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QUARTERLY JOURNAL
OF
MICROSCOPICAL SCIENCE.
EDITED BY
K. RAY LANKESTER, M.A., LL.D., F.R.S.,
Linacre Professor of Comparative Anatomy, Fellow of Merton College, and Honorary
Fellow of Exeter College, Oxford ; Corresponding Member of the Imperial
Academy of Sciences of St. Petersburg, and of the Academy of
Sciences of Philadelphia; Foreign Member of the
Royal Bohemian Society of Sciences.
WITH THE CO-OPERATION OF
ADAM SEDGWICK, M.A., F.RS.,
Fellow and Lecturer of Trinity College, Cambridge ;
AND
W. F. R. WELDON, M.A., F.R.S.,
Jodrell Professor of Zoology and Comparative Anatomy in University College, London ;
late Fellow of St. John’s College, Cambridge.
VOLUME 38.—New Serizs.
With Lithographic Plates and Engrabings on Wood.
LONDON:
J. & A. CHURCHILL, 11, NEW BURLINGTON STREET.
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CONTENTS.
CONTENTS OF No. 149, N.S., AUGUST, 1895.
MEMOIRS : PAGE
On the Variation of Haliclystus octoradiatus. By Epwaxp
T. Browns, B.A., University College, London. (With Plate 1) iL
The Collar-cells of Heteroccela. By Groren Bipper. (With
Plate 2) . F ‘ ' : 9
The Metamorghacts of Hetamodenas: By Henry Bory, M.A,
F.L.S., late Fellow of Trinity College, Cambridge. ee
Plates 3—9) ‘ 45
A Criticism of the Cell- Tiaaey: ; Heine an cere to Mr. Scleminls 8
Article on the Inadequacy of the Cellular Theory of Development.
By Gitpert C. Bourne, M.A., F.L.S., Fellow of New College,
Oxford . : : : : ‘ ‘ ce lod
CONTENTS OF No. 150, N.S., NOVEMBER, 1895.
MEMOIRS :
On the Distribution of Assimilated Iron Compounds, other than
Hemoglobin and Hematins, in Animal and Vegetable Cells. By
A. B. Macatuvm, B.A., M.B., Ph.D., Associate-Professor of
Physiology, University of Toronto. (With Plates 10—12) » 75
On the Structural Changes in the Reproductive Cells during the
Spermatogenesis of Elasmobranchs. (From the Huxley Re-
search Laboratory, Royal College of Science, London.) By
J. E. S. Moors, A.R.C.S. (With Plates 13—16) : . 275
Notes on the Fecundation of the Egg of Spherechinus granu-
laris, and on the Maturation and Fertilisation of the Egg of
Phallusia mammillata. By M. D. Hitt, B.A.Oxon. (With
Plate 17) 5 : : : - , . 315
Further Remarks on the Cell-Theory, with a Reply to Mr. Bourne.
By Apam Szpewicx, F.R.S.. 5 4 : . 331
lv CONTENTS,
CONTENTS OF No. 151, N.S., JANUARY, 1896.
MEMOIR:
The Development of Asterina gibbosa. By E. W. MacBrinz,
Fellow of St. John’s College; Demonstrator in Animal Mor-
phology in the University of Cambridge. (With Plates 18—29)
CONTENTS OF No. 152, N.S., FEBRUARY, 1896.
MEMOIRS :
The Early Development of Amia. By BasHrorp Dezan, Ph.D.
(With Plates 30—32)
On Kynotus cingulatus, a New Species of Earthworm from
Imerina in Madagascar. By W. Briaxtanp Benuam, D.Sc.
Lond., Hon.M.A.Oxon., Aldrichian Demonstrator in Compara-
tive Anatomy in the University of Oxford. (With Plates 35
and 34) .
Notes on the Ciliation of the Ectoderm of the Amphibian Embryo.
By Ricuarp AssHETON, M.A. (With Plate 35) ;
Ontogenetic Differentiations of the Ectoderm in Necturus. Study
IJ.—On the Development of the Peripheral Nervous System.
By Juuia B. Pratt. (With Plates 36—38)
PAGE
339
413
445
465
485
On the Variation of Haliclystus octoradiatus.
By
Edward T. Browne, B.A.,
University College, London.
With Plate 1.
A NorMAL specimen of Haliclystus octoradiatus, Clark,
has eight adradial groups of tentacles; eight adradial genital
bands ; eight colleto-cystophors, one midway between every
two groups of tentacles ; and four internal, interradial septa.
The variation in number, shape, and symmetry of these
organs forms the subject of this paper. The specimens were
collected by the officials of the Marine Biological Association
at Plymouth during November, 1892, and the spring of 1893.
Texamined 154 specimens, and found 120 specimens perfectly
normal and 34 specimens abnormal. Some of the abnormal
forms are beyond doubt good cases of congenital variation,
and others are cases of an imperfect regeneration of organs
damaged or completely destroyed by injury. Congenital varia-
tion is usually shown by an increase or decrease in the number
of organs, which may either vary together or separately.
Only three specimens show a numerical variation in all the
organs. One has six groups of tentacles, six colleto-cystophors,
six genital bands, and three internal septa. Two specimens
have twelve groups of tentacles, twelve colleto-cystophors,
twelve genital bands, and six internal septa. In the last two
specimens the increase in the number of organs is not followed
by a corresponding increase in the amount of tissue. Each
VOL. 38, PART 1.—NEW SER. A
2 EDWARD T. BROWNE.
organ is below the average in size, and the tentacles in each
group are also below the normal number. I have noticed
among the Ephyre of Aurelia aurita, that when a great
increase in the number of arms occurs, the arms are below the
average In size.
Another specimen has seven groups of tentacles, seven
colleto-cystophors, seven genital bands and five septa, an
increase in the number of septa, but a decrease in the other
organs.
A numerical variation of the septa only occurs in seven
specimens (about 44 per cent.) ; four of these are given above,
and the others, which have only three septa, will be described
in another part of this paper. I think in all cases the
numerical variation of the septa may be safely considered to
be congenital. The septa run nearly the whole length of the
body, and are not likely to be affected by an external injury.
In the majority of abnormal specimens the septa have their
normal number, and the groups of tentacles, colleto-cystophors,
and genital bands show variation. Usually each set of organs
shows an independent variation, either in number, shape, or
position. One specimen, however, with the normal number
of septa, has seven groups of tentacles, seven colleto-cystophors,
and genital bands, all of which are symmetrically arranged.
It is now probably the simplest plan to describe the variation
in each set of organs separately, and to commence with the
colleto-cystophors.
The Variation of the Colleto-cystophors.—In the
five specimens already described the colleto-cystophors vary in
number along with the groups of tentacles, and occupy their
normal position on the margin of the umbrella. But many
specimens show that the colleto-cystophors vary independently
of the other organs.
Four specimens with eight groups of tentacles have nine
colleto-cystophors. In two of these (figs. 2, 4, and 5)
the increase is produced by the twinning of one of the colleto-
cystophors. The other two specimens have the additional
colleto-cystophor in an abnormal position, One is on the
ON THE VARIATION OF HALICLYSTUS OCTORADIATUS. 3
margin of the umbrella, very near to a group of tentacles
(fig. 1); the other is adradial and on the aboral side of a group
of tentacles, a little way from the margin (fig. 3).
Five specimens have fewer colleto-cystophors than groups of
tentacles ; in each case one is missing. It is difficult to say
whether the decrease is due to congenital variation or to the
result of an injury.
One specimen (fig. 6) with seven groups of tentacles has
eight colleto-cystophors. An examination of the specimen
shows that two groups of tentacles are united into one group.
The colleto-cystophor, which has been shut from its normal
position by the union of the two groups, is situated close to,
and on one side of, the double group.
Five other specimens show a similar union of groups of
tentacles, but the colleto-cystophors correspond in number to
the groups of tentacles.
Mr. Hornell (1) has examined many large specimens of
Haliclystus octoradiatus taken at Jersey. He states that
33 per cent. show a variation either in the number of colleto-
cystophors or in the groups of tentacles. More than half of
these are cases in which a colleto-cystophor is absent from its
proper position.
Only five of the Plymouth specimens have fewer colleto-
cystophors than groups of tentacles (about 3 per cent.).
Mr. Hornell also examined 118 specimens taken at Jersey,
and found 78 specimens (66 per cent.) with a capitate tentacle
upon the apex of the colleto-cystophor. Some specimens have
only a slight swelling at the apex with a few nematocysts, and
others show various gradations up to a perfect capitate ten-
tacle, just like an ordinary tentacle. The following table
taken from Mr. Hornell’s paper gives the number of colleto-
cystophors with a capitate tentacle in each specimen.
4, EDWARD T. BROWNE.
14 specimens with 1 colleto-cystophor with a capitate tentacle.
15 ” 2 39 ” »
15 ” 3 ” ” 9
9 ” 4: ” » ”
se) 5 ” oy) 23
5 9 6 ” » ”
4 ” 7 9 » ”
8 » 8 ” ” ”
I searched all the Plymouth specimens to see if a similar
variation existed, and only found one doubtful case. This
specimen (figs. 7 and 8) has seven groups of tentacles in the
normal position and one group a little way inside the oral sur-
face of the umbrella. The proper position of this group is
occupied by a capitate tentacle with a swollen base, which may
or may not be an abnormal colleto-cystophor. The specimen
possesses the usual eight colleto-cystophors, normal in shape
and position.
Two specimens have capitate tentacles on the margin of the
umbrella in an abnormal position. One has three tentacles
just above a double colleto-cystophor (figs. 4 and 5), and the
other has three tentacles close to a colleto-cystophor (fig. 10).
I think the Jersey specimens give an excellent illustration
of local variation of a species.
The Variation of the Genital Bands.—In a normal
specimen there are eight adradial genital bands, separated into
four distinct pairs by the interradial septa. Some specimens
show a variation upon this arrangement. One specimen (fig. 9)
has six adradial and two interradial genital bands. The change
in position occurs through the union of two adjacent adradial
groups of tentacles into one interradial group. This union has
reduced the number of groups of tentacles and colleto-cysto-
phors to seven, but the genital bands remain normal in number,
The change in position of the two genital bands is also well
shown by their being separated by an interradial septum
occupying its normal position. I think this may be regarded
as a good case of congenital variation.
Another specimen (fig. 6) has six adradial and two perradial
genital bands, There are seven groups of tentacles. One
ON THE VARIATION OF HALICLYSTUS OCTORADIATUS. 5
group is larger than the others, and has two genital bands
running towards it. This large group is perradial, and repre-
sents the union of two adjacent adradial groups. Two other
specimens show a similar variation. A slight alteration in this
arrangement may take place by the union of two adjacent
genital bands into one broad band (fig. 18). The genital bands
usually start some distance down the body of the medusa and
extend across the umbrella. Two specimens show an exception
by having short bands commencing near the margin of the
umbrella (figs. 19 and 20).
The Variation in the Position of the Groups of Ten-
tacles.—In most cases the change in position of the groups
of tentacles is due to a decrease in number, and usually
affects the symmetry of the umbrella. The decrease is some-
times brought about by the union of two adjacent adradial
groups of tentacles into an interradial (fig. 9) or a perradial
group (figs. 6 and 18).
In a few cases the position occupied by a group is excep-
tionally abnormal. Two specimens have a group of tentacles
on the oral side of the umbrella, some distance from the
margin. One of these (figs. 7 and 8) has seven normal groups
of tentacles, but the eighth group is a little way inside the
margin, which projects beyond the group and has a tentacle-
like colleto-cystophor in the position which 1s under normal
conditions occupied by the eighth group of tentacles. The
other specimen (figs. 12 and 15) has three septa, six colleto-
cystophors, and five normal groups of tentacles. But there is
also an abnormal group of tentacles upon a short stalk, which
rises above the oral surface of the umbrella, and occupies a
position about half-way between the centre and the margin of
umbrella. Opposite this group of tentacles, upon the margin
of the umbrella, there are two other sets of tentacles, close
together, with the tentacles arranged in nearly a single row.
An unique case amongst the abnormal forms is that of a
specimen (figs. 1] and 13) with eight groups of tentacles and
colleto-cystophors in the normal position. One of these gronps
of tentacles is smaller than the others, and has, on its outer
6 EDWARD T. BROWNE.
side, a lateral outgrowth of the umbrella. This outgrowth
contains two colleto-cystophors and two groups of tentacles,
one behind the other. The specimen has altogether ten groups
of tentacles and ten colleto-cystophors.
The Regeneration of Injured or Lost Organs.—
It is evident from the mutilated condition of some specimens
that a considerable amount of injury may happen to the
umbrella without causing death to the medusa. The damaged
or lost organs may be replaced by new ones, which may or
may not resemble the old ones. A new symmetry may even
arise through a decrease in the number of organs, which in
some cases might be mistaken for congenital variation. The
simplest case is the loss of one group of tentacles, well illus-
trated by aspecimen (fig. 21) which has all its organs perfectly
normal except that one group of tentacles is missing. The
prolongation of the umbrella and the genital band suddenly
terminates, as if the tentacles had been cut off with a knife.
Another specimen (fig. 22) shows a similar abrupt termination
of the genital band, but a few short tentacles are present which
may be reasonably regarded as anew growth. The destruction
and regrowth of tentacles are also well shown in a specimen
(fig. 16) with five normal groups and with two groups having
only a few short tentacles. A genital band and a short pyro-
longation of the umbrella marks the position of the eighth
group which is missing.
Two specimens show both congenital variation and an
abnormality due to regeneration. One of these (fig. 14) has
three septa, five genital bands (the sixth is absent, but its posi-
tion is faintly marked), and six colleto-cystophors ; but there
are only four normal groups of tentacles present. The other
two groups have evidently been destroyed and are again budding
out afresh. The other specimen (fig. 17) has three septa,
seven genital bands, six colleto-cystophors, and only four normal
groups of tentacles. One half of the umbrella, containing these
groups of tentacles, is normal in shape, but the other half has
apparently been destroyed, and three new groups of tentacles
are in the process of development.
ON THE VARIATION OF HALICLYSTUS OCTORADIATUS. 7
REFERENCE.
1. Hornet, J., 1893.—* Abnormalities in Haliclystus octoradiatus,”
‘Natural Science,’ vol. iii, p. xxxiil.
DESCRIPTION OF PLATE 1,
Illustrating Mr. E. T. Browne’s paper on “The Variation
of Haliclystus octoradiatus.”
PLATE 1.
Fic. 1.—A portion of the umbrella showing a colleto-cystophor in an ab-
normal position. Aboral side. x 10.
Fie, 2.—A portion of the umbrella showing the twinning of a colleto-
eystophor. Aboral side. x 10.
Fic. 3.—Half of the umbrella showing a colleto-cystophor in an abnormal
position. Aboral side. x 10.
Fie. 4.—Oral view of a specimen with a genital band in an abnormal
position, and a double colleto-cystophor. x 9.
Fic. 5.—Double colleto-cystophor (Fig. 4) with tentacles on the margin of
the umbrella. Aboral side. x 18.
Fre. 6.—Oral view of a specimen showing the union of two groups of ten-
tacles. x 6.
Fic. 7.—Oral view of a specimen showing a group of tentacles inside the
umbrella, and a tentacle-like colleto-cystophor.
Fie. 8.—Lateral view of the abnormal group of tentacles described in Fig. 7.
10.
Fic. 9.—Oral view of a specimen showing the union of two groups of ten-
tacles, and the double genital band separated by an interradial septum. x 6.
Fic. 10.—A portion of the umbrella with tentacles on the margin in an ab-
normal position. Oral side. x 10.
Fig. 11.—Oral view of a specimen with a lateral outgrowth of the umbrella.
x6.
Fie. 12.—Oral view of a specimen with a group of tentacles inside the
umbrella. x 6.
Fic. 13.—Lateral view of the outgrowth of the umbrella (Fig. 11). x 8.
8 EDWARD 'T. BROWNE.
Fic. 14.—Oral view of a specimen showing congenital variation (three septa)
and the new growth of tentacles. x 6.
Fic. 15.—Lateral view of the group of tentacles inside the umbrella (Fig.
2). KG;
Fie. 16.—Oral view of a specimen showing the loss of tentacles by injury
and the growth of new ones. x 6.
Fic. 17.—Oral view of a specimen showing the new growth of tentacles on
the half of the umbrella which has been injured. x 6.
Fie. 18.—Oral view of a specimen showing the union of two groups of ten-
tacles and two genital bands. x 6.
Fics. 19 and 20.—Oral view of specimens showing the commencement of
genital bands near the margin. x 6.
Fic. 21.—Oral view of a specimen showing the loss of a group of tentacles
by injury. x 5.
Fie. 22.—Oral view of a specimen showing the fresh growth of tentacles.
x 6.
THE COLLAR-CELLS OF HETEROCGLA. 9
The Collar-cells of Heteroccela.
By
George Bidder.
With Plate 2.
SUMMARY.
Tue collar-cells are in normal life short and barrel-shaped,
with separated cylindrical collars, which are never united. In
certain pathological conditions, probably connected with suffo-
cation, they elongate very greatly, diminishing in the diameter
of their upper part, or “collum ;” and in some species, though
not in Sycon compressum, the collars may then come into
contact. In certain other pathological conditions the collar is
lost, though apparently it can be regenerated. These meta-
morphoses appear unconnected with the ingestion of food, which
also was not found to induce any migration of the collar-cells.
On the other hand, migration seemed to occur under excep-
tionally unhealthy conditions.
The collar is made up of (in Sycon compressum) about
thirty parallel rods united by a film of some other substance.
The flagellum is intimately connected with the nuclear mem-
brane. There is an interstitial substance between the bodies
of the cells. The area inside the collar appears to be provided
with a sphincter membrane.
Cells preserved and cut by the paraffin method show an
average contraction of 5 : 4 linear in the best sections. In most
preparations this contraction is uneven, producing Sollas’s
membrane and other fictitious appearances.
