certain that they can be considered iden-
tical with the highly refractive granules of the blood-plates and
plasmocytes. The similarity is sufficiently great, however, to
suggest that possibly these granules are not really food granules
stored for future use, but that they are secreted granules, per-
haps a ferment causing the coagulation of the blood when ejected
from their place in the plasmocytes into the blood serum.
The highly organized structure of the blood-plates indicates
that they cannot, as has been supposed by many, be originated
in the blood with great rapidity and precipitated at the very
moment that they are required. The blood-plates must pos-
sess a phylogenetic life cycle, through which they have acquired
structure, increased by growth, accumulated food, and secreted
certain highly refractive matter. In a word, the blood-plates
must be considered an independent element of the blood, of
equal rank with the red and white corpuscles, and of hardly
less importance.
1 Since the above was written I have repeatedly found in the human blood
blood-plates which contained débris of erythrocytes.
652 EISEN. [VoL. XV.
B. THE BLOOD-CORPUSCLES OF AMPHIUMA AND NECTURUS.
XI. GENERAL REMARKS ON THE ERYTHROCYTES.
In the following pages I will confine myself to a description
of the structure of the red corpuscles and to their derivatives,
the fusiform corpuscles. I expect to show that in the blood of
the above-named batrachians we must distinguish between two
different kinds of red blood-corpuscles, differing from each other
in size, form, and structure. In one class (Pl. XXXVI, Figs.
22, 23) we find groups of centrosomes suspended free at the poles
of the cell, far away from the nucleus; while in the other class
(Pl. XXXVI, Fig. 24) we find no such groups, but, on the con-
trary, an archoplasmal structure with centrosomes, situated in a
granosphere close to the nucleus. Corresponding to the two
classes of red corpuscles we find two classes of fusiform cor-
puscles derived from the former. In one class the centrosomes
are seen to be free and far away from the nucleus (Pl. XXXVI,
Fig. 25) ; while in the other the archoplasmal structures lie in
a depression of the nucleus, one at each pole. The structure
of the centrosomes will be described in detail ; and as regards
their nature and function in the first-mentioned class of cor-
puscles, it may be suggested that their function is purely
mechanical, for the purpose of balancing and guiding the
large oblong cells through the capillaries, thus preventing
stagnation and undue obstruction.
XII. Two Kinps oF ERYTHROCYTES AND Two KINDS OF
FusIFORM CORPUSCLES.
Not only in the blood of Amphzwma and Necturus, but in all
other batrachian and reptilian blood examined by me, have
been found two distinct kinds of nucleated erythrocytes. One
is more round than the other, and the two kinds stain some-
what differently. In Amphiuwma and Necturus erythrocytes of
the oblong kind described above possess directing globules,
while the rounder kind have none. The nucleus of the former
is longer and narrower, with an uneven outline ; that of the
No. 3.] BLOOD-PLATES OF THE HUMAN BLOOD. 653
latter is broader, with an even, smooth outline. There is also
another important difference between the two cells. The
nucleus of the rounder cell generally possesses a slight depres-
sion at each pole, and adjacent to this is seen a narrow, differently
staining zone, which under certain favorable conditions resem-
bles the granosphere with centrosomes in the fusiform cor-
puscles. No such zone is found in the longer corpuscles. It
appears, therefore, as if the groups of globules in the longer
corpuscles are morphologically of the same nature as the cen-
trosomal spheres in the round corpuscles.
As might be expected, we also find in the blood of these
species two distinct kinds of fusiform corpuscles. One kind
(Pl. XXXVI, Fig. 25) is undoubtedly a degenerated oblong red
corpuscle, retaining all its characteristics. The nucleus is in
a far advanced state of degeneration, while the groups of glob-
ules and granules are yet in position at the poles. The other
kind of fusiform corpuscle is shorter. The nucleus possesses
a depression at each pole, and outside of this we find the
cytospheres, granosphere, centrosphere, and centrosomes, more
or less distinctly brought out. These latter structures corre-
spond to the plasmocytoblasts in the blood of Batrachoseps. In
the blood of Mecturus and Amphiuma they do not develop into
plasmocytes.
XIII. StructTuRE OF THE OBLONG ERYTHROCYTES.
The oblong red corpuscles of both Amphiuma and Necturus
are so similar that in the following pages I shall refer to them
together, after first having mentioned the difference between
them. Even with a comparatively low power it will be seen
that at each pole, outside of the nucleus, there exist one or
more groups of dark-staining granules. In JVecturus there is
generally only one such group at each pole, while in Amphiuma
there are several, nearly always arranged in pairs of two, four,
or six, at or near the pole. Occasionally we find smaller
groups at the sides, or even isolated globules. In Figs. 22 and
23 I have figured two erythrocytes from Amphiuma, and in
Fig. 29 one from Wecturus ; both under a moderate power
654 EISEN. [Vou. XV.
of magnification. In Mecturus the groups are always smaller
than in Amphiuma, but otherwise there is a great resemblance
between them.
Under the highest systems these groups resolve themselves
into a number of small globules of different sizes and shapes ;
and in the majority of globules we find one or more small, dark
granules. The appearance of these globules and their darker
granules is such that I can only compare them to centrosomes
surrounded by a differentiated and differently staining sphere.
As to the nature of this sphere I am somewhat undecided, but
from analogy am inclined to consider it identical with the somo-
sphere, previously described (Eisen, p. 17, etc.). Especially in
Necturus is it readily demonstrated that these globules are sur-
rounded by or connected by a very thin, foam-like cytoplasm
of irregular form. Whether this is cytoplasm pure and simple,
or whether it partakes of the nature of archoplasm in the
sense of Boveri, is not to be determined at present, as the
hemoglobin in the cells evidently prevents a proper differ-
entiation. The somospheres and centrosomes are, however,
readily stainable, and a study of them is not connected with
difficulties, provided double staining is not attempted. Every
effort at double staining with eosin or with fuchsin mixtures
has proved a failure. With such stains these groups are not
even brought out, but remain unstained. They stain readily,
however, with basic aniline dyes, such as the methylen blues,
toluidines, thionins, gentian-violet, etc. I have found poly-
chromes-methylen-blue one of the best. With this latter stain
we find that the globules are not always as regular as they
appear to be at first. They are sometimes confluent, some-
times send out ramifications and projections, as, for instance,
in Pl. XXXVI, Figs. 27, 28, etc. As a rule, we find one or
more dark granules in each such projection or globule, but in
many instances there are none to be seen. The globules may
be* more or less numerous in each group. In Wecturus there
are seldom more than a dozen, while in Amphiuma I have
sometimes counted thirty in a group. The darker granules
appear to increase in number by budding in a manner similar
to that of centrosomes, as described by Heidenhain, and this
No. 3.] BLOOD-PLATES OF THE HUMAN BLOOD. 655
is my principal reason for identifying them as such. These
granules are sometimes well defined, sometimes with blurred
outlines, which latter, however, may be ascribed to imperfection
in staining, due perhaps to the surrounding haemoglobin.
It is possible that these globules with granules are of a nature
similar to those described by Ludwig Bremer from the red blood-
cells of Zestudo carolina. There are, however, some very great
differences in structure and shape, due perhaps in part to the
methods employed by Bremer, whose preparations were prin-
cipally fixed with osmic acid. When I employed his method, I
was not able to bring out any of the finer structures of these
groups, and I am satisfied that osmic acid, as well as most
other fixatives used, instead of fixing, distorts the structure
of the blood-cells. But there are some characteristics in the
paranuclei of Bremer which are not found in the cells now
considered. His paranuclei are less regularly situated and
often appear to be connected with the true nucleus. I have
at times found structures similar to Bremer’s in Déemyctylus
torosus, in which species I have, however, never seen any
globules similar to those in Wecturus and Amphiuma. Taking
all in all, it seems as if the globules of these genera are dif-
ferent from the paranuclei of 7estwdo, or, if of the same origin,
they must possess a different function.
XIV. FuNcTION OF THE GLOBULES.
In none of my preparations did I succeed in finding any
erythrocytes in mitosis, and therefore cannot decide whether
the globules and granules have any function connected with
cell-division. I doubt their having any such function, as their
great number would rather tend to disarrange karyokinesis than
to assist it. Any other function that may be ascribed to them
is probably of a purely mechanical nature. How this is possible
will, I think, be more clear when we remember, as I have before
pointed out, that there are two kinds of red blood-corpuscles,
one of which is rounder than the other and does not pos-
sess the numerous globules of the longer kind. The unusual
length of the red cells carrying the globules is most striking.
656 EISEN. [Vo. XV.
Such great length must be in certain respects a disadvantage
to the free circulation of the cells, which if turned sideways must
necessarily be impeded in their travel. If, however, the poles
could be loaded and increased in weight, the cells would be more
apt to travel with their pointed ends forward, and their speed
would thus be increased instead of retarded. I believe the func-
tion of the globules is to thus load the poles of the cells and
keep their pointed ends forward instead of sideways. This
opinion is strengthened by the fact that the rounder erythro-
cytes possess no such directing globules.
XV. SUMMARY.
1. We must distinguish between true and false blood-plates,
and between organized and non-organized blood-plates. The
only true blood-plates are those of plasmocytic nature. The false
blood-plates are either fusiform corpuscles, degenerating frag-
ments of leucocytes and erythrocytes, or chemico-mechanical
precipitations of fibrin and globulin.
2. In the blood of Satrachoseps the organized blood-plates
are of two kinds, fusiform corpuscles and plasmocytes, the
latter derived from the former. In the blood of many other
batrachians and reptilia the organized blood-plates are only
fusiform corpuscles, no plasmocytes being found.
3. In the human blood the true blood-plates are true plasmo-
cytes, possessing the same general nature and structure as
those in the blood of atrachoseps. In these blood-plates in
the human blood we may distinguish various zones and spheres.
These are :
a. Three outer cytoplasmic spheres : cytosphere, hyalosphere, and grano-
sphere.
6. Three inner spheres : centrosphere, somosphere, and centrosomes.
c. A centrally or laterally situated, highly refractive granule, which may
be either a food granule, or a secreted product, perhaps a fer-
ment causing the coagulation of the blood. All the true blood-
plates of the human blood have this structure.
4. In the human blood, under certain conditions, there may
also be found false blood-plates, caused by precipitation of glob-
No.3.] BLOOD-PLATES OF THE HUMAN BLOOD. 657
ulin, degenerating cells, etc. ; but these plates do not possess
any constant and differentiated structure, nor any definite and
constant form. In healthy blood all the blood-plates are true
plasmocytes, and even in diseased blood the plasmocytes con-
stitute the only true blood-plates.
5. For true blood-plates I propose to drop the word plates
and substitute the word plasmocytes.
6. In batrachian and reptilian, as well as in bird’s blood, we
must recognize two distinct kinds of red blood-corpuscles
(erythrocytes) and two kinds of fusiform corpuscles. Each
kind of red blood-cell degenerates into a distinct kind of fusi-
form corpuscles.
7. In Amphiuma and Necturus one class of erythrocytes is
oblong, the other more rounded. They possess, respectively,
the following characteristics :
a. Oblong erythrocytes.
Quite oblong ; nucleus oblong without depressions at the poles,
with uneven or warty outline. At the poles of the cells, far
from the nucleus, are found paired or single groups of granules
and globules, probably identical with centrosomes and grano-
sphere, and surrounded by a thin archoplasm.
6. Rounded erythrocytes.
More rounded, some entirely round ; nucleus rounded, with smooth
outline. Generally a depression at each pole, furnished with
the various spheres characteristic of a plasmocytoblast. There
are no separate globules at the poles.
8. The two kinds of fusiform corpuscles partake of the same
general characteristics. They have been derived from the
erythrocytes through degeneration and are generally not pos-
sessed of acell membrane. In the round fusiform corpuscles
the plasmocytoblasts are active, shown by increasing size ; but
in Necturus, Amphiuma, Diemyctylus, Chondrotus, Plethodon,
and many others they do not develop into plasmocytes.
g. The function of the globules and granules (centrosomes
with somospheres) in the oblong erythrocytes is probably a
mechanical one. It consists of so loading the poles as to cause
them to be directed forward, while the erythrocytes travel
through the capillaries. The advantage of such an arrange-
658 EISEN. [VoL. XV.
ment is to prevent a blocking of the capillaries and to cause an
increase of speed in the travel of the long erythrocytes through
them. The rounder erythrocytes do not require this loading
of the poles, as their shape would not interfere with their
movements in the capillaries.
BIOLOGICAL LABORATORY,
CALIFORNIA ACADEMY OF SCIENCES,
San Francisco, November 11, 1897.
No.
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LM)
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3.] BLOOD-PLATES OF THE HUMAN BLOOD. 659
BIBLIOGRAPHY.
AFANASSIEW. Ueber den dritten Formbestandtheil des Blutes, etc.
Arch. f. klin. Med. Bad. xxxv, p. 217. 1884.
ARNOLD, JuLius. Zur Morphologie und Biologie der rothen Blutkérper.
Virchow’s Arch. Bd. cxlv, pp. 1-29. Taf. I und II. 1896.
ARNOLD, JuLIuS. Zur Biologie der rothen Blutkérper. M/dénch. med.
Wochenschrift. No. 18. 1896.
ARNOLD, JULIUS. Ueber die feinere Struktur der hamoglobinlosen
und hamoglobinhaltigen Knochenmarkzellen. Virchow’s Arch.
Bd. cxliv, p.67. Taf.I und II. 1896.
ARNOLD, JULIUS. Ueber die Herkunft der Blutplattchen. Centra/bl.
f. allg. Path. u. path. Anat. Bad. viii, Nos. 8 und 9, p. 289. Mai,
1897.
BizzozEro, J. D’un nouvel élément morphologique du sang. Arch.
Ital. de Biol. Tome ii, p. 345. 1882.
BizzozERo, G. Virchow’s Arch. Bd. xc, p. 261. 1882.
Bi1zzozEro, G. Les petites plaques du sang et lacoagulation. Arch.
Ital. de Biol. Tome ii, p. 276. 1882.
Botkin, EuGen. Zur Morphologie des Blutes und der Lymphe.
Virchow’s Arch. Bd. cxlv, p. 369. Taf. VII. 1896.
Botkin, EuGEN. Leukocytolyse. Virchow’s Arch. Bd. cxli, p. 238.
1895.
BREMER, Lupwic. Ueber das Paranucleark6rperchen der gekernten
Erythrocyten, nebst Bemerkungen iiber den Bau der Erythrocyten
im Allgemeinen. Arch. f. mikr. Anat. Bd. xlv, p. 433. 1895.
BREMER, LupwiG. Ueber die Herkunft und Bedeutung der Blutplatt-
chen. Centralbl. f. d. med. Wiss. No. 20. 1894.
EBERTH UND SCHIMMELBUSCH. Die Thrombose nach Versuchen
und Leichenbefunden. Stuttgart. 1888.
E1seN, Gustav. Plasmocytes ; the Survival of the Centrosomes and
Archoplasm of the Nucleated Erythrocytes, as Free and Independent
Elements in the Blood of Batrachoseps attenuatus Esch. /Pyvoc.
Calif. Acad. Sci. Ser. 3, Zoél., Vol. i.
FRIEDENTHAL, H. Die Funktion der weissen Blutkérperchen. Sod.
Centralbl. Ba. xvii, No. 19, p. 705. Oktober, 1897.
GiGLio-Tos, E. I. Plasmociti di Eisen. Anat. Anz. Bd. xiv, Nos.
2 und 3, pp. 81-88. 1897.
GRAwItTz, Ernst. Klinische Pathologie des Blutes. Otto Enslin.
Berlin. 1896.
Hayem, G. Dusang,etc. Paris. 1889.
ISRAEL, O., UND PAPPENHEIM, ARTHUR. Ueber die Entkernung der
Saugethiererythrocyten. Virchow’s Arch. Bd. cxliii, p. 419. Taf.
IX-XI.
660 EISEN. [Vo. XV.
Ent
83
85
‘87
92
"92
’89
89
‘87
87
65
83
nos
86
LILIENFELD, L. Verhandlungen der physiologischen Gesellschaft in
Berlin, 1891. Du Bots-Reymond Arch. f. Physiol. P.172. 1892.
Lowit, M. Ueber die Bildung rother und weisser Blutkérperchen.
Sitzungsbh. ad. Akad. ad. Wiss. v. Wien. Bad. Ixxxviii, 3 Abtheil,
p- 356. 1883.
Lowit, M. Ueber Neubildung und Zerfall weisser Blutkérperchen.
Sitzungsb. d. Akad. d. Wiss. v. Wien. Bd. xcii, 1 Abtheil, p. 22.
1885.
Lowir, M. Die Umwandlung der Erythroblasten in rothe Blutkérper-
chen. Sztzungsb. d. Akad. ad. Wiss. v. Wien. Bd. xcv, 3 Abtheil,
p. 129. 1887.
Lowir, M. Studien zur Physiologie und Pathologie des Blutes und
der Lymphe. Jena. 1892.
Macactium, A.B. Studies on the Blood of Amphibia. Zvans. Cana-
dian Inst. Vol. ii, Part 2, p. 221. 1892.
MonpiIno, CASIMIRO, ET LuIGI SALA. La production des plaquettes
dans le sang des vertébrés ovipares. Arch. Ital. de Biol. Tome
xii, p. 297. 1889.
MOonpDINo, CASIMIRO. La genése et le développement des éléments du
sang chez les vertébrés. Arch. Ital. de Biol. Tome xii, p. 297.
1889.
Mosso, A. De la transformation des globules rouges en leucocytes et
de leur necrobiose dans la coagulation et la suppuration. Arch.
Ital. de Biol. Tome viii, p. 252. 1887.
PAPPENHEIM, ARTHUR. Ueber Entwicklung und Ausbildung der Ery-
throblasten. Virchow’s Arch. Bd. cxlv, p. 587. Taf. XIII und
XIV.
ScHULTzE, Max. Ein heizbarer Objekt-tisch und seine Verwendung
bei Untersuchungen des Blutes. Arch. f. mikr. Anat. Bd.i, p. 35.
Taf. II, Figs. 17 und 18. 1865.
WEIGERT, C. Fortschritte der Medecin. 1883.
Wuiassow. Ziegler’s Beitrage zur pathologischen Anatomie. Bd. xx,
Pp. 543. 1894.
WOOLDRIDGE. Die intravascularen Gerinnungen. Dx Bots-Reymond
Arch. f. Physiol. P.397. 1886.
No. 3.] BLOOD-PLATES OF THE HUMAN BLOOD. 661
EXPLANATION OF THE FIGURES.
FIGs. 1, 2,17, 18 are from preparations stained with eosin-hemalum. Figs.
3, 16, 19, 21, stained with toluidine. Figs. 23-27, metanil-yellow; polychromes-
methylen-blue. Fig. 20, the same as the last, but with after treatment of weak
oxalic acid in water. Figs. 28-36, metanil-yellow and polychromes-methylen-blue.
Thus-xylol. All were studies with Zeiss, Apo. 3 mm., Apt. 1: 40, Ocs. 8 and 12.
Figs. 1, 2, 22, 23, 26, 27, 30, 33, 36, projection on table. All the other figures are
drawn to a larger scale, in order to be more distinct. Figs. 22-24, 30 drawn from
Zeiss D., Oc. 4.
The only light used was that passed through the light filter previously described;
daylight being very unsatisfactory.
EXPLANATION OF PLATE XXXV.
Plasmocytes, or Blood-Plates, from Human Blood.
Fic. 1. A group of plasmocytes, partly surrounded by free protoplasm of
doubtful nature. Some of the plasmocytes are free, all possess cytoplasmic pro-
jections. The white centers are highly refractive, unstainable granules. The dark
field surrounding them contains the archosomal zones, such as centrosomes, cen-
trosphere, etc. The red-colored parts contain the cytoplasmic spheres, with
longer or shorter cytoplasmic filaments.
Fic. 2. Another group of plasmocytes, or blood-plates, of the human blood.
The plasmocytes are adhering to each other, but there is no diffuse protoplasm.
The details are as in Fig. 1.
Fic. 3. A free plasmocyte. In the two outer spheres we can recognize the
plasmosphere and the unstained hyalosphere. The granosphere is stained dark
violet. The centrosphere is white, and the centrosomes, with presumably a somo-
sphere, are stained dark.
Fic. 4. A plasmocyte with two separate groups of centrosomes, between them
a paler centrosphere.
Fic. 5. A plasmocyte with a large refractive granule, a paler centrosphere
with centrosome. Another centrosome lies outside of the granosphere, and out-
side the latter are the other cytoplasmic spheres, not separable from each other.
Fic. 6. A plasmocyte with four central centrosomes and three centrospheres,
one of which contains a centrosome.
Fic. 7. A plasmocyte with two centrosomes, surrounded by a food, or ferment,
granule. The dark zone is the granosphere; the two outer paler zones, the cyto-
plasmic spheres.
Fic. 8. A plasmocyte with a very large central granule, at the edge of which
is seen a centrosome. The granosphere is dark; the outer cytoplasmic spheres
are pale blue.
Fic. 9. <A group of plasmocytes of different sizes illustrating the variation in
form and size. The largest plasmocyte with two groups of centrosomes, three of
662 EISEN.
which are in each group. The two round, pale bodies are probably food, or
secreted, granules of a highly refractive nature. The central paler body is per-
haps the centrosphere. In é the large body is the granule, also a single centro-
some in a centrosphere. ¢ possesses a single centrosome, and d two groups of
centrosomes, one of which is less defined. The plasmocyte (a) has a diameter
about one-half that of a red corpuscle.
Fic. 10. A plasmocyte with three centrosomes placed inside the granule.
Fic. 11. A plasmocyte with two refractive granules and three free centro-
somes ina group. The fourth centrosome is surrounded by a centrosphere.
Fic. 12. A large plasmocyte which appears as if in division. The large white
fields are refractive granules. The centrosomes are surrounded by a centrosphere.
Fic. 13. One of the largest plasmocytes observed in human blood. Its
diameter equals about two-thirds that of a red blood-corpuscle. There are three
groups, each with several centrosomes and a granule. The very large, paler field
is probably a centrosphere, in the center of which is a somosphere with a single
centrosome. There are two smaller plasmocytes, possibly offshoots of the same
group.
Fic. 14. A plasmocyte with two granules and a centrosphere with a single
centrosome.
Fic. 15. A plasmocyte with a large granule and a centrosome and centro-
sphere lying independently in the granosphere.
Fic. 16. A plasmocyte similar to the former, but with a group containing
three centrosomes.
Fic. 17. A plasmocyte with a very large, highly refractive granule, surrounded
by three darkly stained centrosomes, which again are surrounded by a grano-
sphere.
Fic. 18. A large plasmocyte similarly organized to the last one. The centro-
somes appear to be connected by asomosphere. The large granulated red area
is the granosphere. This, as well as the last plasmocyte, is from a slide stained
with eosin-hemalum, which stain has brought out more definitely the cytoplasmic
filaments.
Fic. 19. A plasmocyte with a large lower refractive granule, an upper centro-
sphere, and two centrally situated centrosomes.
Fic. 20. A plasmocyte with true, distinct centrosomes and a large granule.
Whether the darker areas surrounding the centrosomes are to be explained as
somospheres is uncertain.
Fic. 21. In this plasmocyte we have two large granules of a highly refractive
nature, with a single centrosome between them.
PU XXXV-XX XV.
Eisen,del. | B. Meisel, bith Boston
oH
ve
Journal of Morphology. Vol. XV.
PL XXXV-XXXVIT.
664 EISEN.
EXPLANATION OF PLATE XXXVI.
Amphiuma means.
Fic. 22. An erythrocyte or red blood-cell. Zeiss D., Oc. 4. A large central
nucleus and four separate groups of centrosomes situated at the poles. These
groups are of about the same size, and equidistant.
Fic. 23. Another erythrocyte, in which the four groups of centrosomes are less
equal in size, and not as regularly distributed; still each group is quite distinct.
This and the former figure represent the most common class of red corpuscles, in
which the centrosomes are situated far away from the nucleus and grouped at
the poles of the cells.
Fic. 24. A red blood-corpuscle, or erythrocyte, belonging to a different class
from the last two. The cytoplasm in this class stains with basic stains. There
are no groups of centrosomes at the poles as in the former class, but close to the
nucleus is seen a pale, unstained zone, undoubtedly containing centrosomes and
spheres. These latter are only brought to view in the fusiform corpuscles.
Fic. 25. A fusiform corpuscle derived from an erythrocyte, or red blood-cor-
puscle, of the class shown in Figs. 22 and 23. At the poles are seen centrosomes.
The nucleus is in strong degeneration, or dissolution. The hemaglobin is already
diffused, and the outer membrane of the cell is breaking up. This class of fusi-
form corpuscles is entirely distinct from the following.
Fic. 26. A fusiform corpuscle of the other type, derived from a red corpuscle
of the kind shown in Fig. 24. The nucleus is degenerated, but not to such a
degree as the one represented in Fig. 25. At each pole is seen a zone with differ-
entiated spheres and centrosomes, corresponding to the plasmocytoblasts of the
fusiform corpuscles of Batrachoseps. In this instance, however, they do not
develop into plasmocytes. The paler outer zone corresponds to the plasmosphere
and hyalosphere in the plasmocytes.
Fic. 27. Two groups of centrosomes from a pole of a red blood-corpuscle of
Amphiuma. One or more centrosomes are seen to be surrounded by a proto-
plasmic envelop, which probably can be considered homologous to a somosphere.
Fic. 28. Another group of centrosomes with somospheres. In many of the
smaller somospheres no centrosomes are visible. The group appears to be in
active division and development.
666 EISEN.
EXPLANATION OF PLATE XXXVII.
Necturus maculatus.
Fic. 29. A red blood-cell, or erythrocyte, under a low magnification, showing
the groups of centrosomes at each pole. The following figures show either
entire groups of centrosomes more highly magnified, or isolated centrosomes with
somospheres. As will be seen in the red blood-cell of Vecturus, there is only one
single group of centrosomes at each pole, while in the red blood-cells of Amphz-
uma there are generally two or more.
Fic. 30. A group of centrosomes. Some of the isolated bodies I consider to
be centrosomes surrounded by a somosphere. The pale yellowish zone surround-
ing these bodies in Figs. 27, 28, 30, and 36 may be common cytoplasma, or per-
haps acentrosphere. In Fig. 30 it is seen to have a distinct foam structure. Each
one of the two upper globular somospheres contains two centrosomes. The one
to the Jeft has sent out a bud, in which is seen a rather undefinable centrosome,
perhaps an offspring from one of the larger ones.
Fic. 31. Another group of centrosomes. The large somospheres are rounded,
globular, oblong, or even ring-like. In most of them are darker bodies, which I
consider to be identical with the centrosomes of Heidenhain, or the centriols of
Boveri. In many somospheres no centrosomes are found.
Fic. 32. A somosphere sending out two buds, one of which is globular, the
other oblong. The dark bodies are the centrosomes. Many of these are not well
defined in the drawings, not being very distinctly outlined on the preparations,
which probably was due to optical or other imperfections.
Fic. 33. A whole group of centrosomes and somospheres from the pole of
one of the red blood-corpuscles.
Fic. 34. Another group of somospheres and centrosomes more highly magni-
fied. Oc. 18.
Fig. 35. A group of centrosomes, with somospheres from one of the poles of
a red blood-corpuscle. Some of the somospheres apparently possess no centro-
somes. Oc. 12.
Fic. 36. a@-# represents five whole groups of somospheres and centrosomes
from the poles of the red blood-corpuscles. Figs. e, 4 ¢ represent isolated somo-
spheres with darker stained centrosomes. In the other figures the yellow ground-
substance represents either pure cytoplasm or centrospheres.
STAINS.
Polychromes-methylen-blue, nach Unna. Dr. G. Griibler & Co., Leipzig.
Eosin. James W. Queen & Co., Philadelphia, U.S.A. Already mixed and in
solution, composition unknown.
Toluidine blue, extra. Actien-Gesellschaft f. Anilin-fabrication, Berlin (66,711),
I per cent. watery solution, ro per cent. alcohol.
Metanil-yellow. Actien-Gesellschaft f. Anilin-fabrication, Berlin.
All stains were supplied by C. C. Riedy, San Francisco, Cal.
THE PHOSPHORESCENT ORGANS IN THE TOAD-
KISH SLORICHEEDYS NOLATUS (GIRARD:
CHARLES WILSON GREENE,
ASSISTANT PROFESSOR OF PuysioLoGy, LELAND STANFORD JUNIOR UNIVERSITY.
CONTENTS.