10 GEORGE BIDDER.
PREFATORY REMARKS.
The feeding experiments referred to in this paper were
performed on Leucandra aspera and Sycon raphanus at
the Naples Zoological Station,—some during an occupation of
the Cambridge University table in 1887-8, some during later
opportunities for work there, which I owe to the great kindness
of Professor Dohrn. The observations on living cells were
made chiefly on Sycon compressum at Plymouth; they
were undertaken largely on the stimulus of the paper (19) by
Vosmaer and Pekelharing. Some months during which Mr.
Sedgwick has been good enough to allow me to work in his
laboratory I have devoted to reviewing my permanent pre-
parations of all species. Except where otherwise stated, the
collar-cells of S. compressum are described below, this
species having been preserved with the greatest care and
success.
Sycon raphanus grows abundantly on the walls of the
tanks of the Naples Zoological Station. It differs here from
the varieties ordinarily met in the possession of a very long
fur of fine linear spicules. It has the obvious advantage that
physiological experiments can be made in surroundings natural
to it; on the other hand, it is rather small and soft for free-
hand living sections, and its collar-cells are comparatively
small.
Leucandra aspera (var. gigantea, Vosm.) breeds in the
port of Naples. It has the advantage of great size, large
collar-cells, and a robust constitution habituated to the most
poisonous surroundings; but its huge longitudinal spicules
render free-hand sections practically impossible. It is very
remarkable that in impure water it throws out a fur of fine
spicules like that possessed by S. raphanus (var. aquariensis
nova); it has occurred to me that this may be a filter against
bacteria.
S. compressum grows abundantly on the tidal rocks
within ten minutes’ walk of the Plymouth Biological Station.
THE COLLAR-CELLS OF HETHROCGLA. 11
It appears to be annual, in common with S. ciliatum,
Halichondria panicea (cf. Johnston, 1, p. 92), and Hy-
meniacidon sanguineum. The rocks were covered from
December to March of 1894 with large specimens of these four
species; in September of the same year there were in some
localities crusts of Halichondria, but it was for the most
part difficult to find any sponges, except that careful search
revealed a large number of very minute Sycon. Iam informed
that a general absence of littoral sponges was noticed also in
the autumn of 1893. Carter (No. 2) states that S. com-
pressum breeds in May (larve at Plymouth July 18th,
1895).
This species is the best suited of all I know for examination
under high powers during life. Its collar-cells are among
the largest, if not as large as any known. Its strong radial
spicules give a convenient consistency without impeding the
razor ; they also protect the section from being crushed on the
slide. Such sections are necessarily of great thickness as
compared with paraffin sections, but the chambers of the sponge
are so wide and extensive that rows of collar-cells can always
be found standing out freely either against the light or against
quite transparent tissues. On the rocks above mentioned
S. compressum is habitually left for an hour or two at every
ebb-tide to live on the water contained. in its canal-system ;
the conditions of life under the cover-slip are therefore only
partially unnatural. In experience, unless the slide, razor, or
finger holding the sponge have been dirty, the flagellar motion
will continue two to two and a half hours after covering, though
changes of form, detailed below, become apparent after about
a quarter of an hour.
I have not yet used a gas-chamber, the sections having been
merely placed in sea-water between an ordinary slide and
cover-slip. Using a Leitz ;4, oil immersion with Zeiss oc. 3
(old system) the collars and moving flagella appear with
diagrammatic distinctness. I employed an Abbé condenser
and blue glass, with incandescent gauze light focussed exactly
on the object. About fifty living sections were examined,
12 GEORGE BIDDER.
including two or three of S. ciliatum. Probably in all about
5000 living collar-cells were seen distinctly.
Since in the existing state of our knowledge it appears to be
inconvenient to use names for the tissues of sponges which
connote comparison with other groups of multicellular animals,
I shall, where useful, employ the following terms :
Ectocyte. Any cell forming part of the external surface
of a sponge, including the afferent system of canals.
Mesocyte. A parenchym cell.
Endocyte. Any cell forming part of the surface of the
central cavity of a sponge, including the efferent system of
canals and the flagellate chambers.
It also appears convenient to use the term gonocyte to
designate a generative cell.
In this paper the “ basal width” of a collar-cell is the length
of a line passing through four or five cells side by side, divided
by the number of cells. The “ collar-width” is used shortly
for the diameter of the collar at its origin from the cell. The
“height ” of the cell does not include the collar.
GENERAL STRUCTURE OF THE Livine CoLLAR-CELLS.
The collar-cells of 8S. compressum in normal life measure
about 12 high by 6°6 u extreme basal breadth (basal width) ;
the width of the collar—the most constant dimension—being
about 4°6. A few measurements of S. raphanus in life
give their height 7 , basal width 5; judging from the per-
manent preparations of L. aspera, its cells are about the same
size as those of S. compressum.!
1 At Plymouth, in a sponge agreeing closely in spiculation with Carter’s
Acanthella stipitata (fide Ridley and Dendy), I have met with probably
the smallest collar-cells yet recorded. The chambers (fig. 22) were about
6°7 » to 83 p, the apopyle about 3°3y in diameter; the cells were greenish in
life, about 1*7m high and ‘8 » basal diameter, appearing as a mosaic in which
the apopyle contrasted as a large round white hole. The smallest chambers
measured by Ridley and Dendy are three times this diameter, but Ridley
described the structure of Acanthella pulcherrima (fide Ridley and
Dendy) as “a transparent, almost colourless mass, . . . containing
THE COLLAR-CELLS OF HETEROCGLA. 13
The protoplasm is in life greenish, and in normal condition
of ground-glass appearance. Lach cell contains from four or
five to a dozen spherical granules, up to 1 yw, or rarely 2m in
diameter, rather more refracting than the surrounding proto-
plasm. I have called such granules “ basal spherules ” (18,
p. 476) from their strong tendency to segregation in the base
of the cell.
The cells have nearly the form and relation to each other
of full corn-sacks standing side by side in a granary (v. figs.
1, 3, 9a, 15, and 19); in the normal condition they are dis-
tinctly but not widely separated, appearing to be actually in
contact only at their bases. The generally barrel-shaped
lateral surface of the cell always shows a clear smooth line
in optical section; the circle marking upon it the base of the
collar is also a smooth and sharply defined line. On the other
hand, the convex or irregular area inside the collar (intra-
choanal area) has nearly always a fainter outline, as though it
were less refractive; it is often irregular, often finely punc-
tated, often strongly granular. This was observed also in S.
raphanus.
CoLuar.
The living healthy collar is from 2u to 7y in height, in-
variably an almost perfect cylinder, very little constricted at
its base ; ending sharply above without either rim or expan-
sion (figs. 1, 2,3,and 19). It has no vertical cleft, thereby
differing from the spathiform collar of Choanoflagellata as
described by Franze (19). From observations in life the
thickness of collar or flagellum appeared to be 4 to fu.
Once in 8. compressum, and once in S. raphanus, I
observed in a fresh preparation the free edge of a collar, looked
at from above, to present a “ milling ” or beaded appearance,
as in fig. 7; in each case the cell had been some time under
the cover-slip. Since the accompanying plate was engraved I
have re-examined all my permanent preparations with a Zeiss
nucleoid bodies about ‘007 to (008 mm. in diameter.” The identification of
the chambers in my living specimen was unmistakable.
14 GEORGE BIDDER.
apochromatic 2:0 mm. objective of 1°40 aperture, ocular 8.
With this power the beaded appearance from above is conspi-
cuous in all cells of good sections strongly stained with hema-
toxylin. See subjoined woodcut, 0.
a, 6, Collar-cells over-stained in bulk (Series D), showing in a (profile) inter-
stitial substance, and in J (from above) iris membrane. Same slide as
fig. 15, e, Plate 2.
c. From Series A, stained borax carmine and hematoxylin, extracted with
acid, focussed on flagellum to show connection with nucleus and per-
foration of nuclear membrane.
d. Series D, cleared with olive oil, stained on slide with hematoxylin, extracted
with acid. Showing pear-shaped nucleus and perforation of iris.
From drawings made with Zeiss apochromatic immersion, 2 mm., ap. 1°40,
oc. 8. ;
The “ beads” remain sharply defined while focussing from
top to bottom of the collar, each bead being about '25 « in
diameter, and the less stained interspace between them about
‘15 4. The number seems to be fairly constant; though I
never succeeded in counting exactly, it was in no collar esti-
mated at less than twenty or more than six-and-thirty for S.
compressum. The collar is, in fact, composed of a series of
twenty or thirty parallel rods, or non-vibratile cilia, staining
with hematoxylin, though less darkly than the flagellum, and
united by a thinner film of non-staining substance. The
THE COLLAR-CELLS OF HETEROCGLA. 15
structure can be seen in profile, though generally less easily ;
but in a few cases the fibrils have become separated in the
course of preservation, and stand out like the fringe of a tassel.
I find that in Naples I once observed fresh preparations of
Leucosolena primordialis in which the endocytes showed
no collars or flagella, but appeared as if set with short cilia;
the conditions were probably pathological.
As to Sollas’s membrane, the statements of Vosmaer and
Pehelharing! (19), which I originally went to Plymouth to
confute, I can now only confirm.
The sponges were examined alive from rising tide, from
ebbing tide, from deep tide-pools; after hours in a small vessel,
after days and weeks in the aquarium. Many sections were
watched on the slide until absolute death ensued. In nosingle
instance was Sollas’s membrane observed in a sea-water pre-
paration.
As mere accident it would seem that often two neighbouring
collars must be in contact, yet I only succeeded in observing
with certainty two or three cases of this. There is never any
membrane whatever in a plane at right angles to the axes of
the collars. Neighbouring collars never pass into one another
in a continuous curve. In every case that I have yet ex-
amined, where I had reason to believe that the sponge was
thoroughly healthy, the collar-cells had the form shown in
figs. 1,2, and 19. In perfect health the collar is very little,
if at all, expanded from the cylindrical ; it is never trumpeted.
After suffocation, as detailed below, the collars become coni-
cal, expanding distally ; probably this is the explanation of my
observations on S. raphanus (figs. 114 and 11c,—noted in 8,
p. 630), especially as in this species F. E. Schulze (3) states
that “unter Umstiinden kann eine solche Erweiterung des
1 In their otherwise complete summary of the literature these authors
have omitted Topsent’s statement (9, p. 27) that in Cliona celata “les
cellules sout unies entre elles par les collerettes. Collerettes et cils sont
rétractiles comme les pseudopodes de cellules amiboides.” Quite recently
(24, p. 282) he writes, ‘‘ Les choanocytes d’une méme corbeille peuvent rester
libres de toute adherence entre eux, ou bien ils se soudent, a l’occasion, par
les bords de leurs collerettes,”
16 GEORGE BIDDER.
aiisseren glatten Bautheiles vorkommen dass die benachbarten
Collare sich fast beruhren.” In S. compressum such con-
tact does not occur, either in healthy life or in any of the
morbid conditions I was able to investigate.
Sollas’s membrane occurs, on the other hand, in paraffin
sections of S. compressum (v. figs. 16—18, 20), preserved
by any delicate method except the very best; careful examina-
showing that it is always associated with great distortion of
the cells, and that this is also the case in the drawings by other
authors. Where there is no distortion (fig. 15) the membrane
is not present.
Dendy was right in saying that cells showing the membrane
may also possess flagella, though generally this is not the case.
And the phrase “ portions of flagella and collars irregularly
sticking together” (19) is not descriptive of this very definite
structure as it occurs in many sections. But these same
sections have been prepared with great care (all being osmic
acid preparations) from a sponge which I know in life had all
its collars disunited and normally cylindrical; and in five
cases the same individual was examined partly by a living
section, partly by paraffin sections (cf. figs. 19 and 20). It is
not disproved that union of the collars may occur in some
living sponges—more probably in some dying sponges. But the
evidence of ordinary paraffin sections for its existence must
now be considered valueless, and, with exception of the ob-
servations quoted and explained above, there was no other
evidence for its existence. There remains no reason to believe
that it occurs in nature at all, and I must thank Dr. Vosmaer,
my old friend and master, for yet another lesson in sponge lore.
Some measurements will be found in the note on distortion
of cells at the end of this paper.
It is worth mentioning that in the living larva of S. rapha-~
nus (Naples, June) I found that the transparent ends of
the flagellate cells, lettered by Barrois (5) as “ collier,” are
solid and refractile, as faithfully figured by Schulze, x 5;
the convex distal surfaces are correctly shown by both authors,
THE COLLAR-CELLS OF HETEROCGLA. 17
Flagellum.
In the living 8. compressum, the flagellum may be 304
to 504 long. The movement is certainly asymmetrical, with
a longer rest on one side than on the other.!' In several cases
it was also certain that the motion lay entirely in one plane.
It is rare to see flagella moving more rapidly than about 10
beats to the second. I have guessed the greatest rapidity I
observed to be 15 or 20 beats to the second. The thickness
in life was estimated at tu to +>; it appeared uniform,
except sometimes for a thickening of the part inside the
collar. In paraffin sections this thickening is also found, in
perhaps a third of the cells; it does not extend for more than
about 1 from the intra-choanal surface, the thickness above
this point being uniform, and measured in different flagella from
15. to°3. But in the paraffin sections the flagellum can be
traced inside the substance of the cell to the nucleus (cf. cut, c),
and in the osmic preparations stained in bulk it is not wider
here than in its terminal portion. For greater definiteness I
shall term the part of the flagellum below the general outline
of the cell the radix of the flagellum.
Intra-choanal Area.
It has been stated above that the outline of the intra-choanal
area is in life less definite than that of the sides of the cell.
Vosmaer and Pekelharing mention carmine experiments which
hint that here, as supposed by the early authors, food is taken
in; and it will be seen below that careful examination of my
own permanent preparations is far from contradicting this view.
Tn life, neither ingestion nor egestion were ever witnessed ;
but in a sponge which had been two hours in a basin of sea
water, after two hours’ exposure at low tide, almost every cell
1 Minchin states this very definitely for Leucosolenia coriacea (16,
p. 264). I have also a note that the same holds good for Leucandra
aspera and another sponge (I think S. raphanus). These four are all
sponges with tubular or thimble-shaped chambers ; it is possible that the beat
is symmetrical in short hemispherical chambers where the axis of the collar-
cell is nearly parallel to that of the chamber.
vou. 38, PART 1.—NEW SER. B
18 GEORGE BIDDER.
possessed a globule containing angular dark particles,—some-
times, as in fig. 4, projecting on the surface between collar and
flagellum. These globules were observed and drawn moving
in the distal protoplasm of the cells; there were numerous
bodies of similar appearance (cf. fig. 13a@) floating freely in
the chamber. It is possible that they were some minute
organism with whose appearance I am not acquainted; but
the strong suggestion was that they were ejecta. I have often
suspected, from paraffin sections, that the food vacuoles of
sponges are filled with some gelatinous matter, coagulated in
preservation.
The “vacuoles” in fig. 5 and fig. 9 were also moving in
the protoplasm, but it does not seem impossible that they
were nuclei (a view established since this was in type).
In paraffin sections stained with hematoxylin the free end of
the cell very noticeably appears, with the ordinary immersion
lens, as a dark band (figs. 15, 17, 18). Viewed from above, it
is often seen that this stained area is really annular (cf. cut, d),
the flagellum appearing as the dark centre of a white disc, which
is generally about one third the radius of the intra-choanal area.
And with the apochromatic immersion it can be seen in profile
to be indeed the case that at the focus of the flagellum the
terminal plate of stained matter is interrupted by an unstained
interval, showing that the substance stained is arranged as a
diaphragm, and not a complete disc (cf. cut, d). In the profile
of cells treated with acid alcohol after staining, the hematoxylin
is found to be confined to this diaphragm, the protoplasm be-
neath being comparatively unstained.
In many of the cells viewed from above the stained annulus
shows a radial structure. Though marked in a few cases, in
most cells it is impossible with the magnifying power employed
to make certain whether this exists or not; but generally in
the optical profile the dark line marking the section of the
annulus is to some extent beaded, or broken, especially on
focussing above or below the flagellum. Where the rays were
recognised, their number was never more than ten or twelve
(cf. cut, d) ; on the other hand, the root of the collar, focussed on
THE COLLAR-CELLS OF HETEROCGLA. 19
the surface with the cell in full profile, often seemed to show
beads corresponding in number with the collar fibrils. On the
whole, I believe that the radiation represents a condition exist-
ing in life. Vosmaer (19) figures a ring near the base of the
collar (“at the base ”’ in explanation of plate) in Spongilla,
of which he promises a description ; it is not beaded.
The substance which stains in this annular manner I shall
call the iris, and the aperture in its centre the pupil. It is
a natural suggestion that the iris is a contractile sphincter,
aud the pupil the ingestive and egestive aperture of the cell.
Some sections support the view that the thickening at the base
of many flagella is cell-protoplasm projecting in an ameeboid
cone through the pupil, the true flagellum running in the
axis unthickened to the nucleus.
Nucleus.
There is nothing exact concerning the nucleus to be re-
corded from the observations of living cells. I have above
referred to the “empty vacuoles” of figs. 5 and 9; one of
similar position is shown in fig. 3. If the identification be
correct, these indicate (1) that the nucleus is distal in life,
_ (2) that it moves in the protoplasm. In a drawing made in
life from the same preparation as fig. 13, of cells with very
active flagella, there is a large clear sphere in each cell which
can scarcely be other than a nucleus.
Preparations stained in bulk with borax-carmine show in
the nuclei of collar-cells a well-defined chromatin reticulum
surrounded by a stained nuclear membrane. In the wall of
one chamber was a beautiful karyokinetic spindle; presumably
the rather large cell in which it occurred was a collar-cell
dividing in two.
In 8. compressum hardened for one hour in 1 per cent.
osmic acid, and stained carefully in bulk with hematoxylin, —
the nuclei are almost always spherical; the radix of the
flagellum can be recognised as a refractile thread passing from
the nucleus to the pupil of the iris. The same series of sections,
stained also on the slide with hematoxylin and extracted with
20 GEORGE BIDDER.
acid alcohol, shows more often a fine, stained, tapering point,
forming a distal prolongation to the nucleus, issuing through the
pupil of the iris as the flagellum (cf. cut,d). In the nuclei of a
preparation treated with 4 per cent. osmic acid, stained in bulk
with borax-carmine, and on the slide with hematoxylin, the two
forms are also seen: where the nucleus is spherical the flagellar
radix is seen as a faintly-stained thread piercing the dark
nuclear membrane (cf. cut, c); where the nucleus is pointed,
the point—that is, the radix of the flagellum—can often be seen
to be a protrusion of the nuclear membrane. In either case the
nuclear membrane is interrupted, so that in profile the outline
shows a clear break opposite the flagellum.
In S. raphanus, treated with iodine followed by alcohol
and borax-carmine, there is often a comparatively thick stained
thread passing from the nucleus to the flagellum. In the same
species, preserved in weak alcohol gradually strengthened, and
stained in borax-carmine, very many of the nuclei appear
pear-shaped, the distal half of the nucleus being a cone with
its apex in the centre of the intrachoanal area.
In these last sections many of the cells have the nuclei
filiform and ribbon-shaped, so that they probably do not give
the living form; and in the cell shown in fig. 5, treated with
weak alcohol under the microscope, showed the “ vacuole ”
perfectly spherical, refracting, and absolutely distal. But the
particular form of distortion described, assuming it distortion,
points to a firm mechanical connection between flagellum and
nucleus. It seems likely that the spherical nucleus, with a
filiform radix issuing from it, represents an unaltered living
structure ;! we have then to consider whether the pear-shaped
or bulb-shaped nucleus, which all additional reagents tend to
develop represents the staining of other substances surround-
ing the radix, or a change in form of the nuclear membrane.
All that can be stated definitely is that the flagellum is
firmly and intimately connected with the nuclear membrane,
and that when this is spherical in outline the sphere shows a
break at the point where the flagellum intersects it. The
1 This was found to be true in the fresh tissue.—July, 1895,
THE COLLAR-CELLS OF HETEROCGLA. 91
appearances are consonant with the flagellum being a rod-like
or tube-like process of the nuclear sheath.
Vosmaer (19, fig. 8), figures, without describing, such a con-
nection in Halichondria; and Heider (7), in the larva of
Oscarella, describes the root of the flagellum at the nucleus.
With this structural disposition may be correlated the general
(not invariable) distal position of the nucleus in collar-cells
that are elongated, as shown for Heterocela by myself
(18, fig. 4) and Dendy (20, fig. 24). Leucosolenia is figured
by Minchin (17, figs. 2 and 3) and myself (18, fig. 3) with a
distal vacuole to each cell and a basal nucleus; Spongilla,
according to Vosmaer’s plate, differs in these respects from
Halichondria precisely as Leucosolenia from the Hete-
rocela. A suggestion has been made to me that the nucleus
serves the flagellum as a mechanical fulcrum in the semi-fluid
protoplasm ; and it is obvious that if the whole intra-choanal
area be a cell-mouth the flagellum can have no permanent base
except in the interior of the cell. If this view be correct, the
same function would seem to be performed in certain lines of
descent by the walls of a permanent vacuole, verifying for
Leucosoleuia an alternative suggestion of Minchin’s (1.c.),
who doubted “whether this space represents a ‘ Central-
k6rper,’ or a kind of food-vacuole, or whether it is in some way
connected with the movements of the flagellum and collar.” !
Maas’s embryological work on Silicea (21) seemed to point to
the possibility that the relative size of nuclei might indicate
'T have no intention to discuss the classical literature on connections
described in other groups between nuclei and flagella on cilia. But my
friend Mr. J. J. Lister has kindly pointed out to me the description of
Camptonema nutans (a Heliozoon-like organism) by Schaudinn (25) in
which he describes the axis of each pseudopodium expanding to envelope a
nucleus in a manner most suggestively recalling the condition drawn in my
woodcut at ¢. Schaudinn puts forward tentatively the view “‘ dass der Kern
bei der Bewegung der Pseudopodien eine bedeutende Rolle, vielleicht als
regulatorisches Centrum, spielt.” I think we should first carefully test on
Leucosolenia and Spongilla the hypothesis I have borrowed above before
yielding to the ever-enticing temptation to appeal to the nucleus as cell-
brain,
By) GEORGE BIDDER.
ontogenetic history, particularly as to whether in Sycon also
the lining of the efferent system arises from the granular cells
(with large nuclei) of the larva. Measuring thirty nuclei of each
tissue, near the osculum of S. raphanus, gave the following
average diameters :
Nuclei of collar-cells : : . 215 p.
», Of cloacal epithelium . é Be
»» Of dermal epithelium . : 2 Ge.
The largest cloacal nucleus is 4°7 , and two thirds were over
2°5 uw; the largest nucleus of a collar-cell is 2°64, and there is
no other over 2°54. Between these two classes, therefore, the
difference is very marked ; but on the other hand, three fourths
of the collar-cell nuclei and dermal nuclei are mutually indis-
tinguishable as regards size.
In a borax-carmine preparation of Leucandra aspera all
cells but the gonocytes showed a nuclear reticulum, with the
possible exception of two parenchym cells. Both in Sycon
and Leucandra the gonocytes show the well-known large
vesicular nucleus with nucleolus.
Interstitial Substance.
The interstitial jelly between the collar-cells, the existence of
which I have never suspected from living preparations, proves
in these permanent sections under the apochromatic lens to
have considerable importance. It appears not only in the
best sections of S. compressum, but also in sections made at
Naples from 8S. raphanus. In permanent preparations of the
normal condition it often reaches to the level of the base of
the collar, as drawn by Dendy for Leucosolenia (14, pl. 8, fig. 3),
sinking in a tension-curve between the two.cells (cf. cut, a). In
sections where Sollas’s membrane has been produced, the mem-
brane is seen uniting the tops of the collars, separated from
the surface of the jelly by a vacant space, not being, as Lenden-
feld suggested (10), a misinterpretation of this surface. I
satisfied myself that in fig. 17 the line is not the outline of a
jelly, but actual irregular fusion of collars, the effect being
THE COLLAR-CELLS OF HETEROCGLA. 23
that they have been forced into contact while of natural size,
and then been subject to individual constriction.
I must admit that increased optical definition proves it was
the surface of this substance, coinciding with the upper limit
of the basal spherules and the constriction of the cells, which I
mistook for an intracellular septum in the “ column-and-plinth”
cells (18). It will be shown that it is now probable that the
form of these cells is not connected with nutrition, and that
Dr. Dendy’s surmise with regard to them was nearer the truth
than my own.
Pathological Changes.
Two distinct series of changes in form, due to abnormal
conditions, were noticeable from their constancy of character
and sequence. They appear interesting not only for the light
they throw on the histology recorded in preserved sponges, but
also from the point of view of cell-physiology.
The first were observed in healthy living sections placed in
a drop of sea water under the cover-slip on a glass slide; I
shall call them “suffocation changes.’”? They consist mainly
of the formation and elongation of a transparent neck (collum
of authors) to the normally barrel-shaped cell. Beginning
with increased transparency of the upper (distal) part of the
cell, the transparent region so distinguished soon becomes
elongated and constricted, the spherules remaining in the
wider and opaque base (figs. 5,6, 8). Being narrower, the
distal parts of the cells are obviously more separated than
before. The collars become conical, expanding at the mouth—
possibly in geometrical consequence of the constriction of the
collum (figs. 8c, 9, 10, 11, 12). During these changes the
flagella continue to move, so that the tissue must be consi-
dered living; they become very gradually slower, but after all
motion has ceased it is long before the delicate flagellum and
collar further change their outline. The extreme form drawn
in fig. 12 was from a section that had been under observation
one hour and three quarters ; for another twenty minutes the
cells were motionless, but unaltered.
24, GEORGE BIDDER.
The degree of change differs in different specimens; but
usually after two hours every chamber presents an appearance
it may be convenient to call “ striated,” the lumen being
greatly reduced, the elongated thin cells forming a herring-
bone pattern down the chamber, and appearing (if it be not,
indeed, the fact) as if many of them became free.
Carter (2) draws two collar-cells from teased living prepara-
tions of 8S. compressum, of which his fig. 1 corresponds
exactly to my fig. 10, and his fig. 2 to my fig. lla. He de-
scribes changes on the slide to amceboid forms; but he is
treating entirely of cells “ scratched out from the body of the
sponge,” whereas I have confined my observations to cells in
situ.
Fig. lla is a sketch made from S. raphanus (Naples, Aug.
1892), with the note ‘‘ a tendency to elongation of cells as the
preparation dies ;” while figs. 114 and lic, made at the same
time, bear the note “flagella” [in other parts] “still in
motion, certainly none on these cells.”” I have already quoted
Schulze’s observation (3) as to occasional concrescence of
collars in this species; the cells drawn by him as normal
appear to have mostly entered on the phase of my figs. 6 and
8, that is, to have been twenty or thirty minutes under the
cover-slip; his Taf. 14, fig. 4, is practically in the stage of my
figs. 11 and 12.
The form of cell produced by this series of changes appears
identical with that described by Dendy in his “x ”’ chambers
(12, 20), and is certainly so with the ‘ column-and-plinth ”
cells described by me (18, p. 477). Similar cells in permanent
preparations show the nucleus in most cases at the extreme
distal end of the cell, the granules are in the base. The upper
surface of this base coincides—at least in most instances—
with the upper surface of the intercellular jelly, and the con-
tours of the uppermost enclosed granules lie in the same plane.
With the ordinary immersion objective the appearance of a
septum is in many cases convincing, so that even now it is
only with the apochromatic lens that I find it possible to
resolve it into its component optical elements.
THE COLLAR-CELLS OF HETEROCGLA. 25
The second series of changes I will call “tide changes.”
S.compressum is a tidal sponge, and when removed from
the water will live in a damp atmosphere for two or three days.
The cells become rounded and transparent, they retain their
flagella but lose their collars ; after restoring the sponge to
healthy conditions the collars reappear.
In some specimens gathered from bare rocks about four
hours after the sea had left them, having been one and a half
hours in drizzling rain, the cells were rather short, rather
round, notably granular, and mostly without collars. The
flagella were moving, in one sponge with greater violence than
I have ever seen. In one cell, after ten minutes in fresh sea
water, I thought I saw the collar reappear, but the observa-
tion was open to doubt.
In a sponge twenty-seven hours out of the water (in an
empty corked bottle), the cells were very low, rounded, and
transparent, with bright granules; the flagella were active,
though not on all cells; collars were very rare. From the
same sponge, after twelve hours in sea-water, another section
showed the cells less transparent, and higher (fig. 13), with a
few more collars; after another eighteen hours in sea-water
there were in most parts of the sponge perfectly normal
collared cells, in other parts the curious modification shown in
fig. 14. Both forms of collar may be considered to have been
regenerated, since two or three other “dry” sponges showed
loss of collars from almost all cells, and it appears that few
collars persist after a day’s removal from the water.
While it is obviously impossible from these observations to
point out with certainty the exact stimulus to which the
changes are due, some of the facts available are worth review-
ing. Increased salinity and retention of waste products in
the chambers are common to the conditions producing both
series, but the tidal changes also occurred when the salinity
may be supposed to have been reduced. In all the suffocation
changes the preparation had been brought to the warm tem-
perature of the laboratory, but this was true for a much longer
time of some sponges on which the tide phenomena were
26 GEORGE BIDDER.
observed. The latter were, however, always exposed to a con-
siderable mass of air, and respiration may be supposed to have
been still possible; under the cover-slip this was of course not
the case. On the other hand, the radial chambers under the
cover-slip each contained the excretory products of at the
most two hours, for which time only they had been deprived
of food; in the case of the tidal changes, nothing but gaseous
matters could have been either received or eliminated for one
or two days. It seems, therefore, plausible to suggest that the
characteristic appearance results, in the suffocation changes,
from want of oxygen or presence of carbonic acid ; in the tidal
changes, from starvation or the presence of non-gaseous excreta.
The local suffocation transparency appears to be mere segrega-
tion, the tidal transparency may be due to starvation. It may
possibly be important that the metamorphosis here attributed
to lack of oxygen results in a maximum surface, that attributed
to presence of poisonous products results in a minimum surface.
Tidal changes were never observed to originate under the
cover-slip, nor on the other hand did. cells so metamorphosed
ever give rise to suffocation forms. The elongated suffocation-
cells died extended, the hemispherical tidal cells died hemi-
spherical, neither modification showing any signs of giving rise
to the other. Only in one section (of a sponge twenty-seven
hours out of the water) I found, after an hour on the slide, a
chamber lined with the usual low, round, collarless cells (as in
fig. 13), but with two collared-cells of the extreme suffocation
form (as in fig. 12), 30 long, stretching almost across the
chamber. The contrast was very striking, and seemed to hint
that accompanying the loss of the collar is some change, perhaps
of the lateral walls, which means the loss of power of exten-
sion. These two cells had escaped the tidal modification, and
therefore were able to respond to the stimulus of suffocation.
All appearances suggest that the extension under suffocation
is due to constriction of the lateral wall—whether it be a con-
traction set up by these conditions, or a normal tone which the
enfeebled cell-contents can no longer overcome.
Apparent migration of collar-cells into the parenchym
THE COLLAR-CELLS OF HETEROCGLA. 27
was observed in a sponge (S. compressum) which had been
a month in the circulation of the aquarium, with other
sponges, &c., allowed to decay in the dish containing it. The
living section at first sight seemed to be full of embryos;
these proved, however, to be the remnants of the flagellated
chambers, some parts still exhibiting perfectly normal collared
cells with active flagella and cylindrical separated collars; the
space between the “ Leucon”-like chambers being largely
filled with parenchym. Paraffin sections showed many wide
canals, resembling the normal afferent system. Only a few of
the collar-cells are elongated, and the recognisable collar-cells in
general are comparatively fewin number; in some places they line
only part of achamber; in some places the chambers are shorter
or narrower than in the normal sponge ; in some places they form
small closed chambers, or pseudo-blastule, consisting of as fewas
a dozen cells, lying in a plentiful gelatinous parenchym, into
which appearances suggest that their fellows have migrated.
The condition appears identical with that recognised as
common in winter for Spongilla (Lieberkiihn, Metschnikoff,
Weltner). It becomes a question whether we are not to
ascribe the metamorphosis of Halisarca as described by
Metschnikoff (6), and that of S. compressum described by
Masterman (23), to conditions unfavourable to general vitality,
rather than to the inception of nutritious sive innutritious
particles.
Nutrition.
Vosmaer and Pekelharing (19) find carmine and milk, after
one hour’s feeding, in the choanocytes and in the lumen of the
chamber, especially frequently in the collars themselves. After
a longer time the particles are chiefly in the cell-bodies, rarely
free or in the collars; after a still longer time they are found
in the parenchyme.
Masterman (28) recently published an account of nutrition
in S. compressum in which he describes an extraordinarily
rapid cycle of events. It has been suggested above that he
may have been deluded by pathological metamorphoses uncon-
28 GEORGE BIDDER.
nected with nutrition, as I was formerly (18) in my hypothesis
as to changes of cell-form accompanying digestion.
Of my own experiments I printed shortly the main results
in February, 1888. Omitting the passage (quoted in 19) on
Sollas’s membrane, I reprint the statement.!
“Tn Leuconia aspera I find that carmine granules are
taken in freely by the collared cells, not appearing in the
mesoderm, and only infinitesimally in the other epithelia. .. .
“T observed that during four hours a Leuconia plentifully
supplied with carmine ejected none in its oscular stream, which
was powerful and continuous. Its flagellate cells proved to be
heavily charged with carmine grains. Such complete filtration
would be uneconomical, if not impossible, were the carmine
arrested merely by the ingestion of cells laterally bounding
the current.
“‘T believe, from a consideration of the observations of
others and the above facts, that the collared cells primitively
both ingest and digest for the sponge; the function of diges-
tion being in some sponges, but not in Leuconia, passed to
cells situated in the mesoderm. I think that probably only
under exceptional necessities of structure do other cells of a
sponge ingest food in valuable quantity.
“My experiments were suggested by a recognition of the
fact that in the current through a sponge the region of slowest
motion, and therefore of greatest deposit and easiest arrest, is
in ‘the flagellate chambers, where the transverse area of the
total channel for the water is greatest. This fact also explains
1 Extracted from the ‘ Proceedings of the Cambridge Philosophical Society,”
vol. vi, pt. iv, ‘Preliminary Note on the Physiology of Sponges.” Fifty
copies only were printed in full through a mistake owing to change of
editorship by which an abstract of ten lines was substituted in the ‘Proceedings *
as issued; for this reason I print a “ Preliminary Note ” of work still, alas!
unfinished. I hope soon to publish a discussion of the mechanical conditions
here referred to. The lamellar forms of sponges are naturally independent of
oscular velocity, since the stream of foul water is 180° from the stream of
fresh water. It is the increase of this angle which leads to the number of
stalked forms, from which are usually evolved the flabellar species and
varieties.
THE COLLAR-CELLS OF HETEROCGLA. 29
the persistent union of nutritive with motor functions in the
cells lining these chambers, since the flagella have their highest
efficiency where the velocity is least. The healthy nutrition
of a sponge (excepting lamellar forms) depends on the energy
of the current from the osculum being high; the economy of
its motor apparatus depends on the velocity of the water in its
chambers being low. All transition from more to less primi-
tive canal-systems exhibits an increase in the ratio between
these quantities.”