PAGE
Mis Wratroed ctor erect se sesee ve co soci oe ceases hee oreo sos oc case pasesboveabs sat 3c5 ectubacsennseeeastscd 667
II. Distribution of the Phosphorescent Organs and the Lateral-Line Sense
OT BANS) soe ccsececcpscacve cas sey oncnsssaseascovectetsatssancocctatcsedsvasdchaas cassacoctpentasessoseenees -. 669
1. Lines of Organs on the Body ............. -2 670
2. Organs of the Lower Jaw and Head 7073
Pit) Structure of these hosphorescent) Organs seen cose essences ctor ecesscse sete easeaereasaces 677
Day MCE Gen Set cee nar careers sar accvesesscen ss es cscsee sce scsesctectesstoviccesnnncietasveeastusesen 677
2. The Gland .. = ... 678
3. The Reflector.. ... 678
4-, The Pigment est sets ae seet ects nests ... 679
IV. Nerve Supply of the Phosphorescent Organs ............-..s-scsceccssesseseeseseseeess 679
V. Orientation of the Organs with Reference to the Surface of the Body.. 680
VI. Development of the Phosphorescent OrgansS.............::.ssececesssesseeeeneseseeceeses 681
WEI Skunction of the PhosphorescentiOxpansiecerccsceteesocreeree perce neces 684
I. INTRODUCTION.
FisuEs described as possessing phosphorescent organs belong
almost without exception to the deep-sea fauna. They live at
depths where the light of the sun rarely if ever penetrates, and
this fact is supposed to account for the process of evolution
which has brought about the phylogenetic development of light-
producing organs. It lent interest, therefore, to the study of
phosphorescent organs in fishes when in 1889 a paper appeared,
describing phosphorescent organs in Porichthys, a shore fish.
The study of these organs was made on unfavorable material,
as the author states, and as the structures described were so
obviously different from phosphorescent organs in other fishes,
it seemed desirable to reinvestigate the question.
668 GREENE. [VoL. XV.
It is my purpose in this paper to present the distribution,
structure, and development of the phosphorescent organs in
Porichthys, together with their surface relations to the lateral-
line system of sense organs.! In the future I hope to follow
with a second paper on the morphology of the lateral-line
system, work now partially completed.
The results presented in this paper were obtained chiefly
from the study of Porichthys notatus Girard. But I have
also some material from Porichthys nautopedium Jordan and
a new species of Porichthys kindly furnished me by Prof.
Gy He Gilbert:
Porichthys notatus is found abundantly along the Pacific
coast from Sitka to Panama. It is taken in early spring and
summer at tide water where it comes to spawn. The eggs are
cemented in a single layer to the under surfaces of stones, and,
as the male remains with the brood until the young become
free-swimming (when they are about one inch in length), it
is comparatively easy to secure adults together with young.
Adults are also taken in large numbers in the trawls on the
fishing banks north of San Francisco in spring and summer.
In winter they are very scarce on the banks.
In a surface view, the phosphorescent organs appear as
bright silvery spots distributed in lines or rows over the surface
of the body of the fish. The average number of organs is about
350 on each side of the fish, 700 organs inall. Of these, 275 on
either side are located in eight lines on the ventral and ventro-
lateral surface of the body. In this region the rows are very
conspicuous (Pl. XX XVIII, Figs. 1 and 2), but more obscure
1 The material for this study was collected and the work begun at the Hopkins
Seaside Laboratory, Pacific Grove, Cal., in the summers of 1892 and 1894. Iam
deeply indebted to the directors, Dr. O. P. Jenkins and Dr. C. H. Gilbert, for the
privileges of the laboratory and for advice and encouragement. I wish also to
express my indebtedness to Dr. C. O. Whitman for the privileges of a table at
the Marine Biological Station, Woods Holl, Mass., during the summers of 1896
and 1897; also to thank the members of the instructing corps for suggestive
criticism and advice. Miss Clapp has kindly allowed me to examine her manu-
script on the Lateral-Line System of Opsanus tau, and has criticised my manuscript,
as well as given me many suggestions as to methods of making preparations. And,
finally, I am especially indebted to my wife, whose invaluable criticism and enthu-
siastic interest have been my constant support.
No. 3. PHOSPHORESCENT ORGANS. 66
9
or sometimes entirely absent over the dorsal aspect of the body.
Each organ, viewed from the surface, appears as a silvery spot
more or less circular in outline and varying in size from a point
scarcely visible with the unaided eye to .8 mm. in diameter.
The larger organs are found on the ventral surface of the
fish, the less conspicuous ones on the dorsal surface —a fact to
be again referred to later in the paper. They are often sur-
rounded by, or bordered on one side by, an increased amount
of pigment. This is especially noticeable in organs on the sides
of the body where the pigment is much increased in amount on
the side of the organ away from the mid-ventral line.
The structures, indiscriminately designated by Test as “lateral-
line organs,” “slime glands,” ‘mucous pores,” or “ pores,” are
in reality lateral-line sense organs of the kind designated as
nerve hillocks by Merkel. Such an organ, viewed from the
surface, presents a point on the epidermis free from pigment,
the end of the sense organ itself with its immediately surround-
ing supporting cells. It is too distinct and characteristic in
appearance to be mistaken by even the most casual observa-
tion.
The lines of phosphorescent organs and lateral-line organs
are so closely associated in their distribution in the skin of the
fish that they may be most economically mapped out together.
Test did not discriminate between these two sets of organs.
Terms he introduced, referring to lines of organs, will be
indicated by quotation.
II. DistRIBUTION OF THE PHOSPHORESCENT ORGANS AND
THE LATERAL—LINE SENSE ORGANS.
The organs of both systems are arranged in lines or rows
and are very constant in their relation to each other in any
given row, also the rows are constant in their relative positions
in different individual fishes. The number of organs in a given
line of either phosphorescent organs or sense organs may, how-
ever, vary much in different individuals (see Table). The
average number only will in most instances be given in the
general description.
670 GREENE. [VoL. XV.
The lateral-line organs are located free on the surface with
the exception of certain rows on the head which are in canals.
These lines of canal organs form only a small part of the lateral-
line system in Porichthys. Unless otherwise specified, surface
organs are meant.
It is impossible to determine the homologies of the parts of
the complex lateral-line system without a knowledge of the
development of the sensory Anlage and of the innervation of
the system, including the origin of the nerves in the central
nervous system. Such a study has been made in part for only
a few fishes, while the great mass of fishes remain unknown
in this regard. It is necessary, therefore, while awaiting the
development of our knowledge of the origin and distribution of
the lateral-line nerves, to designate the groups of lateral-line
organs in particular species by some sort of descriptive terms.
The intention in this paper is not so much to name the groups
in Porichthys as to describe their location by short descriptive
terms that will serve temporarily, z.e., until we have a surer
foundation for establishing homologies between the parts of
the lateral-line system in different species of fishes, when a
permanent nomenclature may be adopted.
1. Lines of Organs on the Body.
The lateral row, /a (Pl. XXXVIII, Figs. 1-3), begins
on the side at a point posterior to the upper border of the
pectoral fin and directly below the third dorsal ray. It runs
straight back along the side to the upper third of the base of the
caudal, and contains both kinds of organs. This line contains
an average of thirty-six sense organs with a phosphorescent
organ immediately below and generally one above each sense
organ. In the lower series the phosphorescent organs are well
developed and large, but the organs of the upper series are
quite small and rudimentary; in fact they are often wanting,
there being only an average of twenty-two in the fifteen speci-
mens tabulated. The organs in this line correspond very nearly
with the segments of the part of the body along which they lie.
They are found above the grooves which mark the boundary
between the myomeres.
No. 3.] PHOSPHORESCENT ORGANS. 671
The “pleural” row, f/, consists of parallel lines of phos-
phorescent organs and sense organs. The row of phosphores-
cent organs begins at a point posterior to the middle of the
base of the pectoral and below the anterior ray of the dorsal
fin. The line curves backward and downward to a point back
of the lower edge of the pectoral and above the first anal ray,
thence straight back along the side, ending usually above the
twenty-third anal ray. The organs of this line vary in number
from forty-three to sixty-two, or an average of fifty-three, and
have no relation to the body segments. The row of sense
organs is located immediately below the row of phosphorescent
organs and follows the same general course. This row usually
extends backward only to the thirteenth anal ray, but in three
specimens from Alaska (see Table, Nos. 8—1o) the row extends
to the base of the caudal fin. There is an average of thirty-
two sense organs in the row, excluding the three exceptions
mentioned. In these three there are sixty, bringing the general
average up to thirty-six. ,
There are two caudal, ca, lines of sense organs on each side
the fin, located on the upper and lower thirds, respectively.
These rows contain only sense organs, which are well devel-
oped at the base of the caudal, but become smaller toward the
extremity of the fin. There are as many as twenty-five in each
line in the oldest specimens, but the number varies greatly,
increasing with the age of the specimen. These rows are in
line with the lateral and pleural rows of sense organs, but they
are not continuous with them.
The “anal” row, a, runs along the body on either side of
the base of the anal fin from opposite the interspace between
the second and third anal rays to the base of the caudal. The
phosphorescent organs of this line correspond in position to
the anal rays and are in pairs, with exceptions to be mentioned.
The first organ is usually single; the next twenty-eight or
twenty-nine, paired; the last six or seven, single. The last
four or five organs are situated under the base of the caudal
and are arranged in a markedly compact row. There is an
average of thirty-six organs in this row, counting the pairs but
once. The outer organs of the pairs are apparently larger than
672 GREENE. [Vor. Xv.
the inner, but this appearance is in part due to the fact that
the inner organs are more deeply buried in the angle of the
base of the fin. The posterior two or three pairs are closely
united. The anal row contains a single line of sense organs.
There are thirty-four of these organs in the line. The first
is placed just in front of the second pair of phosphorescent
organs, and each successive one bears the same relation to its
corresponding phosphorescent organ, except the last two, which
are placed external to and just above the four or five phos-
phorescent organs along the base of the caudal.
The “ventral rows, v, form a parenthesis on the stomach,”
extending from the side of the anus three-fourths the distance
to the ventral fin. The anterior ends of the two rows are
usually continuous with each other, and comprise thirty-four
phosphorescent organs on either side. The organs present a
clear, circular outline without apparent increase of pigment
around them. These rows are not accompanied by sense
organs.
The “gastric” line of phosphorescent organs, ga, begins a
little below the middle of the front of the base of the pectoral,
curves forward, downward, then backward, around the lower
edge of the pectoral; then extends straight back along the
side of the belly to a point dorsal to the anterior edge of the anal
papilla. The line contains an average of thirty organs, about
ten in the curved portion and twenty in the straight part of the
line.
The “gular” line, gw, begins a little back of the isthmus
and runs parallel with its fellow backward to the posterior and
ventral side of the ventral fin, then curves outward and back-
ward to a point below the anterior end of the straight portion
of the gastric line. There is an average of twenty-seven organs
in this row. The gular line gives off a short spur of seven
organs running forward along the external border of the
ventral fin.
The gastro-gular line, ga gw, of sense organs begins at the
isthmus somewhat anterior to the end and toward the median
ventral side of the phosphorescent row, runs posteriorly parallel
to the phosphorescent line to its end, then backward parallel to
No. 3.] PHOSPHORESCENT ORGANS. 673
the gastric row, curves upward around its posterior end, and
terminates in close relation to the pleural row of sense organs
(see Pl. XXXVIII, Fig. 1). There are fifty organs in the
entire row.
The “scapular” row, sc, begins just back of the posterior
pore of the temporal canal, runs straight back above the pec-
toral fin, then curves in toward the base of the dorsal fin, where
it is continued into the dorsal row at the base of the third
dorsal ray. There are seventeen sense organs in this row, with
an average of ten phosphorescent organs alternating with them.
The straight part of the scapular row is accompanied by a
scapular accessory row, sc ac, of three to five sense organs, with
a small phosphorescent organ above each.
The dorsal row, d, extends along the dorsal surface of the
body at the base of the dorsal fin from the third dorsal ray to
the caudal peduncle. This row contains an average of seventy-
one sense organs. Rarely phosphorescent organs are found
between the first three or four organs of the row, but in such
exceptional specimens they are always small and rudimentary.
Outside the dorsal row is an irregular line, or accessory row,
d ac, which contains sense organs and rudimentary phosphores-
cent organs arranged as in the lateral line. The number of
organs in this row is quite variable, an average of sixteen in
five specimens (see Table).
2. Organs of the Lower Jaw and Head.
The branchiostegal row, 67, begins in front of the isthmus
and extends outward over the membrane of the gill-cover to the
base of the lower branchiostegal ray ; then along the membrane
between the first and second rays almost to the edge of the
gill-cover. There are thirty-four phosphorescent organs in this
line and no sense organs.
The ‘“ mandibular’ row, md, of phosphorescent organs ex-
tends around the inner rim of the ridge formed by the dentary
bones. It contains twenty-two organs on either side.
The operculo-mandibular row consists of surface organs and
canal organs. It begins on the side of the head at the anterior
pore of the temporal canal. It runs downward on the surface
674 GREENE. [VoL. XV.
to in front of the opercular spine, where it sinks into a canal.
This canal extends along the posterior border of the preopercle,
around the angle of the mouth, and forward on the ventral sur-
face of the mandible to near its anterior end, where the line
comes again to the surface and ends at the symphysis of the
jaw (Pl. XX XVIII, Fig. 2). There are eight pores or openings
to the canal portion. This line contains twenty-one organs,
seven at the upper free end, seven in the canal, and seven at
the lower free end. At the upper free end there are occa-
sionally rudimentary phosphorescent organs between the sense
organs — an average of one.
There are two “opercular” rows, an upper and a lower.
The upper row, z of, contains ten phosphorescent organs and
fourteen sense organs. These are arranged in parallel lines,
the phosphorescent organs above and the sense organs immedi-
ately below them. The line begins posterior to the second
pore from above of the operculo-mandibular canal, and extends
in a curve backward and upward, over the operculum, toa point
posterior to the opercular spine. The row of sense organs is
the longer of the two.
The lower row, / of, contains from two to ten phosphorescent
organs, on an average four, and an average of seventeen sense
organs, the phosphorescent organs occurring between the sense
organs at the base of the line. The row begins opposite the
third operculo-mandibular pore and extends backward and up-
ward to near the posterior angle of the opercular flap. The
curvature of the lower row is a little greater than that of the
upper, the two ending near each other.
The infraorbital consists of two portions, a preorbital and a
suborbital, each of sense organs. The preorbital or nasal
portion is made up of a row of four to six organs from the
posterior nasal opening to the base of the anterior nasal papilla,
two pairs of organs between the anterior nasal papillae, and two
to three more in a transverse line at the posterior base of the
tube. The subdrbital extends from the posterior nasal open-
ing around under the eye to the anterior pore of the temporal
canal. It contains fourteen sense organs and one large phos-
phorescent organ below the posterior border of the eye.
No. 3.] PHOSPHORESCENT ORGANS. 675
The temporal canal (Pl. XX XVIII, Fig. 3, ¢) corresponds to
the squamosal of Allis in Amia. It contains a single canal organ.
Above this canal are two free organs. The infraorbital, temporal,
scapular, and dorsal form a continuous series of sense organs.
A short maxillary canal, mx, with two organs, extends along
the maxillary bone below the posterior nasal opening. At its
lower end are two free organs with a small phosphorescent
organ above each in very old specimens.
A malar row of sense organs, ma, extends downward from the
suborbital across the cheek to the angle of the mouth, thence
along the mandible over the operculo-mandibular canal to its
anterior end. There are thirty-one organs in this row, thirteen
in the malar portion and eighteen in the mandibular. Two
spurs are connected with this line, a short anterior spur of two
organs from the middle of the cheek portion of the line and
a second posterior one of four to five organs from the angle
formed at the mouth.
The supraorbital line, sp 0, is enclosed in a canal which
begins by a pore at the median border of the posterior nasal
opening, runs backward and inward toward the median line of
the head, where it anastomoses with its fellow of the opposite
side at a point in a transverse line drawn through the posterior
border of the lens of the eye. Here the canals diverge and
each runs to a point behind the eye a distance equal to half the
diameter of the latter. This canal opens at its ends only.
Five sense organs are found in the canal —three in the anterior
part and two in the posterior limb.
The “frontal” group, /7, consists of two rows of organs.
It is located over the posterior end of the frontal bone, about
midway from the median line of the head to the outer edge of
its flat top. The outer row of each group consists of an aver-
age of six sense organs, with five phosphorescent organs, alter-
nating in a longitudinal row. The inner row is a shorter one,
parallel with the posterior part of the outer row, and consists
of from four to five sense organs with phosphorescent organs at
the inner and outer edge of each sense organ. The phospho-
rescent organs of this group, like those of other dorsal groups,
are especially variable in number and rudimentary.
676 GREENE, [Vou. XV.
The “occipital” row, oc, contains from nine to twelve sense
organs, sometimes with a pair of rudimentary phosphorescent
organs on the inner and outer side of each sense organ, some-
times with no phosphorescent organs. The row begins near
the posterior edge of the spinous dorsal, curves first inward
toward the median dorsal line, then outward and forward (see
Pl. XX XVIII, Fig. 1).
In a species described from the Galapagos Islands, Porich-
thys nautopedium Jordan, the arrangement of phosphorescent
organs and sense organs corresponds, group for group, with
that given above. There are only slight variations from the
average number of organs, except in the pleural row, which,
like the pleural row of three specimens from Alaska (Table,
Nos. 8-10), is continued back to the base of the caudal.
In the new species of Porichthys previously referred to,
obtained recently at Panama by Dr. Gilbert, the location of the
lines of phosphorescent organs and sense organs corresponds
very closely with that in Porichthys notatus. Also the num-
ber of both kinds of organs, as will be seen by a reference
to the appended table, Nos. 16 and 17, is very similar to the
number in the common form. It may be noted, however,
that on the dorsal surface, where in Porichthys notatus rudi-
mentary organs are found, phosphorescent organs are wholly
absent. The phosphorescent organs on the ventral surface
of this species are not more than half as large as in the
common species. :
In general, we may say that the phosphorescent organs of
the three species of Porichthys studied are always well devel-
oped and prominent along the ventral and ventro-lateral sur-
faces of the body, while along the dorsal surface they are
markedly small and rudimentary, and are very variable in
their development in the different specimens of the same
species.
The sense organs, on the other hand, are quite constant both
in their presence and the extent of their development in the
different regions. The sense organs are accompanied by der-
mal papillae, two for each organ. These dermal papillae differ
very much in the extent of their development, being most
NE SENSE ORGANS.
southern Alaska coast. Specimens 16 and 17,
| Tem- ; Supra- | Outer liner =
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TABLE SHOWING VARIATIONS IN THE NUMBER OF PHOSPHORESCENT ORGANS AND OF LATERAL—LINE SENSE ORGANS.
Specimens 1 to 15, Porichthys notatus. Numbers 1 to 7 and 11 to 15, inclusive, off the California coast. Numbers 8 to 10, off the southern Alaska coast. Specimens 16 and 17
. ’
Porichthys, sp. indsc., from Panama.
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33 | 42
No. 3.] PHOSPHORESCENT ORGANS. 677
marked in the nasal, dorsal, anal, and mandibular lines. In
these lines they often reach a length of 2 mm., and are three
and four parted at their ends.
III. SrrRucTURE OF THE PHOSPHORESCENT ORGANS.
Organs from different parts of the body have a common
general structure, differing only in minor details. It will,
therefore, be sufficient to describe a typical organ in detail and
later compare with it those organs which have a specialized
form or structure.
The epidermis of Porichthys has no scales and is richly
supplied with large, club-shaped mucous cells in all stages of
development. The dermis is quite a thick layer of dense con-
nective tissue bearing blood vessels, nerves, and pigment cells.
The phosphorescent organs are imbedded in the deeper portion
of this dermis.
Each organ consists of four parts (Pl. XX XVIII, Fig. 4, and
Pl. XX XIX¥ Figs. 5 and 9), the lens, the gland, the reflector,
and pigment.
1. Lhe Lens.
In a typical organ, from the anal or ventral row, for example,
the outer or more superficial portion of the organ consists of a
group of cells, the lens (Pl. XXXIX; Fig. 5, 7). The surface
of the lens directed toward the exterior, the distal portion, is
oval or spherical in outline, while the deeper or proximal portion
is projected into a more or less pronounced subconical form.
The cells of the lens are polygonal in form in the center of the
structure, becoming flattened toward the surface. At the distal
surface the cells are quite regular and form a pavement-like
layer (Pl. XXXIXY Figs. 5 and 9), but in the deeper conical
portion the cells are very irregular in form, often with processes
which interlace in a confused mass at the extreme proximal part.
The lens cells have a small oval nucleus which is very sharply
defined in contrast with the modified cytoplasm. The cell body
is very dense, homogeneous, and highly refractive. The co-
agulation of this dense substance by reagents often slightly
678 GREENE. [VoL. XV.
separates the cells, thus rendering their outlines in sections
very distinct.
The lens has no capillaries distributed to its substance. In
Golgi preparations nerve fibers were found distributed to the
superficial part of the lens and terminating among its cells in
small free varicose ends. The number was, however, not greater
than that found in the surrounding connective tissue of the skin
and seems, therefore, of no special significance (Pl. XXXIX;
Fig. 8).
2. The Gland.
The gland forms a shallow cup surrounding the proximal
two-thirds of the lens. It is composed of cells varying greatly
in size and shape (Pl. XXXIX¥ Figs. 5-7). The gland cells
are held in a mesh of connective tissue and capillaries and in
part by processes from the cells of the base of the lens.
They have their long diameters placed vertical to that portion
of the surface of the lens along which they lie. These cells
have large round nuclei which are often vacuolated. The
cytoplasm of the cells is very granular and is stained with
the greatest difficulty. In alcoholic material these gland cells
present an appearance which indicates that their granular con-
stituents are largely dissolved out. In fact it is impossible
to gain a true idea of the character of these cells from such
material, as I found after many trials. On the other hand, the
cells are beautifully preserved in Flemming’s fluid and present
in such preparations the structure shown in Pl. XX XIXY Figs.
6 and 7.
The gland is richly supplied with blood vessels, which enter
around the distal border and also by puncturing the reflector
below. The capillaries form a network among the gland cells
and are in sections generally filled with red and white corpuscles.
3. The Reftector.
The reflector forms one of the most striking structures of
the organ. It also is a cup-shaped mass which encloses the
gland and the proximal portion of the lens, extending up around
No. 3.] PHOSPHORESCENT ORGANS. 679
the latter for over two-thirds of its surface (Pl. XX XVIII, Fig.
4, and Pl. XX XIX¥ Fig. 5). The reflector is composed of con-
nective tissue, the matrix of which is modified into peculiar fine
strands or fibrils, called spicules. These spicules very strongly
reflect light. This property is very manifest even in the thin-
nest of sections where the reflector is dark gray or brown by
direct light but bright silvery by reflected light. The spicules
form a dense mass of fibrils somewhat regularly parallel with
the surface of the lens. Small oval nuclei are scattered
throughout the reflector, also a certain number of ordinary
connective-tissue fibrils are scattered among the spicules, espe-
cially toward the periphery of the cup. That these fibrils are
not ‘calcareous spicules,” as von Lendenfeld describes for
phosphorescent organs of Scopelus and other deep-sea forms,
is evident, since they are not altered by nitric acid, nor, in fact,
by any of the numerous fixing reagents used in their preparation.
Small blood vessels are found in the reflector, but only as
they pierce the structure to reach the gland within.
4. The Pigment.
The pigment mass is composed of the large, many-branched
type of cells characteristic of the pigment cells of the skin of
fishes and amphibia generally. The cells are located around
the outer and deeper surface of the reflector and vary in number
in different organs. They are sometimes so numerous as to
form dense masses, and again, as in the ventral or anal rows,
there may be only three or four such cells to the organ.
IV. Nerve Supply OF THE PHOSPHORESCENT ORGANS.
A most diligent and persistent effort was made to demon-
strate the presence of a special nerve supply to the phospho-
rescent organs. Numerous preparations of the skin containing
the organs were prepared by the methods which give specific
nerve staining. By the iron-haematoxylin method and by the
gold-chloride method, no nerves could be distinguished in the
organs. By the Golgi method beautiful preparations were
680 GREENE. [Vou. XV.
obtained showing the distribution of nerves to the skin and
to the epidermis, but in only two sections were nerves shown
to have direct relation to the phosphorescent organ. The
better one of these sections (Pl. XXXIX/ Fig. 8) showed
nerves branching over the surface of the lens. Whole mounts
of the skin were made by the methylen-blue method of Bethe.
These showed the most detailed network of nerve fibers lying
in the connective tissue of the skin. Preparations containing
both phosphorescent organs and lateral-line sense organs
showed branches of the lateral-line nerve coming off the main
stem and ending in the sense organs with almost diagrammatic
regularity. Nerve fibers or bundles of nerve fibers were found
in the skin above or below the phosphorescent organs, but no
nerve bundles penetrated the phosphorescent organs and ended
there. Two organs out of a very large number prepared con-
tained each a single nerve fiber which entered the organ and
terminated there. In one or two others single fibers seemed
to terminate in the organ, but the fact could not be determined
with certainty.
The facts set forth above, based on very favorable prepara-
tions, justify the conclusion that the phosphorescent organs of
Porichthys possess no specific nerve supply. The few individual
nerve fibers demonstrated to enter the organ may be considered
as branches from the general nerve supply of the skin.
V. ORIENTATION OF THE ORGANS WITH REFERENCE TO
THE SURFACE OF THE Bopy.
A line passing through the middle of the three parts of the
phosphorescent organ may be taken as its axis, and the position
of this axis with reference to the body surface as the direction
or position of the organ. In the ventral rows of the body the
organs are directed downward, that is, the axes of the organs
are almost or quite vertical with the surface of the body of the
fish. The organs on the sides of the body and the lower part
of the head are directed downward and slightly outward, and
the axis of each organ thus makes quite a wide angle with the
vertical to the surface, In the pleural row this angle is from
No. 3.] PHOSPHORESCENT ORGANS. 681
30° to 40°, while in the lower series of the lateral row it is
often 80° or more ; in fact the axis is sometimes quite tangent
to the surface of the body at that point. In this class of
organs a variation from the typical arrangement of the parts
may be noted. In a transverse section of such an organ, say
from the pleural row, the deeper part of the cup of the reflector
extends inward and upward (Pl. XXXIX% Fig. 10) ; that is,
toward the dorsal part of the fish. It forms a somewhat deeper
pocket than is formed by organs on the ventral surface. In
such specimens the pigment is amassed around the upper or
dorsal portion of the reflector. In these organs on the side
of the body the conical base of the lens is relatively longer and
is always in the axial line of the organ. The cells of the gland
in such organs are arranged radially around the conical part of
the lens.
The organs on the dorsal surface of the body and head have
their axes vertical to the surface of the body. All these
organs are small and rudimentary and irregular in their develop-
ment, as shown by the fact that the lens is small and irregular
in form, that the gland and reflector have more ordinary
connective tissue in their structure, and especially by the
inconstancy in the presence of the dorsal organs in different
individual fishes.
VI. DEVELOPMENT OF THE PHOSPHORESCENT ORGANS.
The phosphorescent organs arise quite late in the develop-
ment of the embryo. In skins of embryos 8.5 to 8.9 mm.
in length, and in a transverse serial section of an embryo not
measured but probably of about the same length, I find the
first or incipient stages in the development of the rows of
phosphorescent organs on the ventral surface of the body.
The organs appear first in.the ventral, gastric, branchiostegal,
and apparently also in the anal rows at the same time. In the
anal row it is not easy to determine their first appearance, owing
to the changes accompanying the development of the fin.
In embryos 8 mm. in length the sensory Anlage of the lateral-
line system is complete and the sense organs are sufficiently
682 GREENE. [Vou. XV.
well differentiated to be counted in skins. But the ventral and
branchiostegal rows are not accompanied by sense organs, and,
therefore, furnish a crucial test as to the origin of the organs.
In the serial sections of the embryo mentioned above, the basal
layer of cells of the epidermis of the ventral side of the body is
slightly thickened in the region occupied by the adult ventral
line (Pl. XL¥ Fig. 13). The thickening is produced by a more
rapid increase in the number of the epidermal cells in the
particular area.
In the gastric row of the same specimen there is a similar
multiplication of cells just above the accompanying sensory
Anlage (Pl. XL} Fig. 14). In this case the multiplication
of cells shows a sort of center toward which the surrounding
nuclei perceptibly converge. This is the beginning of a cell
aggregation which soon proliferates into well-marked centers or
groups, the antecedents of the individual organs of the line.
In this incipient stage of the gastric organs the cells lie imme-
diately against the cells of the sensory Anlage of the gastro-
gular row of sense organs, but they show no other evidence of
origin from it. In skins it happened in one instance that the
sensory Anlage of this row was torn free of the epidermis, and
the cells along its upper border were apparently undisturbed.