The mechanism of filtration we now know to have nothing
to do with Sollas’s membrane; the cardinal fact of filtration
was very striking, and remains to be explained.!
As to the locality of ingestion and digestion, my permanent
preparations available are in all from five specimens of S.
raphanus, eight of Leucandra aspera, and one of Leu-
cosolenia clathrus. The intervals between the first applica-
tion of suspended particles (carmine, starch, &c., rubbed up in
the sea-water), and that of the preserving fluid were respec-
tively 5, 10, 10, 11, 14, 21, 27, 50, 60, 77 minutes, 44 hours,
18 hours, 22 hours, and 3 days. Most of the sponges were
placed in clear sea water for various periods before killing ;
but the accumulations on the spicules, &c., render this of
doubtful value.
Re-examining anew all these preparations very carefully
with Zeiss’s apochromatic immersion lens, I can support my
old conclusions, and make some additions. Ingestion com-
mences freely at once; on the whole, evidence is in favour of
it taking place within the collar of the cell. After twenty
minutes the foreign particles are often found enclosed in a
vacuole, and they are more generally in the basal parts of the
collar-cells.
Carmine is found here in S. raphanus which had been
in pure sea water eighteen hours, after feeding for twenty
minutes; only very fine particles are present, in the bases of
1 [ should warn anyone repeating the experiment that carmine is often
soluble to a considerable extent in sea water. That which I used at Naples
in 1887 was not soluble in the sea water of the aquarium.
30 GEORGE BIDDER.
the cells, and mostly in vacuoles. The cells containing foreign
particles do not lose their collars, and the column-and-plinth
appearance occurs independently of the amount of carmine
contained. Nor do the collar-cells show any tendency to
migration, even after being fed (L. aspera) for four and a half
hours, when many are filled to their very outlines with carmine.
In the sponge here referred to about 1 per cent. or fewer of
the glandiform ectocytes contain a grain or two of carmine.
This may be excretion, but there is no evidence against it
being casual ingestion. In most recently fed preparations
there are one or two canal ectocytes containing a grain of
carmine.
Examination confirmed the statement (18) that there are a
number of gonocytes connected by processes or pseudopodia
with the basal surfaces of the collar-cells, and containing, in
both body and process, spherules precisely resembling the
basal spherules of these cells. I still believe, therefore, that
the gonocytes nourish themselves on the basal spherules at the
expense of the collar-cells; and in the hypothesis (which I
think I owe to an oral suggestion of Miss Greenwood in 1888)
that these spherules are stores of digested food. The prepara-
tions mainly examined are of the 8S. raphanus eighteen hours
after feeding, where the carmine lies among the basal spherules.
A large number of the gonocytes are in contact with collar-
cells which contain plentiful carmine; in only two of them I
found carmine- grains, and it is tempting to deduce that vacuoles
and undigested food do not pass into the gonocyte.
In L. aspera and S. raphanus migration of the collar-cells
into the parenchym certainly does not take place after satiation
to any degree for any period with carmine; nor in L. aspera
when a large proportion of the collar-cells contain completely
ingested starch grains;! nor after fourteen minutes’ feeding with
carminate of alumina, freely ingested; nor after one hour’s
feeding with Indian ink, freely ingested. There is one clear
1 The use of the polariscope for recognising starch grains is easily
practicable with the highest powers. Without it vacuoles of the same size
are often difficult to discriminate.
THE COLLAR-CELLS OF HETEROCGLA. 31
case (S. raphanus, eighteen hours after feeding) of carmine
in the parenchym jelly among similar sized brownish particles,
giving vividly the impression that they have been discharged
from the collar-cell above. There is one apparently certain
case (Li. aspera) of a starch grain apparently enclosed between
mesocytes in the parenchym near an afferent canal. I have
seen no other instances, and there is nothing which leads me to
suppose that as a rule undigested food ever passes into the
parenchym, nor have I any observations which indicate the
means of nutrition of the parenchym otherwise than as con-
cerns the gonocytes. And it is worth stating that the few
carmine-grains observed in ectocytes were never enclosed in
vacuoles.
Though there are many cells containing carmine in S. ra-
phanus after eighteen hours’ feeding, the particles are fine
and the mass small. lL. aspera, twenty-one and a half hours
after twenty-one minutes’ feeding, shows no carmine.
As to the natural food and feeding of the sponge, S. com-
pressum killed directly from the sea shows in the protoplasm
of its collar-cells, besides and among the basal spherules,
numerous minute irregular particles, often highly refractive ;
sometimes three or four in a vacuole-like structure (cf. cut, a),
Many appear to be bacilli, being rod-like bodies 1p to 1°84
long by ‘1uto*2 broad. In another specimen there are lying
freely in the chambers several specimens of what appears to be
an alga, one a sphere of four cells, one probably of sixteen;
also lying inside the collars of different collar-cells are several
isolated spheres, of about the same size as the individual cells
of the larger spheres, and similarly stained. In this preparation,
and another of L. aspera, there are in the chambers several
larger nucleate cells, possibly Protozoa, partly enveloped by
the distended collars, sometimes more than one cell converging
on them. I have not hitherto witnessed any similar phenomena
in life, nor do I know of any such being recorded.
In several instances in the carmine preparations there are
grains inside a collar, as Vosmaer and Pekelharing describe,
and the evidence certainly so far points to ingestion by the
32 GEORGE BIDDER.
intra-choanal area, however difficult it may be to understand
how the food is brought there. It is also obvious that where
there is an interstitial substance the water cannot pass over
the surface of the cell, as I formerly supposed. Therefore
until direct evidence is obtained we must consider it probable
that the pupil of the iris is the aperture both of ingestion and
egestion. I have never witnessed in life anything suggesting
pseudopodial action of the collar (except possibly change of
length), but it is difficult otherwise to see how cells can ingest
through the intra-choanal area starch-grains as wide as them-
selves.
It is commonly stated that sponges can be easily starved by
filtering the water. Fig. 3 represents collared cells from S.
raphanus which had been four days in water passed entirely
through filter-paper ; there was no difference apparent from
sponges which had been detached on the same day and re-
placed in the water from which they had been gathered.
In L. aspera and 8S. raphanus the current is not stopped
by the application of carmine,—which, as stated above, is in-
gested from the first. The current was stopped (L. aspera)
after a few minutes by the carminate of alumina employed,
which may have had with it some soluble poison producing
this effect; but the sponge was preserved within fourteen
minutes from first administration, and the collar-cells were
found to have ingested the carminate freely. Far from the
dermal pores closing for hours against suspended matter,
powdered charcoal (L. aspera), and starch (S. raphanus),
in sponges killed after seven minutes and five minutes res-
pectively, were found solidly filling the afferent canals. With
the starch the prosopyles were also filled, and widely open,
and there was starch free in the flagellated chambers and even
in the cloaca; the starch grains (and still more the particles
of charcoal) were too large for easy ingestion, but they were
adhering to and certainly occasionally ingested by the collar-
cells.
Topsent (9) finds that with the parasitic Cliona “ méme d’y
THE COLLAR-CELLS OF HETEROCGLA. 33
mettre en suspension des granules de carmin, provoque l’occlu-
sion relativement rapide des papilles.” It is not clear from
the words whether this is due to the presence of particles or
only to stirring the water; but it is well known that these
papille are exceedingly sensitive. For other sponges, and
especially S. raphanus, Lendenfeld makes repeated state-
ments (11, pp. 588, 592, 675, &c.) as to closure of pores
against carmine (and not against milk). They are contradicted
by the experiments of every other worker; and notwithstanding
their picturesque elaboration, and the dramatic deductions for
which these statements are responsible, the 149 experiments
that he records include no evidence that the narrative is based
on even erroneous observation.
DistorRTION OF CELLS IN PRESERVATION.
The following results may be of some interest to those who
study histology on preserved material from other groups as
well as sponges, though the measurements are too few to
profess to be more than suggestive.
Measurements were made of the collar-cells in six series of
sections, A, B, C, D, E, F, in order to compare their dimen-
sions with those of life. The series were from five specimens
(S. compressum), D and E being from one sponge; and in
the case of all but A the collar-cells from a closely adjoining
portion of the same individual were examined and measured
during life.
All the sponges were preserved in osmic acid for one hour ;
followed by alcohol, benzol, and paraffin. In C, D; and F the
change from water into absolute alcohol was effected by dia-
lysis; in all but B the change from absolute alcohol into
benzol was made in the same way; all were transferred by
gradual changes of temperature and percentage through soft
paraffin to hard paraffin of a temperature not exceeding 65°C.,
generally 62° C.
A was the only sponge preserved in + per cent. instead of
ai per cent. osmic acid, it alone was decalcified (1 per cent.
nitric acid in 90 per cent. alcohol), it alone was stained in
voL. 38, PART 1.—NEW SER. C
34. GEORGE BIDDER.
bulk with borax carmine, and alone was cut by the ribbon
method, all the other sections being made with the oblique
razor.
The distal expansion and fusion of the collars known as
Sollas’s membrane (fig. 18) appeared plentifully in the paraffin
sections of A, B, C, and F; scarcely at allin Dand E. It was
not present in the living sections examined from any of the
sponges; all alike showing the characters described in the
previous paper.
It was found that the average cubical contraction of the
cells is about to one half of their living dimensions:
Average volume of living collar-cell . . 270 cubic p.
S5 » Of collar-cell in balsam = 25
3
This was calculated from the linear measurements, which
contract unequally in different directions :
Height! from 28 living cells . , o AD
a 86 balsam cells . : Se at ist
Basal width from 34 living cells ; Gas?
is », 203 balsam cells i . 5°6p.
Collar width from 50 living cells : . 46p.
* » 126 balsam cells : . ody.
The best series of sections (D, drawn in fig. 15) and the
worst series (A, drawn in figs. 17 and 18) show respectively
the following ratios in their linear dimensions to those of life:
in Series D. in Series A.
Collar width ; : : 83 : "5
Basal width 4 P ; ‘88 ‘ ‘
Height . 4 : j 8 : "5
Height of collar. ; 10 , 1:0
Deduced ratio of volume of cell to that
in life 4 : "55 5 2
Deduced mean linear eantrantion ratio . 82 ; 6
1 **Collar-width ” is measured at the origin of the collar from the cell ; “basal
width ”’ is the length of a row of cells divided by the number of cells in the
row; “height” is the distance between two parallel lines at right angles to
the axis of the cell, and tangential to its apical and basal surfaces respectively,
Contraction is here measured by the ratio of the fiual to the original magni-
tude, referred to briefly as the ‘‘ contraction ratio,”
THE COLLAR-CELLS OF HETEROCGLA. 35
The difference of the best two series of sections from all
the others is in the uniformity of their contraction. It will
be seen from the drawings that while the living form of the
cell is barrel-shaped (figs. 1, 2, 3, 19), the tendency of preser-
vation is to produce a sphere (figs. 17, 18, 20, 21; Dendy’s
figs. 24 and 25, plate 14, vol. xxxv, and fig. 38, plate 4, vol.
xxxii, of this journal, &c.). This necessarily produces a highly
disproportionate contraction at the base of the collar and it
results that the measurement of the ratio of this dimension to
the greatest width of the cell affords a fair index of the distortion
which the preparation has suffered. Thus the artifact nature
of Sollas’s membrane is concisely demonstrated by the follow-
ing figures, averaged from all the measurements :
Basal width in living cells ‘ . Orap.
in balsam with separated sila, e aaoy cs
s in balsam showing Sollas’s membrane 3 AO: Gps:
Collar-width in living cells : vs xs Olbs
35 in balsam with separated eallare. 2, Aeon.
in balsam showing Sollas’s membrane nye ah pee
In life, as in the preparations where collars are separated,
the collar-width—that is, the apical width of the cell—averages
three fourths of the extreme width. Where Sollas’s membrane
is present the collar-width ranges from two thirds to one third
of the extreme width of the cell.
The change can be best followed by comparing figs. 19 and
20 (series B), which are drawn from the same sponge to the
same scale,—the one in life, the other from a paraffin section
mounted in Canada balsam.
The nett result of the measurements may be seen in the
averages of three series of paraffin sections, D, C, and A:
cubic p. B
Cell-volume in life (ef. figs. 1, 19) . 270 ~ Collar-width in do. . 4°6
is in balsam, Series D (ef. fig. 15) . 170 5 B 4°3
% i elie. . 185 i i 3-0
B 3 3 Aneities.. 17,
18) . 865 K ‘ 2-9
There are, therefore, two principal phenomena due to the
36 GEORGE BIDDER.
transference of cells through osmic acid, alcohol, and benzol,
into paraffin, and finally Canada balsam :
(1) There is a reduction in the total volume of the cell,
which apparently cannot be avoided, corresponding to a mean
linear contraction of about 5: 4 in the best preparations, and
5 : 3 in the worst.
(2) Independently of the extent to which this takes place
there is generally a change of form. It appears possible (cf.
figs. 1, 15) almost entirely to avoid this, but by most methods
the rectilinear and angular outlines of life (figs. 1, 2, 3, 19)
are replaced by pyriform (figs. 20, 21), ovoid (fig. 17), spherical
or even oblate (fig. 18) contours in the permanent preparations.
Thus, taking from the averages of the last table the con-
sequent ratios of the linear dimensions to those of life, we
obtain :
Mean linear contraction ratio Contraction ratio of collar-
in— width in same sections.
Series D (fig. 15) . 82 : 2 360
Series C : Sas) : . 68
Series A (figs. 17,18) . ‘6 ; . 46
It was experimentally shown that the extreme changes of
cell-form were not produced in alcohol. Bringing part (E) of
a sponge in four minutes through 30 per cent. and 50 per
cent. into 70 per cent. alcohol, the cells were compared in
paraffin sections with the part (D) of the same sponge
treated uniformly by slow dialysis. The collar-width (4:0 ,)
and the basal width (5°44 to 5°7u) in E retain their normal
proportions to each other, and the collars are not united. It
is true, however, that the mean contraction is greater (ratio
‘74) than in D, and the height of the cells is disproportionately
diminished (7°8 « as against 9°5 w in D and 12:0 w in life).
It was also experimentally shown (fig. 16) that in some
sections of the best series (D, cf. fig. 15) stained on the slide
in the ordinary way through turpentine and four grades of
alcohol into Grenacher’s hematoxylin, the cells suffered con-
siderable distortion, and in many cases developed Sollas’s
THE COLLAR-CELLS OF HETEROCGLA. 37
membrane. Similar results were obtained on clearing the
sections in benzol and in olive oil.
I am inclined to consider the chief engine of distortion to
be the passage from alcohol into benzol, chloroform, or turpen-
tine, and vice versa. The cells of fig. 15 probably escaped,
not only because the passage into benzol was effected by very
gradual dialysis, but because they were first hardened in
alcohol between 85 per cent. and absolute strength for
some eighteen hours. It may be noted that the tendency
of all the cells to assume a drop-like form proves that the
force effecting their distortion is surface-tension.
It does not seem unlikely that the reduction in volume is
due to the abstraction of water and soluble matters by the
alcohol. It is not due to shrinkage of the paraffin block, for
from the standard tables contraction through 45° C. would be
in wax to ‘96, and in paraffin not more than to ‘99 of the
original linear dimensions. I have no reason to suppose that
there was any appreciable compression in cutting the sections ;
and since the nuclei remain spherical, and the collars are un-
altered in length, this cannot be assumed. But it must be
pointed out that the mean contraction-ratio is less certain than
the amount of distortion, since it involves the measurement of
the living cell-height, which can only be done accurately in
fortunate instances.
The collar rarely contracts in length; this may either be
due to its thinness, or to the nature of the rods which com-
pose it. Sollas’s membrane may be due to either a local con-
striction of the collar or the forcible contraction of its base
throwing out the free lip; it should be noticed, however, that
in such a section as is drawn in fig. 18, the chamber has so far
contracted as a whole, that where the free ends of the collars
remain of their living diameters, they must be pushed into
contact.
By the definition of “ basal width” employed, it will be
seen that this measurement expresses the linear contraction of
the wall of the chamber as a whole. There is generally least
contraction in this plane, the tendency of the cell to become
38 GEORGE BIDDER.
spherical increasing the breadth in proportion to the height.
In Series C the measurements give no evidence of contraction
in this dimension ; but the cells are spherical and even orange-
shaped, showing that the absence of change in anatomical
dimensions is no guarantee against the most profound cellular
distortion.
MertuHobps.
The main practical conclusion was that cell-form tends to be
profoundly modified in the passage between alcohol and paraflin
solvents, and that this may unfortunately be the case even in
the process of staining on the slide. It seemed likely that the
dangers of the embedding process are modified by very gradual
dialysis from alcohol into benzol, and largely guarded against
by super-hardening in 1 per cent. osmic acid and in absolute
alcohol. For osmic acid even the sponge tissue requires to be
cut in the smallest practicable pieces and repeatedly shaken,
otherwise the inner chambers are not thoroughly hardened ;
the exposure used was one hour in the dark. Dialysis from
water into absolute alcohol, or from alcohol into benzol, each
took from six to twelve hours; they were left up to fifteen
hours with good results. The best preparation (Series D) was
stained in bulk with equal parts of Grenacher’s hematoxylin
and 70 per cent. alcohol, being brought into this solution from
40 per cent. alcohol by four equal changes of strength; no acid
was used, and the result was a very valuable overstaining of
the collars and iris membranes. The sections were fixed with
water, the paraffin cleared in chloroform. It will be found
convenient to have in a pipette a thin solution of balsam in
chloroform, so that it can be squirted instantly on the sections
after removal from the chloroform, to prevent drying before
the thicker balsam has time to spread.
It will be seen that I am greatly indebted to the methods of
Vosmaer and Pekelharing (19), which were closely followed
up to the stage of embedding in paraffin; but I am convinced
that staining on the slide is highly destructive of cell-form,
unless the transference from benzol to alcohol be effected with
THE COLLAR-CELLS OF HETEROCGILA. 39
the tedious care used for the tissue in mass. I believe the
form of the cells in Vosmaer’s drawings has been influenced
by this process, though the oval outlines of nuclei and vacuoles
in the sections is probably attributable to the razor. Passage
into glycerine is of course attended with the same necessity
of preliminary passage into alcohol, but comparison with
similar sections stained on the slide and mounted in Canada
balsam show that the cells in glycerine are only equally dis-
torted or coutracted, and, as these authors state, the collars
and flagella are more visible, and the preparation very brilliant.
REFERENCES.
. Jounston, G.—1842, ‘A History of British Sponges and Lithophytes.’
. Carter, H. J.—1875, ‘Annals and Mag. Nat. Hist.,’ vol. xvi, p. 1.
. Scuuize, F. E.—1875, ‘ Zeitschr. wiss. Zool.,’ vol. xxv (suppl.), p. 247.
. Barrois, C.—1876, ‘ Ann. Se. Nat.,’ ser. 6, vol. iii, art. 11.
. Scuvize, F. E.—1878, ‘ Zeitschr. wiss. Zool.,’ vol. xxxi, p. 262.
- Metscunixorr, E.—1879, ibid., vol. xxxii, p. 349.
. Herer, K.—1886, ‘ Arb. Zool. Instit. Wien,’ vol. vi, p. 175.
. Broper, G.—1888, ‘ Proc. Phil. Soc. Cambridge,’ vol. vi, pt. 4.
.
. Topsent, E.—1888, ‘“‘Théses presentées 4 la faculté des Sciences de
Paris,” ‘Contributions données par la faculté,’ Poitiers, Typographie
Oudin.
10, LENDENFELD, R.—1889, ‘ Zool. Anz.,’ xii, p. 361.
11. LENDENFELD, R.—1889, ‘ Zeitschr. wiss. Zool.,’ vol. xlviii, p. 406.
12. Denpy, A.—1890, ‘ Quart. Journ. Micr. Sci.,’ vol. xxxii, p. 1.
13. Cuatin, J.—1890, ‘Comptes Rendus,’ p. 889.
14. Denpy, A.—1891, “A Monograph of the Victorian Sponges,” part 1,
Melbourne, ‘ Trans. Roy. Soc. Vict.,’ vol. iii, p. 1.
15. BippER, G.—1891], ‘ Quart. Journ. Micr. Sci.,’ vol. xxxii, p. 625.
16. Mincuin, E. A.—1892, ibid., vol. xxxiii, p. 251.
17. Mincutn, E. A.—1892, ‘ Zool. Anz.,’ xv, p. 180.
18. Bipper G.—1892, ‘ Proc. Roy. Soe.,’ vol. li, p. 474.
19.—VosmakER, G. C. J., and Pexetnarine, C, A.—1893, ‘ Tijdschr. Neder.
Dierk. Ver.,’ (ii), Deel. 4, p. 38.
San oar dOndD &
40 GEORGE BIDDER.
20. Drenpy, A.—1893, ‘Quart. Journ. Mier. Sci.,’ vol. xxxv, p. 159.
21. Maas, O.—1893, ‘Z. Jahrb. Morph. Abth.,’ Bd. vii, p. 331.
22. Franzb, R. H.—1893, ‘Zool. Anz.,’ xvi, p. 44.
23. Mastrermay, A. T.—1894, ‘ Annals and Mag. Nat. Hist.,’ vol. xiii, p. 485
24, Torsent, E.—1894, ‘ Arch. Zool. Exp.,’ vol. ii, p. 283.
25. Scuaupiny, F.—1894, ‘ Sitz. Akad. Wiss.,’ Berlin, lii, p. 1277.
EXPLANATION OF PLATE 2,
Illustrating Mr. G. Bidder’s paper on “The Collar-cells of
Heteroceela.”’
Figs. 1 to 14 and fig. 19 are from living cells. All the drawings except
figs. 3 and 21 are multiplied about 1000 times linear; figs. 1, 2, 15, 16, 17,
18, 19, and 20 being drawn with the camera lucida,} figs. 9, 11 ce, 12, and 22
drawn free-hand and scaled from micrometer measurements, the remaining
figures are free-hand drawings approximately to the same scale. All drawings
were made with Leitz =, oil-immersion, Zeiss oc. 3 old system, rarely oc. 4.
Figs. 3 and 11 are from Sycon raphanus, fig. 21 from Leucandra
aspera, fig. 22 from Acanthella pulcherrima, the remainder from 8.
compressum,
Fic. 1.—Drawn from living 8. compressum, forty minutes after it was
taken froma tide-pool. These collar-cells were pressed against the cover-slip,
hence they appear closer together and more in one place than in the other
figures. Cf. fig. 15.
Fie, 2.—Another part of the same sponge, drawn immediately after the
section was placed on the slide. The flagella were so active that only their
bases could be drawn.
Fie. 3.—S. raphanus, living collar-cells (Naples, 1889), prob. x 2500.
The shaded spherules were stained with Bismarck brown; the full number is
not drawn in all the cells.
Fic. 4.—T wo cells with distal globules (excreta ?), alive, flagella very active;
from 8. compressum two anda half hours exposed by the low tide, two
’ The small numerals at the side of fig. 16 show the distortion found to
exist in drawing with the Nachet camera when all adjustments are made with
apparent accuracy.
THE OOLLAR-CELLS OF HETEROCGLA. Ad
hours in sea water after gathering. Very satisfactory preparation; all over it
could be seen tall cylindrical cells, wide apart, with stiff cylindrical collars and
flagella very active until two and a half hours after the preparation was made.
These cells drawn in the first half-hour. Part of the same sponge placed
when this preparation was made into osmic acid for an hour and a quarter,
and dialysed through alcohol and benzol, showed in sections stained on the
slide spherical or oblate collar-cells with a flat Sollas’s membrane and few
flagella (possibly due to imperfect dialysation in benzol).
Fie. 5.—From same sponge as fig. 19, six hours in a small saucer of sea-
water; flagellar movement languid.
Fic. 6.—S. compressum, flagella moving.
Fic. 7.—S. compressum. Edge of collar showing beaded or milled-edge
appearance, flagellum in optic section ; same preparation as fig. 5.
Fic. 8.—S. compressum, living section; a, soon after the preparation
was made; 4, twenty minutes after, the flagella in very violent action; c, one
hour forty minutes after (the two left-hand cells of 4), the right flagellum
was gone, the left still working; ¢, two hours twenty minutes after, the tops
of the same cells, the bodies being hidden. Flagella were still moving in
many of the chambers two hours thirty-five minutes from the time the prepa-
ration was made; many of the collar-cells were elongated to six or seven times
their width.
Fic. 9.—T wo successive drawings of a cell from the same sponge as fig. 4,
but an hour and a half after the preparation was made. Part of the section
was dead; the flagellum of this cell was moving well. Note the very long
collar.
Fics. 10 and 12.—S. compressum gathered under a moist rock, placed
for three hours in the circulation of the Biological Station. The first drawings
from the living section present nearly the same appearance as fig. 1, the cells
being short and more closely packed than usual. After three quarters of an
hour the appearance is much as in fig. 8a, and the flagella are growing slack.
Fig. 10 was drawn one hour and twenty minutes, and fig. 12 one hour and
fifty minutes after preparation; the flagella were still moving in fig. 10,
motionless in fig. 12. No further change was observed two and a quarter
hours after preparation. Paraffin sections formed Series C of the text.
Fic. 11.—S. raphanus, some time under the cover-slip. There were
flagella still moving in the preparation, though there were none visible on the
cells drawn in 4 and ec.
Fie. 12.—See fig. 10.
Fic. 13.—S. compressum, ten hours in sea water after twenty-seven
hours absence from it; flagella moving actively. This is the typical form of
cell, though there are a few with collars of the normal form. As noticed also
42 GEORGE BIDDER.
in other sponges there were in the chambers large masses containing hundreds
of transparent globules (fig. 13 a) laden with small detritus. While their
individual size and appearance strongly suggest ejecta from the cells (cf. figs.
4 and 10), their large aggregate mass makes this supposition difficult without
stronger evidence.
Fic. 14.—From the same sponge after one day more in sea water. Most
chambers showed perfectly normal collars and flagella; this (transitional ?)
form occurred in several places. Flagella active.
Fig. 15.—Series D of paraffin sections, preserved in osmic 1 per cent. at
the time fig. 1 was drawn from the same sponge. ‘The cells are very unvary-
ing throughout the preparation, fusion of collars being rare and difficult to
find; it occurs in a few cells. (See also woodcut a, 4).
Fic. 16.—A typical set of cells from another slide of the same series of
sections as fig. 15; fixed with water, cleared in turpentine, passed through
absolute, 90 per ceat., 70 per cent., 50 per cent., and 30 per cent. alcohol
into Grenacher’s hematoxylin; after two minutes back in the reverse order,
half a minute in 30 per cent. and some minutes in each of the other alcohols,
mounted through turpentine in Canada balsam and chloroform. Perhaps a
quarter of the collars in this preparation are unaltered in form, most are either
shortened or constricted, some of the cell-bodies are contracted.
Fic. 17.—S8. compressum. A Sollas’s membrane halfway up the collars,
shown by careful focussing with the immersion lens to consist, as here drawn,
of a series of bars and bands. With a dry lens it is seen as a strongly-
stained line quite continuous round the chamber.
Fics. 17 and 18 are from Series A; in about half the chambers the collars
are separated, in about half united. Preservation as in text, except that the
passage into alcohol was by 10 per cent. changes every ten minutes, and the
tissue was eighteen hours in paraflin at 63° C. beforeembedding. These two
sections stained on the slide in Grenacher’s hematoxylin and mounted in
glycerine.
Fic. 18 (v. supra).—Typical Sollas’s membrane, very frequent. The roughly
shaded portion indicates the basal parts of cells above the focus, the under
surface of the membrane being seen.
Fic. 19.—S. compressum, living cells, flagella in movement. See fig. 20.
Fic. 20.—Typical part of a section (Series B) made from the sponge from
which fig. 19 was drawn; after preservation at the same time in osmic acid
1 per cent. eighty minutes, 10 per cent. changes of alcohol every eight minutes,
10 per cent. or 15 per cent. changes of benzol every quarter of an hour;
stained on slide, Grenacher’s hematoxylin.
Fie. 21.—Sollas’s membrane from a paraflin section of L. aspera; pre-
served osmic acid 1 per cent., brought gradually through alcohols and decalci-
fied with 1 per cent. formic acid in 90 per cent. alcohol, embedded through
THE COLLAR-CELLS IN HETEROCGLA. 45
chloroform. The outlines of cells in the adjoining chamber are shown; the
dark spot and the black dots are carmine, with which the sponge had been fed
for four hours and a quarter. The preparation is unstained ; the light shading
of the spherules is due to osmic acid.
Fie. 22.—Living flagellate chamber from Acanthella stipitata, Carter,
drawn to the same scale as figs. 1, 2, 11 c, 12, &c. The shaded dots are the
bases of collar-cells, the white space the apopyle.
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THE METAMORPHOSIS OF ECHINODERMS, 45
The Metamorphosis of Echinoderms.
By
Henry Bury, ™.A., F.L.S.,
Late Fellow of Trinity College, Cambridge.
With Plates 3—9.
INTRODUCTION.
Acassiz’s view, put forward more than thirty years ago
(1, p. 61), that the actinal and abactinal surfaces of Echino-
derms (at least of Echinids, Asterids, and Ophiurids) are
formed from the right and left enteroccel pouches (‘ water-
tubes,” as he calls them) respectively, has met with very
general acceptance ; but surprisingly little has been done to test
it by modern methods.
Agassiz’s observations were, of course, made on the whole
larvee, without any assistance from sections. Gd6tte (9) in
1876 applied the section method to Antedon, and found
Agassiz’s view to hold good there; yet, on the other hand,
Ludwig (15) in 1882 failed altogether to trace this symmetrical
arrangement of the enteroccls in Asterina, while he showed
conclusively that Agassiz was mistaken in supposing that
the left enteroccel gives rise to nothing but the hydroccel.
No one has ever attempted, so far as I know, to show how
Holothurians can be included in Agassiz’s scheme; and the
conclusion of those who have studied their development has
generally been (21, 31, 32) that in retaining the dorsal mesen-
tery of the larva as a longitudinal mesentery in the adult (not
a transverse one, as it isin Antedon, and should be on Agassiz’s
46 HENRY BURY.
hypothesis) they exhibit a primitive approach to a worm-like
ancestor.
With a view to clearing up some of these differences of
observation and opinion, I commenced in the spring of 1888
an examination of the metamorphosis of all available Echino-
derm larve, paying special attention to the behaviour of the
mesentery, in the belief that this would afford an important
clue to the difficult question of the origin of Echinoderms and
the relation of the radiate adult to the bilateral larva.
The solution of so wide a phylogenetic question cannot be
hoped for, and should not be attempted as often as it is, from
the narrow standpoint of the ontogeny of one or two forms;
nor should too much reliance be placed upon the accounts of
other observers, however careful they may be, whose attention
has not specially been directed to the points at issue. As far
as possible, therefore, I have worked out for myself the meta-
morphic changes of at least one form of larva in each of the
five classes of Echinoderms.
I very soon found, however, that to understand these changes
properly it was necessary to go back in almost every case to
much earlier stages—in some cases right back to the earliest
formation of the body-cavities from the archenteron. It would
have been most undesirable to burden the pages of the present
paper with the preliminary results thus obtained; and I there-
fore in 1888 (5) published an introductory paper on the sub-
division of the enteroccel and the origin of the skeleton, which
I intended to follow up as soon as possible with the present
paper on the metamorphosis and phylogeny; but ill-health
altogether stopped my work for a long period, and even now
the difficulty of obtaining material has prevented me from
carrying out my scheme as fully as I had hoped. It did not
seem desirable, however, to withhold any longer the numerous
facts bearing on the subject embodied in the subsequent pages.
Most of the material for these studies was obtained at the
Zoological Station at Naples in the spring of 1888, but a second
visit there in 1893 was necessary to complete my studies of
Auricularia ; while for most of my larve of Asterias rubens
THE METAMORPHOSIS OF ECHINODERMS. 47
I am indebted to the kindness of Professor McIntosh, who
most generously placed at my disposal the resources of the
Marine Laboratory at St. Andrews, where these larve are ex-
ceedingly abundant.
Of course it is not possible within the limits of this paper
to give a detailed account of the development of every larva ;
and indeed so much is already known of most of them as to
render this unnecessary; I have therefore, in most cases,
given only such a brief outline of the facts as would render
the metamorphosis intelligible and bring into prominence
those features to which I attach theoretical importance. This
method has indeed the disadvantage of making my work appear
somewhat sketchy and superficial, but this seemed preferable
to loading the pages with a number of facts having no con-
nection with the theoretical views expressed in the second part
of this paper, or with a mere recapitulation of observations
originally made and recorded by other writers. In the case
of Holothurians, however, it has been necessary to describe in
some detail the metamorphosis of Synapta, as almost all the
points which seem to bear on the origin of this class have been
overlooked by previous observers.
To this alone is due the prominent position of the Holothu-
riaus in the ontogenetic portion of this paper—the order in
which the various groups are taken being throughout purely
a matter of convenience, and wholly independent of all phylo-
genetic considerations.
Part I.—OnvToceny.
A. HOLOTHURIANS.
Metamorphosis of Synapta.
The earliest stage of Auricularia with which we need here
concern ourselves is one in which the full size is already
attained, and the eleven pouches of the hydroccel (five large
tentacles, five as yet small radial vessels, and one polian
vesicle) distinctly visible. The right and left body-cavities
are still small, and have hitherto been symmetrically arranged
48 HENRY. BURY.
and about equal in size; but now a small finger-like process
is pushed out from the anterior end of the left cavity (fig. 1)
which very soon meets the posterior end of the hydroceel, and
grows forward along its ventral surface. This process, which
has no fellow on the right side, was recognised by Metschni-
koff (21, pl. ii, fig. 11), but seems to have escaped Semon’s notice.
While it is forming, the two body-cavities begin to -grow
towards one another on the ventral side, a little behind the
cesophagus, the growth both of this part and of the anterior
process of the left cavity being marked by great pseudopodic
activity of the cells forming the walls of the cavities (fig. 1).
About twenty-four hours after the appearance of this pro-
cess of the left body-cavity, we reach the stage represented
‘in fig.2. The body- cavities have come close together ventrally,
but not symmetrically, a portion of the left cavity overlapping
the anterior edge of the right (fig. 3). It is further to be
noticed that while the left cavity now extends anteriorly all
along the ventral face of the hydrocel (“ oral cavity,” fig. 3)
it scarcely extends so far posteriorly as the right cavity. In
sections (with the help of which I have confirmed all the
observations embodied in the figures) the external wall of the
left body-cavity exhibits a marked thickening in the region in
which the two cavities approach one another; occasionally,
though rarely, there seems to be already a fusion of the two
cavities at this point.
The Auricularia has now reached its latest stage of develop-
ment, and in a very few hours the whole aspect of the larva is
changed, and the metamorphosis from the bilateral into the
radial form has begun.
As several points in this change have been overlooked both
by Metschnikoff (21) and by Semon (82), we must follow it
in considerable detail. The onset of metamorphosis is marked
externally by the breaking-up of the ciliated bands, the col-
lapse of the stomach, and the growth of the hydrocel round
the cesophagus; and as far as possible these various groups of
phenomena will be dealt with separately, though it is impos-
sible to keep them wholly apart.
THE METAMORPHOSIS OF ECHINODERMS. 49
CruiateD Banvs anp Ruines.—Even before the collapse of
the stomach the ciliated band of Auricularia shows signs of
breaking up, at first only by a thinning of the band at certain
points (fig. 2), and later by its complete separation into seg-
ments. Many of the changes which follow have already been
correctly described (21 and 32), but as much has been misun-
derstood (especially by Semon) it will be better to describe
the phenomena in full. ‘To simplify this description as far as
possible I shall begin at the posterior end of the larva, where
the changes are least complicated ; and with the help of figs.