They were slightly increased in number, however, indicating a
stage comparable to that from the ventral line figured in cross-
section in Pl. XL} Fig. 13, or in the corresponding line in
JPL GL 1g, Tak.
Although it would seem impossible to affirm that the origin
of the phosphorescent organs in the gastric row is independent
of the sensory Anlage which has arisen by migration of cells,
yet Iam convinced that such is the case. The above facts,
especially the independence of the origin of the rows above
mentioned not associated with sense organs, form the basis
of my belief that the phosphorescent organs arise by local pro-
liferation of cells from the epidermis in the region which they
permanently occupy.
The further progress in the development of the phosphores-
cent organ consists in the rapid multiplication of cells, giving
rise to a distinct nodule which projects as a small papilla from
No. 3.] PHOSPHORESCENT ORGANS. 683
the inner surface of the epidermis (Pl. XL¥ Figs. 15 and 16).
The orientation of the organ is determined in this very early
stage. Organs of the ventral line are vertical to the surface
of the skin, while organs of the ventro-lateral surfaces are
oblique (Pl. XL¥ Figs. 15-20). In embryos 13 mm. long this
papilla, in vertical section of ventral organs, presents the
general outline of a finger-tip. It has a diameter of about six
cells and is four to five cells deep. Pigment cells now appear
in the connective tissue beneath the organ and are found in
almost every section. In skins they show as the much-branched
type of pigment cell spreading over the inner surface of the
papilla. In specimens 14 mm. long there are from three to
six such cells around each organ.
The next stage consists in the gradual separation of this
papilla from the epidermis. The papilla becomes constricted
where its sides are continuous with the general epidermis, the
constriction continuing until complete separation occurs. At
the same time a new layer of columnar cells forms in the
epidermis and all evidence of the former union is obliterated.
The separated mass now has a diameter of about .o4 mm. and
is found in embryos 18 to 20 mm. in length.
Soon after separation from the epidermis occurs, in fact
before in some instances (Pl. XXXIXj Fig. 12, and Pl. XL;
Fig. 21), the structure elongates slightly in the line of the
axis of the developing organ and a differentiation occurs near
the base, enabling one to distinguish in the mass two parts, an
outer to become the lens and an inner the gland. No separa-
tion occurs between the two portions, yet the cells of each
become more and more specialized in the direction of the cells
of the adult lens and gland, respectively. By the time the
embryo becomes free swimming, a length of about 25 mm.,
the organs possess the general characters of adult organs.
Later growth consists in a great increase in size, due to the
multiplication of the cells in the lens and gland, respectively.
Accompanying the differentiation of the lens and the gland
there is a corresponding differentiation of the capsule. The
connective-tissue cells of the dermis first form a cup-shaped
aggregation around the base of the epidermal portion of the
684 GREENE. [Vou. XV.
developing organ (Pl. XL Figs. 21, 23, and 24). The matrix
of these connective-tissue cells is gradually converted into
the modified fibrils which characterize the adult reflector
(Pl. XX XIX? Figs. 9-11, and Pl. XL; Fig. 24). These fibrils
are at first intermixed with a large amount of ordinary con-
nective-tissue strands but ultimately form almost the entire
mass. The pigment cells likewise increase in number and
form masses of cells about the reflector, especially in organs
on the side of the body.
Organs in different rows do not appear at the same time.
Those on the mandible and ventral surface of the body appear
first, and since they reach the highest development are taken as
types. The lower series of the lateral line at 20 mm. is not
farther advanced than organs of the ventral line at 11 to 12
mm. Those organs above the lateral line appear later and are
always very rudimentary. In fact, such organs as I have des-
ignated rudimentary are never present above the lateral line
and on the dorsal surface of the body up to the time when the
embryos become free-swimming, a length of at least 25 mm.
VII. Funcrion oF THE PHOSPHORESCENT ORGANS.
I have kept specimens of Porichthys in aquaria at the Hop-
kins Seaside Laboratory, and have made numerous observations
on them with an effort to secure ocular proof of the phospho-
rescence of the living active fish. The fish was observed in
the dark when quiet and when violently excited, but, with a
single exception, only negative results were obtained. Once
a phosphorescent glow of scarcely perceptible intensity was
observed when the fish was pressed against the side of the
aquarium. Then, this is a shore fish and quite common, and
one might suppose that so striking a phenomenon as it would
present if these organs were phosphorescent in a small degree
would be observed by ichthyologists in the field, or by fisher-
men, but diligent inquiry reveals no such evidence.
Notwithstanding the fact that Porichthys has been observed
to voluntarily exhibit only the trace of phosphorescence men-
tioned above, still the organs which it possesses in such num-
No. 3-] PHOSPHORESCENT ORGANS. 685
bers are beyond doubt true phosphorescent organs, as the
following observations will demonstrate.
A live fish put into an aquarium of seawater made alkaline
with ammonia water, exhibited a most brilliant glow along the
location of the well-developed organs. Not only did the lines
of organs shine forth, but the individual organs themselves
were distinguishable. The glow appeared after about five
minutes, remained prominent for a few minutes, and then for
twenty minutes gradually became weaker until it was scarcely
perceptible. Rubbing the hand over the organs was followed
always by a distinct increase in the phosphorescence. Pieces
of the fish containing the organs taken five and six hours after
the death of the animal became luminous upon treatment with
ammonia water.
Electrical stimulation of the live fish was also tried with
good success. The interrupted current from an induction coil
was used, one electrode being fixed on the head over the brain
or on the exposed spinal cord near the brain, and the other
moved around on different parts of the body. No results fol-
lowed relatively weak stimulation of the fish, although such
currents produced violent contractions of the muscular system
of the body. But when acurrent strong enough to be quite
painful to the hands while handling the electrodes was used,
then stimulation of the fish called forth a brilliant glow of light
from apparently every well-developed organ in the body. All
the lines on the ventral and lateral surfaces of the body glowed
with a beautiful light, and continued to do so while the stimu-
lation lasted. The single well-developed organ just back of
and below the eye was especially prominent. No luminosity
was observed in the region of the dorsal organs previously
described as rudimentary in structure. I was also able to
produce the same effect by galvanic stimulation, rapidly making
and breaking the current by hand.
The light produced in Porichthys was, as near as could be
determined by direct observation, a white light. When pro-
duced by electrical stimulation it did not suddenly reach its
maximal intensity, but came in quite gradually and disappeared
in the same way when the stimulation ceased. The light was
686 GREENE. [VoL. XV.
not a strong one, only strong enough to enable one to quite
easily distinguish the apparatus used in the experiment.
An important fact brought out by the above experiment is
that an electrical stimulation strong enough to most violently
stimulate the nervous system, as shown by the violent con-
tractions of the muscular system, may still be too weak to
produce phosphorescence. This fact gives a physiological
confirmation of the morphological result stated above that no
specific nerves are distributed to the phosphorescent organs.
I can explain the action of the electrical current in these
experiments only on the supposition that it produces its effect
by direct action on the gland.
The experiments just related were all tried on specimens of
the fish taken from under the rocks where they were guarding
the young brood. Two specimens, however, taken by hooks
from the deeper water of Monterey bay, could not be made to
show phosphorescence either by electrical stimulation or by
treatment with ammonia. These specimens did not have the
high development of the system of mucous cells of the skin
exhibited by the nesting fish. My observations were, how-
ever, not numerous enough to more than suggest the pos-
sibility of a seasonal high development of the phosphorescent
organs.
Two of the most important parts of the organ have to do
with the physical manipulation of light — the reflector and the
lens, respectively. The property of the reflector needs no
discussion other than to call attention to its enormous develop-
ment. The lens cells are composed of a highly refractive
substance, and the part as a whole gives every evidence of
light refraction and condensation. The form of the lens gives
a theoretical condensation of light at a very short focus. That
such is in reality the case, I have proved conclusively by exami-
nation of fresh material. If the fresh fish be exposed to direct
sunlight, there is a reflected spot of intense light from each
phosphorescent organ. This spot is constant in position with
reference to the sun in whatever position the fish be turned
and is lost if the lens be dissected away and only the reflector
left. With needles and a simple microscope it is comparatively
No. 3.] PHOSPHORESCENT ORGANS. 687
easy to free the lens from the surrounding tissue and to examine
it directly. When thus freed and examined in normal saline, I
have found by rough estimates that it condenses sunlight to a
bright point a distance back of the lens of from one-fourth to
one-half its diameter. I regret that I have been unable to make
precise physical measurements.
The literature on the histological structure of known phos-
phorescent organs of fishes is rather meager and unsatisfactory.
Von Lendenfeld describes twelve classes of phosphorescent
organs from deep-sea fishes collected by the Challenger expe-
dition. All of these, however, are greater or less modifications
of one type. This type includes, according to von Lendenfeld’s
views, three essential parts, z.e., a gland, phosphorescent cells,
and a local ganglion. These parts may have added a reflector,
a pigment layer, or both ; and all these may be simple or com-
pounded in various ways, giving rise to the twelve classes.
Blood vessels and nerves are distributed to the glandular
portion. Of the twelve classes direct ocular proof is given
for one, 2.¢., ocellar organs of Scopelus which were observed
by Willemoes Suhm at night to shine “like a star in the net.”
Von Lendenfeld says that the gland produces a secretion, and
he supposes the light or phosphorescence to be produced either
by the “burning or consuming” of this secretion by the phos-
phorescent cells, or else by some substance produced by the
phosphorescent cells. Furthermore, he says that the phos-
phorescent cells act at the “will of the fish” and are excited
to action by the local ganglion.
Some of these statements and conclusions seem insufficiently
grounded, as, for example, the supposed action of the phos-
phorescent cells, and especially the control of the ganglion
over them. In the first place, the relation between the ganglion
and the central nervous system in the forms described by von
Lendenfeld is very obscure, and the structure described as a
ganglion, to judge from the figures and the text descriptions,
may be wrongly identified. At least it is scarcely safe to
ascribe ganglionic function to a group of adult cells so poorly
preserved that only nuclei are to be distinguished. In the
second place, no structural character is shown to belong to the
688 GREENE. [Vou. XV.
“phosphorescent cells’ by which they may take part in the
process ascribed to them.!
The action of the organs described by him may be explained
on other grounds, and entirely independent of the so-called
“ganglion cells’’ and of the ‘“ phosphorescent cells.”
Phosphorescence as applied to the production of light by a
living animal is, according to our present physiological-chemical
notions, a chemical action, az oxidation process. The necessary
conditions for producing it are two —an oxidizable substance
that is luminous on oxidation, z.e.,a photogenic substance on the
one hand, and the presence of free oxygen on the other. Every
phosphorescent organ must have a mechanism for producing
these two conditions ; all other factors are only secondary and
accessory. If the gland of a firefly can produce a substance
that is oxidizable and luminous on oxidation, as shown as far
back as 1828 by Faraday, and confirmed and extended recently
by Watasé, it is conceivable, indeed probable, that phosphores-
cence in Scopelus and other deep-sea forms is produced in the
same direct way, that is, by direct oxidation of the secretion of
the gland found in each of at least ten of the twelve groups of
organs described by von Lendenfeld. Free oxygen may be
supplied directly from the blood in the capillaries distributed
to the gland which he describes. The possibility of the regula-
tion of the supply of blood carrying oxygen is analogous to,
what takes place in the firefly and is wholly adequate to account
for any “flashes of light” “‘at the will of the fish.”
In the phosphorescent organs of Porichthys, the only part
the function of which cannot be explained on physical grounds
is the group of cells called the gland. If the large granular
cells of this portion of the structure (Pl. XXXVIII, Fig. 4,
and Pl. XXXIX?¥ Figs. 5-7) produce a secretion, as seems
probable from the character of the cells and their behavior
toward reagents, and this substance be oxidizable and luminous
1 The cells which von Lendenfeld designates “ phosphorescent cells’? have as
their peculiar characteristic a large, oval, highly refracting body imbedded in the
protoplasm of the larger end of the clavate cells. These cells have nothing in
common with the structure of the cells of the firefly known to be phosphorescent
in nature. In fact, the true phosphorescent cells are more probably the “ gland
cells” found in ten of the twelve classes of organs which he describes.
No. 3.] PHOSPHORESCENT ORGANS. 689
in the presence of free oxygen, 7.¢., photogenic, then we have
the conditions necessary for a light-producing organ. The
numerous capillaries distributed to the gland will supply free
oxygen sufficient to meet the needs of the case. Light pro-
duced in the gland is ultimately all projected to the exterior,
either directly from the luminous points in the gland or reflected
outward by the reflector, the lens condensing all the rays into
a definite pencil or slightly diverging cone. This explanation
of the light-producing process rests on the assumption of a
secretion product with certain specific characters. But com-
paring the organ with structures known to produce such a sub-
stance, z.e., the glands of the firefly or the photospheres of
Euphausia, it seems to me the assumption is not less certain
than the assumption that twelve structures resembling each
other in certain particulars have a common function to that
proved for one.only of the twelve.
I am inclined to the belief that whatever regulation of the
action of the phosphorescent organ occurs is controlled by the
regulation of the supply of free oxygen by the blood stream
flowing through the organ; but, however this may be, the
essential fact remains that the organs in Porichthys are true
phosphorescent organs.
STANFORD UNIVERSITY, CAL.,
August 13, 1898.
690 GREENE.
"84
89
96
90
‘81
64
'72
85
89
‘87
95
LITERATURE.
Emery, E. Phosphorescent Organs in Scopelus. Mittheil. a. d.
Zool. Station zu Neapel. Bd. v.
EIGENMANN, C. H. AND R. I. On the Phosphorescent Spots of
Porichthys Margaritatus. West. Amer. Scient. Vol. vi. 1889.
GIESBRECHT, W. Ueber den Sitz der Lichtentwicklung in den Photo-
sphaeren der Euphausiiden. Zool. Anz. Bd. xix, 486.
LANGLEY, S. P., AND VERY, F. W. On the Cheapest Form of Light.
Amer. Journ. Sci. Third Series. Vol. xl, No. 236.
LreypiG, F. Die Augenahnlichen Organe der Fische. Bonn. 1881.
LEuUCKART, R. Ueber muthmassliche Nebenaugen bei einem Fische.
Bericht a. d. Versamml. deutsch. Naturf. zu Giessen. 1864.
PANCERI. (Paperson Phosphorescence.) Azz. des Sct. Vat. Tome
xlv. 1872.
Sars, G. O. Schizopoda. Challenger. Vol. xiii.
Test, F. C. New Phosphorescent Organs in Porichthys. 2z//.
Essex Inst. Vol. xvi.
VON LENDENFELD, R. The Structure of Phosphorescent Organs of
Fishes. Challenger. Vol. xxii, p. 276.
WartTAsE, S. Physical Basis of Animal Phosphorescence. Bod. Lect.
Marine Biological Laboratory, Woods Holl. 1895.
692 GREENE.
EXPLANATION OF PLATE XXXVIII.
Fics. 1 and 2. Lateral and ventral view of the lines of phosphorescent organs
and lateral-line organs in the adult of Porichthys notatus. The representation of
organs isdiagrammatic. Small circles show the location of phosphorescent organs,
and the dots the lateral-line organs. Iam indebted to my friend, Edward Hughes,
for the outlines upon which these two figures are constructed.
a anal. md mandibular.
ér_branchiostegal. mx maxillary.
ca caudal. oc occipital.
d@ dorsal. of m operculo-mandibular.
dac dorsal accessory. pl pleural.
Jr frontal. se scapular.
ga gastric. sc ac scapular accessory.
gagu gastro-gular. sup o supraorbital.
gu gular. ¢ temporal.
Za lateral. u op upper opercular.
2 op lower opercular. vy ventral.
ma malar.
Fic. 3. Lines and canals on the head. For explanation, see Figs. 1 and 2.
Figs. 4 to 24 are drawn with camera lucida. Figs. 4 to 8 represent the
structure of adult phosphorescent organs, and Figs. 9 to 24 show the stages in
the development of the phosphorescent organs of Porichthys notatus. The
orientation of each organ with reference to the dorso-ventral plane of the fish is
indicated by a short arrow pointing ventrally. Every section of epidermis shows
numerous large mucous cells, many of which are empty.
Fic. 4. Cross-section of an adult organ in a new Porichthys from Panama.
The gland shows several capillaries, all in cross-section but one. /, lens; g/, gland;
y, reflector; 2, pigment; 4/, blood vessel.
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694 GREENE.
EXPLANATION OF PLATE XXXIX?
Fic. 5. Cross-section of a ventral organ in Porichthys notatus Girard. 7, lens;
g/, gland; ~, reflector; 4/, blood vessels. In this section only a small amount of
pigment shows to the left.
Fic. 6. Section of the deeper portion of a phosphorescent gland highly mag-
nified to show the character of the cells. Flemming preparation.
Fic. 7. A somewhat oblique section of the deeper portion of a gland a little
to one side of the center of an organ. Flemming preparation.
Fic. 8. The nerves distributed around the lens. Lens in outline. Golgi
preparation.
Fics. 9-11. Sections through a ventral, a pleural, and a lateral organ, respec-
tively, of an embryo just before becoming free-swimming. The orientation of
the organ with reference to the epidermis varies according to the position on the
body. These three figures should follow in stage of development that shown
in Fig. 24.
Fic. 12. Section of an embryonic organ just separating from the epidermis.
This organ shows a differentiation even before it is cut off from the epidermis.
Compare with Figs. 21 to 23.
Pl. XXX
Journal of Morphology. Vol. XV.
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696 GREENE.
EXPLANATION OF PLATE XL?
Fic. 13. Cross-section of a ventral organ at its first recognizable stage. Only
a slight aggregation of cells in the basal layer of the ectoderm has appeared.
This row is not associated with a lateral-line sense-organ Anlage. Embryo about
9 mm. long.
Fic. 14. Cross-section of gastric organ associated with sense-organ Anlage,
san. From the same embryo as Fig. 13.
Fic. 15. Cross-section of a pleural organ of an embryo 14 mm. long. The
sensory Anlage shows to the left of organ.
Fic. 16. Section of a gular organ just external to the base of the ventral fin.
Same embryo as Fig. 15.
Fic. 17. An organ from the same region as Fig. 16, but associated with a
sense-organ.
Fic. 18. A pleural organ slightly older but from the same embryo as Fig. 15.
s an, sensory Anlage.
Fic. 19. An inner anal organ beginning to separate from the epidermis. Same
embryo as Fig. 15.
Fic. 20. Section through a pair of anal organs, showing a different degree of
development. Same embryo as Fig. 15.
Fics. 21 and 22. Organs in process of separation from the epidermis. Com-
pare with Fig. 12.
Fics. 23 and 24. Organs separated from the epidermis. The lens, 7, and the
gland, g/, are distinguishable, and the reflector, 7, is forming from the connective
tissue. Pigment cells, #, are arranged around the outside of the reflector.
l Morphology. Vol. XV.
Journal of Morphology 0 Pi XESS
ON THE SPECIES CLINOSTOMUM
HETEROSTOMUM.
W. G. MacCALLUM,
Jouns Hopkins HospriTat,
RUDOLPHI (1) in 1809 described a worm found by one Andreas
Jurine in the oesophagus of Ardea purpurea, and named it
Distomum heterostomum. The main points in his description
were briefly as follows: the worm measured 3 lines in length
by 1 in width; the anterior fourth of its body formed a distinct
neck; the anterior sucker, which was large, with a triangular
aperture, was ventrally situated and subterminal —a swollen
margin surrounded it. The ventral sucker, situated near the
anterior, was smaller and deeper, with a more longitudinal aper-
ture tending to be triangular. A third orifice, situated about
one line behind this, was thought to give exit to the hidden
cirrus. The body appeared transversely striated in preserved
specimens: cirrus situated behind the ventral sucker.
In 1819 (2) he outlined this description in his Syxopszs.
In 1845 Dujardin (3) gave a description evidently based on
this one of Rudolphi, for he added no single point in the anat-
omy, and probably merely translated Rudolphi’s words.
Diesing (4) in 1850 very briefly repeated this description
without any further observations. He, however, gave another
reference as to its habitat, — Rosa (5), — where it is said to be
found under the tongue of Avdea purpurea as well as in the
oesophagus.
R. Ramsay Wright (6) in 1879 described specimens from the
mouth of Botaurus minor and gave a figure. He discussed the
close relationship of D. heterostomum with D. complanatum, D.
hians, and D. dimorphum, and suggested that the relative size of
the suckers, on which much stress was laid in the differentiation
of these species, may have been inaccurately observed, the promi-
nent border about the mouth being taken for the anterior sucker ;
and in this suggestion one must agree with him entirely. He
698 MacCALLUM. [Vou. XV.
corrected the old statement of the relation in size of the suckers
in this form, giving the measurement of the anterior at 0.3 mm.,
and of the ventral ato.8 mm. The pigmentation of the intes-
tinal coeca and the existence of diverticula were described and
a figure given. The main canals and caudal pore, as well as
the subcuticular network of the excretory system, were described
and the communication with the interstices of the parenchyma
emphasized. The genital apparatus was partly worked out —
vitellogens, testes, ovary, and a receptaculum seminis were men-
tioned, together with another structure situated anterior to the
testes of unknown function. This was undoubtedly the cirrus.
The measurements of the eggs were given at 0.099 mm. by
0.066 mm.
Von Linstow (7) in 1883 gave a brief description of the worm,
making the measurement of the anterior sucker slightly larger
than that of the ventral. He described the quadrangular sac
filled with eggs which lies behind the acetabulum, and gave the
measurements of the eggs at.11 mm. by .079 mm. He also
mentioned the position of the two lobulated testes, the ovary
and yolk-gland. His specimens were found in Ardea nycticorax.
Again (8), in 1886, in his description of some of the Fedts-
chenko material collected in Turkestan, he described and figured
the worm. His description adds nothing to what was known of
the anatomy, however, and the figure shows only the suckers,
eggs, testes, ovary, part of the intestinal coeca, and a suggestion
of the subcuticular excretory canals.
Stossich (9) in 1892 gave von Linstow’s description.
Since this, Parona, in 1896, has mentioned the worm, but
otherwise it has not been noticed.
My own observations on the anatomy of this form are as
follows: Ten specimens were found adhering to the floor of
the mouth and sides of the tongue of a great blue heron (Ardea
herodias) shot on the Grand River at Dunnville, Ontario, Can-
ada. They measured 6-10 mm. in length and 1-2 mm. in
breadth during life, although, of course, the measurements vary
greatly with the degree of contraction. They are of a dull
reddish color, the intestines showing through as brownish black
lines, one on each side of the body, and the testes and uterus,
No. 3.] CLINOSTOMUM HETEROSTOMUM. 699
with its contained eggs, as whitish masses. The body is flat-
tened and hollowed ventrally, the neck being more terete and
narrower, but also capable of being flattened and hollowed
ventrally.
The anterior sucker is not large, but appears large from
being surrounded by a mobile lip made up of a protrusion of
the body wall. It is situated ventrally, slightly behind the
anterior extremity. The acetabulum, situated some distance
behind this, at the junction of the first and second thirds of
the body, is larger than the anterior sucker in my specimens.
At about the middle of the portion of the body behind the
ventral sucker, the genital openings are seen close together,
that of the female apparatus being directly in front of the male
opening. The skin is unarmed.
The anterior sucker is situated on a slight eminence, and is
entirely surrounded by the lip, which seems to assist in the
physiological action of the sucker. The cavity runs upward
and backward, and narrows suddenly into the very short
oesophagus, which is provided with a muscular pharynx. The
musculature of the sucker is composed of three layers, meridi-
onal, radial, and equatorial, or arcuate. The pharynx, similarly
constructed and provided with a thick cuticular lining, opens
directly dorsally into the much pigmented intestine, which
branches almost immediately, sending the two coeca to the
posterior end of the body. These coeca remain simple, but
become much sacculated by the development of folds in the
walls. The musculature of the intestine is limited to a single
longitudinal layer. It is the arrangement of the mucosa with
its extensive pigmentation, however, that offers one of the most
striking characteristics of this species. The wall of the coe-
cum is thrown up into high folds resembling somewhat the
valvulae conniventes of the human intestine, and giving on
section, when the coecum is cut tangentially, a ladder-like
appearance. The epithelium is in a single layer, and consists
of very tall cells tapering somewhat toward the lumen of the
tube. These stain deeply at the base, where the rounded
nucleus is situated (Pl. XX XIX, Fig. 6), but not so well centrally
— possibly this appearance is partly due to their being thinner
700 MACCALLUM. [VoL. XV.
there. They are densely loaded toward the free margin with
granules and larger masses of a brown pigment.
The ventral sucker is larger than the anterior, and is situated
somewhat deeply in the body of the worm, the margins appear-
ing rather depressed. The musculature is as usual made up of
three sets of fibers—an internal running in a somewhat tri-
angular course parallel to the inner surface of the sucker, and
provided with a triradiate fibrous intersection (Pl. XXXIX,
Fig. 1) —a set of radial fibers making up the bulk of the sucker,
and an external layer disposed in fasciculi over the surface of
the sucker. The radial fibers sometimes cross in their course
from the outer to the inner wall. In the external layer there is
a fibrous point near the center, from which the fasciculi radiate
backward; in front of this, however, they arch irregularly in
a transverse direction.
There are no peculiarities in the arrangement of the body
musculature ; longitudinal, transverse, and two diagonal layers
are present in their usual relations. There is, however, a reén-
forcement of the transverse layer in the neck region. The
skin is quite unarmed and smooth, and I have been able to
observe none of the transverse striations spoken of by some
authors (Rudolphi (1), etc.). It is probable that this appear-
ance was given by the delicate canals of the watervascular
system, which form a rich network in the subcuticular tissues.
The gland-like cells which lie under the superficial musculature
seem more than usually numerous in this species, and form a
thick layer. The parenchyma in the anterior portions of the -
body is made up of large oval cells with large round nucleus
provided with a deeply staining nucleolus (Pl. XX XIX, Fig. 5).
The protoplasm of these cells is very granular, the granules
taking on a sharp eosin stain. In the posterior parts of the
body the structure of the parenchyma is less apparent, and the
definite cell bodies give place to a vacuolated reticular formation,
with smaller scattered deeply staining nuclei.
The watervascular system is made up in this form of four
parts —the ciliated funnels giving rise to fine tubules, the
ciliated canals forming the subcuticular network, the lateral
canals, also ciliated, and the paired caudal reservoir with the
No. 3.] CLINOSTOMUM HETEROSTOMUM. 701
caudal pore. The subcuticular canals joining one another for
the most part at right angles, the transverse canals being much
wider than the longitudinal, form a most conspicuous network,
which may be injected with carmine by certain methods (R. R.
Wright (6) ). A connection exists between this network and
the lateral longitudinal canals which run at the sides external
to the intestinal coeca. These lateral canals arise from two
branches on each side anteriorly, the smaller joining the larger
at about halfway, and posteriorly they run into pear-shaped
dilatations which open by a common sphinctered terminal ori-
fice. These dilatations near the opening are lined by rather
high club-shaped epithelial cells.
The nervous system (Pl. XX XIX, Fig. 4) consists of a large
commissure running dorsal to the muscular pharynx, and con-
necting two ganglionic swellings. From this are given off
several small nerves to the anterior extremity, and backward
there run four main trunks, two dorsal arising from the com-
missure, and two lateral running from the ganglionic swellings
ventral to the coeca toward the posterior end of the body. A
short distance behind the bifurcation of the intestine these
ventral cords give off a branch which runs marginally back-
ward. The finer ramifications of these nerves have not been
traced. :
We have left for consideration the genital apparatus. The
male portion of this apparatus consists of two median testes
with their vasa efferentia and a complicated cirrus.
The testes are large, and situated one behind the other in
the median line, the left being anterior; their outline is not
simple, but they tend to be lobulated. The vasa efferentia are
wide and dilated at points with spermatozoa; they unite im-
mediately before joining the cirrus sac. The penis (Pl. XX XIX,
Fig. 3) consists of a long convoluted, rather thick-walled sac
filled with spermatozoa, into which the vasa efferentia, or rather
the short vas deferens formed by the union of these tubes, opens.
This sac runs directly into a narrow, very muscular tube, which
shows active peristaltic movements during life. At the end of
this there is, apparently, a differentiated portion of the muscu-
lature which acts asa sphincter, making a dividing line between
702 MacCALLUM. [VoL. XV.
this part of the tube and the dilated portion which follows, and
which has a peculiarly modified cuticular lining. This dilated
portion of the tube is surrounded by a thick layer of deeply
staining cells very closely resembling those found in the general
subcuticular tissues. The cuticular lining is furnished with
large, highly refractive polygonal masses of cuticular substance.