3, 4, and 5, the formation of the two posterior (fourth and
fifth) ciliated bands ought to be easily intelligible.
It is convenient to divide the ciliated band of Auricularia
into three regions: (1) an anterior ventral, (2) a posterior
ventral loop, and (3) a pair of longitudinal bands uniting
these loops. These will at once be recognisable in any figure
of Auricularia. In fig. 3 the whole band is outlined, but
only those parts which persist and form the ciliated rings of
the pupa are shaded—the dorsal parts (seen through the trans-
parent tissue) being left lighter than the ventral. It must be
understood that this arrangement is purely diagrammatic, and
that the persistent parts of the band do not admit of accurate
delimitation.
Fifth (posterior) Ciliated Ring.—This is usually
formed from the two lateral (right and left) pieces of the
ciliated band, which form the junction of the longitudinal
bands with the posterior ventral loop (fig. 3, V) ; sometimes,
however (for a short time time only), each piece is further
divided into a dorsal and a ventral portion. ‘The two halves
(right and left) soon acquire a more transverse arrangement
(figs. 4 and 5), and finally completely encircle the posterior
end of the body, the anus having meanwhile assumed a terminal
position.
Fourth Ciliated Ring.—This is invariably formed from
four pieces (figs. 3 and 4, 1V). The dorsal and ventral por-
1 It is convenient to distinguish between the ciliated band of Auricularia
and the ciliated rings of the pupa or “ barrel” stage.
vou. 38, PART 1.—NEW SER. D
50 HENRY BURY.
tions on each side unite first (fig. 5), then the two sides unite
ventrally, and at a later period their union on the dorsal side
completes the ring.
Mouth and Atrium.—Before describing the formation of
the three anterior rings, we must turn for a moment to the
behaviour of the mouth, and the formation of the atrial cavity.
Quite early in the metamorphosis four pieces of the ciliated
band group themselves round the mouth and there form a ring
(figs. 3 and 4; and 21, pl. ii, figs. 14—16). The fate of
the “nerve bands” (fig. 1) I have never been able to deter-
mine.
In fig. 4. it will be noticed that the mouth is pushed over to
the left side of the larva, while the apex of the latter is turned
considerably to the right. The latter change is partly visible
in fig. 3, but varies considerably in different larve, being
always most marked in spirit specimens in which some
shrinkage has occurred.
In the next stage the mouth retreats into the interior, and
an atrial cavity is formed, the external opening of which narrows
rapidly and passes over to the left side (fig. 5); the apex of
the larva has meantime nearly regained its original position,
though it is still much to the right of a line passing through
the longitudinal axis of the stomach.
The portions of the ciliated band which, as above described,
encircle the mouth, now lie at the bottom of the atrial cavity,
where they form, as Metschnikoff described, the epithelium
of the tentacles. Though they form a ring, I have not included
it among the ciliated rings of the pupa, of which there are five
outside the atrium.
Figs. 4.and 5 represent such well-marked stages in the meta-
morphosis that it will be convenient to refer to them in future
as marking, respectively, stage A (before the atrium is formed)
and stage B) with the aperture of the atrium not yet terminal
and the five ciliated rings still very incomplete). We will now
return to these ciliated rings, to assist the study of which I
have given in fig. 6 a diagrammatic view of the anterior
pole of Auricularia, constructed on the same lines as fig. 3.
THE METAMORPHOSIS OF ECHINODERMS. 51
Third Ciliated Ring.—This is formed as simply as any,
from one piece on each side (occasionally two on the left side,
the ventral one being small, and soon joining the dorsal one).
In stage B (fig. 5) they join on the ventral side, but remain
apart much longer on the dorsal side, where they eventually
unite in front of the water-pore.
First and Second Ciliated Rings.—Figs. 3 and 6 will
show that we have now four pieces of the ciliated band with
which to construct the two anterior rings. At first they form
two calliper-like loops, one on each side (fig. 6), and this
arrangement is still partly retained in stage A, of which fig. 7
is a polar view.
In stage B (figs. 5 and 8) the opening of the atrium has
moved into the left loop, and it seems as if this loop were
about to form the first ciliated ring. Fig. 9, however, shows
that only the ventral portion of the loop is concerned in form-
ing this ring, while three pieces (II a, 6, and ¢ in figs. 7,
8, and 9) form the second ring. The two pieces of the right
side (II 6 and c) usually unite first (occasionally II ¢ is
absent), and a little later the right and left sides unite
dorsally ; lastly they unite on the ventral side, and the ring
is complete.
The investigation of the phenomena just described requires
a good deal of care and patience. The rapidity of the changes
is one of the most serious difficulties, necessitating a rigorous
search each day among one’s specimens for signs of the im-
pending change. At the time when most of my observations
were made (Naples, March and April, 1888) the stage shown
in figs. 2 and 3 was usually reached about ten o’clock on one
morning, stage A late that evening, and the stage shown in
fig. 9 about ten o’clock the next morning; after that the
changes were slower. Development was much slowed down
by keeping the larva in cold water, and in this way I was
able to obtain all the intermediate stages without resorting
to twenty-four hours’ consecutive watching.
Another difficulty lies in the shape of the larva, which
makes it hard to obtain prolonged views of any but the dorsal
Fy HENRY BURY.
and veutral surfaces ; and in their delicacy, which renders it
almost an impossibility to preserve and embed them without
losing much of the original form by shrinkage. I have suc-
ceeded fairly well by carefully embedding in celloidin, but
the tissues are never so clear as in the living anima]. Almost
equally good results, with greater economy of material, may
be obtained by balancing the living larva in a watch-glass
with one hand, making a rough sketch with the other, and
gradually correcting and adding details with the help of
repeated observations. No doubt this requires practice, but
it has the advantage that several stages can be followed on the
same larva. All my polar diagrams (figs. 6—9) were made
in this way, and though not drawn quite to the same scale,
are accurate enough for my present purpose.
Semon has drawn attention to the marked diminution in
size which accompanies metamorphosis, and has given some
measurements (32, p. 29), but these can only be regarded as
approximate, the variations in size being great.
The discrepancies between Metschnikoff’s account and
Semon’s are so very large, that I naturally looked out carefully
for any abnormalities which might help to reconcile them ;
but though a good deal of variation was noticeable (some
of it possibly due to specific differences), none of it
threw any light on this point. Metschnikoff’s description
and figures are on the whole extremely accurate, and my
account is rather an addition to his than a correction of it.
It is curious, however, that he overlooked the asymmetrical
position of the mouth and atrium, since two of his figures show
it (21, pl. ii, figs. 16 and 18), one of them representing the
water-pore and atrium on the same side of the body, which
could not happen if they occupied the positions he assigns to
them. His failure to understand the formation of the two
anterior ciliated rings was probably due partly to the difficulty
of obtaining polar views, which alone render the changes
intelligible ; and partly to his having missed that stage, which,
in my experience, usually occurs at night.
Semon’s account I am wholly at a loss to understand, so
THE METAMORPHOSIS OF ECHINODERMS. 53
entirely does it differ from anything which has come under
my notice. We can hardly have obtained different larve,
since we both worked at the same place (Naples) and at about
the same time of year (January to the end of May, 1888, in my
case; November, 1885, to October, 1886, in his—but few
larvee found after March) ; and although during my subsequent
visit to Naples in 1893 I met with many abnormal specimens
(obviously pathological, as the condition of the whole ecto-
derm showed) yet none of them ever approached those figured
by Semon as intermediate between Auricularia and the pupa
(82, pl. i, figs. 5 and 6). Stranger still is the fact that while
these figures are wholly irreconcilable with Metschnikoff’s, he
does not even allude, in this connection, to any differences
between their accounts.
* Hyprocawzt, &c.—On a previous occasion (5, p. 11) I
described the formation of a cavity which I regarded as the
homologue of the left anterior body-cavity in the Echino-
derms. As Ludwig (17, p. 609) seems to think this homology
disproved by his observations on Cucumaria, it will be well
to review briefly the grounds on which my suggestion was
based.
When Ludwig first noticed this cavity (which he calls the
“‘ Madreporenblase ”) in Cucumaria it had the appearance of
a simple swelling on the water-tube; and as he could not
find any trace of it on the previous (fourth) day, he concludes
that it is altogether secondary.
Whatever may be the case in Cucumaria, this conclusion is
not justified in Auricularia. There, after the separation of
the posterior body-cavities, the anterior portion of the coelom
forms a small, pear-shaped, thin-walled vesicle, from which a
short tube with thicker walls (“ pore-canal”’) runs to the
exterior. Then the left wall of the vesicle thickens and
presently produces the rudiments of the radial canals and
tentacles; but the walls of the dorsal portion, into which the
pore-canal opens, still remain thin. Shortly before metamor-
phosis this thin-walled portion becomes constricted and divided
into two, the smaller of which remains in connection with
54 HENRY BURY.
the thick-walled portion and forms the inner wall of the
hydroceel ; while the other one (the dorsal), which is Ludwig’s
“‘ Madreporenblase,”’ is, in my opinion, the homologue of the
anterior body-cavity in other Echinoderms (see figs. 1 and 2;
and 5, figs. 22—25). The two cavities are rapidly pushed
asunder by the formation of a second thick-walled tube, which
almost immediately becomes continuous on one side of the
“ Madreporenblase ” with the pore-canal. This new tube I
regard as the true water-tube, though Ludwig, under the name
of * Steinkanal,’’ confuses it with the pore-canal (for the dis-
tinction see 5, p. 21).
No one from the study of Auricularia alone can say that
one of these two cavities (“ Madreporenblase ” and hydroceel)
is more primitive than the other—they are parts of the same
primary coelomic pouch. Moreover, had Auricularia been
opaque—had I been forced to rely on sections alone, it is
very probable that I should have overlooked this division (as
previous observers had done), so rapidly does it take place, and
regarded the ‘“ Madreporenblase ” as a later outgrowth of the
water-tube. With all regard to Ludwig’s admirable care in
research, I cannot at present feel satisfied that he has not
overlooked similar changes in Cucumaria.
To sum up, we find in Auricularia two cavities and a tube
connecting them having precisely the structure and relations
of the anterior body-cavity, hydroccel, and water-tube in other
Echinoderms ; and to my mind it is far easier to believe that
the ‘‘ Madreporenblase ” of Auricularia is the anterior
body-cavity, than to admit that a cavity which is present in
all other Echinoderm larve yet examined is totally absent in
Holothurians.
In the fully-formed Auricularia (fig. 1) the hydroccel, which
is flattened dorso-ventrally, lies with its posterior end slightly
nearer the ventral surface than its anterior end. Its inner
face (that nearest to the cesophagus) is strongly concave, while
its outer face, from which spring the radial vessels and tenta-
cles, is convex.
The position of the water-tube, which enters on the inner
THE METAMORPHOSIS OF ECHINODERMS. 15)
side, is certainly not easy to determine with relation to the
radial vessels, which are on the outer side; but I have fully
satisfied myself by means of sections of this and later stages
that it was correctly given in my previous paper (5, p. 22),
and I cannot admit the plea of variability with which Semon
meets my criticism of his figures (33, p. 9).
The whole hydroccel is pushed more and more towards the
ventral surface of the larva by the elongation of the water-
tube; but the obliquity which brings its posterior end even
nearer this surface than its anterior becomes more marked,
while the anterior end bends over at the same time more
towards the middle line than the posterior (fig. 3).
The formation of the water-vascular ring round the ceso-
phagus follows rapidly on the stage shown in figs. 2 and 8.
The posterior end of the hydroccel bends round on the ventral
side of the oesophagus (close to its junction with the stomach)
about as far as the middle line. The anterior end, on the
other hand, crosses over on the dorsal side of the cesophagus
to the right side of the larva, and then, bending posteriorly
and ventrally, passes round to the ventral surface, where it
eventually joins the other end of the hydroccel, and completes
the water-vascular ring.
It will be remembered that according to Metschnikoff the
most posterior pouch of the hydroccel Auricularia is the rudi-
ment of the polian vesicle, while the most anterior becomes
one of the radial canals. Semon expresses some doubt on this
point ; but a series of sections through the larva during stage
A (before the closure of the water-vascular ring) sets this
question at rest. The posterior pouch (which at the close of
this stage may even be to the right of the middle line, though
it seems to vary somewhat) turns slightly inwards, and pushes
before it the wall of the left body-cavity (see figs. 10 and 18,
in which, however, but little more than the peritoneal covering
of the vesicle is visible). None of the other pouches ever
“ project into the body-cavity in this way; and from this and
later stages it is abundantly clear that Metschnikoff was right
in identifying this posterior pouch with the polian vesicle. It
56 HENRY BURY.
is clear also that the closure of the water-vascular ring is
effected on the ventral side, just at the base of the polian
vesicle. How near to the middle line this junction occurs is,
however, difficult to determine. I have failed to observe the
polian vesicle in external views, owing, no doubt, to its being
turned inwards towards the body-cavity ; and in sections so
much distortion almost inevitably occurs that the exact middle
line is hard to determine. Moreover it will be noticed (figs.
4 and 5) that, owing to the asymmetry of the larva in stages
A and B, the middle line of the stomach does not correspond
with that of the larva as a whole. The polian vesicle, how-
ever, is certainly to the right of the middle line in many larve
at the close of stage A (fig. 13), though there is some reason
to think that in later stages it moves back again somewhat to
the left: at avy rate, the closure of the water-vascular ring,
whether to the right or left of this line, is not far removed
from it.
In Cucumaria, Ludwig (17, p. 607) thinks that this closure
occurs on the right side, not in the interradius of the polian
vesicle ; and in view of the remarkable discrepancies existing
among Echinoderm larve on this point, it must be admitted
that he is possibly right, but until every stage has been traced
(Ludwig admits that he has missed the actual completion of
the ring), it would be rash to assert that Cucumaria and
Synapta differ in this respect.
With the commencement of stage B and the fornatien of
the atrial cavity (to be further described later on) the hydro-
col ring, which is only completed at the commencement of
this stage, assumes a new position. In stage A its oral sur-
face was directed nearly towards the ventral surface of the
larva, and only slightly towards the anterior end; now, how-
ever, it faces (approximately) the aperture of the atrium, and
as this moves towards the anterior end, the water-vascular ring
comes to lie more and more nearly at right angles to the
longitudinal axis, a position which it finally assumes in the pupa
or “barrel”? stage. In stage B, however (fig. 5), the aperture
of the atrium is not yet polar, and the oral surface of the
THE METAMORPHOSIS OF ECHINODERMS, 57
hydroceel ring, facing it, is directed decidedly towards
the left side.
This arrangement is of brief duration and easily overlooked,
but, as will be shown in the second part of this paper, it affords
an important clue to the probable phylogeny of the Holo-
thurians.
In stage A the radial vessels and tentacles lie nearly in the
same plane; but in stage B the latter turn up alongside the
outer wall of the atrium, while the former bend back parallel
to the long axis of the stomach, and grow rapidly towards the
posterior pole of the larva. On the outer side of each runs a
two-layered prolongation of the wall of the atrium; but this,
and the ectodermic covering to the tentacles, are so well
known from previous descriptions that they need not be dwelt
on here.
Before describing the positions occupied by the parts of the
hydrocel in the fully-formed pupa, it will be well to come to
some conclusion as to the nomenclature of the rays.
In adult Holothurians it is usual to speak of the rays as
right and left dorsal, right and left ventral, and median
ventral; the water-tube being in the dorsal interradius.
Ludwig applies these terms to the young Cucumaria; but in
the young Synapta their employment might lead to some con-
fusion. Up to the end of stage B, and sometimes even in the
pupa, the original right and left sides of the larva are distin-
guishable by means of the groups of calcareous discs at the
posterior end, and this enables us to see that the water-pore
remains, as in Auricularia, decidedly to the left of the middle
line ; and though its exact position in older stages (fig. 9 and
later) is rather hard to determine (and perhaps variable), it is
never truly median.
Again, the radial vessel which is nearest to the ventral
median line in stage B (fig. 5) is certainly not the median
vessel of the adult, but (as I believe) the left ventral. Since,
then, the two planes of symmetry (larval and adult) do not
correspond, it may be better to apply that nomenclature to the
rays which has been adopted on morphological grounds in other
58 HENRY BURY.
Echinoderms. The animal is looked at from the aboral pole,
with the interradius of the water- pore directed away from the
observer, this interradius being regarded as anterior. The
ray immediately to the left of thisis designated No. I, the next
beyond it No. II, and so on, that immediately to the right of
the anterior interradius being No. V. The interradii may be
conveniently marked by the letters A, B, C, D, E—A being
the anterior interradius, and the order of succession being the
same as in the case of the rays. This system of numbering
and lettering is adopted in fig. 14, in which the arrangement
of the hydroccel in the “pupa” is diagrammatically shown. As
there seen, the water-tube is adradial, being nearer to radius
V than to radius I, from which it is separated by one of the
tentacles. The polian vesicle is also adradial, lying in inter-
radius B, close to radius II.
Semon’s figure (82, pl. ii, fig. 2) of these parts is extra-
ordinarily inaccurate; not only, as I pointed out before (5,
p. 22), is it out of harmony with his statement of the position
of the water-tube in Auricularia, but this tube is represented
on the wrong side of the polian vesicle ; seen from the side, as
he has drawn it, it should be on the right of this vesicle (com-
pare fig. 14), but he has represented it on the left !
In none of my specimens can I detect that curious relation
of the tentacles to the radial vessels which Ludwig describes
in Cucumaria (16, p. 183) ; but if, as is probable, it obtains in
Synapta also, in later stages than I have examined, it may
help to explain one curious discrepancy between Synapta
(and probably Holothurians as a whole) and other Echino-
derms. In all Echinoderms, at any rate in young stages,
the water-tube is adradial, but in most forms it is nearest
to the left side of the interradius in which it lies; in the
larva of Synapta, however, and of Cucumaria (16, pp. 187, 188 ;
17, p. 611) it lies, as shown in fig. 14, on the right of this
interradius. Now if the tentacle which separates it from
radius I belongs to this radius (as Ludwig says), it may be
that the precocious development of this tentacle has pushed
the water-tube (in appearance at least) somewhat out of its
THE METAMORPHOSIS OF ECHINODERMS. 59
true position; but even this does not wholly account for this
peculiarity.
AurmmentTARY Canat.—Although in stage A the mouth is
much narrowed, yet it retains throughout this stage its ventral
aspect. With the beginning of stage B, however, it follows
the inward movement of the hydrocel ring, and turning
slightly inwards from the ventral surface, is bent upwards and
towards the left side; in effecting this change it drags in not
only the thick ectodermic ring derived from the ciliated band
(21, pl. ii, fig. 16) but also some of the transparent tissue
surrounding it, and an atrium is formed, lined by ectoderm.
The aperture of this atrium is at first fairly large, but it
rapidly narrows, and passes up the left side of the larva towards
the anterior pole; in doing so it gets further and further
away from the mouth, and the atrium in consequence increas-
ing in size, more of the transparent tissue becomes involved
in its formation.
The stomach is sharply contracted at the onset of meta-
morphosis, diminishing to about half its original diameter ; and
its internal surface is consequently thrown for a time into
strong folds. Otherwise it undergoes no important changes.
The intestine in Auricularia occupies a position which
is nowhere else met with in Echinoderm larve, being directed
downwards and backwards (figs. 1 and 2), instead of, as in
all other cases, running forwards along the ventral surface of
the stomach.
In stage A a small cecal pouch usually (but not invariably)
appears at the base of the intestine, running forward from the
junction of the latter with the stomach. larly in stage B
this part of the intestine widens rapidly, and its opening into
the stomach is shifted to the left side. In this way the cecum
of the previous stage is transformed into a short transverse
intestine (fig.5; and 21, pl. u, fig. 16), from the right side
of which the original, posteriorly directed intestine runs back
to the anus, which is still median.
This transverse intestine rapidly increases in size, and
becomes slightly bowed, its anterior margin being strongly
60 : HENRY BURY.
convex, and extending quickly forward along the ventral sur-
face of the stomach nearly in the middle line. Although the
concavity of its posterior margin is not so marked as this
anterior convexity, yet it is for a short time sufficiently
evident to enable us to distinguish an ascending portion
running up on the left side from the junction with the
stomach, and a descending portion, continuous with the
remainder of the intestine, on the right. (It is curious that
Semon in all his figures represents the ascending portion as
being on the right side.)
The forward growth of the convex margin of the intestine
reaches its limits in the young pupa, in which it extends
nearly as far forward as the water-vascular ring, the polian
vesicle being just internal to it (fig. 15). The combined width
of the ascending and descending portions of the intestine,
just where it bends over, very nearly equals that of the
stomach.
After this the importance of this part rapidly declines ; and
before the ciliated bands of the pupa have wholly disappeared,
the intestine is almost completely straightened out (see 21,
pl. iii, fig. 23).
Catom.—The question of the existence of an anterior
enteroccl has already been discussed; and we have only here
to consider the behaviour of the posterior body-cavities, which
we left in Auricularia just meeting (but not uniting) on the
ventral surface of the stomach, the left one having a tubular
prolongation forward on the ventral side of the hydroccel.
This is as far as the cavities can be traced in the living
animal, the collapse of the stomach and increased opacity of
the tissues rendering further observations, except by means
of sections, almost impossible. Even in sections the changes
are sufficiently hard to trace, owing to the rapidity with which
they occur and to the excessive delicacy of the tissues. All
through stage A there should not be a greater difference than
three hours between the ages of the larve examined, while
to avoid shrinkage the greatest care must be taken in every
stage of preservation ; embedding is best done first in celloidin
THE METAMORPHOSIS OF ECHINODERMS. 61
and then in paraffin—the latter alone, in my experience, gives
no satisfactory results with any of the pelagic forms of Echino-
derm larve.
The “ oral cavity ”’ (as I propose to call the anterior prolon-
gation of the left body-cavity) closely follows the movements
of the hydrocel. Its anterior end is thus bent round the
dorsal surface of the cesophagus, and down on the right side ;
while its posterior end, where it joins the left body-cavity, is
brought down by the movements of the hydroccel nearer to the
ventral surface and to the middle line. This end of it, however,
does not correspond with the extreme posterior end of the
hydrocel, but passes on to the surface of this organ at first
between the polian vesicle and the second tentacle, and after-
wards, when the polian vesicle has about reached the middle
line, between the second tentacle and the first radial vessel.
At this point, towards the end of stage A, it separates com-
pletely from the left body-cavity. Its two ends then come
nearer and nearer together, and, as I believe, fuse together
early in stage B, so as to form a complete ring on the oral
surface of the water-vascular ring (figs. 11 and 15). It is just
possible, indeed, that the two ends do not fuse, but that a thin
mesentery remains separating them; but I have not been able
to find anything of the kind.
The subsequent history of this cavity I have not traced. It
may be identical with the cavity figured by Semon in the same
position at a later stage (82, pl. iv, fig. 9), but none of my
specimens are old enough to determine this.
Besides this oral cavity, we noticed in Auricularia another
process of the left body-cavity overlapping the anterior end of
the right body-cavity on the ventral side (fig. 3). This process
(hardly recognisable in Auricularia) increases rapidly in size
during stage A, and may be called the “ventral horn” of the
left body-cavity. It grows rapidly round the right side of the
cesophagus, on to the dorsal surface (figs. 10 and 11). The
wall separating it from the right body-cavity very soon breaks
down, starting from the ventral middle line; but up to the end
of stage A a small portion of it remains (figs. 11—18), which
62 HENRY BURY.
enables us to trace the true relations of this cavity, which
without the greatest care in tracing its origin might be thought
to be a diverticulum of the right, instead of the left, body-
cavity. When it reaches the dorsal side it continues its growth
past the middle line, till it reaches the water-tube, at the
level of which it lies. Beyond this I have not been able to
trace it with certainty; but since at this stage the most anterior
portion of the right body-cavity is posterior to the water-tube,
there is scarcely room for doubt that the ** ventral horn” forms
one side of the mesentery (very short in the pupa) which sup-
ports this tube—the other side being formed by the anterior
part of the main mass of the left body-cavity, which gradually,
during stage A, grows up the left side of the csophagus
(fig. 12).
By the time stage B is reached, all trace of separation be-
tween the right body-cavity and the ventral horn of the left
cavity has disappeared; so that the mesentery of the water-
tube (formed, as it seems, between two parts of the left
cavity) is continuous with the main dorsal mesentery (formed
between the right and left body-cavities), with which, indeed,
Semon has confused it. The junction of the two is, however,
still marked by a change in position—the water-tube and its
mesentery being adradial (fig. 14), while the true dorsal mesen-
tery is strictly interradial. It will be seen that the dorsal
mesentery is even more on the left of the original middle line
than the water-tube—not, as Semon considers it, in the median
plane. Its posterior end is, however, nearer to this plane than
its anterior.
This dorsal mesentery is first formed early in stage A by
the meeting of the body-cavities about two-thirds of the way
down the stomach, and from this point spreads rapidly forwards.
Nearly at the posterior end of the stomach it passes sharply round
to the left side, almost on to the ventral surface (fig. 16), thus
forming a short transverse mesentery, which we shall see again
in the pupa.
Very early in stage A the two body-cavities fuse ventrally
along the ventral surface, at the point at which they so closely
’
THE METAMORPHOSIS OF ECHINODERMS. 63
approach one another in fig. 8. From this point the line of
fusion runs obliquely forward, as we have seen, along the
posterior edge of the “ventral horn” of the left body-cavity.
Posteriorly the fusion usually (but perhaps not invariably)
extends to the anterior margin of the intestine, apparently
nearly in the middle line, but of this I cannot be quite sure.
Over the transverse portion of the intestine the left body-
cavity (which at this level is much smaller than the right)
pushes further across to the middle line (fig. 16) than the
right body-cavity, and then passes even on to the right side,
ending altogether just before the level of the posterior end of
the stomach is reached. Its margin thus takes an oblique
course, from left to right; but as the margin of the right
body-cavity is still far removed from it at this stage, we cannot
speak of a definite mesentery at this point. The right body-
cavity extends somewhat further posteriorly than the left ; but
very little behind the posterior end of the stomach it also
comes to an end.
Of stage B I have obtained but few examples, and none of
them, I regret to say, show the mesenteries satisfactorily,
In the fully-formed pupa the dorsal mesentery remains very
much as we left it at the close of stage A, except that, as
already mentioned, it is now continuous with the mesentery of
the water-tube. Posteriorly, at about the level of the junction
of the intestine and stomach (but in this stage a long way
from the posterior end of the latter), it passes sharply round
to the ventral side—in other words (since the original dorsal
and ventral surfaces are no longer evident), it passes from the
middle of interradius A into interradius B, thus forming the
short transverse mesentery above mentioned. From this point
it is continued into a mesentery which runs along the left edge
of the ascending portion of the intestine, curves round the
anterior margin of the latter, external to the polian vesicle
(fig. 15, ‘‘ mesentery”), and then descends for a short distance
along the right edge of the descending portion of the intestine.
How far this intestinal mesentery marks the division of the
two body-cavities, and how far it is a new growth, the absence
64 HENRY BURY.
of satisfactory sections of stage B prevents me from deciding ;
but the fact that the polian vesicle certainly hangs into the left
cavity in stage A (figs. 10 and 138), as well as the appearance
of certain specimens in which the fusion of the two cavities
does not seem to take place, make it quite possible that this
mesentery arises very nearly along the line of fusion, which,
owing to the growth of the curved part of the intestine, has
assumed rather a peculiar course.
This, however, is only conjecture; and I must admit that,
except in the region of the esophagus, I am quite unable to
determine the exact limits of the body-cavities of the larva in
the adult Synapta. It is, however, obvious that here, as in
Cucumaria (16 and 17), it is by no means the simple matter
which Semon represents it to be.
The descending part of the intestinal mesentery (lying in
interradius D, close to radius III) is not continued, in the
early pupa, on to that part of the intestine which lies posterior
to the stomach—this part of the intestine being at this stage
completely surrounded by the ccelom, though whether by the
right and left cavities combined, or by the former alone (as
certain early sections suggest) I cannot with accuracy deter-
mine. In later pupe the intestine is tied to the body-wall by
nnmerous threads of protoplasm; and only in the young
Synapte which have completely lost the ciliated rings does the
fully-developed mesentery, continuing the above-mentioned
descending mesentery, appear.
B. ASTERIDS.
Two very distinct types of development occur among
Asterids, without, so far as I know, any connecting links
between them. The most marked difference lies in the be-
haviour of the larval cesophagus, which survives in the adult
in one form, but is replaced by an entirely new one in the
other; there are, however, other important points of dissimi-
larity as well.
I propose to take first the type in which a new cesophagus
is formed, since it has hitherto been but little investigated ;
THE METAMORPHOSIS OF ECHINODERMS. 65
the other type, which includes the larva of Asterina gib-
bosa, is well known from Ludwig’s researches (15), and has
recently been further studied by MacBride (19).
1. Bipinnaria asterigera.
Under this head I propose to describe the more salient
features in the development of a large Bipinnaria found in the
Mediterranean, which closely resembles, if it is not identical
with, “B. asterigera,” described by Sars (29). My material
was in part collected by myself at Naples in 1888 and 1893, and
partly obtained through the kindness of Professor Kleinenberg
from Messina. A fairly complete series has thus been secured,
the only important gap being due to the entire absence of
really early stages; but as it would be beyond the scope of the
present paper to describe in detail all the stages observed, I
must content myself for the present with calling attention to
those features which have a direct bearing upon my theoretical
conclusions.
I have been uvable to discover from what adult form this
larva springs, though there are some reasons for thinking that
it may be from Luidia; all my specimens were obtained from
the plankton, and the youngest of them had already acquired
the long preoral lobe, terminating in a double fin-like ex-
pansion, which is so characteristic of this larva.
The arrangement of the body-cavities at this stage is
as follows :—On the right the usual prolongation of the ante-
rior body-cavity runs into the preoral lobe, and fuses with the
left anterior cavity in front of the mouth; posteriorly it is
continuous with the right posterior cavity, which lies beside
the stomach and has a much greater dorsal and ventral ex-
tension than the anterior cavity. The left anterior body-
cavity extends forwards like the right one, but its posterior
end is not continued directly into the posterior body-cavity,
but into the rudiment of the hydrocel; this again opens into
the posterior cavity, a deep constriction marking the point of
junction. The arrangement is, in fact, almost exactly the
VoL. 38, PART 1,—NEW SER. E
66 HENRY BURY.
same as in Brachiolaria (belonging to the second type of
Asterid development), as shown in fig. 17. Dorsally and
ventrally the left posterior body-cavity curves forwards in
two horns, which, as it were, embrace the hydrocel, both
horns being much longer than in fig. 17; the dorsal horn
extends nearly to the water-pore, at the junction of stomach
and oesophagus, while the ventral one runs alongside the in-
testine, and almost at the level of the anus curves over
(between the stomach and intestine), and fuses with the right
body-cavity. This fusion makes it impossible to determine
the exact limits of the two cavities (right and left), but we shall
probably not fall into any important error if we regard the
point of junction as median.
Dorsally the right and left cavities already form a me-
sentery of no great thickness; but ventrally they are still
widely separated over the intestine, except at its extreme
posterior end.
The water-pore is, as usual, situated at the junction of
cesophagus and stomach, rather to the left of the dorsal middle
line. Slightly in front of the pore and in the median plane a
cavity of irregular shape, absolutely unconnected with the
body-cavities, lies over the base of the esophagus. I men-
tioned it in a previous paper (9, p. 31) as probably of schizoceel
origin, and this is rendered the more probable by the fact that
the apparently homologous cavity in Asterias rubens and in
Echinid Plutei arises in this way. In the present paper I
propose to refer to it as the “ dorsal sac.”
The larva just described measured about 1:7 mm. in length.
The next larva obtained measured nearly 4 mm. (exclusive of
the arms), and is shown in ventral view in fig. 18. In it the
posterior end of the hydroccel is completely shut off from the
body-cavity, and its anterior end, where it joins the anterior
enteroceel, is further constricted. The dorsal horn of the left
posterior cavity has fused with the anterior enterocel from the
pore to the commencement of the hydroceel ; and the right
cavity extends across the anterior end of the stomach, on the
dorsal side, rather to the left of the middle line, so that the
Fk
THE METAMORPHOSIS OF ECHINODERMS. 67
anterior end of the dorsal mesentery is left rather than median,
though not so much so as the water-pore. In this way a
portion of the right posterior body-cavity comes to lie close
behind the dorsal sac, with which, owing to the thinness of the
intervening wall, it is rather liable to be confused in sections.
The two, however, are absolutely distinct.
The hydroceel has thicker walls than before, especially in the
five primary pouches, which now appear ; and the rudiments of
the water-tube and dorsal organ are also distinguishable,
though I do not propose to deal with the details of their
development in the present paper.
At this stage also the first rudiments of the skeleton appear
—namely, five terminal plates, lying parallel to the mesentery,
on its left side ; two are dorsal, one is at the extreme posterior
end, and two are ventral; the latter are shown in fig. 18.
There are also a few plates, not shown in this figure, lying over
the hydroccel. The madreporite appears very soon after the
terminals; but except for the fact that all the terminals lie
over the left body-cavity, the skeleton has no special interest
in our present inquiry.
The completion of the water-tube follows closely on the
stage just described. It runs from the hydrocel up to the
immediate neighbourhood of the water-pore (where it remains
permanently open to the anterior enteroccel) almost exactly
along the line occupied at any earlier stage by the mesentery
separating the anterior and posterior body-cavities. It is accom-
panied throughout its course by the dorsal organ, which now
almost encircles the hydroccel, and ends anteriorly under-
neath the dorsal sac, as we shall presently see is the case in
other Asterid larve as well (see fig. 19). Its tubular nature is
well marked at this point, as well as round the hydroceel (fig. 20),
but where it accompanies the water-tube it is usually very
narrow, and its lumen, if present, is very hard to detect. With
its further development we have no present concern.
Almost simultaneously with the completion of the water-
tube, the separation of the hydroccel from the anterior body-
cavity is effected. Fig. 21 is a lateral view of a larva at this
68 HENRY BURY.
stage, from which it will be seen that this separation occurs
in the same interradius as the water-tube. Already the
pouches of the hydroccel have increased in number to twenty-
five (they are much more conspicuous in sections than in ex-
ternal views), but there is no pause in this condition, fresh
pouches being rapidly and continuously formed between the
terminal one and the next adjoiningit. In this way the hydro-
coel spreads over the surface of the larva until its five main bran-
ches come in contact with and fuse with the five excrescences
(see figs. 20 and 22) which contain the terminal plates ; but it
is to be noted that this is effected without that rotation of the
two series of organs noticed by Ludwig in Asterina (15) ; the
anterior dorsal pouch of the hydroccel becomes enveloped by
the anterior terminal plate, and the pouch nearest the anus by
the corresponding terminal excrescence.
Fig. 21 will also serve to illustrate the relation of the anterior
and posterior body-cavities on the left side in later stages. It
will be seen that the “ ventral horn ”’ of the posterior cavity
has there grown so far round that it forms a long thin mesentery “/
with the ventral wall of the anterior cavity. The dorsal wall
of the latter is marked, as we have seen, by the line of the
water-tube ; and along this line, at the close of larval life, a
secondary separation of the two cavities is effected by the forma-
tion of a septum. It will at once suggest itself that in the
remnant of the anterior cavity thus enclosed we have the
equivalent of the ‘‘ axial sinus”’ of the adult (see 5, p. 37), and
a study of its subsequent history shows that this is the case.