In sections these are sometimes detached. The subcuticular
circular musculature is disposed much as it is in the general
body surface. Narrowing again, this dilated portion passes to
the external orifice, which is a crescentic slit immediately behind
the opening of the female apparatus.
In the testes there is a somewhat alveolar structure, and the
formation of spermatozoa seems to take place by the production
of a morula-like mass from the dividing cell, the division prod-
ucts of the nucleus being peripheral; by the disintegration
of this mass, as each nuclear particle is provided with a
certain portion of the protoplasm, a number of spermatozoa
are formed.
The female apparatus consists of an ovary, yolk-gland,
Laurer’s canal, and uterus, with their connecting ducts. The
ovary is triangular and situated between the two testes on
the right side. It is quite small, being less than one-fourth the
size of either testis, and has a median position dorsoventrally
in the body. It is somewhat lenticular in cross-section. The
ciliated oviduct runs upward and forward from its anterior por-
tion to join the other ducts leading to the uterus. The vitel-
larium, beginning at the level of the middle of the acetabulum,
and extending back to the posterior end of the body, is made
up of separate acini which send ductlets to form a transverse
duct on each side, and these transverse ducts uniting in the
center form a somewhat ventrally placed dilatation or reservoir.
The nuclei of the cells making up the yolk-gland stain deeply
with haematoxylin, while the yolk-granules stain brightly with
eosin.
From the yolk-reservoir there are given off two tubes, one
of which, running laterally, is thick-walled and widens into the
uterus. The other tube runs forward and upward (this also
is thick-walled and ciliated), and at a point where it becomes
No. 3.] CLINOSTOMUM HETEROSTOMUM. 793
dilated receives the oviduct, which has curved on itself and
now runs backward and downward. The dilatation contains
yolk-granules, spermatozoa, and occasional ova, and is continued
into a third ciliated canal which runs forward and upward to
open dorsally into the median line by a circular aperture. This
is Laurer’s canal.
The walls of the first dilatation of the uterine tube are very
mobile, and may be considered to form an odtyp as the egg
there receives the shell. It is surrounded by a number of large
cells with large round nucleus and nucleolus, and deeply stain-
ing granular protoplasm which probably function as a shell
gland, although such a relation could not be directly deter-
mined.
In many specimens the proximal portion of the uterine tube
contains great numbers of spermatozoa which lie in the inter-
stices between the eggs. The uterine tube is rather thin-walled
and short, and after making a few convolutions in the area
between the testes it runs forward to join at its left posterior
angle the large quadrangular sac, which forms, when full of eggs,
so conspicuous a feature in the living worm. The walls of the
sac, which occupies the whole space between the acetabulum
and the anterior testis, and the intestinal coeca laterally, are
rather thicker than those of the uterus proper. They are like
the rest of the uterine wall, underlaid by a layer of deeply
staining cells, probably of a secretory nature. At its posterior
end, directly in front of the anterior testis, this sac passes by a
sphinctered opening into a muscular bulb-like vestibule, which
opens again in the median line ventrally by the elongated
sphinctered external orifice which lies directly in front of the
male genital pore (Pl. XXXIX, Fig. 3).
The connections of these various ducts is made plain by the
figure (Pl. XX XIX, Fig. 2) and need not be further described.
I have not been able to make out any trace of a receptaculum
seminis such as is described by Wright.
The eggs are large and numerous, and are provided with a
thick shell and a well-marked operculum.
I have before me, through the kindness of Dr. Hassall, the
type specimens of two forms which, as he has suggested, are
704, MACCALLUM. [VoL. XV.
closely related to D. heterostomum. These were collected in
various places, and from several different hosts. They are:
Clinostomum gracile Leidy.
Distomum galactosomum Leidy.
In addition to these, I have the specimens described by R. R.
Wright, under the name Distomum gracile, from the branchi-
ostegal membranes and fins of Perca flavescens. Another
specimen, which I labeled D. gvaczle, I found encysted in the
pectoral muscles of a frog in 1895. Other specimens found
encysted ina trout have been determined by Stiles and Hassall
as Clinostomum heterostomum. Finally, I have a number of
specimens of an immature distome found encysted under the
skin of /ctalurus dugesi, collected in Mexico. These, also, I
have received from Dr. Hassall. All of these are immature
forms.
In his original description of Clixostomum gracile (10), Leidy
describes the prominent margin about the mouth-sucker, which
is smaller than the acetabulum. The length is 3 lines, breadth
I line. Habitat — intestine of Zsox, and encysted in gills, fins,
and muscles of Pomotis vulgaris and Micropterus dolomieu.
R. R. Wright (6) later identifies this form with the D. gracile
of Diesing (12), which he describes from the perch. He gives
a detailed description of the large acetabulum with its trian-
gular aperture, but saw no trace of genital organs, and suggests
that the sexually mature form might be found in some larger
fish or piscivorous bird. Attention is called to the large pig-
mented intestinal coeca, and more particularly to the subcu-
ticular meshwork of the watervascular system communicating
with a wide median stem. Diesing’s own description is not
at all detailed.
Leidy’s description of D. galactosomum (11) is briefly as fol-
lows: Habitat —Roccus lineatus. Anterior sucker surrounded
by a prominent margin — body unarmed — ventral sucker sessile
with triangular aperture; larger than anterior. Size 6-12 mm.
by 2-2.5 mm. Intestines extend from the small pharynx to
the tail, tortuous.and sacculated. The animal has a reticular
appearance, due to a network of opaque white lines communi-
No. 3.] CLINOSTOMUM HETEROSTOMUM. 705
cating with the lateral vessels running to a caudal vesicle;
the white is due to granules of calcium carbonate, as it is
removed by immersing in acetic acid. Generative apparatus is
undeveloped.
On comparing the descriptions of these two forms, they are
seen to differ in no respect whatever, unless it be in their hosts,
which are still very similar.
Although I have not the type specimens for comparison, the
description given by Looss (13) of Destomum reticulatum found
encapsulated in the musculature of Sz/wrus glanis, for which
we have also the synonym D. dictyotus (Monticelli, '93), applies so
exactly in every particular to the forms we have just considered,
that I have not the least hesitation in concluding that they
are the same. Indeed, this identity has been pointed out by
Leuckart (14), who considers it one with Leidy’s C/xostomum
gracile,
The other specimens before me are identical forms, although
none are definitely diagnosticated except those labeled by Stiles
and Hassall, Clinostomum heterostomum. In short, there seems
to me to be no doubt that all the specimens are individuals of
one and the same form, and that that form is in reality a devel-
opmental stage of D. heterostomum.
My own observations on these specimens give the following
additional points.
There is a slight variation in the stage of development of the
different specimens, as may be judged from the differences in
the maturity of the genital organs, but in all the two testes may
be discerned with the ovary lying between them (Pl. XX XIX,
Fig. 7), beside the coils of the very immature uterus. The
lumen of the portion of the uterine tube running forward is
quite narrow in proportion to the thick layer of deeply staining
cells which surrounds it. Anteriorly it bends on itself, and
enters the sac described in D. heterostomum, which is here a
long, narrow, empty sac whose walls, furnished like the uterus
with a thick layer of cellular tissue, seem relatively very thick.
The cirrus is still very immature, although in one of my speci-
mens (that from the pectoral muscle of the frog) the differen-
tiation of its parts is fairly complete.
706 MACCALLUM. [VoL. XV.
The intestinal coeca are not pigmented in any of these speci-
mens, but the sacculated form is precisely that seen in the adult
D. heterostomum.
In the specimens marked C/:nostomum heterostomum, by Stiles
and Hassall, the skin is provided with a very delicate armature
of spines, but it seems to me, in view of the entire identity of
structure in other respects, that this may be a variation due to
the difference of hosts, or merely a temporary characteristic,
and that this specimen is a different stage of the same form.
In every other respect these forms are entirely identical with
the adult D. heterostomum, and it will be readily seen that the
genital organs as described are precisely what would be expected
in the developmental stages of this worm. It is on the ground
of this identity in anatomical structure that I should class all
these immature forms as developmental stages in the life his-
tory of D. heterostomum.
Several authors have suggested a close relationship between
the adult D. heterostomum and the following other forms:
D. hians Rudolphi.
D. complanatum Rudolphi.
D. dimorphum Diesing.
: D. aquilae Leidy.
Of these, D. kians and D. complanatum are readily distin-
guished by their having the genital pore in front of the acetab-
ulum, and the ovary in front of the anterior testes.
D. dimorphum is described by Diesing (15) from two hosts,
it being found in the muscle, body cavity, and intestine of certain
fishes, and again, evidently in a more mature condition, in the
mouth of Ardea cocot and the oesophagus of Ciconta americana.
The acetabulum is said to be smaller than the anterior sucker ;
the mouth terminal; genitalia are absent in the early stage.
In the more adult the genital pore is not far from the caudal
extremity. From this description, which seems to be the only
one extant, and from his figures it is evident that this worm is
closely related to D. heterostomum ,; indeed, it cannot be sharply
distinguished, but as the original material is not accessible to
me, I can go no further than this.
No. 3.] CLINOSTOMUM HETEROSTOMUM. 707
As to D. aquilae, one can judge but little from the descrip-
tion (11): ‘‘Spatulate, cochleariform, widest behind, obtuse at
both ends; mouth circular, unarmed ; acetabulum sessile about
as large asthe mouth. Length, 3 lines; width in front, }a line;
behind, 4 a line. From the trachea of the Bald Eagle.” The
type specimen, however, shows a worm similar in every respect
to that described as D. heterostomum, with the exception that the
anterior sucker seems to be surrounded by no elevated border,
and it is on this ground that I should hesitate to consider them
identical without further examination of fresh material.
Up to this point I have employed the generic name, Dzszo-
mum, in speaking of the form under consideration, by way of
avoiding confusion, but according to the rules of nomenclature
now generally adopted, this generic name can no longer stand,
because (Railliet (16)) it is antedated by Dzstomus Gaertner,
applied to a genus of tunicates, and further, as Stiles points
out, because Retzius, in proposing it, arbitrarily created a syno-
nym for the existing generic term Fasczola.
In the subdivisions generally adopted as a substitute for the
genus Distomum, this form would fall into the section mes-
ogonimus (Monticelli (17), '88), but Stiles and Hassall have
pointed out that this, in turn, is antedated by the generic name
Clinostomum (Leidy (10), '56), which was proposed to include
a group of which the type species was Clinostomum gracile,
a form identical with the D. reticulatum of Looss, on which
Monticelli’s genus was based.
Hence, in order to concur with the modern nomenclature,
we can only name this form Clinostomum heterostomum, and it
will now include the larval forms formerly known, respectively,
as Clinostomum gracile, D. galactosomum, D. reticulatum Looss,
and possibly also the adult D. dimorphum and D. agqutlae.
In adopting this generic name, however, it is necessary that
I should amplify the generic diagnosis given by Leidy (10) as
follows (as suggested by Stiles) :
Family, Fasciolidae.
Genus, Clinostomum Leidy ('56).
Hermaphroditic flukes: Genital pore with contiguous male
708 MacCALLUM. [Vov. XV.
and female openings situated posterior to the acetabulum, either
about halfway between the acetabulum and posterior extremity
(as in the type Clinostomum gracile), or zmmediately posterior to
the acetabulum, as in DD. Westermanni, Kerbert. Oral sucker
without tentacles or spines: Caudal extremity without retractile
appendage. Intestine simple, bifurcate.
Thus there are contained in this description two groups of
flukes, which will probably later be parted into separate genera.
I wish to express my thanks for literature, etc., to Prof. R.
Ramsay Wright, of Toronto University, whose readiness to aid
me in my work has been unfailing; also, to Drs. Stiles and
Hassall, of Washington, whose library and specimens I have
made use of, and who have been very kind in advising me with
regard to the questions of nomenclature.
BALTIMORE, October 10, 1897.
No. 3.] CLINOSTOMUM HETEROSTOMUM. 709
Sy An hens
LITERATURE.
RuUDOLPHI. Entozoorum Historia. 1809. ii, p. 381.
RuDOLPHI. Entoz. Synops. 1819. pp. 102-388.
DuJARDIN. Hist. nat.d. Helm. 1845. p. 400.
DIESING. Syst. Helm. 1850. p. 353.
Rosa. Lettere Zoolog. v, Nr. 4.
R. R. Wricut. American Helminthology. No.1. 1879.
Von Linstow. Arch. f. Naturgeschichte. Bd. xlix. 1883. p. 306.
Von Linstow. Viaggi Fedtschenko. Vermi. 1886. p. 30, Fig. 49.
StossicH. I distomi degli Uccelli. Trieste. 1892.
Leipy. Proc. Acad. Sci. Philadelphia. 1856. Vol. viii, p. 45.
. Leipy. Proc. Acad. Sci. Philadelphia. 1887. p. 24.
. DresinG. Sitzungsber. d. k. Akad. Wien. 1858. Bd. xxxii, p. 336.
. Looss. Zezt. f. wiss. Zool. Bad. xli.
LEUCKART. Parasitend. Menschen. 1889. Bd. i, 40 Anm.
. Drestnc. Neunzehn Arten Trematoden. XB. Denkschr.d.k. Akad.
Wien. 1856. Taf. III.
. RAILLIET. Nomenclature des Parasites. Recuezl de méd. vétérinaire.
15 mars, 1896.
. MONTICELLI. Saggio di una Morfologia dei Trematodi. Napoli.
1888. p. 92.
710 MacCALLUM.
DESCRIPTION OF PLATE XXXIX.
Fic. 1. Clinostomum heterostomum — adult seen from ventral surface; cz.,
cirrus sac; ov., Ovary; ex., excretory vesicle ; 4.4, testes; go., opening of uterine
tract; #., uterine sac ; v7¢., yolk gland.
Fic. 2. Female genital apparatus, seen from dorsal surface; Zc., Laurer’s
canal; od., oviduct ; yd., yolk ducts; yv., yolk reservoir.
Fic. 3. Cirrus (from ventral surface); v.e., vasa efferentia; s, sac containing
spermatozoa; c, muscular tube; Z, extrusible portion opening at external genital
pore; go., external female genital pore.
Fic. 4. Scheme of nervous system ; nervous system in blue, alimentary tract
in red.
Fic. 5. Parenchyma cells in anterior portion of the body, with strands of body
musculature.
Fic. 6. Intestinal coeca in longitudinal section.
Fic. 7. Genital apparatus of immature form (D. gracile, etc.).
Journal of Morphology Vol. XV. PLXAXIX
a
Lith. Werner &Winter Frankfort 26.
MITOSIS IN NWOCTILUCA MILIARTS AND ITS BEAR-
ING ON THE NUCLEAR RELATIONS OF
THE PROTOZOA AND METAZOA.
GARY N. CALKINS.
TABLE OF CONTENTS.
IPT.) "OBSERVATIONS ioc copccecesesese-coenct Sa eee arin tenn sanc Poceasadcane gesseter sack descadcssecsvecaccéisacce
B. THE SPHERE IN THE RESTING CELL
C. THE NUCLEUS DURING DIVISION
1. The Chromatin..
a. Prophase.... if
BANUC tapas © teereratte te ctacacea cee neces cose e ects scat sore a texas Coseentaedeecucieape eestone fact
EAM AD ASC Yerterte ara tec ce dacs esctrct scadanarane oaasanta teen cst ca tata tanaeseecabesasce ae 722
d. Telophase ............. 723
2. The Nuclear Membrane 725
Se ANS Sy oN) spares een GE
Ana lines Mian tlesfi bers esse secscee tots coe essere aces tees cote ater ante tet cncect aecce caresses 729
Gee Cen trOSOMe eases tecwereetece sere eee tee at ecec astra cete tac ceae onc a oee enc se aston 731
D. THE MECHANISM OF MITOSIS IN NOCTILUCA..............:::c200ceececeeeseee0s 737
III. The NucLEAR RELATIONS OF NOCTILUCA TO METAZOA AND PROTOZOA 738
PAPE TATIONS (LOM MLE TAZ O Avecesscascestearesecsconedstancatnecamsrarseateteveneasatzedsctereencraswe 738
BO IRELATIONS! TO) PROTOZOA ccec acces con ossscecscteaesnaestasedscescetestecazosee loecscsesectcace 739
Ken Origin’ Of (ChrOMOSOMES |osrcecececsncaresccccavaecseassncstecosaveqpuevesecesontecsnsnsevosees 739
2 Originiof Centrosome and’ Spheres. 22 aceca-ccscscs-eccsseceseuenc-eenenacetececsers 744
TAVieuiS UMMA Ve OF | OBSERVATIONS ec ccecesescseccsszsisasceosecsecensasvescsrescreasccsstceroresaesteseser 757
In view of the probable origin of the Metazoa from the
colony-forming Protozoa, many observers have attempted, more
or less definitely, to trace the phylogenetic development of
special parts of metazoan cells from analogous parts in Protozoa.
Prominent among such attempts have been those of Biitschli
(91), Heidenhain ('94), Hertwig (96), and Lauterborn ('96), and
the two last-named writers have shown that, from the least
differentiated parts of the most primitive protozoan nuclei, a
progressive series may be traced which culminates in /Voerz-
7i2 CALKINS. [VoL. XV.
luca miliaris, where the nucleus and the mitotic figures are
quite as complex as in the Metazoa. Ishikawa (94) had already
shown the similarity between JVoctz/uca and the Metazoa in
this respect, although his account of mitosis in the former was
incomplete. Voctz/uca holds so important a place in the series,
especially in relation to the process of mitosis, that its nuclear
division should be fully and accurately known, and it has been
my aim during the last two years to clear up, if possible, the
points still left obscure.
The wide distribution of Moctz/uca and the large size of its
nuclear elements have made it a favorite subject for research,
although it has seldom been studied from a cytological stand-
point. Huxley’s description (55) was purely morphological, as
were those of De Quatrefages ('50), Krohn ('52), and Busch
(55). Brightwell (57), describing the external phenomena of
cell division, undoubtedly saw the sphere,! but called it a
secondary nucleus. Cienkowsky (71 and '73) described the
external features of spore-formation, but without reference to
the nucleus or the accompanying structures. Robin (7g) in
his description of the spore-formation seems to have mistaken
the sphere for a nucleus, and his accurate illustrations tell more
about the nuclear processes of division than the text.
It was Ishikawa (94) who first gave accurate details of
mitosis in /Voctz/uca. His work can best be reviewed by pre-
senting his own summary (pp. 324-326) as follows :
1. The division of the animal is preceded by the loss of the peristome,
teeth, and the tentacle, the last of which is not thrown off, as Robin is
inclined to think, but is redrawn into the body of the animal. The mouth
and the “ Staborgan ” are, however, always present (Robin).
2. The spore-forming individuals differ from the dividing ones in not
possessing the mouth and Staborgan in addition to the organs above men-
tioned, and by the excessively empty appearance of the cell interior
(Cienkowsky).
3. The division of the nucleus is always preceded by a concentration of
a part of the cytoplasm in the form of a spherical or oval granular body,
1T use the term “sphere” to designate the large, clearly outlined cytoplasmic
mass which is probably to be identified as an attraction sphere. During the pro-
phase of mitosis it divides and forms an amphiaster with connecting central-spindle
fibers, and in the anaphase a centrosome is found within it.
No. 3.] THE PROTOZOA AND METAZOA. Fag
mostly close to the nucleus. This is the archoplasm or kinetic center of
division, and corresponds most probably to the “ Nebenkern” of Von la
Valette St. George.
4. In living animals at the stage of (3), the nucleus appears more or less
homogeneous and transparent, and is not so distinctly to be seen as the
archoplasin, But treated with reagents the chromosomes come into view
distinctly.
5. Each chromosome consists of a row of disc-shaped microsomes
irregularly scattered in the nucleoplasm. The number of the chromosomes
is not clear, but in most cases has been counted to be ten.
6. The chromatin substance of each of the microsome-discs collects at
the periphery and forms a microsome-ring.
7. In the nucleus of a dividing animal, each microsome-ring splits into
half-rings, thus dividing a chromosome in halves, while in that of the spore-
forming animals two successive divisions of a microsome-ring take place,
so that a single chromosome is directly divided into four daughter ones.
8. The chromosomes collect on the side of the nucleus which is nearest
to the archoplasm and spread out towards the other pole. The pole where
the archoplasm lies thus corresponds to Rabl’s “ Polfeld,” and the other
pole to his “ Gegenpol.”
g. The archoplasm divides and forms a very large spindle which first
lies tangential to the surface of the nucleus. This division of the archo-
plasm is succeeded by the separation of the chromosomes into two groups,
each attracted (?) by its respective archoplasm.
1o. The archoplasmic spindle thus formed pushes in the nuclear wall on
which it lies, and the nucleus assumes in consequence a half-ring form.
11. By the separation of the archoplasms a spindle is produced which in
all essential characters appears like the form known as the “ dyaster stage,”
with a large archoplasmic mass at each end of the spindle.
12. The fibers of this spindle are therefore continuous from one pole to
the other, and, lying outside the nuclear wall, become in no way connected
with the chromosomes. But there is seen at this stage another set of fibers
running from the center of the archoplasm to the polar ends of the chromo-
somes. This structure of the spindle corresponds exactly with that of the
spermatocyte of Sa/amandra maculata, as investigated by Hermann, with
only the difference of the persistence of the nuclear wall in Wocté/uca, and
the necessary modification in consequence of this fact. The optical appear-
ance of these two kinds of fibers is different, just as in Sa/amandra.
13. Besides these two sets of fibers, the Verbindungsfaden are clearly to
be recognized extending between the separating chromosomes.
14. The central-spindle fibers originate from the archoplasm, the radial
fibers, probably from both the cyto- and nucleoplasms, and the Verbin-
dungsfaden from the linin substance.
15. In the spore-buds the archoplasm is to be seen lying close to the
nucleus up to the time of full development of the spore, just before its
714 CALKINS. [Vou. XV.
detachment from the mother-animal, and a part of it becomes transformed
into the flagellum, just as in many vegetable swarm-spores, as Strasburger
shows.
16. In the center of the archoplasm is generally seen a centrosome,
which often shows a dumb-bell form. Sometimes, however, two centro-
somes are found in the archoplasm of the spore-forming cells. In many
cases, again, there is found in the center of the archoplasm a number of
small oval rod-shaped or curved bodies staining exactly like centrosomes,
instead of one or two centrosomes. These may represent the group of
centrosomes of Heidenhain.
17. The origin and the fate of the centrosome are not known. Ina few
instances it appears to be formed from the nucleus.!
Section 5 is rather obscurely worded, but, from the detailed
description given elsewhere, it appears that the chromosomes,
which are of various lengths, consist of irregular rows of micro-
somes lying scattered throughout the nucleus. ‘The chromo-
somes, except a few, do not seem to lie in definite order, but to
be scattered more or less irregularly in the nucleus.”
In the descriptive part of Ishikawa’s paper section 7 is
stated in a more conservative manner: ‘ While the chromo-
somes of the nuclei of the dividing individual are represented
by a double row of microsomes, those of the nuclei of the
spore-forming individuals appear to consist of four rows.” He
explains this difference as follows: “In division the nucleus
has to divide only once, and hence the chromosomes require
only once to divide, while in the spore-formation divisions of
the nucleus take place rapidly one after the other, and two
divisions take place almost simultaneously ” (/. ¢., p. 304).
It may be pointed out, however, that in spore-forming indi-
viduals the nuclei continue to divide rapidly and without inter-
vening resting periods, until there are from three to five
hundred descendants of the original nucleus. What reason,’
therefore, can there be for quadruple division of the original
chromosomes ?
The details regarding formation of the nuclear plate and the
separation of the chromosomes are not given.
1 The killing agents used by Ishikawa were Flemming’s stronger solution,
picro-acetic, and acetic acids. The material was stained with Bohmer’s haema-
toxylin, acid fuchsine, methylin blue, and methylin green.
No, 3.] THE PROTOZOA AND METAZOA. 715
Ishikawa speaks of ‘radial-fibers”’ (section 12), but as these
are analogous in every way to similar fibers in ordinary mitosis,
I see no reason for adopting this term in preference to the cur-
rent name ‘ mantle-fibers.”
The present paper deals almost exclusively with the points
touched upon by Ishikawa in sections 3-14, 16, and 17. The
details of chromosome-formation are found to differ consider-
ably from Ishikawa’s account, and for the first time the forma-
tion of the nuclear plate in Voctzluca is described. Ishikawa’s
observations in regard to the sphere and centrosome are, in the
main, confirmed.
Before giving the results of my observations I wish to express
my obligation to Professor Wilson, not only for the material
upon which most of my work was done, but also for his kind
advice and unceasing interest.
I. MATERIAL AND METHODS.
The material for my work was collected partly by Professor Wilson at
Beaufort, N.C., in June, 1895, and partly by myself at Port Townsend,
Puget Sound, in July, 1896. In the latter place it was found in great abun-
dance, often covering hundreds of square yards, its presence indicated by
brilliant phosphorescence by night, or in daylight, when wind and tide were
favorable, by patches of brilliant orange.!_ They were collected early in the
morning and preserved at intervals during the day and succeeding night, and
all stages of vegetation, division, and spore-formation were thus secured.
Five different killing agents were used, vzz., corrosive sublimate (saturated
in normal salt solution), sublimate-acetic (10 per cent acetic), picro-acetic
(Boveri’s formula, with 1 per cent acetic for the Beaufort material, satu-
rated picric with 5 per cent acetic for that from Puget Sound), Hermann’s
fluid, and Flemming’s stronger solution.
The material was prepared for study either by mounting zz fofo or by
imbedding in paraffin and sectioning. Considerable difficulty was expe-
rienced in handling such small objects until the following method was
devised. The alcohol containing /Vocti/uca was poured into a glass cylin-
der two inches long and one-half an inch in diameter, covered at the lower
end with bolting cloth of such fineness that the /Voctz/uca were held back
while the alcohol passed through. They were then readily handled by car-
1 Noctiluca was observed and collected in Alaskan waters as far north as Juneau
and Sitka, and even in Glacier Bay, where the water is at a constant low temper-
ature. The water of Puget Sound is deep and cold (about 51° at all depths), but
at Sitka it is shallow and perceptibly warmer.
716 CALKINS. [VoL. XV.
rying the tube from one staining pot to another. In all cases they were
cleared in xylol and mounted in balsam. For sectioning, either large quan-
tities of Voctz/uca were imbedded in bulk, or individuals were selected and
imbedded separately. In most cases the latter method gave the most satis-
factory results, especially for dividing nuclei, where it was often desirable
to section in definite planes. Heidenhain’s iron haematoxylin, the Biondi-
Ehrlich mixture, and Reinke’s modification of the Flemming triple method
gave the best results in staining. Bordeaux red, orange, and eosin were
used as secondary stains with the iron haematoxylin.
II. OBSERVATIONS.
A. Tue Restinc NucCLeEvs.
The nuclei of (Voczzluca belong to the so-called vesiculate
type. They are spherical, oval, or elliptical in shape, and vary
considerably in size, the largest measuring about 50m in
diameter (Fig. 6), the smallest about 30m, while those in the
various stages of spore-formation are still smaller. They are
always enclosed by a distinct, often thick, membrane, which,
although it disappears at certain regions during mitosis, is, as
a whole, retained through all phases of nuclear change.
The interior of the nucleus consists mainly of two distinct
substances. One of these is granular and stains with acid dyes,
while it is so abundant that it gives a massive appearance to
the nucleus. The staining reactions of this substance show it
to be the same, probably, as the ‘‘oxychromatin” described by
Heidenhain ('94) in the nuclei of leucocytes. Unlike the latter,
however, the oxychromatin granules in /Vocti/uca appear to be
isolated bodies of large size and not suspended in a colorless
network, the “linin’’ of Heidenhain. The other substance
stains intensely green with the Biondi-Ehrlich mixture and
represents the “chromatin” of Flemming (80) or the ‘ basi-
chromatin” of Heidenhain. In the resting nucleus the basichro-
matin is invariably collected in from eight to eleven, or more,
great chromatin reservoirs which, for the sake of brevity, may
be called the karyosomes in place of the earlier and more mis-
leading term, —nucleoli. By this use of the term, however, it
must not be understood that the karyosomes are local thicken-
ings of a general basichromatic reticulum. There is no such
No. 3.] THE PROTOZOA AND METAZOA. Vales,
network in JVocteluca, and ‘‘karyosomes’’ here represent the
entire chromatic reticulum of metazoan cells. Karyosomes are
characteristic of many Protozoa, and the nuclei containing them
are classed together by Gruber ('84) as the “vesiculate”’ type
(“Blaschenformige”’). In all such nuclei a membrane, nuclear
ground substance, or “sap,” and one or more central granules
of chromatin can be distinguished.
In WNoctzluca the two nuclear substances differ widely in their
affinity for dyes, the oxychromatin being stained a clear and
intense red by the Biondi-Ehrlich mixture, while the basichro-
matin is stained a brilliant green. This difference in color is
so marked, and the limits of the two substances so distinct,
that the smallest particles of green basichromatin can be easily
detected on the red background of oxychromatin. This stain
is, therefore, invaluable in following the chromatin changes
during nuclear activity.
In addition to the basichromatin and oxychromatin, a small
spherical body (x) surrounded by a hyaline area (Figs. 3, 4, 6,
and 7) is usually to be found in the nucleus. With iron haema-
toxylin and orange it takes an even more brilliant haematoxylin
stain than the chromatin, remaining black or blue while the
karyosomes are a deep gray. I have not succeeded in staining
it successfully with the Biondi-Ehrlich mixture. In most rest-
ing nuclei only one such body is present. From the subse-
quent changes in the nucleus, and from analogous bodies in
other Protozoa, there is some reason to regard this corpuscle
as the centrosome.
B. Tuer SPHERE IN THE RESTING CELL.
In the cytoplasm, outside of the resting nucleus, lies a large
cytoplasmic mass which was first pictured by Allman ('72) and
later described by Robin ('78) as a nucleus. From its relation
to the nucleus and its behavior during cell division this cyto-
plasmic mass may be called the “attraction-sphere” or simply
the “sphere,” although in its resting stage no centrosome can
be found in it. It is often as large as or larger than the nucleus
(Figs. 1 and 8), but differs in appearance at different stages. It
718 CALKINS. [Vou. XV.
is always in contact with the nucleus, sometimes having the
form of a sphere, but more often that of an irregular mass with
a darker peripheral and a lighter central portion. The latter
appearance is caused by the accumulation of microsomes around
the periphery, and by their absence in the central portion. In
specimens fixed with Hermann’s or Flemming’s fluid the sphere
is seen to have pseudopodia-like processes extending from it
into the surrounding cytoplasm and passing imperceptibly into
the reticulum (Fig. 9).
In addition to the microsomes in the sphere other deeply
staining, but larger, granules are frequently seen. Sometimes
only one of these can be found ; again they are quite numerous,
even forming a small group of deeply staining bodies. Similar
granules are also found in the cytoplasm, distributed through-
out the reticulum. These granules are often strikingly similar
to centrosomes and might easily be mistaken for them. Care-
ful comparison of many spheres, however, both in sections and
in total preparations, shows that they cannot be centrosomes.
Their position is often eccentric (Fig. 5), sometimes even in
the periphery of the sphere, while the presence of similar gran-
ules in the cytoplasm shows that they cannot be “centrosomes ”
in Heidenhain’s sense. It is not improbable that the centro-
somes described by Ishikawa, which were found sometimes
single, sometimes double, and sometimes in groups, were cyto-
plasmic granules of this kind. Ishikawa apparently saw the
‘sphere’ only during division : “The archoplasm, as we have
stated, comes to be first seen at the stage a little before the
division of the spore-formation” (/.c.). My observations, how-
ever, leave no doubt that it persists as an extra-nuclear mass
throughout all periods.
C. Tue Nuc Leus purine Division.
The phenomena of nuclear division in octzluca are so com-
plicated and apparently so different from typical mitosis in the
Metazoa that the following general summary will help to make
the details more clear.
During the early stages of nuclear activity the sphere divides
into two similar halves connected by fibers which here, as in
No. 3.] THE PROTOZOA AND METAZOA. 719
some Metazoa, may be called the “central-spindle” (Figs. 9
and 11). The central-spindle then sinks into the nucleus
which forms a trough to receive it (Fig. 13). The trough
deepens and the lips of the nucleus approach each other over
the central-spindle until the latter finally occupies the position x
of the Fig. @, the daughter-spheres lying partly in the groove
and partly exposed at the ends (Fig. 13). The nucleus then
divides in a plane at right angles to the central-spindle,
each half being accompanied by one of the daughter-spheres.
The anaphase-stage presents the well-known striated appear-
ance of protozoan mitotic figures —the striations being formed
by the daughter-chromosomes which are directed towards the
separating spheres (Fig. 18). In the final stage of division
the daughter-nuclei become completely separated, the furrow
in which the central-spindle had lain becomes obliterated, and
the sphere resumes its normal appearance in the resting cell.
In spore-forming divisions the process is in the main the
same, but the mitoses follow each other in quick succession
and without intervening resting stages. Here the daughter-
chromosomes of an anaphase without further change form the
chromosomes of the ensuing prophase. The spheres again
divide and the process is continued until the original nucleus
is divided into as many parts as there will be spores (from 300
to 600).
1. Zhe Chromatin.
a. Prophase.— Nuclear division is preceded by a concentra-
tion of the cytoplasmic microsomes in the sphere. The hyaline
area disappears, the entire mass diminishes in size and becomes
more homogeneous, and by these changes the sphere becomes
more dense and more clearly defined, so as to be even more
conspicuous than the nucleus. It was probably this stage
which misled Brightwell (57) and Robin ('78) into mistaking
it for the nucleus.
Within the nucleus, meantime, the large basichromatic
karyosomes gradually break up into smaller pieces, apparently
in the same way as the chromatin in Actznosphaerium accord-
ing to Gruber’s ('83) and Brauer’s ('94, '94a) descriptions. The
disintegration of the karyosomes seems to be accomplished bya
720 CALKINS. [Vou. XV.
progressive process of division, and from a study of the various
modes of aggregation of the chromatin fragments in different
nuclei, it appears that they divide first into two nearly equal
parts, these into four, the four into eight, etc. In most cases
the parts thus formed soon become scattered so that it is
almost impossible to follow them ; but in some favorable cases
the parts remain in groups in the places occupied by the origi-
nal karyosomes until as late as the eight-part stage (Fig. 4).
I have never been able to follow the fragmentation beyond this
stage. The chromatin-granules thus formed leave their origi-
nal positions and become concentrated at the side of the
nucleus which lies away from the sphere (Fig. 5). But even
here it can be made out that the larger pieces continue their
fragmentation into smaller and smaller portions. The final
result of this disintegration is the formation of a great num-
ber of minute chromatin granules similar to those of the
nucleus of Actznosphaertum (Brauer).
As they break up, the chromatin masses often, but appar-
ently not always, give rise to beaded fibers which have no
definite arrangement in the nucleus (Figs. 6, 7,12). Ishikawa
(94) in his second paper describes these fibers as chromosomes
and pictures them in his Figs. 33, 38, 40, 44, and 47. He
thought they were formed from the parts (‘ microsome-discs’’)
of the disintegrated nucleoli (karyosomes) which were put
together ‘one after the other like chains of mammalian blood
corpuscles.” I am convinced, however, that although these
fibers appear in the manner described by Ishikawa, they are
not the actual chromosomes. They do not pass directly into
the nuclear plate, nor do they split longitudinally. This stage
is a prophase of division, as shown by the condition of the
sphere (Fig. 12). It is antecedent to the stage of the nuclear
plate, and later than that of the resting nucleus. It must,
therefore, correspond to the spireme-stage of mitosis, though
it cannot be interpreted as a monospireme, for in total prepa-
rations the ends of the figures are in full view and are in no
way connected with each other (Figs. 6, 7).
After the above changes the granules which compose the
fibers of the spireme break down into still more minute
No. 3.] THE PROTOZOA AND METAZOA. 721
granules —the ultimate chromatin elements (Figs. 9, 10, and
36). These are so fine that one can distinguish them only
after the most careful differential staining. At first they
are distributed equally throughout the entire nucleus, but even
before all of the chromatin of the karyosomes is converted into
chromomeres, those which have already formed begin to col-
lect in lines extending from the side of the nucleus which lies
nearest the sphere, towards the opposite side (Figs. 9, 10, 32,
35, and 36). These lines of granules are the beginnings of
the chromosomes. The Biondi-Ehrlich solution and the iron
haematoxylin with orange G shows them best; the bright lines
of chromatin granules show distinctly in contrast to the broad
lines of oxychromatin lying between them. In sections
stained by the first method (Fig. 10)! the minute green chro-
matin elements can be traced through all stages of chromo-
some-formation, from isolated granules of incredibly small size,
distributed throughout the nucleus, to the compact chromo-
somes directed towards the sphere. The chromosomes next
increase in thickness at the peripheral ends, probably by
aggregation of the granules, and taper towards the center of
the nucleus until at the inner end the threads are no thicker
than the single granules (Fig. 11). The tapering of the chro-
mosomes is, however, but a passing phase ; later they gradu-
ally thicken throughout their entire length and finally become
of uniform thickness (Fig. 38).
Throughout all stages of nuclear activity there is no evidence
to show that oxychromatin is being changed into basichromatin
or vice versa. The chromatin granules are derived from the
karyosomes, and at all stages can be distinguished from the
oxychromatin granules by their intense green chromatin stain.
The oxychromatin granules, meantime, do not change in size
or in staining reaction. They are many times larger than
the chromomeres, and during chromosome-formation they are
arranged in lines parallel with the chromosomes (Figs. 9, 32, 36).
1 These minute granules are difficult to represent in black and white and Fig. 1o
represents but poorly the actual preparation. The intense black dots distrib-
uted throughout the nucleus and among the larger granules of oxychromatin give
a fairly accurate picture.
722 CALKINS. [VoL. XV.
When the latter are completely formed, these granules fill the
remainder of the nucleus, the basichromatin of the chromo-
somes occupying no more space apparently than when in the
form of karyosomes. ‘
6. Metaphase.— At this period the chromosomes are single,
thick rods or fibers of chromatin and distinctly granular,
although each granule is much larger than one of the original
elements. Shortly afterwards, however, the chromosomes
become double, consisting then of two rows of granules
formed by a longitudinal cleft down the middle line (Fig. 38).
The main part of the chromosomes does not lie in the center
of the nucleus as usual in other Protozoa (Actinosphaerium,
Euglypha, etc.). On the contrary, the chromosomes lie ina
linear group with their ends close against the membrane of
the nucleus at the side nearest the sphere (Figs. 8, 11, 26).
At this stage, or even before (Figs. 12 and 14), the nucleus
begins to elongate in a direction which for convenience may
be called the “primary axis,” the ends of the chromosomes
being arranged in the median line of the nucleus, that is, along
the primary axis (Fig. 14). The nucleus thus assumes the
appearance of a cylinder with rounded ends, the chromosomes
extending from the margin nearest the sphere towards the
opposite side (Fig. 14). This stage is rarely seen, however,
for the elongate nucleus soon bends around the central-spindle
to form a C-like figure (Figs. 12, 13). In many cases the
curvature along the primary axis and the elongation of the
nucleus are simultaneous and not separate actions. The curva-
ture continues until the extremities of the primary axis become
more or less closely approximated (Fig. 13).
By the curvature of the nucleus the chromosomes are carried
around the central-spindle until they form an incomplete ring.
They become focused at the point x of the Fig. @, which is
formed by the curvature of the nucleus. The axis in which the
central-spindle lies may be called the “secondary axis” (Fig.
13).
c. Anaphase.— The structure of the chromosomes in the
nuclear plate and their subsequent changes can be studied only
in sections. These may be cut either transversely or longitu-
No. 3.] THE PROTOZOA AND METAZOA. 723
dinally, z.e., in planes which pass through the secondary or the
primary axis. In transverse section the chromosomes are thick,
double fibers which tend to converge in the median line and to
spread out from here like the ribs of a fan (Figs. 22, 26). At
the proximal ends they are so compact that it is difficult to
make out the double nature, but towards the middle the parts
become more separated, and at the distal ends the granules in
the chromosomes can hardly be distinguished from the remain-
ing chromatin elements, which are still free in the nucleus.
Separation of the daughter-chromosomes begins at the proxi-
mal ends, z.e., at the ends nearest the spindle. Each chromo-
some splits down the line of original longitudinal cleavage (Fig.
27, A), and the parts move in opposite directions, proximal ends
first (Figs. 28, 29). Fig. 38 represents a section through the
primary axis, showing the chromosomes in the nuclear plate in
the metaphase. Fig. 27 shows the same stage in transverse
section. In Fig. 27 the chromosomes have just begun to
separate (A), while in Figs. 28, 29 the division is further
advanced. During the separation which follows, some of the
daughter-chromosomes, moving in opposite directions, pass each
other, and a curious crossed appearance results (Figs. 28, 29).
Fig. 30 is from a vertical section through the secondary axis,
showing an anaphase; the chromosomes are turned in opposite
directions towards the spheres. Fig. 31 represents the same
stage in a section cut horizontally through the secondary
axis.
As the daughter-chromosomes separate, the nucleus again
elongates, but in a direction at right angles to that of its
original elongation, z.e., in the direction of the secondary axis
(Figs. 5-20, 30). During this secondary elongation the nuclear
lips are pressed more closely together above the central spindle
until in some cases a mere slit is all that is left of the former
opening (Fig. 17).
ad. Telophase. —The description of nuclear division given
above may apply equally to vegetative division or to that occur-
ring in spore-formation. The telophase is, however, essentially
different in the two cases. In the former, after the chromo-
somes are completely separated at the distal ends, the nucleus
724 CALKINS. [VoL. XV.
itself begins to divide. At first this is indicated by a slight
depression in the center of the nucleus (Fig. 20). The daughter-
nuclei then move further apart until the connecting-piece is
reduced to a mere thread, which soon breaks. The membrane
reforms, and all traces of the groove are obliterated as each
daughter-nucleus finally rounds out. The chromatin of the
daughter-chromosomes fuses to form again the great karyosomes
of the resting nucleus (Fig. 25), probably by a reversal of the
process of disintegration described above. The distal elements
of the daughter-chromosomes are the first to disappear, while
the thicker parts remain as the last evidence of division (Fig.
25). In some instances the karyosomes begin to form before
the daughter-nuclei are separated, as in Fig. 20.
In the spore-forming divisions, on the other hand, which
follow each other in quick succession, the chromosome-structure
is not altered, and the daughter-chromosomes pass directly into
the nuclear plate of the next division figure. This process is
repeated nine or ten times until all of the spores are formed.
Fig. 21 represents the beginning of a second division before
the first is quite complete, and with a relatively large “ Verbin-
dungsstiick””’ left between the nuclei. The daughter-chromo-
somes which are to form the nuclear plate of the second division
are single (Fig. 30), and yet just before the second division they
are double (Fig. 38). This division must take place while the
nuclear plate is forming. Ishikawa states that chromosomes
are quadruple in nuclei destined to form spores, but I have
never found the chromosomes in the nuclear plate otherwise
than single or double. Even were they quadruple in the parent
nucleus it would not suffice to explain the eight or nine subse-
quent divisions which the nuclei undergo before the spores are
formed. The elements composing the chromosomes must
divide longitudinally before each division of the nucleus. As
there is no time for growth of the chromosomes between
‘divisions, the quantity of chromatin in the nucleus is constantly
reduced by half, and the nuclei become smaller and smaller.
No. 3.] THE PROTOZOA AND METAZOA. 725
2. The Nuclear Membrane.
In the resting cell the nuclear membrane is comparatively
distinct and thick, but in the active nucleus it becomes very
much thinner and more plastic. Except in one region it does
not disappear during mitosis, but persists as a permanent
portion of the nucleus. In one region, however, it does dis-
appear during mitosis. When the nuclear plate is formed and
the chromosomes are ready for division, it disappears in the
region between the nuclear plate and the central-spindle. The
nuclear plate is thus left as a free ring around the central
spindle (Figs. 13, 29, 31). As the daughter-chromosomes
separate, the membrane is reformed between them (Fig. 30),
but it always remains broken in the region between the
daughter-chromosomes and the spheres. The position of the
gap which is thus formed in the membrane can be clearly seen
from Fig. 31. Here the membrane is complete in all places
except where the mantle-fibers pass from the centrosomes
to the chromosomes. During the telophase the membrane
reforms in the broken places and again becomes continuous.
The above-described gap in the membrane during mitosis is
strikingly similar to a temporary stage in metazoan mitosis
which has been described by many observers. In all such cases
the nuclear membrane disappears first at the poles in front of
the developing spindle-fibers. In some instances (e.g., in Tha/-
lassema Griffin ('96) ) the membrane persists in this stage for
some time, and only at a late period in mitosis does it completely
disappear.
3. The Sphere.
We may now consider the history of the sphere more in
detail. As previously stated, the approach of division is first
recognized by the concentration of cytoplasmic microsomes in
the sphere. During disintegration of the karyosomes and’
formation of the chromosomes a structure is formed consisting
of two daughter-spheres connected by a ‘“‘central-spindle,” to
which, from analogy with metazoan mitoses, the name of
amphiaster may be given (Figs. 9, 11). As the nucleus elon-
71} 26 CALKINS. [VoL. XV.
gates and bends in the plane of the primary axis, the central-
spindle sinks into the depression which is thus formed, until it
finally occupies the position + in the Fig. @. The spindle thus
lies in the secondary axis of the nucleus which encircles it, the
spheres alone remaining outside (Figs. 13-15). The nuclear
plate is wrapped around the spindle like a ring, the chromosomes
lying midway between the two poles. The central-spindle
fibers are at first not straight, but run from pole to pole in
curved lines (Fig. 11). This curvature is caused by the spheres
moving around the nucleus through an arc of go°, the fibers
becoming straight only when the central-spindle finally lies in
its definitive position (Fig. 13). In some cases the bend of the
central-spindle is much exaggerated until an acute angle is
formed. During this time the growing chromosomes are
entirely separated from the central-spindle, but when the
nuclear plate is complete the intervening membrane disappears,
and then, as in the metazoan spindle, the chromosomes lie
directly upon the central-spindle. After the preliminary
arrangement of the elements of the mitotic figure, the central-
spindle undergoes a considerable elongation (Figs. 17-19, 30),
while at the same time the nucleus is drawn out in the direction
of its secondary axis in the form of a hollow cylinder with a
ring of chromosomes at either end (Figs. 17-19). The dumb-
bell-shaped amphiaster lies in the hollow with the spheres
projecting at either end (Fig. 13). After division of the vege-
tative nucleus the sphere returns to its resting condition (Fig.
20). It becomes less dense and less homogeneous, and the
central part more or less hyaline, while the peripheral portion
still retains its granular aspect. The transformation may begin
even before the nuclei are completely separated. Such a con-
dition is indicated in Fig. 20, where the daughter-spheres, having
lost their densely granular spherical condition, have become
more diffuse, and have acquired the characteristic appearance
of the resting spheres. On the other hand, after spore-forming
divisions, the daughter-spheres do not return to the resting
condition. They retain their spherical shape and dense granu-
lation (Figs. 17-19), and soon divide again for the ensuing
mitosis. It frequently happens that this secondary division of
No. 3.] THE PROTOZOA AND METAZOA. EG)
the daughter-spheres takes place before the daughter-nuclei of
the preceding mitosis are completely separated (Fig. 21). Fig.
21 also shows that the plane of the second division of each
sphere is at right angles to the plane of the first division. The
same thing is shown in an earlier stage in Fig. 18, where the
primary axis of the daughter-nucleus is bending around the
sphere ; and again in the sections represented in Figs. 24, 39.
In some cases, particularly after fixation with Hermann’s
fluid, thick but short fibrous processes may be seen passing
from the sphere into the cytoplasm (Figs. 9 and 11). These
are analogous to the astral rays of metazoan cells, and like
the central-spindle fibers they are formed from the substance
of the sphere, being made up of granules or microsomes
similar to those found in the sphere.
According to the foregoing description, the sphere in /Voctz-
luca apparently corresponds to Boveri’s archoplasm (’88). It
is a persistent cytoplasmic structure, of a definite size and
shape in resting and active cells, and appears to consist of
a specific substance. Astral rays and central-spindle are
formed from its substance, while at certain stages a centro-
some lies within it (see p. 731).
The main observations by. contemporary writers on the
sphere fall into one or other of two groups, according as they
agree with Van Beneden’s or Boveri’s conception of the origin
of this structure. Van Beneden thinks that the sphere, while
it consists of the same substance, is morphologically different
from the cell plasm. Boveri originally considered it (and
with it all spindle and astral fibers) as not only different
from the cell plasm morphologically, but as composed of an
independent substance —the archoplasm. Among botanists
the ‘“‘archoplasm idea” is generally accepted, and is expressed
by Strasburger’s term ‘“kinoplasma,” while among zodlogists
this theory has not been generally approved, most observers
supporting either Van Beneden’s theory or some modification
of it. There is, however, the greatest latitude in this support.
Many interpret the sphere as formed anew from the cyto-
plasmic reticulum at each mitosis. (See among others Heiden-
hain ('94), Reinke (94), Wilson ('95), Eismond ('95), Kostanecki
728 CALKINS. [VoL. XV.
(96), v. Erlanger ('97), etc.) Others, however, while regarding
the sphere as a differentiation of the cytoplasmic reticulum,
find that it is permanent in the cell, dividing by fission like
the centrosome. (Van Beneden '87, Meves '95, vom Rath
'95.) Here the two views stand rather close together; on
the one hand, it is maintained that there is a distinct structure
permanent in the cell, but composed of modified cytoplasmic
reticulum ; on the other hand, it 1s held that the distinct struc-
ture is composed of a distinct substance. The difference here
is certainly not great.?
The fact that in some cases the sphere appears only during
mitosis is perhaps the most serious obstacle to Boveri’s inter-
pretation. He avoids it, however, by maintaining (96) that
the archoplasm is not necessarily restricted to a definite body,
but may exist throughout the cell in the form of minute
granules, which during mitosis are attracted around the centro-
somes, forming spheres. He now (’97) considers that the eggs
of Ascaris, upon which his idea was based, differ from other
cells in regard to the presence and form of the archoplasm,
the general rule being that fibers run directly from the centro-
some into the cytoplasm. He also says that with the excep-
tion of NVoctzluca (Ishikawa) he knows of no other form of
cell where a solid archoplasmic substance exists around the
centrosome.”
Schaudinn’s Paramoeba must now be included with Voctz-
luca. Kostanecki’s ('96) work on Ascaris does not sustain
Boveri’s conception, since he finds that, as in other forms, the
spindle-fibers pass directly from the centrosome into the cyto-
plasm; and his work shows that in regard to archoplasm this
form cannot be classed with Woctzluca and Paramocba.
1 Ina number of cases the term “archoplasm” has been used to designate
portions of the cell which are not obviously homologous with the parts described
by Boveri. Thus Foot ('96) calls everything in the cell which stains with Lyons
blue “ archoplasm,” and Moore (’94) apparently restricts the term to the residual
spindle-fibers (Nebenkern).
2« Ja, mit Ausnahme von JVocti/uca, wo das Archoplasma nach Ishikawa
genau den Eindruck jener kornigen Substanz des Ascaris-Eies macht, wiisste
ich keinen anderen Fall zu nennen, wo zunichst im Umkreis des Centrosoms
eine dichte kornige Kugel besteht, die sich allmahlich in das Strahlensystem
umwandelt” (/. ¢., p. 40).
No. 3.] THE PROTOZOA AND METAZOA. 729
It appears, therefore, that the archoplasm-theory in its
original form is far from being definitely established. Heiden-
hain ('94,'97), one of the chief opponents of the archoplasm
idea, has, however, described a substance connected with the
centrosome which is of considerable interest in relation to
that theory. In resting cells this substance is described as
follows : “ Diese Centralkérper sind innerhalb des Microcen-
trums durch eine Zwischenmasse miteinander verbunden,
welche meist recht gut sichtbar ist, und im giinstigen Fall
durch das Rubin der Praparate stark gefarbt erscheint. Diese
Zwischenmasse entspricht ganz genau der Materie der ‘pri-
maren Centrodesmose’ beim Leukocyten”’ ('97, p. 231). It is
composed of minute granules, and from it are developed the
fibers of the central-spindle which form the secondary “ Centro-
desmus.” This substance would seem to agree to a certain
extent with the “archoplasm” of Boveri's theory, and I believe
that it may be compared to the substance of the sphere in
Noctiluca. Like the centrodesmus, the sphere is made up of
granules surrounding the centrosome, and during mitosis the
central-spindle fibers are formed from it. Like archoplasm,
it is a specific substance in the cell, surrounding the centro-
some and giving rise to the spindle-fibers. While Boveri's
original conception of archoplasm as a distinct substance in
the cell may not hold for the Metazoa, it is, I believe, entirely
consistent with the facts in Protozoa, as will be shown in the
comparative part of this paper.
4. The Mantle-fibers.
It has been shown above that the fibers of the central-spindle
have no connection with the chromosomes, but pass without
interruption from pole to pole. In the metaphase, and after
the disappearance of the membrane, a second set of spindle-
fibers is formed, passing from the sphere to the ends of the
chromosomes. These are the mantle-fibers or “radial-fibers ”’
of Ishikawa. He speaks of them as follows: “Of the origin
of the mantle-fibers in MocteJuca, I can say but a few words,
Cf. p. 755.
730 CALKINS. [VoL. XV.
since the whole problem remains obscure and requires a thor-
ough study with better methods and optical instruments. In
sections given above, these fibers which are formed within
the nucleus and probably attached to the chromosomes appear
to come into close juxtaposition, but not to be continuous with
those without, z.¢., those seen within and without the nucleus
appear to be different from each other, the former originating
from the nucleoplasm and the latter from the cytoplasm, just
as Brauer thinks concerning the formation of the spindle-fibers
of Ascaris megalocephala bivalens”’ (p. 323). My observations,
while not conclusive, show, I believe, that the mantle-fibers
(or ‘radial-fibers” of Ishikawa) are connected with the ‘nucleo-
plasm,”’ but lie completely outside of the nucleus in the sub-
stance of the sphere. The mantle-fibers in mitotic figures of
most Metazoa are usually first visible just before or during the
time of disappearance of the nuclear membrane. So it is with
Noctiluca, but here, as in the first stages of spindle-formation
in various eggs, the membrane, instead of disappearing entirely,
fades away in one part only.
Much has been written during the last few years on the ori-
gin of the spindle-fibers, the main question being whether they
are of nuclear or cytoplasmic origin. In the different cells of
Metazoa the fibers arise sometimes from the nucleus, sometimes
from the cytoplasm. In other cases, z.e., when the mitotic fig-
ure contains a central-spindle and mantle-fibers, they arise from
both nucleus and cytoplasm, in which case the central-spindle
usually comes from the cytoplasm (Hermann ('91), Meves ('96),
Flemming (95), Heidenhain (94), Ishikawa ('94) ).1. In many
cases, however, no central-spindle can be distinguished, and in
such forms the fibers sometimes arise in the cytoplasm (Stras-
burger (92, '97), Mottier ('97), Osterhaut (97), and botanists
in general; Griffin (96), Wheeler ('95), Mead ('97), etc.), or,
in many cases, from the nucleus (Weismann and Ishikawa ('89),
Brauer (93, '94), Riickert ('94), Korschelt (95), Wilson ('96),
v. Erlanger ('97) ).
I have already shown that the fibers of the central-spindle
1 An exception is found in Ascaris megalocephala univalens where the central-
spindle is intra-nuclear (Brauer ('93) ).
No. 3.] THE PROTOZOA AND METAZOA. By
in Woctiluca are formed from the substance of the sphere, or
archoplasm. The mantle-fibers are probably nuclear in origin,
as indicated by Ishikawa. They are focused in the centrosome
and connect with the chromosomes. The microsomes compos-
ing them are at first only loosely strung together (Fig. 38), but
later they become more closely packed, forming solid fibers (Figs.
30-39). Since the chromosomes lie in a ring around the cen-
tral-spindle the mantle-fibers also surround it, and thus complete
the similarity to some mitotic figures in Metazoa (Figs. 1, 28,
and 29).
When fully formed, the mantle-fibers seem to be stiff fila-
ments of constant length and thickness. They do not shorten
as mitosis progresses, nor do they become thicker, but in the
telophase they appear the same as in the earlier anaphase
(Fig. 24).
5. The Centrosome.
It is with some hesitation that I undertake a description of
the centrosomes in Voctzluca, for they are so minute and are
accompanied by so many large cytoplasmic granules that, except
in mitosis, their identification is not only difficult, but sometimes
impossible. I am certain that Ishikawa, in many cases at least,
mistook some of the cytoplasmic granules for centrosomes, for
I have been entirely unable to find centrosomes of the size and
form described by him. Nevertheless, his general conclusion
that a centrosome in JVoctiluca does exist is correct ; for its
identification when connected with the mantle-fibers presents
no difficulty whatever (Figs. 23, 24, 28, and 38). Inregard to
its origin Ishikawa had little to say, but he surmised that, in
some cases at least, it comes from the nucleus.
While fully appreciating the difficulties in the way of deter-
mining the origin of a body so minute and so easily confounded
with other granules, there are, nevertheless, several phenomena
connected with the prophase of nuclear activity which give, I
believe, an apparent basis for the conclusion that the centro-
some is a minute granule lying inside the nucleus of the resting
cell, and that it migrates out into the attraction-sphere during
division.
732 CALKINS. [VoL. XV.
In the metaphase the centrosome can easily be identified in
all nuclei, whether undergoing vegetative or spore-forming divi-
sion, and in the latter it can be distinguished at all stages. In
spore-forming mitoses it divides early in the anaphase so that
single centrosomes are rarely seen. I have not observed its
division in the anaphase of vegetative mitoses ; it probably
remains undivided until the prophase of the next division. In
the metaphase the double centrosome lies in the inside of the
sphere, but usually in an eccentric position (Fig. 38). In Fig.
38 (a mitosis in a spore-forming cell) the two daughter-centro-
somes lie close together and separation would take place later
in a direction at right angles to the plane of the paper. The
preceding division is just finished, and the daughter-centrosomes
have divided for the ensuing mitosis.
The daughter-centrosomes divide in the early anaphase, after
which they lie more nearly in the center of the sphere (Figs. 28
and 31), and, as a rule, they remain near the center until the
division is complete (Figs. 23 and 39). They may lie eccen-
tricly, however, even in the telophase (Fig. 24), but in this
case it is away from the nucleus. Except for the mantle-fibers,
the centrosome is in no way the center of a radial system ; the
sphere lies around it as an inert undifferentiated mass.
The centrosome appears to vary somewhat in form and in
size. Insome cases it is comparatively large and round (Fig.
24); in others it is round but much smaller (Figs. 23, 39);
while in still other cases it is irregular in outline or even tri-
angular (Fig. 38).
In the resting cytoplasm I have failed completely to find a
centrosome in the sphere. In the resting nucleus, however,
there is a minute body which stains intensely with the iron
haematoxylin and which differs considerably in appearance from
chromatin granules (see p. 7). This body disappears during
the chromosome-formation and is not seen again until the
nucleus reforms after division (compare Figs. 3, 4, 6, 7 with 8,
Q, 10, II, etc., where chromosome-formation has begun). It can
always be distinguished from the chromatin granules by its luster
and small spherical form. It takes no part in spireme-formation,
but remains a separate element (Figs.6 and 7). At the begin-
No. 3.] THE PROTOZOA AND METAZOA. 733
ning of nuclear activity it generally lies in that half of the
nucleus which is nearest the sphere, and often quite close to it.
I have been unable to trace it further than this, when it disap-
pears. At about the same time—ze., at the beginning of
chromosome-formation — the nuclear membrane, lying just
below the sphere, shows distinct undulations or wrinkles, while
it is intact and uniform at all other points. These can be
seen only in sections, but I have found them in all sections
of nuclei in this stage (Figs. 32-36), while in sections of
nuclei in resting stages the membrane is as smooth at this
point as at all others. At the points where the undulations
appear, spaces are formed by the elevation of the membrane.
These spaces are, as a rule, free from the basichromatin
and oxychromatin of the nucleus (Figs. 32 and 33). In nu-
merous cases, however, two minute, deeply staining granules
were found in the spaces thus formed. The granules were
always found in pairs, and in many instances no other granules
could be seen (Figs. 32-34). In other cases, and, I am obliged
to admit, in the majority of cases, other granules were found.
The latter always had the appearance of chromatin, and it
is not surprising that chromatin granules should be found
here, for the nucleus at this stage is full of them. The two
granules in question, however, have a distinct brilliancy, or
luster, and usually have a definite position in relation to the
elevations of the membranes (Figs. 32 and 33). Fig. 34
represents a section where the two granules lie just outside of
the nuclear margin, which is distinctly indented at this point,
and where the nuclear membrane could scarcely be distin-
guished. They are not in the substance of the sphere, but
lie in a hyaline space between it and the nucleus. A similar
stage is represented in Fig. 35. In this case only two other
granules were visible, and these are represented on the left of
the figure near the membrane. They are similar in form and
appearance to other cytoplasmic granules found in the proto-
plasm of Woctzluca. Finally, in Fig. 36, the two granules lie
in the sphere just outside of the nuclear membrane and the
latter is rapidly assuming its regular contour. Other granules
are pictured in the cytoplasm and in the sphere.
734 CALKINS. [Vot. XV.
R. Hertwig (96) doubts Ishikawa’s account of the centrosome
in WVoctzluca, and, in accordance with his theory regarding this
body, he would probably consider the sphere in JVoctz/uca as
an enlarged centrosome. He also regards the centrosphere in
the sea-urchin egg as an enlarged centrosome, in opposition to
Boveri (95), Hill (95), and Kostanecki ('96), who have found cen-
trosomes within it.1 From his experiments with strychnine on
developing eggs he also concludes that the centrosome may
have the form of pole-plates, as in some Protozoa. He supposes
the centrosome to be, originally, an intra-nuclear structure (as
in Euglena, Spirochona, etc.), which becomes extra-nuclear in
Noctiluca, Paramoeba, and the majority of Metazoa. The intra-
nuclear pole-plates of most Protozoa, the sphere in JVoctzluca
and Parvamoeba, and the sphere in Echinoderm eggs are, there-
fore, regarded by Hertwig as homologous structures, each of
which he would call the centrosome. At first sight this is a
most plausible theory, but many indubitable facts stand in its
way. Boveri, Hill, and Kostanecki have found centrosomes in
the sphere of sea-urchin eggs; Balbiani ('95) has described less
substantially a centrosome in the pole-plate of Spzvochona ; and
Ishikawa demonstrated the centrosome within the sphere of
Noctiluca, an observation which I can fully confirm.?
As there is no trace of the centrosome in the sphere during
nuclear rest, the conclusion is apparent that its presence here
during nuclear activity can be accounted for either by forma-
tion de xovo or by migration from some other part of the cell.
This alternative involves a question in cellular biology which
is far from settled — vzz., is the centrosome a permanent ele-
ment of the cell?
In the first place, a number of investigators, especially
among botanists, deny entirely the presence of a centrosome
in certain dividing cells (Strasburger (97), Osterhout ('97),
Mottier (97), etc.), while the remarkable observation made by
Juel (97) seems to indicate that the centrosome is not neces-
sary in the formation of a spindle-figure. Juel found that a
single chromosome was parted from its fellows and formed a
1 Wilson also (’97) finds unmistakable centrosomes in 7oxopneustes and Arbacia,
which are derived from the middlepiece of the spermatozoa.
2 See appendix, p. 49.
No. 3.] THE PROTOZOA AND METAZOA. 735
separate small cell around itself. It then passed into the
resting stage, from which it emerged to form a mitotic-figure
in which all of the elements, save centrosomes, were present.
In the second place, a number of observers have maintained
that, for each mitosis, the centrosome arises de movo, as in the
well-known theories of Biirger ('92), Watasé ('93), Reinke ('94),
and others. The results which Hertwig ('95) and Morgan ('96)
obtained by treating the cell with dilute poisons, etc., are evi-
dence in the same direction. Foot ('97) also holds that the
centrosome in the first cleavage-spindle of A//olobophora foetida
is the physiological effect of the spermatozoén upon the cyto-
plasm of the egg, and not a permanent morphological element
brought in by the spermatozoon. To this list must be added
a great number of observers who have followed the history of
the centrosome up to a certain point in cell changes, after
which they found no further trace of it. Finally, many
observers have followed the centrosome from one generation
to another and maintain it to be a permanent cell organ in
accordance with the original view of Boveri (87) and Van
Beneden (87). Brauer ('93) and the majority of spermatologists
have traced the centrosome from generation to generation, in
some cases up to a certain definite structure in the mature
spermatozoon (Moore ('93), Meves ('96), Hermann (92), Cal-
kins ('95)), and many observers have followed the centrosome
in the same way in the cleavage of the egg (Boveri ('87), Van
Beneden (’87), Henneguy ('91), Mead ('95),! Griffin ('96)?).
From this review of the subject it is plain that the question
of permanency of the centrosome has no immediate prospect
of settlement. Repeated examinations of my sections, the
evidence of which I have given above, I believe, justifies the
conclusion, in a provisional sense at least, that the centrosome
of Nocttluca is a permanent element ; that it exists during the
1 Mead later ('97) comes to quite an opposite view. He says: “The fore-
going observations convince me that the asters and centrosomes in the Chaetop-
terus ovum arise by modification of the cytoplasmic reticulum ” (p. 394).
2 Wilson ('97) has traced the sperm-centrosome through the first cleavage and
into the 2-celled stage in Avdacia. Contrary to the view of Doflein ('97), he finds
that the centrosome is not represented by the entire middlepiece of the spermato-
zoon, but that it is contained within the middlepiece which is thrown off as a
shell after the spermatozo6n enters the egg.
7306 CALKINS. (Vou. XV.
resting stages as a deeply staining granule in the nucleus (see
p. 7); that it divides and gives rise to the two granules
whose history I have given; that these finally come to lie in
the cytoplasmic sphere where they separate as the amphiaster
is formed, one going to each daughter-sphere, where mantle-
fibers connect them with the chromosomes.
The nuclear origin of the centrosome has been observed in
a number of forms. Thus, in all Protozoa hitherto described,
with the possible exception of Euglypha, Noctiluca, and Para-
moeba, the centrosome, or its equivalent, is intra-nuclear in
origin. In the Metazoa the well-known results which Brauer
obtained in the case of Ascaris megalocephala univalens have not
been duplicated for other forms, although a number of obser-
vers have been led to think that, as in Ascaris, the centrosome
is intranuclear. Among these may be mentioned Balbiani
(93), Julin ('93), Mathews ('94), and Carnoy and Lebrun ('97),
O. and R. Hertwig have long maintained that the centro-
some is primitively intra-nuclear, having been differentiated
originally from part of the nuclear substance. There is a
very important difference, however, between the intra-nuclear
centrosome of Metazoa and Protozoa. Both Brauer and
Mathews, for example, describe the centrosome as surrounded
by a sphere, whereas in /Vocti/uca the sphere lies permanently
outside of the nucleus.! If my observations are correct, sphere
and centrosome in JVoctz/uca must, therefore, have an independ-
ent origin. As already indicated, however, there are so many
chances for error in tracing the centrosome in JVoctiluca
through resting stages that this conclusion must remain an
open question until further research throws more light upon it.?
1 Mathews’s statement of this process is as follows: “At maturation the cen-
trosomes are first accurately to be distinguished as two (at a very early stage
apparently as one) deeply staining, small, but distinct and characteristic, granules
lying side by side either in the nuclear membrane or immediately without it,
and invariably on that part of the vesicle nearest to the surface of the egg. Occa-
sionally one of the granules appears before the other and migrates some distance
from the nucleus before the second appears. In cases where they both lie clearly
outside of the nucleus, the nuclear membrane is invariably broken behind them”
(p- 324)-
2 Except for its decided staining qualities this intra-nuclear body might be con-
sidered a nucleolus and so be used as evidence in support of views of Karsten
No. 3.] THE PROTOZOA AND METAZOA. 737
D, Tue MeEcHANISM oF Mitosis IN NOCTILUCA.
The peculiar type of mitosis in Mocté/uca may perhaps
throw some light on the mechanism of mitosis in general.
Here the entire absence of astral rays passing from the sphere
into the surrounding cytoplasm excludes all contractility
hypotheses in so far as they are based upon active physio-
logical contractility of these fibers (Van Beneden, Boveri,
Flemming, Reinke, etc.), and the same remark applies to
Heidenhain’s view that mitosis is affected through elastic
tension of the rays. Furthermore, there is no morphological
evidence to show that division is affected by contraction of
the mantle-fibers (Hermann), for, as shown above, these do
not shorten and thicken, but remain the same throughout
mitosis. The possibility that their substance is taken up by
absorption into the sphere, as Wilson has suggested for Zoro-
pneustes ('96), is removed by reason of two facts, vzz., the
daughter-spheres do not enlarge in the anaphase (spore mitosis)
and the mantle-fibers can always be traced to distinct points
in the sphere, z.e., to the centrosomes. It must be, therefore,
that the separation of the daughter-chromosomes is caused
either by an active divergence of the spheres, or by growth of
the central-spindle fibers and the consequent passive separa-
tion of the spheres (Driiner). The former is improbable
because of the entire absence of the antipodal cones or astral
rays, leaving the second as the only mechanical hypothesis
which agrees with the facts.
My conception of the process is, then, as follows: the central-
spindle lying within the ring of chromosomes is advantageously
placed for exerting the necessary dividing force. The nuclear
membrane disappears and mantle-fibers connect the ends of
the chromosomes with centrosomes in the spheres. The cen-
tral-spindle elongates, causing separation of the spheres ; the
mantle-fibers, remaining firm, move with the spheres, dragging
('94), Lavdowsky ('94), Carnoy and Lebrun ('97), and others who regard the
nucleolus as the seat of the centrosome. On the contrary, it is a body which
much more resembles the so-called “ nucleolus-centrosome” described by Bal-
biani in Sfzrochona, and should be, I think, placed with the latter structure as
one of the primitive forms of the sphere. See appendix, p. 49.
738 CALKINS. [Vou. XV.
the ends of the chromosomes with them. As the central-
spindle becomes longer, the chromosomes are more and more
separated, until finally the distal ends are separated and chro-
mosome division is completed.
R. Hertwig (95) has already pointed out that this view of
the mechanics of mitosis is perfectly consistent with the facts
of nuclear division in the Infusoria. No mechanical hypothe-
sis, however, can fully explain the different phenomena in-
volved in the mitosis of (Voctiluca. The action of the central-
spindle cannot explain the first elongation of the nucleus in
the primary axis, nor the later ring-form. Neither does it
offer any explanation of the forces which cause the in-sinking
of the central-spindle into the position of the secondary nuclear
axis. The ultimate cause of these phenomena remains unex-
plained, and there seems to be little doubt that something
deeper than mere mechanical force is necessary to explain
mitotic activity. This is strikingly confirmed by Juel’s obser-
vation on the isolated chromosome of Hemerocallis fulva and
by Boveri’s recent ('97) experiments on sea-urchin eggs in which
he found that blastomeres are incapable of dividing when
chromatin is absent.
Ill. THE NUCLEAR RELATIONS OF NOCTILUCA TO METAZOA
AND PROTOZOA.
A. RELATIONS TO METAZOA.
The similarity of the mitotic-figures of /Voctz/uca to those of
the Metazoa has already been indicated by Ishikawa. The
comparison can now be carried still further. They agree in
the following points: (1) the central-spindle fibers end in
spheres which contain centrosomes ; (2) the central-spindle
occupies a position in the center of the nuclear plate; (3) the
chromosomes lie freely around it without an intervening nuclear
membrane ; (4) the central-spindle fibers are not connected
with the chromosomes ; (5) mantle-fibers connect the chromo-
somes with the centrosomes ; (6) the chromosomes in Vocézluca,
like those of the Metazoa, are composed of granules which are
at first separate, then unite to form a segmented spireme,
No. 3.] THE PROTOZOA AND METAZOA. 739
and after division again become disintegrated ; (7) as in the
Metazoa the chromosomes divide by longitudinal division ; (8)
the centrosomes, finally, are equivalent ; they are the focal
points of the mantle-fibers ; they lie in the spheres during
activity, and they divide during the anaphase in preparation for
the ensuing division. There is some evidence that, as in Ascaris
megalocephala untvalens (Brauer), they come from the nucleus.
In only one respect does the mitotic-figure in Woctzluca differ
from that of the Metazoa—the nuclear membrane does not
entirely disappear. In all other respects its description would
answer for that of any ordinary metazoan mitotic-figure,.
B. RELATIONS TO PROTOZOA.
Mitosis in JVoctzluca is so similar to that in Metazoa that in
itself it throws little light on the origin of the process. The
rapid increase of our knowledge of indirect division in the
Protozoa has, however, made it possible to draw an accurate
comparison between JVoctz/uca and other Protozoa in which the
phenomena of mitosis appear in a still simpler form ; and here
we find some light on the possible origin of mitosis.
1. Origin of Chromosomes.
In many primitive forms of protozoan nuclei, the chromatin
is compressed into a single homogeneous sphere (U7vog/ena,
Dinobryon, Eudorina, etc.), and with no indication of ‘achro-
matic’’ substances in the form of ‘ ground-substances’’ or
“nuclear-sap.’ In other forms of nuclei closely allied to
these, the chromatin in the resting stage is similarly collected
into a homogeneous sphere, but it lies imbedded in the nuclear
ground-substance, the whole surrounded by a nuclear mem-
brane (Actinophrys sol, Gruber ('83), Heterophrys, Acanthocystis,
Artodiscus, etc., Penard (89), and many Flagellates). Penard
and Gruber found that in those cases where the chromatin
forms a single mass it becomes divided into two, three, or
more separate portions. Rhumbler (90) agrees with Gruber
and Penard in such an arrangement of the chromatin in
Rhizopods and Heliozoa, and Wolters (91), Labbe ('97), Clarke
740 CALKINS. [VoL. XV.
(95), and others find similar results in the Sporozoa. Similarly
Schultze (66) described the nucleus of Gvomza as variable ; one
type containing very large granules; another, granules of
medium size ; and still a third type with very fine granules.
Other observers on different forms of Protozoa have given
similar descriptions.
From what we now know about the chromatin changes in
Protozoa, it is probable that the different types of nuclei which,
like Gromia, have been described for the same organism are,
in reality, developmental stages in preparation for division.
Gruber ('83) first showed that in Actinosphaerium disintegration
of the central chromatin-mass is the earliest indication of
mitosis. He found that it first divides into two portions, then
into four, later into eight, and so on until a great number of
minute chromatin elements fills the nucleus. The nuclear
plate is then formed, and division of the nucleus ensues. This
observation was confirmed by Hertwig (84); and Brauer (95)
gave the same general result.!
In other Protozoa the chromatin is permanently in the form
of small chromatin elements. This is the case in Amoeba
proteus, Amoeba binucleata, and Amoeba crystalligera, Euglena
viridis, Cryptomonas, and the macronuclei of the Infusoria.
From Schaudinn’s description ('94) the chromatin elements in
Amoeba crystalligera do not change during mitosis but are
simply separated into equal parts by nuclear division. Similar
results were obtained by Blockmann ('94) and Keuten ('95) in
Euglena viridis.
According to the latter’s account, the nucleus of this flagellate consists,
as in Amoeba crystalligera, of a peripheral ring of elongate chromatin
bodies, and a central “nucleolus.” In division the nucleolus first elongates
while the chromatin is arranged in radial lines. Elongation of the nucleolus
continues until the connecting-piece is reduced to a thin fiber. The chro-
matin is not formed into chromosomes, but is divided into two equal masses,
1 Brauer’s account of the resting nucleus differs in detail from that of Hertwig
and Gruber. He showed the nucleus to be similar to that of a metazoan cell, con-
sisting of chromatin in the form of a reticulum and “ achromatin” in the form of linin.
I have examined many hundred nuclei of Actinosphaerium in the vegetative state
and after fixation with sublimate acetic and Hermann’s fluid, but in none of
them could I find the mononucleolate nucleus described by Gruber and Hertwig.
The chromatin reticulum was invariably present with from two to four net knots.
No. 3.] THE PROTOZOA AND METAZOA. 741
and the daughter-chromatin elements gather around the daughter-nucleoli,
with connecting or ‘‘ Zwischenfaden” elements, which form parallel lines
from pole to pole. These give the striated appearance which is so charac-
teristic of protozoan mitosis. The daughter-nucleoli finally become totally
separated, and the chromatin, which has become massed at the distal ends
of the striae, but which still consists of separate units, forms two clumps
around them.
In these forms the chromatin appears to be perpetually
ready for nuclear division, and the many elements never fuse
to form a homogeneous chromatin-mass. Brauer (95) in
describing the formation of the chromatin elements carried
the analysis a step further in Actznosphaerium.
He found that, as in the metazoan nucleus, the first stage in division is
the disappearance of the net knots, the reticulum becoming more distinct.
The latter then disintegrates and the nucleus is filled with minute chromatin
elements. “Auf einem etwas spateren Stadium ist der ganze Kernraum
mit isolirten K6rnen, den Chromosomen, erfiillt, deren Zahl nicht zu bestim-
men ist” (p. 203). These are not chromosomes, as Brauer states, but
elements which fuse later to form the chromosomes extending across the
middle of the nucleus. They are next transversely divided, no longitudinal
division taking place. It is probable that such an arrangement of the chro-
matin in the nuclear plate of Actinosphaerium represents a primitive stage
in chromosome-formation.
The chromatin in metazoan nuclei passes through a similar
stage before each mitosis, and similar elements are welded
together to form chromosomes of definite shape and size for
each species. A long step in the direction of this metazoan
condition is taken by Voctz/uca, where chromatin elements are
formed as in Actinosphaerium, but become more compactly
fused to form chromosomes of a certain distinct character, and
where these chromosomes are divided longitudinally.1
1 An entirely opposite view of the significance of chromosomes has been main-
tained by Mitrophanow. According to him, the chromatin in the nucleus of
Collozoum is a single compact mass: “la chromatine, en forme d’une petite masse
arrondie, et l’achromatine, que a l’aspect de deux appendices coniques” (p. 625).
He insists that division here is a simplification of mitosis, which, “deviendra
claire, si nous considérons la masse de chromatine comme une chromosome
unique” (p. 626). This interpretation stands alone in the literature of protozoan
mitosis, and instead of simplifying the problem makes it more complex. Mitro-
phanow’s material was fixed with nitric acid and stained with an aqueous solution
of safranin, and it seems probable, therefore, that a better technique will give quite
different results in Collozoum.
742 CALKINS. [Vo. XV.
A spireme-stage is wanting in the prophase of Actznosphae-
rium, and I have been unable to demonstrate a typical spireme
in JVoctzluca, although, as previously pointed out, the chro-
matin passes through a stage which simulates the metazoan
spireme (Figs. 6, 7, and 12). There are, however, certain Pro-
tozoa in which a true spireme seems to occur. Karawaiew
(95) describes spireme-formation in the Radiolarian Aulocantha
scolymantha. In the resting stage, this nucleus has a large
“spongy” nucleolus, consisting of a dense central mass with
numerous branches. When preparing for division the “ spongy ”
mass breaks up into threads, until finally the entire chromatin-
mass becomes a tightly wound spireme. Sooner or later the
spireme-threads undergo a longitudinal division, at the same
time becoming granular. Karawaiew saw no nuclear plate, the
next stage after the spireme being a late anaphase in which
two striped regions were found at the poles. Each stripe con-
sisted of a row of chromatin-granules. The account is by no
means complete, and it is probable that more careful examina-
tion will show a nuclear plate.
Our acquaintance with the chromatin-changes in the micro-
nuclei of ciliates is more satisfactory. The exceedingly dense
structure of the micronuclei has led Hertwig and Gruber to
class them as “massive nuclei.” During division they swell
considerably, a change invariably preceded by a spireme-forma-
tion (Maupas ('g9) and Biitschli (76) ). The spireme gives rise
to granular chromatin-threads which as in Actinosphaerium,
become thicker in the central portion, where they are finally
divided by transverse division.
In the great nuclei of Dinoflagellates also, Biitschli (85) and
Lauterborn (95) have shown that the chromatin-reticulum
becomes “increased in size” until a “much-twisted Kniauel ”
results. After this the chromatin becomes arranged in more
or less regular parallel fibers which are divided transversely.
Zacharias describes for the same form a much more complex
process of mitosis, but his results are denied by Lauterborn and
others.
A much more complicated mitosis, including spireme and
chromosome-formation, was described by Pfitzner ('g6) in the
No. 3.] THE PROTOZOA AND METAZOA. 743
case of Opalina ranarum. Schewiakoff (88) gives a somewhat
similar description of mitosis in Euglypha alveolata.
Here the chromatin network of the nucleus gradually becomes thicker,
especially in the region of the net knots, until a coarse spireme appears.
Threads composed of many small particles — the “ chromatin granules ” of
Pfitzner — are finally formed from the spireme; these are ragged at first, but’
become homogeneous and smooth, after which they arrange themselves
around the periphery of the nucleus. The threads then shorten and become
bent into loops, the angle being turned towards the axis of the spindle. At
this stage each chromatin-loop is divided longitudinally and the daughter-
chromosomes separate. After the formation of a daughter-spireme the
nucleus returns to the reticular state.
From this examination of the different changes undergone
by the chromatin in the various forms, it is possible to get an
idea of the probable development of chromosomes, although the
many gaps in the series and the often incomplete observations
make it far from conclusive. The most primitive structure
would seem to be the mononucleolate forms in which the
nucleus has no ‘cell sap,” and where division is possibly
“amitotic.”” An advance is shown in forms where the mono-
nucleolate chromatin-mass breaks up into smaller elements
during division, as in some Rhizopods and Heliozoa, and in
many Protozoa just before spore-formation. A still higher
type is shown in forms where the chromatin exists permanently
in the form of small granules (many Amoebae, Euglena, etc.).
In Euglena and Amoeba these chromatin-granules do not fuse
to form definite structures (chromosomes) ; they simply separate
half from half, and they are clearly equivalent to the minute
elements, which in other cases are formed by the breaking
down of chromatin-masses. The aggregation of chromatin
elements into more or less definite chromosomes is shown in
a primitive way in Actinosphaerium and the micronuclei of
Infusoria. In WVoctiluca the aggregates become more compact,
definite, and chromosome-like, while for the first time they are
divided longitudinally. A still more metazoan-like chromosome
structure is shown by Euglypha alveolata, where the chromatin
elements are not distributed throughout the nucleus, but unite
at once to form a tightly wound thread — the spireme — from
which the chromosomes are later derived by transverse division.
744 CALKINS. [VoL. XV.
These chromosomes, as in WVoe?z/uca, divide longitudinally, and
daughter-spiremes are formed, after which the chromatin passes
again into the resting reticulum. In Luglypha, therefore, the
chromatin-changes seem to be practically the same as in the
Metazoa, and the elements are even more highly differentiated
than in WVoctz/uca, for the latter has no chromatin-reticulum nor
definite spireme. The aggregation of the chromatin elements
after division to form karyosomes in JVoctz/uca is another indi-
cation of the more primitive nature of this form. JVoctzluca and
Euglypha, therefore, may justly be considered as connecting
links, so far as chromatin is concerned, between the conditions
in Metazoa and the simplest Protozoa.
2. Origin of Centrosome and Sphere.
The origin of the metazoan centrosome and attraction-sphere
from simpler elements in Protozoa has been the subject of a
number of interesting theories. The most recent and the most
important of these have been maintained by Heidenhain (94),
Lauterborn (96), and Hertwig (96). According to Heidenhain
the “ central-spindle,”’ as described by Hermann ('91), is identical
with the spindle formed by the micronucleus of the Infusoria
after the latter has undergone a loss of chromatin and has
acquired a differentiated center —the centrosome. He regards
the nucleus of the metazoan cell as derived from the infusorian
macronucleus, while the mantle-fibers are new formations. Not
only does he compare the nucleus and centrosome with the
macro- and micronuclei of Infusoria, but he even makes this
comparison the basis of a theory in which he derives the
Metazoa from the Infusoria. The comparison of centrosome in
Metazoa with the micronucleus is not original with Heidenhain.
Biitschli (91) had already proposed it, and Hertwig and Lauter-
born had adopted the same view. Lauterborn’s recent attempt
to derive the ‘‘achromatic”’ structures of Metazoa from elements
in the Protozoa is even more ingenious than that of Heiden-
hain. The essential difference between the two theories is that
in the one case (Heidenhain) the centrosome has been derived
directly from the nucleus, while in the other (Lauterborn),
No. 3.] THE PROTOZOA AND METAZOA. 745
centrosome and micronucleus are supposed to have had a
common origin. Lauterborn thinks that centrosome and micro-
nucleus may have had a common ancestor in one of the nuclei
of some primitive binucleated Protozoén, and that intermediate
stages are represented bycertain existing Protozoa. Schaudinn’s
Paramoeba furnishes an hypothetical early stage in differentia-
tion of the centrosome, which there is represented by the
Nebenkoérper. Voctzluca and the diatoms represent a more
advanced stage toward the metazoan centrosome. On the other
hand, the micronucleus of the Infusoria represents a differentia-
tion of the same primitive nucleus in a different direction.
Hertwig’s theory deals more specifically with the develop-
ment of the ‘‘achromatic” parts of the mitotic figure. He
expresses it in one paragraph as follows: ‘“ Bei den Protozoen
finden wir alle Uebergange von der gewohnlichen Durchschnii-
rung des Kerns bis zu komplicirten Karyokinesen. In vielen
Fallen —z. B. den Hauptkernen der Infusorien—ist unzweifel-
haft wahrend der Theilung ein achromatisches, dicht mit
Chromatinkérnchen beladenes Netzwerk allein der Sitz der
treibenden Krafte; es bilden sich weder Spindelfasern noch
Polplatten. Bei Cevatium hirundinella ordnet sich das achro-
matische Kernnetz schon zu Spindelfasern an, auf denen die
Chromatinkérnchen gleiten, um auf die Tochterkerne vertheilt
zu werden. LEinen weiteren Fortschritt macht Sfzvochona
durch die Entwickelung von Polplatten. Unzweifelhafte Kary-
okinesen endlich treffen wir bei Actinosphaerium, Actinophrys,
Amoeba binucleata den Nebenkernen der Infusorien. Bei
Paramoeba Eilhardi und Noctiluca scheint sogar der letzte
Schritt der Vervollkommnung, die Ausbildung von Centroso-
men, gemacht zu werden. Wir wiirden daher drei verschiedene
Ausbildungsstufen in der Entwickelung der Centrosomen
aufstellen kénnen: (1) Die achromatische Substanz ist im
ruhenden Kern zwar noch gleichmiassig vertheilt, liefert aber
wahrend der Theilung Polplatten als Aequivalente von Centro-
somen. (2) Die achromatische Substanz ist dauernd zu einem
intra-nuclearen Centrosoma umgebildet. (3) Sie ist zur Bildung
eines extra-nucledren Centrosoma aus dem Kern herausgetreten”’
(pp. 77; 78).
746 CALKINS. [Vow. XV.
While agreeing with Hertwig’s general conception of ‘ achro-
matic’’ structures in Protozoa, I believe that he has left out
of account a number of facts which have an important bearing
on the general problem. The “sphere” in MVoctzluca is not
the centrosome and must be distinguished from it ; the centro-
some in Actinosphaertum (Brauer) cannot be the same as the
“pole-plate,” and the “intra-nuclear” granule described by
Schaudinn in Amoeba, and the so-called “centrosome” de-
scribed by Balbiani must have some significance apart from the
“achromatic”’ structures which accompany them. If we take
these various structures into consideration, the problem becomes
much more complex, and the possible differentiation of sphere
(archoplasm) and centrosome must be sought for much earlier
in phylogeny.
The most primitive nuclei in which a differentiated “achro-
matic’’ body occurs are found in Euglena viridis and Amoeba
crystalligera. In both of these the nucleus consists of a nu-
cleolus-like body with surrounding chromatin. Keuten ('95)
describes this body, which up to this time had been called a
“ nucleolus,’ as similar to a nucleolus in its staining reactions,
but as playing a different réle in nuclear division. He there-
fore calls it a “ nucleolus-centrosome.”
”
My own observations on Euglena viridis, and on a species of Cryfio-
monas which has a similar central body, confirm every stage given by Keu-
ten, but I give a different interpretation to the staining reactions of this
so-called “ nucleolus-centrosome.”” The color reactions which he describes
for this body are not those of a centrosome. It becomes “ orangegelb”
when stained with Orange G ; with carmine solution it stains more intensely
than the chromatin, while with haematoxylin it takes only a faint stain. In
my preparations it takes a haematoxylin stain, remaining black or blue
when the rest of the cell is stained with Orange G.
These reactions are characteristic of archoplasm or of the centrosphere,
and for this reason, if for no other, the name “nucleolus-centrosome ”’ is
not entirely appropriate. Instead of comparing it with the centrosome of
the Metazoa, it would be much more accurate to compare it with the
sphere in Vocti/uca or with the pole-plates of other Protozoa. A true cen-
trosome has not been found in it.
1 Considerable confusion has arisen because of the indiscriminate use of the
term “nucleolus” in connection with protozoan nuclei. It has been applied
to the chromatin-masses (karyosomes) and to various “achromatic ’”’ structures.
No. 3.] THE PROTOZOA AND METAZOA. 747
A similar central nuclear-body has been described by Schau-
dinn in Amoeba crystalligera. With high powers he was able
to make out a certain alveolar (‘‘wabige’’) structure of the
“nucleolus” (achromatic body’’), and, in addition, he occa-
sionally found in it a granule or granules which stained with
chromatin dyes. He included all of these granules in the
nucleolus. The chromatin undergoes no preparatory changes
before division, and the nucleus divides first, the deeply stain-
ing granules having meantime disappeared.
Schaudinn considers this a confirmation of F. E. Schultze’s earlier
description of the division of Azoeba as amitotic, although he seems to
realize the significance of the nucleolus when he says: “ Der als Nucleolus
bezeichnete Theil des Kerns scheint bei Durchschniirung des Kerns, wie Fig.
II 4 u. III 4 zeigen, die Hauptrolle zu spielen” (p. 1035). The presence of
granules within the “nucleolus” is interesting, and may be taken as a pos-
sible indication of an early stage in the differentiation of a centrosome.
The intra-nuclear “achromatic body” plays a more impor-
tant réle in the nuclei of Euglypha, Spirochona, and Kentro-
chona. In the former the bodies at the poles of the spindle
are considered by Schewiakoff the same as the “ Polkorperchen ”
(centrosomes) of metazoan nuclei. Their history is not clearly
made out, but his description seems to indicate a nuclear
origin, though he himself draws a different conclusion. His
account is as follows : “ The polar cytoplasm develops radial rays
(‘Polstralen’) which converge at the ‘Polkorperchen’ (pole-
plate) in a small invagination of the nuclear membrane. At
the same time spindle-fibers make their appearance inside of
the nucleus, and he concludes, therefore, that the ‘ Polkor-
perchen’ is derived from the pole-rays by the coalescence of
the cyto-microsomes lying in them. There is no mistaking
Schewiakoff’s meaning ; the pole-bodies are derived from the
cytoplasmic granules. There are, however, some significant
features in his account of the division which throw consider-
able doubt on this inference. In the first place, at the time
when the spireme is well formed, and just before the formation
of the ‘ Polkérperchen’ the so-called ‘nucleolus’ disappears.
There is, in reality, no occasion for using it at all, for the meaning is much better
expressed by the terms “chromatic body” (or karyosome) and “achromatic body.”
748 CALKINS. [Vou. XV.
In the second place, during the anaphase ‘the chromatin-loops
become reduced to thick threads and the ‘ Polkérperchen’ is
redrawn into the nucleus.’ Finally, after the stage just
described, the chromatin regains first its coarse and then its
fine mesh-like structure, and the ‘nucleolus’ reappears in the
nucleus. If the ‘Polkorperchen’ is withdrawn into the nucleus,
and if the nucleolus reappears shortly afterwards, it is probable
that the ‘nucleolus’ is derived from the pole-bodies. Also, if
the ‘nucleolus’ is thus derived from the ‘ Polkorperchen,’ and if
it disappears when the new pole-bodies are formed, an equally
possible inference is that the pole-bodies are actually derived
from the ‘nucleolus.’’” There is reason, therefore, to believe
that in Euglypha, also, the “achromatic bodies” are intra-
nuclear in origin.
The nuclear division of Spzrochona was first described by
Hertwig (77). Since then it has been described by a number
of observers, Balbiani ('95) being the most recent. Hertwig
describes the resting-nucleus as consisting distinctly of two
parts, one finely granular, and the other homogeneous in
structure. Balbiani (’95) has, in the main, confirmed Hertwig’s
description of the Sfzvochona resting-nucleus. He finds, how-
ever, that the two parts of the nucleus are separated by a
fissure or ‘“‘fente,” which he thinks is the “ Kernspalt”’ of the
ciliate macronuclei. The “ homogeneous” part, which undoubt-
edly corresponds to the “achromatic body ”’ of other Protozoa,
is, therefore, within the nuclear membrane, but, as in Woctiluca
and Paramoeba, it is completely separated from the chromatin.
Hertwig describes a small central granule in the homogeneous
part, while Balbiani’s description shows that other structures
of a fibrous nature are also present before this body appears.
He finds in stained preparations that the achromatic part
(which appears homogeneous during life) contains numerous
short fibers which form a network. These fibers do not stain
with ordinary chromatin dyes and become arranged in a certain
definite manner about the “nucleolus” (central granule in the
“homogeneous”’ or achromatic body), which appears later.!
1 “La partie dite homogéne du noyau ne merite pas non plus cette qualification
dans le noyau fixé par les reactifs. Elle aussi présente un continu filamenteux
No. 3.] THE PROTOZOA AND METAZOA. 749
The exact history of the achromatic body of Spzvochona
is not given either by Hertwig or Balbiani. The former says
that at times of division the homogeneous part enlarges, and a
small central granule, which he calls the “nucleolus,” appears.
This gradually changes by sending out amoeboid processes,
after which it becomes more and more indistinct until it can
no longer be made out. The granular chromatic portion breaks
into smaller pieces until the entire nucleus appears homogene-
ous. After this two masses of homogeneous substance, which
he calls the “end plates,” appear at the two extremities, and
these he regards as the same substance as the original homo-
geneous portion. Balbiani also thinks that the “end plates”
or pole-plates (Calottes) are the same as the homogeneous part
of the resting-nucleus.!
In all the larger Spzvochona nuclei the homogeneous part
(z.e., the “achromatic body’’) encloses that granule which
Balbiani with Hertwig calls the “nucleolus.” In smaller
nuclei, on the other hand, an analogous body is found in the
granular and not in the homogeneous part. Hertwig shows
no connection between these two granules, but claims that the
“nucleolus of the homogeneous part is the homologue of the
metazoan nucleolus.” Plate ('86) gives a somewhat different
account, and admits a possible identity of the two. The
“nucleolus’’ of the homogeneous part, he thinks, is formed
from particles of chromatin which penetrate, while in solution,
from the granular into the homogeneous portion, and there
reform into the solid refringent corpuscle. Biitschli regards
the corpuscle as a condensation of the chromatin-reticulum of
comme la partie granuleuse, mais les filaments sont plus courts, plus fins, beau-
coup moins nombreux, et au lieu d’étre placés parallelment les uns aux autres, ils
s’entrecroissent diversement dans la substance homogéne et transparente (suc
nucleaire) dans laquelle ils sont plongés. De plus ils ne se colorent pas, ou fai-
blement, par les colorants de la chromatine, notamment le vert de methyle, et se
montrent des lors comme formes d’achromatine. Cette structure de la partie
homogéne ne s’observe que dans les noyaux qui ne contiennent pas encore un
nucleole (centrosome) ou dans ceux oti le nucleole se trouve encore dans la partie
granuleuse, lorsqu’il est parvenu dans la partie homogéne les filaments achroma-
tiques prennent une autre disposition que nous decriron par la suite ” (p. 248).
1 “ Nous pouvons done conclurer a4 une identité morphologique et chimique com-
pléte entre les masses polaires du noyau en division et la partie achromatique du
noyau au repos ” (p. 292).
750 CALKINS. [Vor. XV.
the homogeneous portion. Balbiani agrees with neither. He
thinks that the “nucleolus”’ which is found in the granular
part of the nucleus is not an initial but a final step in division
(telophase), the corpuscle or ‘nucleolus’ forming as follows :
The ends of all the chromatin filaments converge at the pole
during the telophase. These ends, which at first are closely
pressed together, separate later, and a space or vacuole is made
containing several granules. The latter collect together at a
later stage to form a single corpuscle. This corpuscle is the
“nucleolus,” and from here it passes through the granular and
into the homogeneous part of the nucleus, where it undergoes
the changes so carefully described by both Hertwig and
Balbiani.
The peculiar changes which this corpuscle undergoes led
Balbiani to regard it, quite independently of the “ pole-plates,”
as the “centrosome.”
He says: “Le globule central participe 4 la fois des caractéres d’un
nucleole vrai, et d’un centrosome; comme nucleole, il disparait par resorption
dans la substance achromatique au debut de la division, pour se regenerer
chez les deux nouveux noyaux par les processus indiqué plus haut; comme
centrosome il condense autour de lui la substance environnante son forme
d’une petite sphere attractive intra-nucleaire, que ne passe pas du noyau
dans le protoplasma pour y jouer le réle d’un centrosome ordinaire pendant
la division de la cellule” (p. 309). He denies Hertwig’s homology of “end
plates ” to “ Polk6rperchen ” or centrosomes, because they are nuclear in
origin, whereas the latter are cytoplasmic, and he attempts to explain them
“comme des accumulations, aux deux poles du noyau en division, de sa
substance achromatique, et leur destinée est de ramener les noyaux nouveaux,
au type normal du noyau au repos chez le Spirochone ” — which explains
nothing at all.
From Balbiani’s description of the “achromatic” parts in
Spirochona it is evident that this nucleus presents one achro-
matic element separated from the chromatin by a “fente,’’ and
similar to the sphere in Woctz/uca; and a second element, —the
so-called “centrosome ”’— which is derived from the chromatic
portion of the nucleus. This body passes from the chromatin
into the ‘‘achromatic”’ part of the nucleus, possibly in the same
manner as the centrosome in JVoctz/uca is supposed to pass
from the nucleus into the sphere. It cannot be identified with
No. 3.] THE PROTOZOA AND METAZOA. 751
the ‘“nucleolus-centrosome” of Ezglena, for the latter is
“achromatic”? and probably equivalent to the pole-plates of
Spirochona.
A very similar description has been given in the case of
another parasitic ciliate, Kextrochona Nebaliae. According to
Rompel’s ('94) description, the nucleus of Kentrochona develops
at one pole an “achromatic’”’ mass, which extends outwards
and bears two centrosomes at its extremity. After this mass
is well developed a similar structure appears at the opposite
pole.
R6mpel then proceeds as follows: “ Wie wird der Gegenpol gebildet ?
Genauer, in welchem Lageverhaltniss stehen Chromatin und Kernspindel
(achromatic portion) wahrend der Ausbildung des Gegenpols? Nach den
vorliegenden Praparaten diirften iiberhaupt nur zwei Méglichkeiten in
Betracht kommen. Entweder wird das von Anfang an ring- oder cylinder-
férmige Chromatin von der Kernspindel central durchbohrt, und diese wird
so zur Achse des Chromatinhohlcylinders, oder die Kernspindel zieht sich
unter dem ventral eingebuchteten Chromatinhohlcylinder her.” Rémpel’s
Fig. 4 é represents the latter alternative, which certainly suggests the action
of thesphere in Voctz/uca ; forin Kentrochona the “achromatic Kernspindel,”
like the “‘central-spindle ” of /Voctz/uca, is apparently sunk in the nucleus
and stretches from pole to pole.
Rompel’s account of the division of Kentrochona is very
faulty, and the different stages described are separated by wide
gaps. His “centrosomes,” too, have been questioned by Hert-
wig (96), Balbiani (94), Flemming (94), and others, most of
whom think he has mistaken micronuclei for centrosomes.
Their criticisms are upheld by Doflein (96, '97), who has recently
reéxamined mitosis in Kentrochona. According to him the
process of division is very similar to that of Spzvochona.
“ Die Theilung des Hauptkerns stammt in den grossen Ziigen mit derjeni-
gen bei Sfzvochona iiberein, wie sie besonders eingehend R. Hertwig und
Balbiani geschildert haben” (p. 363). The chromatin is sharply defined
against the achromatin which “in seitlicher Ansicht erscheint sie als
Anlage der Polplatte” (p. 364).
Doflein mentions a distinct granule in the pole-plate during
the metaphase, which must be analogous to the so-called
“nucleolus”? or ‘centrosome” of Spirochona according to
Balbiani.
752 CALKINS. [VoL. XV.
In Actinosphaerium the centrosome has been described by
only one observer, Brauer ('94), and its presence has been since
denied by Hertwig (96). Here as elsewhere, however, one
positive observation by a good authority must bear more weight
than a number of negative statements. Both Hertwig and
Brauer are agreed that the pole-plates in Actznosphaerium are
derived from the intra-nuclear ‘‘achromatic’”’ body of the resting
nucleus. Brauer describes an accumulation of cytoplasm out-
side of the pole-plates which he calls the cytoplasmic “ Kegel.”
The nuclear membrane is never lost at any point, nor is there
any apparent connection between the protoplasmic ‘“ Kegel”
and the inside of the nucleus, for they are separated by the
pole-plates which “ machen den Eindruck, als wenn es Verdick-
ungen der Membran waren, die stets wahrend des ganzen Kern-
theilungsprocesses erhalten bleibt” (p. 204). The cytoplasmic
accumulation appears only during mitosis. ‘ Ausserhalb des
Kernes an seinen Polen beginnt sich schon, wenn die ersten
Veranderungen im Kern erkennbar werden, feinkérniges Proto-
plasma anzusammeln” (p. 204). In certain cases centrosome
and radiations were seen in this mass, and from Brauer’s figures
of these stages it appears that the pole-plates at this time are
either absent (Figs. 44, 46) or else much reduced (Fig. 45).
Brauer is inclined to think that the centrosome is within the
pole-plate during prophase and metaphase, and that it finds
its way thence into the protoplasm during the anaphase. ‘So
scheint mir der Schluss unabweisbar, dass das Centrosom vorher
nicht im Protoplasma ausserhalb des Kernes, wie die Figuren
es zeigen, gelegen haben kann” (p. 207).
Thus in Actinosphaerium the substance of the pole-plates,
or a part at least, becomes extra-nuclear for a short time. In
Paramoeba (Schaudinn, '96) and in Woctzluca it is permanently
so. Schaudinn describes it in Pavamoeba as follows: ‘ Dicht
neben dem Kern liegt stets das bereits zu Anfang erwiahnte,
stark lichtbrechende, scharf construirte Gebilde. Ich will
dasselbe zunachst mit einem ganz indifferenten Namen, etwa
als ‘Nebenkorper’ bezeichnen. Bei den kleinsten Amoeben
ist es kugelig und ungefahr von derselben Grésse wie der
Kern.” During spore-formation, parvz fassu with a progressive
No. 3.] THE PROTOZOA AND METAZOA. 753
division of the nucleus, this ““Nebenkérper”’ breaks up into
smaller masses, each of which becomes associated with one of
the daughter-nuclei. Here it forms a central-spindle, and the
spore-nuclei divide by mitosis, in the same way, apparently, as
in Woctzluca. A centrosome is not described.
A review of the foregoing facts shows that different forms
of Protozoa present a nearly complete series in which may be
traced the possible development of the complex extra-nuclear
centrosome and sphere from an intra-nuclear ‘‘achromatic’’
body. This, —the homologue of the attraction-sphere, —
apparently, first appears as an axial nuclear rod in Amoeba
crystalligera and Euglena viridis. In Spirochona, Kentrochona,
and the micronuclei of Infusoria it is seen as aggregations of
‘“‘achromatin ’”’ —pole-plates—at the spindle poles. In Actino-
sphaerium the pole-plates remain intra-nuclear for the greater
part of mitosis, but according to Brauer a portion of them at
least becomes extra-nuclear for a brief period ; and during this
period spindle-fibers are formed. The “achromatic” body remains
extra-nuclear in Pavamoeba, in Noctiluca, and in the majority of
Metazoa. In Woctiluca and the Metazoa a portion of the “achro-
matic’? body, — the central-spindle, —although extra-nuclear,
comes to lie during mitosis in the center of the nuclear plate,
where it occupies the same position, morphologically, that it
occupies in Amoeba crystalligera or Euglena viridis.
The centrosome is not so easily traced, and it may be found
that in the majority of Protozoa the intra-nuclear so-called
“‘centrosome”’ is quite a different structure from the centro-
some of the Metazoa, although even here the centrosome has
been traced to an intra-nuclear position by many observers.
Its nuclear origin in Protozoa is made out by Balbiani in
Spirochona and by Doflein in Kextrochona, while Schaudinn
describes a body in Amoeba crystalliigera which must be homol-
ogous. In one case at least — Spzvochona (Balbiani) — its
direct morphological connection with the chromatin has been
traced, although not conclusively.!
1Jn this case the centrosome relation can be questioned on account of the
place of origin of the supposed centrosome. In most cases the centrosome is
found in the region of the spindle poles, but in Sfzrochona, according to Balbiani,
754 CALKINS. [Vou. XV.
In Spzrochona the so-called “centrosome” passes from the
nuclear chromatin into the achromatic body, which, however,
still lies within the nuclear membrane. It is possibly nuclear
in origin in Actznosphaerium, becoming extra-nuclear during the
metaphase of division. It appears to be nuclear in origin in
Noctiluca, but is found in the cytoplasm in the achromatic
body in the early metaphase and remains permanently in the
cytoplasm in spore-forming individuals. Finally it is perma-
nently cytoplasmic in the Metazoa, although occasionally nuclear
in origin even here (Ascaris megalocephala univalens).
The important position that (Voc¢z/uca must hold in all theo-
ries of mitotic development has not hitherto been sufficiently
emphasized. Much stress, indeed, has been laid on its simi-
larity in mitosis to metazoan cells, but little has been done to
show its relations to other Protozoa and the origin of its mitotic
structures. When considered alone, Woctz/uca can throw but
little light on the origin of the complex elements of the meta-
zoan mitotic-figure, but when considered in connection with
other Protozoa the origin of its own elements is seen, and so,
indirectly, the probable origin of the elements in Metazoa.
Noctiluca thus holds an intermediate position in the probable
development of mitosis, and the inference may be drawn that
the origin of the mitotic elements in Metazoa is the same as in
Noctiluca.
The position which WVoctiluca holds in the development of
chromatic structures has been sufficiently pointed out. It has
been shown, first, that /Voctzluca has probably the most primi-
tive form of true chromosomes ; and, second, that Voctzluca
presents probably the most primitive ring-like arrangement of
the chromosomes and nuclear plate around the central-spindle.
In regard to the “achromatic” structures the position of
Noctiluca is not quite so definite, and to be understood must be
considered in connection with lower forms. It has been main-
it originates from chromatin in the region of the “ ZwischenkGérper.” Further
work on this questionable structure must be done before its proper position can be
determined; that it is an important element in the cell and has a rdle to play in
nuclear division is established beyond question by the independent observations
of Hertwig, Plate, Biitschli, and Balbiani.
No. 3.] THE PROTOZOA AND METAZOA. 755
tained by numerous observers that the so-called “achromatic
structures’’ of the mitotic-figure are primitively nuclear in
origin. (See O. and R. Hertwig ('93 and '96), Heidenhain ('94),
etc.) Furthermore, in the various kinds of Protozoa which have
been carefully studied the ‘“‘achromatic”’ portions of the mitotic-
figures are developed from permanent, definite bodies which
are independent of the chromatin, although contained within the
cell-nucleus. As shown elsewhere in this paper, two apparently
similar elements of the metazoan cell have been described,
either or perhaps both of which may be comparable to these
intra-nuclear achromatic bodies in Protozoa. The first of these
is the “‘archoplasm”’ of Boveri; the second is the substance
which forms the ‘“centrodesmus’”’ of Heidenhain, which is
described as a specific substance of the cell, as playing a
certain definite réle in both resting and active phases, and as
distinct from the centrosome, from the nucleus, and from the
cytoplasm. Whether we consider this substance in the form
of ‘‘centrodesmus”’ or as archoplasm it appears, therefore, that
the cells of some Metazoa have a permanent specific substance
which may be considered the homologue of the ‘achromatic
body” of Protozoa.
Boveri does not consider a nuclear origin contrary to the
conception of archoplasm, and with many others regards the
“‘achromatin”’ in many protozoan nuclei as archoplasm (97). He
even holds it possible that there may be nuclei in Metazoa
which show a return to this primitive condition. Heidenhain
does not mention the derivation of the ‘‘centrodesmus”’ from
any other structure in more primitive forms. The only stand-
ards of identification are: its relation to the centrosome, the
formation of the spindle from its substance, and its permanency.
These same points may be used equally well for determining
the more primitive forms of archoplasm.
In Noctzluca the so-called “sphere” surrounds the centro-
some at the period when the latter is unmistakable. Here,
also, the central-spindle is formed from the substance of the
sphere, and the latter is permanent in the cell, persisting as a
specific substance throughout all stages and quite distinct from
the cytoplasmic reticulum. The sphere in /Voctz/uca, therefore,
756 CALKINS. [VoL. XV.
appears to be equivalent to the archoplasm of Boveri and the
centrodesmus of Heidenhain. In the more primitive forms,
unfortunately, the behavior of the intra-nuclear “achromatin”’
during division is not well enough known to warrant definite
conclusions, although the fragmentary evidence which has been
gathered from various sources is sufficient to show, I believe,
that structures possessing all of the attributes of archoplasm
are present in the various forms. Its history is well known,
indeed, in Exglena, but here we are confronted with the assump-
tion, by Keuten and others, that the intra-nuclear element is a
centrosome or its equivalent. There is reason to believe, how-
ever, that the two poles of this achromatic body represent the
pole-plates of other Protozoa, and that the connecting rod repre-
sents the central-spindle. If this hypothesis is correct, the
achromatic body in the nucleus of Azg/ena must be considered
equivalent to the sphere in /Voctz/uca and to the archoplasm of
Boveri or the centrodesmus of Heidenhain.
With our present knowledge it is impossible to go farther
back than Euglena for the development of archoplasm, and the
conclusion which may finally be drawn from our present knowl-
edge of this difficult question seems to be that primarily there
was a specific substance of the cell (archoplasm in Boveri’s
sense) connected in some way with the mechanism of cell divi-
sion, and forming a definite intra-nuclear body (£uglena).
Secondly, that this body became permanently extra-nuclear, but
still connected with nuclear division (JVoctzluca, and Metazoa
with “centrodesmus”’), and finally that it became lost in the
cell and indistinguishable from the cytoplasmic reticulum (cells
without archoplasm or centrodesmus, most egg cells).
The close similarity of mitosis in (Voctz/uca and Metazoa
does not necessarily indicate any phylogenetic connection. Nor
do the various Protozoa, which in this analysis are necessarily
brought together, show phylogenetic characters. We are at
present unable to develop any phylogenetic theory from the
facts of nuclear division. All that can be maintained is that
mitosis, in its many complicated phases, may have passed
through stages of development which are to-day represented
by many different and unallied types of Protozoa.
No. 3-] THE PROTOZOA AND METAZOA. 757
IV. SUMMARY OF OBSERVATIONS.
1. The resting nuclei of Moctzluca miliaris are large, round,
or oval structures, containing (a) chromatin in the form of karyo-
somes, and (0) “achromatin”’ in the form of large granules.
It is enclosed by a nuclear membrane which persists in part
throughout nuclear division.
2. A cytoplasmic substance, corresponding to the centro-
sphere of many metazoan cells, is invariably present. It is a
permanent organ of the cell, often as large, or larger, than the
nucleus ; it divides to form an amphiaster, consisting of two
asters with connecting mantle-fibers, the central-spindle.
3. During the division of the sphere the nucleus elongates
and bends to form a figure like the letter C ; the central-spin-
dle sinks into the opening thus formed, and is finally almost
enclosed by the nucleus.
4. As division progresses, the central-spindle becomes three
or four times as long as it is in the metaphase.
5. The karyosomes break up, by repeated division, into
innumerable chromatic elements. In some cases beaded fibers
are formed by the linear arrangement of larger chromatin
particles. These fibers are the only indication in Woctzluca
of a spireme-stage. The chromatin elements begin to form
the chromosomes, which, at first, are lines of single granules
extending from the nuclear membrane on the side next the
sphere towards the opposite side. At this stage they appear
like radial-fibers extending around the central-spindle and
forming a nearly closed ring. This incomplete ring of chro-
mosomes is the nuclear plate.
6. The chromosomes next become thicker, especially at the
ends next the central-spindle, and, probably, by the aggrega-
tion and fusion of the granules. This enlargement continues
towards the opposite ends, until, finally, the chromosomes are
of uniform thickness.
7. The chromosomes divide longitudinally and while lying
in the nuclear plate. The halves then separate, beginning at
the proximal ends.
758 CALKINS. [Vou. XV.
8. While the chromosomes are forming, the nuclear mem-
brane disappears from between the chromosomes and the cen-
tral-spindle. This leaves the chromosomes in contact with
the spindle-fibers.
g. The central-spindle fibers have no connection with the
chromosomes, but pass without interruption from pole to pole.
The chromosomes are connected with the spheres by another
set of fibers, —the mantle-fibers, — which pass from centro-
somes in the spheres to the ends of the chromosomes.
10. The nucleus, during the anaphase, elongates in the
direction of the central-spindle. The chromosomes are pulled
apart, the final division taking place at the distal end of each.
As they separate still more, they form two sets of oppositely-
directed striations in the nucleus, and the daughter-chromo-
somes again thicken at the proximal ends.
11. The nucleus, finally, divides in the center, often with a
very large connecting-piece between the daughter-nuclei. The
furrow is obliterated, the nuclear membrane reforms, and the
nucleus rounds out. The sphere loses its densely compact
appearance and becomes more expanded, although its granular
structure is retained.
12. In spore-forming divisions the nuclei do not return to
the resting state. The daughter-spheres divide and form sec-
ondary or tertiary, etc., amphiasters ; the daughter-chromo-
somes form the nuclear plate of the next mitosis without
change; and split again longitudinally. This process is
repeated eight or nine times.
13. A centrosome is always found in the sphere during
metaphase and anaphase stages as the focal point of the man-
tle-fibers, but is not found during resting stages. It divides in
the early anaphase in anticipation of the next mitosis.
14. The centrosome, possibly, comes from the nucleus,
where, during the resting stages, a small, deeply staining
granule can be easily distinguished from the chromatin. This
granule disappears during the early stages of chromosome-
formation. At the same period the nuclear membrane, just
below the sphere, shows distinct undulations, or wrinkles,
which form a clear space below the membrane. In numerous
No. 3.] THE PROTOZOA AND METAZOA. 759
cases two distinct granules were seen in this space. These
granules occupy various positions in relation to the membrane,
and in some cases they were found outside of the nucleus and
in the sphere. The inference is therefore drawn that the
centrosome is a permanent cell organ, which is not found in the
resting sphere, but in the nucleus, from whence it becomes
extra-nuclear by a rupture in the nuclear membrane. The
observations on this head are not conclusive, owing to the
smallness of the objects and to the presence of many similar
cytoplasmic microsomes.
COLUMBIA UNIVERSITY,
November, 1897.
APPENDIX.
Since the above was written a number of forms have been
carefully examined in the hope of finding Protozoa in which the
archoplasm may be traced back to a type still more primitive
than Euglena viridis. It was found that Trachelomonas hispida,
T. volvocina, Microglena punctifera, and Synura uvella have
nuclei similar to that of Euglena viridis; t.e., with a central
body and chromatin in the form of small granules. In another
set of forms, including Zvachelomonas lagenella, T. hispida
(variety), and Chzlomonas cylindrica, the chromatin is in the
form of granules and, as in Awg/ena, surrounds a central body,
but, unlike Euglena, there is no nuclear membrane. Finally,
in a species of Zetramitus, not only is the nuclear membrane
absent, but the granular chromatin is distributed throughout
the cell, and only during division are the granules collected
around a central body. This central body corresponds evidently
to the Nebenkorper of the flagellate-stage of Schaudinn’s Para-
moeba Eilhardt, but in Tetramitus, except during cell division,
it is cytoplasmic, no morphological nucleus being present. The
relations of this body to the chromatin are the same as the
relations of the sphere to the chromatin in Voctzluca, although
the former is far more primitive because of the distributed
nucleus (cf. Bacteria). It would seem, therefore, that archoplasm
was originally cytoplasmic; that it attracts (?) the chromatin
about it during cell division (Zetvamitus); that, in somewhat
760 CALKINS. [VoL. XV.
higher forms, its attractive force keeps the granules together
during resting phases as well as during division (Chz/omonas,
Trachelomonas, etc.); and, finally, that in still higher forms a
nuclear membrane is formed about the whole (Euglena, Trach-
elomonads, Synura, ciliates, etc.). But another line of develop-
ment may have arisen from this primitive type. In some forms
the archoplasm may have remained outside of the aggregate of
chromatin granules, becoming secondarily centralized in the
manner described by Schaudinn (96) for the division of the
flagellate-stage of Paramoeba, or in the same way as the central-
spindle in Woctiluca becomes centralized. In this way the
sphere in metazoan cells may have arisen without any connec-
tion with the intra-nuclear body of most Protozoa, while the
centrosome, as Flemming ('97) suggests, is an organ of second-
ary importance in the cell and which may or may not be
present.!
May 24, 1898.
1G. N. Calkins, The Phylogenetic Significance of Certain Protozoan Nuclei.
Ann. N.Y. Acad. Sci., vol. xi, no. 16, 1898.
No. 3.] THE PROTOZOA AND METAZOA. 761
LITERATURE.
'72 ALLMAN. Note on Noctiluca. Q./.4.S. 1872.
'57 BADDELEY, CoL. See BRIGHTWELL.
'93 BALBIANI, E. G. Centrosome et “ Dotterkern.” Journ. de l’ Anat. et
de la Phys. Tome xxix. 1893.
'95 BALBIANI, E.G. Sur la Structure et division du noyau chez le Spiro-
chone gemmipara. Ann. de Micrographie. Tome vii. 1895.
'84 VAN BENEDEN, Eb. AND JULIN. La Spermatogenése chez 1’Asca-
ride megalocéphale. Azxl/. Acad. Roy. Belgique. 3me Sér., Tome
iii. 1894.
'87 VAN BENEDEN ET NeEyT. Nouvelles Recherches sur la fécondation
mitotique chez l’Ascaride megalocephale. Bxdl/. Acad. Roy. Bel-
gigue. 1887.
’'86 BerGu, R. S. Ueber den Theilungsvorgang bei den Dinoflagellaten.
Morph. Jahr. No.2. 1886.
94 BLOCKMANN, F. Ueber die Kerntheilung bei Euglena. SBzo/. Cent.
Bd. xiv. 1894.
’'87 Boveri, Tu. Zellen-Studien. Heft 1. Jen. Zezt. Bd. xxi. 1887.
’88 Boveri, TH. Zellen-Studien. Jen. Zezt. Bd. xxii. 1888.
95 Boveri, TH. Ueber das Verhalten der Centrosomen bei der Befruch-
tung des Seeigel-Eies. Verh. d. physik.-medic. Gesell. in Wiirs-
burg. N.F., Bd. xxix. 1895.
'97 BoveR!I, TH. Zur Physiologie der Kern- und Zelltheilung. Sztz.-Ber.
der physik.-medic. Gesell. zu Wiirzburg. Bd. xiv. 1897.
'93 BRAUER, AuG. Zur Kenntniss der Spermatogenese von Ascaris mega-
locephala. A.M. A. Bad. xliii. 1893.
'94 BRAUER, AuG. Zur Kenntniss der Reifung des parthenogenetisch sich
entwickelnden Eies von Astemia salina. A.M. A. Bd. xliii. 1894.
‘94a BRAUER, AuG. Ueber die Encystierung von Actinosphaerium Eich-
hornii. Z. W.Z. Bad. lviii. 1894.
‘57 BRIGHTWELL. On Self-division in Noctiluca. Journ. Mic. Sct. Vol. v.
1857.
‘92 BURGER, O. Was sind die Attractionsspharen und ihre Centralkér-
per? An. Anz. Bd. vii. 1892.
‘55 Buscu. Beobachtung uber Anatomie und Entwickelung einiger wir-
bellosen Seethiere. Berlin, 1851. Translated in part in Journ. Roy.
Soc. Vol. iii. 1855.
‘76 BUTSCHLI, O. Studien tber die ersten Entwickelungsvorgange der
Eizelle, die Zelltheilung und die Konjugation der Infusorien. 4d/.
der Senckenb, Naturforscher-Ges. Bd.x. 1876.
‘77 BUTSCHLI, O. Bemerkungen tiber die Knopsung von Spirochona
gemmipara, Stein. Z.W,Z. Bd. xxviii. p. 59. 1877.
762 CALKINS. [VoL. XV.
’85 BUrscHLI, O. Einige Bemerkungen iiber gewisse Organizationsver-
haltnisse der sog. Cilioflagellaten und der Noctiluca. Morph. Jahro.
Bd. x. 1885.
'87 BUTSCHLI, O. Protozoa. Bronn’s Kl. und Ord. des Th. Ba. iii.
91 Burscuui, O. Ueber die sogenannten Centralkérper der Zelle und
ihre Bedeutung. Verh. des naturhist-med. Vereins zu Hetdelberg.
N.F., Bd. iv. pp. 535-538. 1891.
95 CALKINS, G. N. The Spermatogenesis of Lumbricus. Journ. Morph.
Vol. xi, No. 2. 1895.
94 CaRNOY, J. B. La Biologie cellulaire. Liége. 1894.
'97 CARNOY AND LEBRUN. La Fécondation chez l’Ascaris megalo-
cephale. La Cel/ule. Tome xiii, No.1. 1897.
’71 CIENKOWSKI. Ueber Schwarmerbildung bei Noctiluca miliaris.
Arch. f. mtk. Anat. Bd. vii. 1871.
'73 CIENKOWSKI. Ueber Noctiluca miliaris. Avch. f. mik. Anat. Bd.
ix. 1873.
'95 CLARKE, J. J. Observations on Various Sporozoa. Q./. M.S. Vol.
xxxvii, No. 3. 1895.
'96 DoFLEIN. Ueber Kentrochona Nebaliae. Zool. Anz. Bd. xix. 1896.
'97 DoOFLEIN. Karyokinese des Spermakerns. Arch. f. mik. Anat. Bd.
xl. 1897.
'95 DRUNER, L. Studien uber den Mechanismus der Zelltheilung. Jew.
Zeit. Bd. xxix. 1895.
'95 E1smonpD, J. Einige Beitrage zur Kenntniss der Attractionssphare und
der Centrosomen. Avxat. Anz. Bd.x. 1895.
'97 ERLANGER, R.v. Beitrage zur Kenntniss der Struktur des Proto-
plasmas, etc. Arch. f. mik. Anat. Bad. xlix.
'97a ERLANGER, R.v. Zur Kenntniss der Zell- und Kerntheilung. Bzo/.
Cent. Bd. xvii, No. 20.
‘82 FLEMMING, W. Zellsubstanz, Kern- und Zelltheilung. Leipzig, 1882.
'95 FLEMMING, W. Zur Mechanik der Zelltheilung. A. 17. A. Bad. xlvi.
1895.
'77 FoL, HERMANN. Sur le commencement de l’hénogenie chez divers
animaux. Arch. zool. Expér. Bd.vi. 1877.
'96 Foot, KATHERINE. Yolk-Nucleus and Polar Rings. Journ. Morph.
Vol. xii. 18096.
'97 Foot, KATHERINE. The Origin of the Cleavage Centrosomes. Journ.
Morph. Vol. xii, No.3. 1897.
'96 GRIFFIN, B. B. The History of the Achromatic Structures in the
Maturation and Fertilization of Thalassema. TZyvans. V. Y. Acad.
Scz. 1896.
'83 GRUBER, A. Ueber Kerntheilung bei einigen Protozoen. Z. W. Z.
Bd. xxxviii. 1883.
'84 Gruber, A. Ueber Kern- und Kerntheilung bei den Protozoen.
Z.W.Z. Vol. xl. 1884.
94
ES
‘97
'97
‘91
'93
"16
'77
84
'88
'89
"95
'96
'95
55
194
94
'93
95
3] THE PROTOZOA AND METAZOA. 763
GUIGNARD, M. L. Sur l’existence des ‘‘sphéres attractives”’ dans les
cellules végétales. Comp. Rend. Tome cxii. 1891.
HEIDENHAIN, MARTIN. Neue Untersuchungen iiber die Centralkér-
per und ihre Beziehungen zum Kern- und Zellenprotoplasma.
Arch. f. mik. Anat. Bad, xliii. 1894.
HEIDENHAIN, MARTIN. Cytomechanische Studien. Arch. Entwick.
Bd. i, No. 4. 1895.
HEIDENHAIN, MARTIN. Ueber die Microcentren mehrkerniger Rei-
senzellen, sowie iiber die Centralk6rperfrage im Allgemeinen.
Morph. Arbetten. Bd. vii, No. 1. 1897.
HERA, V. Etude des variations de la mitose chez Il’Ascaride megalo-
cephale. Arch. de Biol. Tome xiii. 1893.
HERMANN, F. Beitrage zur Lehre von der Entstehung der karyoki-
netischen Spindel. 4.7. A. Bd. xxxvii. 1891.
HERTWIG, O. Die Zelle und die Gewebe. Jena, 1893.
HERTWIG, R. Beitrage zu einer einheitlichen Auffassung d. verschie-
denen Kerne. Morph. Jahrb. Bd. xi. 1876.
HERTWIG, R. Bau und Entwickelung der Spirochona gemmipara.
Jen. Zett. 1877.
HERTWIG, R. Ueber die Kerntheilung bei Actinosphaerium Eich-
hornii. Jen. Zeit. 1884.
HERTWIG, R. Ueber Kernstruktur und ihre Bedeutung fiir Zellthei-
lung und Befruchtung. /enx. Zeit. 1888.
HERTWIG, R. Ueber die Konjugation der Infusorien. Adz. der bayr.
Akad. d. Wiss. 11. Cl. Bd. xvii. 1889.
HERTWIG, R. Ueber Centrosoma und Centralspindel. Sztz.-Ber.
Ges. Morph. u. Phys. Bd. xi, Heft 1. Miinchen, 1895.
HeErTWIG, R. Ueber die Entwickelung des unbefruchteten Seeigel-
Eies. Festschrift fiir Carl Gegenbaur. 1896.
Hitt, M. D. Notes on the Fecundation of the Egg of Sphaerechinus
granularis and on the Maturation and Fertilization of the Egg of
Phallusia mammillata. Q.J. M.S. Vol. xxxviii. 1895.
Hux.ey, T. H. On the Structure of Noctiluca miliaris. /Journ. Roy.
Mic. Soc. Vol. iii. 1855.
IsHIKAWA, C. Studies on Reproductive Elements: II. Woctiluca
miliaris, Sur, its Division and Spore-formation. /ourn. Coll. of Sci.
Imp. Univ. Japan. Vol. vi. 1894.
IsHIKAWA, C. Ueber die Kerntheilung bei Noctiluca miliaris. Ber.
d. Naturf-Geselischaft zu Freiburg t. B. Festschrift fiir A. Weis-
mann. 1894.
JuLin, Cu. Le corps vitellin de Balbiani et les éléments de la cellule
des Metazoaires qui correspondent au macronucleus des Infusoires
ciliés. Bull. Sci. de la France et de la Belg. Tome xxv. 1893.
KARAWAIEW, W. Structure and Multiplication of Aulacantha Scoly-
mantha (Radiolarian). Zool. Anz. Vol. xviii. 1895.
764 CALKINS. [VoL. XV.
'96
"95
'78
95
96
52
'97
95
96
194
95
89
95
'97
'91
194
"95
'93
94
'96
KARSTEN, G. Die Beziehungen der Nucleolen zu den Centrosome
bei Psilotum triquetrum. Ber. d. deutsch. Bot. Ges. Bd. xi.
1894.
KEUTEN, J. Die Kerntheilung von Euglena viridis. Z.W.Z. Bd.
lx. 1895.
KLEIN, E. Observations on the Structure of Cells and Nuclei.
Q. J. M.S.. Vols. xviii, xix. 1878.
KorscHELT, E. Ueber Kerntheilung, Eireifung und Befruchtung bei
Ophryotrocha puerilis. Z.W.Z. Bd. Ix.
KOSTANECKI AND A. WIERZEJSKI. Ueber das Verhalten der sogen.
achromatischen Substanzen im befruchteten Ei. A.M. A. Bad.
xlvii. 1896.
Kroun, A. Notizen tber die Noctiluca miliaris. <Avch. f. Matur-
gesch. 1852.
LABBE. Recherches sur les Coccidies. Arch. zool. Expér. 3me Sér.,
Tomes ii, iii, iv. 1896.
LAUTERBORN, R. Protozoen Studien: I. Kern und Kerntheilung bei
Ceratium hirundinella. 2. W.Z. 1896.
LAUTERBORN, R. Untersuchungen iiber Bau, Kerntheilung und Be-
wegung der Diatomeen. Leipzig, 1896.
Lavpowsky, M. Von der Entstehung der chromatischen und achro-
matischen Substanzen in den thierischen und pflanzlichen Zellen.
Anat. Hefte. 1. Abth., Bd. iv. 1894.
MATHEWS, A. AND WILSON. See WILSON.
Maupas, M. Le rajeunissement karyogamique chez les ciliés. Avch.
zool. Expér. 2me Sér., Tome vii. 1889.
MEAD, A. D. Some Observations on Maturation and Fecundation in
Chaetopterus pergamentaceus. /ourn. Morph. Vol. x. 1895.
MeEap, A. D. The Origin of the Egg Centrosomes. Journ. Morph.
Vol. xii, No. 2. 1897.
MEvEs, F. Ueber amitotische Kernteilung in den Spermatogonien
des Salamanders, und das Verhalten der Attraktionsphare bei der-
selben. Anat. Anz. 1891.
MevES, F. Ueber eine Metamorphose der Attraktionsphare in den
Spermatogonien von Salamandra maculosa. A.J. A. Bad. xliv.
1894.
MITROPHANOW, PaAuL. Note sur la division des noyaux de l'état
végétatif chez le Spherozoaire. Arch. zool. Expér. 3me Sér.,
Tome ili. 1895.
Moors, J. E.S. Mammalian Spermatogenesis. Amat. Anz. Bad.
viii. 1893.
Moors, J. E. S. Some Points in the Spermatogenesis of Mammalia.
Intern. Monat. f. Anat. u. Phys. Bd. xi. 1894.
MorGan, T. M. On the Production of Artificial Archoplasmic Centers
Rep. Am. Morph. Soc. Sczence. Vol. iii. 1896.
3] THE PROTOZOA AND METAZOA. 765
Mortier, Dav. M. Beitrage zur Kenntniss der Kerntheilung in den
Pollenmutterzellen einiger Dikotylen und Monokotylen. /ahro. /f.
wiss. Bot. Bd. x, Heft 2. 1897.
OsTERHAUT. Cytologische Studien aus dem Bonner botanischen
Institut. Jahrb. f. wiss. Bot. Bd. xxx, Hefte 2 and 3. 1897.
PENARD. Etude sur quelques Heliozoaires d’eau douce. Arch. de Biol.
Tome ix. 1889.
PFITZNER, W. Zur Kenntniss der Kerntheilung bei den Protozoen.
Morph. Jahr. Ba. ii. 1886.
PLATE. Untersuchungen einiger an den Kiemenblattern des Gam-
marus pulex lebenden Ektoparasiten. Z.W.Z. Bd. xliii. 1886.
DE QUATREFAGES. Observations sur les Noctiluques. Am. des Sci.
Nat. 3™e Sér. Zool. Tome xiv. 1850.
REINKE, Fr. Zellstudien. 4A.1/.A. Bd. xliv. 1894.
RHUMBLER, L. Beitrage zur Kenntniss der Rhizopoden. 2. W. Z.
Bd. lii. 1892.
Rosin, CH. Recherches sur la reproduction gemmipare et fissipare
des Noctiluques. Journ. de l’Anat. et de la Phys. Tome xiv.
1878.
ROMPEL. Kentrochona Nebaliae. Z. W.Z. Bd. lviii, Heft 4. 1894.
Rickert, J. Zur Eireifung bei Copepoden. Avxat. Hefte. Bd. iv.
1894.
SCHAUDINN, F. Ueber Kerntheilung mit nachfolgender Kérperthei-
lung bei Amoeba crystalligera. S7tz-Ber. ad. k. Ak. ad. Wiss. in
Berlin. Math-Phys. Kl. Bd. xxxviii. 1894.
SCHAUDINN, F. Ueber die Theilung von Amoeba binucleata, Gruber.
Sitz.-Ber. Ges. naturf. Freunde. Berlin. 1895.
SCHAUDINN, F. Ueber den Zeugungkreis von Paramoeba Eilhardi.
Sitz.-Ber. Ges. naturf. Freunde. Berlin. 1896. ;
SCHEWIAKOFF, W. Ueber die karyokinetische Kerntheilung der
Euglypha alveolata. Morph. Jahrb. Bd. xiii. 1888.
ScHULTZE, O. Reichert und der Gromien. A. /. A. Ba. ii.
1867.
STRASBURGER, E. Zellbildung und Zelltheilung. 3ded. 1880.
STRASBURGER, E. Ueber Kern und Zelltheilung. Jena, 1888.
STRASBURGER, E. Cytologische Studien aus dem Bonner botanischen
Institut. Jahrb. f. wiss. Bot. Bd. xxx, Hefte 2 and 3. 1897.
Vrypovsky, F. Reifung, Befruchtung und Furchung des Rhynchel-
nies-Eies. Prag, 1888.
vom RATH, O. Ueber den feineren Bau der Driisenzellen des Kopfes
von Anilocra Med., etc. Z.W.Z. Bd.lx. 1895.
Warask, S. Homology of the Centrosome. /ourn. Morph. Vol. viii,
No. 2. 1893.
WEISMAN UND ISHIKAWA. Weitere Untersuchungen zum Zahlenge-
setz der Richtungskérper. Zool. Jahrb. Bd. iii. 1889.
766 CALKINS. [Vou. XV.
'95 WILson, E. B. Archoplasm, Centrosome and Chromatin in the Sea-
Urchin Egg. Journ. Morph. Vol. xi. 1895.
’96 Witson, E. B. The Cell in Development and Inheritance. New
York, 1896.
'97 Witson, E. B. Centrosome and Middlepiece in the Fertilization of
the Egg. Sczence. N.S., Vol. v, No. 114. March 5, 1897.
’95 WILSON AND Matuews. Maturation, Fertilization and Polarity in
the Echinoderm Egg. Journ. Morph. Vol. x, No.1. 1895.
791 WoLTERS, MAx. Conjugation und Sporenbildung bei Gregarinen.
Arch. f. mik. Anat. 1891.
768 CALKINS.
DESCRIPTION OF PLATE XL.
Figs. 11, 16-19, 21, 22, 24, 26-31, 38 represent nuclei in the process of spore-
formation ; Figs. 1-10, 12-15, 20, 23, 25, 32-37, 39 are various stages of the
vegetative nucleus. Picro-acetic was used for individuals represented in Figs. 1, 5,
8, 15, 19, 20, 24; sublimate-acetic (weaker solution) for Figs. 6, 7, 10, 13, 17, 18, 21—
23, 25, 32-37, 39- Stronger solution for Figs. 26, 27, 29-31, 38; Hermann solu-
tion for Figs. 9, 11, 12, 14, 16, 28; and corrosive sublimate for Figs. 2, 3, 4.
The iron haematoxylin was used in all cases save Fig. 10, where the Biondi-
Ehrlich mixture was used. Figs. 1-9, 11-18, 20, 21 are from preparations zm Zofo ;
Figs. 10, 19, 22-38, from sections. 2 = supposed intra-nuclear centrosome.
Fic. 1. Resting stage of a vegetative nucleus showing g karyosomes and
granular oxychromatin. Sphere on the outside of the nuclear membrane presents
characteristic granular cortex and hyaline central part.
Fic. 2. Nucleus showing early stages in fragmentation of the karyosomes.
Fic. 3. Nucleus showing possible method of karyosome fragmentation. x2 =
the supposed intra-nuclear centrosome.
Fic. 4. Later stage in karyosome fragmentation. The granules are gathered
in groups, each group representing a previous karyosome.
Fic. 5. A still later stage in karyosome fragmentation. The sphere appears
homogeneous.
Fic. 6. A nucleus in the so-called “spireme-stage.” The chromatin granules
are still large, but are arranged in fibers. The supposed centrosome (x) is dis-
tinct from the chromatin.
Fic. 7. Same as Fig. 6.
Fic. 8. A nucleus in the prophase of division. The achromatin granules are
arranged in fibers parallel with the chromosomes. The chromosomes are forming
with the thickened ends towards the sphere. The sphere has become more
dense, and the hyaline center is disappearing. Some of the karyosomes are not
yet fragmented.
Fic. 9. A prophase of division. The granules are beginning to form fibers,
—the chromosomes. The sphere is dividing. Numerous processes stretch out
from the sphere into the cytoplasm. These are probably equivalent to astral
fibers.
Fic. ro. The samestage in section. The nucleus is filled with granules which
are forming chromosomes.
Fic. 11. Stage of the amphiaster. The chromosomes are all formed and are
represented in optical section in the nuclear plate.
Fic. 12. An abnormal nucleus. The sphere is bent into an acute angle.
The nucleus shows elongation in the primary axis before chromosome-formation.
Journal of Morphology Vol. AV
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770 CALKINS.
DESCRIPTION OF PLATE XLI.
Fic. 13. The nucleus and amphiaster during metaphase. The latter lies in
the secondary axis. Central-spindle, nuclear plate, and asters are shown. Mantle-
fibers not represented.
Fic. 14. Metaphase showing position of the nuclear plate and the dumb-bell-
shaped amphiaster.
Fic. 15. Metaphase stage viewed from the opposite side, z.e., the side away
from the central-spindle. The chromosomes are seen in optical section.
Fic. 16. Late anaphase seen from side opposite the spindle. The chromo-
somes are completely separated and show secondary thickenings at the ends
towards the spheres. The nucleus has elongated in the direction of the secondary
axis. -
Fic. 17. Late anaphase showing the position of the central-spindle; the
chromosomes are completely separated.
Fic. 18. Telophase showing the formation of the secondary nuclear plates
and just previous to the division of the daughter-spheres. The plane of the next
division is therefore clearly indicated.
Fic. 19. Late anaphase showing final division of the chromosomes. The
nucleus and the central-spindle were curved so that only one pole (the upper) was
sectioned.
Fic. 20. Late anaphase in vegetative division. The chromatin of the chromo-
somes is reforming into large karyosomes. The daughter-spheres are beginning
to expand and to lose their densely granular appearance, while the characteristic
hyaline portion of the central part is plainly indicated, resembling the sphere in
Fig. 1.
Fic. 21. Late telophase and beginning of the secondary divisions. The
daughter-spheres have formed amphiasters, although the parent nucleus is not
completely divided as shown by the large “ Verbindungsstiick.”
Fic. 22. Section of nuclear-plate stage showing nuclear plate and fan-like
* arrangement of the chromosomes.
Fic. 23. Late anaphase showing the collection of chromatin into karyosomes.
A centrosome (C) is plainly indicated at the focus of the mantle-fibers. There is
no nuclear membrane between chromosomes and sphere.
Fic. 24. Similar anaphase showing aggregation of daughter-chromosomes
as in Fig. 18. No karyosome-formation taking place. Centrosome and mantle-
fibers present.
Fic. 25. Late telophase in vegetative division. Remnants of the daughter-
chromosomes can still be made out, although most of the chromatin is now
collected in a number of karyosomes.
Journal of Morphology Vol.Xv.
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772 CALKINS.
DESCRIPTION OF PLATE XLII.
Fic. 26. Transverse section showing the double nature of the chromosomes
and compact arrangement of the proximal ends.
Fic. 27. Similar section showing one chromosome (A) beginning to split at
the proximal end.
Fic. 28. Later stage. Mantle-fibers connect double centrosomes with the
dividing chromosomes. Two separate bundles of daughter-chromosomes can
already be made out. $
Fic. 29. Division of chromosomes is carried still farther; the distal ends
are not yet divided. In both figures the characteristic crossed appearance of this
stage is plainly visible. The nuclear membrane persists except where mantle-
fibers connect centrosome and chromosomes.
Fic. 30. A later stage in chromosome division. The distal ends are now
separated. The nuclear membrane has reappeared between the central-spindle
and the chromosomes.
Fic. 31. Horizontal section of similar stage. The central-spindle lies through
the middle of the figure, the nuclear membrane is intact except at the poles.
This is about the same stage represented in Fig. 17, and would represent a section
cut in the plane of the paper.
Fic. 32. Prophase of vegetative division showing slight undulation in the
membrane below the sphere, a hyaline space below the undulation, and two dis-
tinct granules in the hyaline space.
Fic. 33. Prophase of vegetative division showing very marked undulation of
the membrane below the sphere, a distinct hyaline sphere, and again two granules
within the space.
Fic. 34. Prophase of vegetative division. A distinct depression is now
formed at the point where undulations appeared in the other cases. The two
distinct granules are now seen in the space just outside of this depression.
Fic. 35. Prophase of vegetative division. The undulations are very marked
in the region of the sphere, and are not found elsewhere. The two distinct granules
are outside of the membrane.
Fic. 36. Asimilar stage. The two granules now lie in the sphere, the chro-
matin is in the so-called spireme-stage, and chromosomes are forming.
Fic. 37. The two granules are now very distinct and lie in the sphere. The
nucleus is elongated in the primary axis, but the chromatin is still widely dis-
tributed.
Fic. 38. Section through primary axis of a daughter-nucleus in about the
same stage as that shown in Fig. 18. The chromosomes are double, the mantle-
fibers are distinctly granular, and the centrosome is double. The sphere has not
yet divided.
Fic. 39. Late anaphase in vegetative division. The nuclear membrane is not
yet reformed, the mantle-fibers are disappearing, and the chromosomes are disin-
tegrating.
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Journal of Morphology Vol.
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