It is also important to notice that this sinus is bounded on
both sides by the left body-cavity. This is important for my
theory, and though confirmed by MacBride (19, p. 433) is
opposed to the statements of some other observers; Ludwig
(15, p. 87) states that in Asterina the ventral wall of the sinus
is part of the original mesentery separating the right and left
cavities, while Semon (82, p. 37) regards the whole mesentery
containing the water-tube as a part of the dorsal mesentery
of Synapta.
No portion of the posterior body-cavity ever intervenes
THE METAMORPHOSIS OF ECHINODERMS, 69
between the outer body-wall and the hydroccel, or between the
centre of the latterand thestomach. Soon after the separation
of the hydroccel from the anterior enteroccel a small circular
depression appears in the outer wall of the former, as shown in
fig. 20. As it isnot invariably present even at this stage, and
seems to be generally absent in the next, I am inclined to regard
it as due to shrinkage after death; but even so it marks a
special weakness of the wall at this place, which is not un-
important as the first indication of the position of the adult
mouth. Inthe next stage an outgrowth of the stomach presses
against the inner wall of the hydroccel, and a very little later
pierces right through this organ and fuses with the body-
wall beyond ; probably it forms a part of the permanent ceso-
phagus, but most of the latter seems to be derived, at a much
later stage, from the ectoderm (fig. 23). By this piercing of
the hydroccel the water-vascular ring is formed, as Metschnikoff
observed. This, at least, is the conclusion I draw from my
sections; but this method of formation is unknown in other
Echinoderms, and it is just possible that an invagination of the
side of the hydroccel (such as occurs in Echinids) may take
place ; it must, however, be formed and obliterated with re-
markable rapidity to have entirely escaped notice, and I am
not disposed to believe in its existence. After all, this un-
usual mode of formation of the water-vascular ring is no
more remarkable than the extreme variation in the point of
closure of this ring exhibited by other Echinoderms (see 5,
fig. 28).
With the help of fig. 22, which is a dorsal view at about
the same stage as figs. 20 and 21, the general course of the
longitudinal dorsal mesentery can be followed ; it starts rather
to the left of the middle line, under the posterior margin of
the madreporic plate, and runs somewhat obliquely backwards
on the right side of, and parallel to, the line of the terminals.
This obliquity increases in later stages; but as at the same
time the centre of the hydroccel pushes itself further and
further back, the plane of this organ is always (as in fig. 22)
parallel to the plane of the mesentery ; while the latter, as will
70 HENRY BURY.
be readily seen, is at right angles to the plane of the water-
tube.
This obliquity of the mesentery causes the right body-cavity
to appear, in sections through the extreme posterior end of the
larva, somewhat smaller than the left; but this is not the case
in sections through more anterior regions (fig. 20), and indeed
the total bulk of the right posterior body-cavity (deducting the
anterior body-cavity, which may be said to end near the
anterior margin of the stomach) is, at the time of the formation
of the water-vascular ring, decidedly greater than that of
the left posterior cavity.
The later stages of development do not call for any detailed
description in this paper. After the junction of the radial
tubes of the hydroccel with the excrescences of the body-wall
which contain the terminals, the two grow out together and
form the arms, into which, in the larva, only the left body-
cavity extends. By this means, as well as by increased growth
within the disc, the left body-cavity gradually exceeds the right
in size, though even in the oldest larva I have obtained (with
fifteen pairs of tube-feet to each arm, and a total diameter of
3-9 mm.) there is still, within the disc alone, not very much dif-
ference between them (fig. 23).
In this latest stage the former protuberance of the stomach
in the centre of the water-vascular ring has been pushed back
by the formation of an ectodermic stomodeum ; but the adult
cesophagus is not yet complete. The stomach is bound to the
body-wall not only by the mesentery, but also by five interra-
dial septa, one of which, as we saw, forms one wall of the
axial sinus. Radially the stomach has five pairs of pouches,
as seen in fig. 24, and the relation of these to the mesentery is
shown in section in fig. 25; as will be seen, the right body-
cavity is dorsal to the stomach, no portion of it being visible
in fig. 24. The anus is, as shown, still in the same interradius
as the water-tube. As I have never succeeded in rearing a post-
larval specimen from this Bipinnaria, I am quite unable to
trace the migration of the anus into the position which it occu-
pies in the adult; but it is probable that the larval anus is
THE METAMORPHOSIS OF ECHINODERMS. 71
entirely obliterated, since in a young Luidia in which the disc
measures about 5 mm. in diameter, I find no trace of any
anus at all.
This Luidia presents certain other features which merit a
brief description. The dorsal sac is very conspicuous on the
aboral side of the water-pore, and a considerable portion of the
dorsal organ projects into it; it is quite distinct from the
ampulla, as well as from the rest of the celom, and is evidently
identical with the space which Ludwig found (18, p. 159)
containing the aboral termination of the dorsal organ. Imme-
diately below this sac, at the level of the original dorsal
mesentery (persisting at this stage transverse to the axis of
the cesophagus and stomach), the genital cord starts from close
by the dorsal organ (though I cannot from my own observa-
tions assert the connection of the two structures) ; but it is
still very short, and cannot be followed far round the disc.
For the most part the original mesentery is fragmentary (at
any rate in my sections), but enough of it remains to indicate
that it follows the growth of the hepatic ceca into the arms—
these ceca being derived from the ten stomachic pouches seen
in fig. 24 (compare figs. 25 and 26). This extension, however,
of the right body-cavity into the arms is probably to be re-
garded as secondary, as I shall explain later. There is much,
in my opinion, to point to the conclusion that the more primi-
tive line of division of the two cavities is marked in the adult
by the ring of the genital cord; but the proof of this is
indirect, and cannot be entered upon here.
2. Brachiolaria.
I have selected Brachiolaria as the representative of the
second type of Asterid development because it is the form
which I have had most opportunity of studying; but the same
general plan of development is found in many true Bipin-
nariz, as well as in the larva of Asterina, which is, as it seems,
only a modified Brachiolaria (1, p. 62; 15, p. 154),
The general external features of Brachiolaria have been
admirably figured by Agassiz (1) ; while the internal anatomy
2 HENRY BURY.
of the larva of Asterina is not only the subject of Ludwig’s
well-known paper, but has recently received further attention
from MacBride(19), whose full account may be expected shortly.
- It is unnecessary, therefore, for me to spend time on a detailed
description, and I may confine my account entirely to those
points which bear most on my theoretical views, and those
which the absence of the intestine in the larval Asterina tends
to obscure in that form.
As already mentioned, most of my material was obtained at
St. Andrews; but the earlier stages have been more especially
studied in larva, probably of Asterias glacialis, obtained at
Naples.
There is no need to describe here the earliest stages of all,
as the few points in which my observations are at variance
with Field’s (8) have no special connection with the present
paper.
The hydroccel seems to be always marked out before the
left posterior body-cavity is separated off, but the exact form
of the ccelom at this stage is subject to much variation;
fig. 17, however, may be taken as a fairly typical example.
In the next stage, in all cases examined by me, the separation
of the posterior enteroccl from the combined anterior en-
teroccel and hydroceel is complete. The posterior cavity then
pushesa “dorsal horn” forwards on the dorsal side, close upto the
water-pore, while a “ ventral horn” growsrapidly forwards along-
side the intestine, and then, at the point at which the latter
(closely pressed against the intestine throughout most of its
course) curves outwards towards the anus, this “ ventral horn”
of the left body-cavity crosses over to the right side. Meeting
with no opposition from the right body-cavity, which at this
stage only reaches the ventral surface at its posterior end, the
“ventral horn” widens out, and passes down the right side of
the intestine, near the posterior end of which it meets the
right body-cavity, and forms with it an oblique mesentery,
shown in fig. 27. All the stages of this movement can, in
perfectly healthy larvee, be easily followed in the living animal,
from which fig. 27 is drawn; but I have fully confirmed the
THE METAMORPHOSIS OF ECHINODERMS. 73
facts shown in this figure by means of sections, one of which is
shown in fig. 28.
The anterior end of this mesentery soon breaks down, but
before it does so the rudiments of the terminal plates appear
(fig. 27) lying over the left body-cavity, though one of them, as
the figure shows, is very much to the right of the intestine.
This constitutes one of the most striking distinctions between
the two types of Asterid larve (compare fig. 18); the other
principal differences may be briefly summed up as follows:
1. The larval cesophagus persists in Brachiolaria, and is not
replaced by a new one, though it loses its functional activity
for some time after the metamorphosis.
2. Up tothe moment the metamorphosis begins, the hydrocce:l
is still open to the anterior body-cavity, but this opening is not
(for the most part, at least) in the same interradius as the
water-tube and water-pore—one of the pouches of the hydroccel
lying (see 9, pl. xxvi, fig. 22) anterior to the pore.
The closing of the hydroccel ring I have not satisfactorily
followed in Brachiolaria, but it is certainly in the interradius
indicated by Ludwig (15, p. 169, and pl. vii, fig. 95), and
probably in every way the same as in Asterina.
3. As in Asterina, the most anterior hydrocel pouch of the
larva does not unite with the most anterior of the dorsal
arm-rudiments, but with the one just behind the anus—the
whole hydrocel ring being rotated (viewing the animal from
the oral surface of the adult—left side of the larva) in a
direction opposite to that of the hands of a watch ; or in other
words, the hydroceel is, so to speak, unscrewed slightly from
the rest of the body.
4, In the later stages of Bipinnaria asterigera the future
oral surface is turned rather towards the posterior end of the
larva; in Brachiolaria, however, it is the future aboral surface
which is directed backwards (compare 24, pl. ii, figs. 1 and 5).
The growth of the left body-cavity round the hydroceel, and
the formation of an axial sinus between the two horns of this
cavity, takes place as in Bipinnaria asterigera.
The ‘dorsal sac” appears at a very early stage as a space
74 HENRY BURY.
of schizocceel origin, lying over the posterior end of the
cesophagus. I have cut numerous sections to satisfy myself
that it has no connection with the celom, from which, indeed,
in the earlier stages it is somewhat widely separated. In later
stages the right body-cavity pushes its way up close to it, but is
at no time connected with it. Field (8, p. 118) found the
same cavity in Asterias vulgaris, and gives figures of its
schizoceel origin; he seems, however, unaware that I had
already drawn attention to it (5, p. 31). MacBride (19) does
not mention it in Asterina, but describes a cavity, derived
from the anterior enterocel, in apparently very nearly the
same situation; this he regards as the homologue of the right
collar-cavity in Balanoglossus ; but until a similar cavity has
been found in other Echinoderms—and especially in one which
conforms more nearly to the usual plan of development—this
conclusion appears to be somewhat rash.
In very old Brachiolariz, just before metamorphosis, I have
in a few cases observed a faint pulsation in this region—appa-
rently in the floor of this cavity ; but the opacity and activity
of the larva make it very hard to study, and it is moreover
much slower and less regular than in Echinid Plutei.
Immediately under the ‘‘ dorsal sac” in older Brachiolariz
lies the termination of the “ dorsal organ,” as seen in fig. 19;
but this, and the ultimate fate of the dorsal sac, have been
already described in connection with Bipinnaria asterigera.
Metamorphosis is ushered in, in the larva of Asterias
rubens, by the fixation of the larva by the knobbed arms of
the Brachiolaria, and the ciliated pit which they surround;
and this fixation, at first voluntary, very soon becomes as com-
plete as in Asterina (19, p. 433). Then the preoral lobe
shrivels up, and all that remains of it is a small stalk lying in
the interradius of the adult anus (interradius E). The larval
intestine and anus soon disappear, but the latter, when last
seen by me, still lay nearly in the interradius of the water-pore
(judging the interradii by the arm-rudiments, not by the
hydrocel pouches), or perhaps rather more nearly opposite
radius V,
THE METAMORPHOSIS OF ECHINODERMS. 75
c. ECHINIDs.
My studies of the internal anatomy of Echinids (both larve
and adults) have been chiefly made on Echinus micro-
tuberculatus; but so far as other forms have been examined
all Regular Urchins follow much the same plan of develop-
ment.
It is unnecessary to repeat here the description which I
gave in a previous paper (9) of the origin and general arrange-
ment of the parts of the cclom in this group; but attention
may be called to the migration of the hydroccel along the
left side of the stomach. Originating at the junction of ceso-
phagus and stomach, it pushes its way backwards over the
surface of the latter till it comes to lie about in the middle of
the left side, where it forms a ring through which (at a later
stage) the cesophagus of the adult grows—the larval cesophagus
not being permanent. Round this hydrocel ring the left
posterior body-cavity grows in the form of a crescent, the
dorsal and ventral horns of which eventually unite and fuse
together anteriorly. I believe that this fusion occurs along
the line of the water-tube, which, as in Bipinnaria, lies close
against the wall of the stomach ; but I have not yet been able
to obtain decisive proof of this.
The limits of the body-cavities in older larve are extremely
difficult to determine, but they appear to be separated by a
mesentery starting just behind the water-pore, and running
back along the middle dorsal line to the extreme posterior
end, where it turns and runs forward along the ventral surface
of the intestine. I have not been able to trace any communi-
cation between the cavities between the anus and stomach,
such as occurs in Asterids, though there are some indications
that it occurs just before metamorphosis.
The general arrangement of the left anterior enteroccel and
the organs adjacent to it was described and figured by me in
a previous paper (9, p. 18, fig. 9); but some further details
may be added here.
The “ pulsating vesicle ” arises at a fairly early period from
76— HENRY BURY.
a group of cells situated over the middle line of the esophagus,
to the right and rather in front of the water-pore. A schizoccel
space soon appears in this mass, and rapidly increases in size
(fig. 29). It is certainly distinct from the ccelom in its origin,
and, to the best of my belief, throughout larval and adult life.
The similarity of its origin and position, as well as its relation
to the dorsal organ (to be described later) lead me to regard it
as homologous with the “ dorsal sac” of Asterids, and by this
name I shall in future refer to it.
In late Plutei its floor projects far into its cavity (fig. 30),
but the extent to which this is seen in sections varies consider-
ably in different specimens, apparently depending on the
methods used in preservation. As far as I can judge from a
careful study of the living animal under high powers of the
microscope, it is this part, and not the cavity as a whole, which
pulsates. This pulsation, I may mention, is certainly continued
in the earliest post-larval stages, though whether it occurs in
the adult I am unable to say.
The left anterior enterocel forms at a fairly early stage a
large ampulla where the pore-canal and water-tube open into
it (fig. 31). In later stages it extends backwards for some
distance alongside the water-tube—further than in fig. 30, but
exactly how far I am still doubtful.
The dorsal organ is rather difficult to trace with accuracy,
but subsequent stages make it almost certain that a mass of
cells (usually filled with yellow granules), projecting into the
dorsal sac on the one hand (fig. 80) and into the ampulla on
the other (fig. 31), is the rudiment of this organ. Besides
this, a cord of cells is frequently noticeable (fig. 80) lying
alongside the water-tube (on the ventral side of this structure
in the larva), which may be a part of the dorsal organ; but I
am unable to distinguish it clearly in transverse section, or to
trace it into connection with the above-mentioned mass of cells
with yellow granules. The aspect of all these parts varies
much with the methods employed, and great care should be
taken to kill the animal in an extended condition. Where
this is not done the great mass of transparent tissue projecting
THE METAMORPHOSIS OF ECHINODERMS. ee
into the dorsal sac (fig. 30) often shrivels up and forms, with
the adjacent mass of yellow cells, a compact knot of tissue
seemingly composed mainly of nuclei; even this, however, is
instructive, since it presents a close resemblance to the dorsal
organ as it projects into the dorsal sac and axial canal in post-
larval stages.
The water-tube les throughout its course close against the
stomach, and finally enters the water-vascular ring adradially—
being, when the animal is viewed from the right (future aboral)
side, on the left side of its interradius.
When the time for metamorphosis is reached, the hydroccel
seems to have moved even beyond the middle of the left side,
and the ambulacral surface is directed somewhat backwards,
as in Bipinnaria asterigera; but at the same time the
original apex is pushed over to the right, and the mesentery
separating the right and left body-cavities becoming somewhat
oblique, its plane still continues to be about parallel to that
of the ambulacral surface, and at right angles to the axis of the
adult cesophagus.
The larva at this stage creeps about on the bottom by
means of its five primary tentacles, the larval arms elevated,
and the thin membranous “amnion ” spread out like an um-
brella, supported on the spines of the young Echinid.
The actual metamorphosis is accomplished very rapidly ;
the “amnion” contracts and is absorbed, while the spines
which were embedded in it become erect ; then the larval cwso-
phagus is absorbed, and the spicules of the larval arms are
(usually) broken off by the force of the accompanying con-
tractions of the ectoderm—so that in less than an hour a
perfect Pluteus is transformed into a small rounded Echinid,
in which radial symmetry entirely replaces the bilateral sym-
metry of the larva. This young Kchinid is usually rendered
extremely opaque by a species of histolysis, which begins in
the Pluteus with the proliferation of cells into the cavity of
the stomach, and afterwards extends to other tissues, render-
ing the examination of the internal organs extremely difficult
Exactly the same thing usually occurs in the larva of Antedon,
78 HENRY BURY.
as well as, in aless degree, in Ophiurid Plutei and Brachio-
laria; but the fact that one or two of my KEchinid Plutei
hardly showed it at all, and that Seeliger was not troubled with
it at all in Antedon, indicates that, as that author suggests
(30), it is probably pathological. In all larve kept under
satisfactory conditions it soon clears off, and the tissues return
to their normal condition.
Although this histolysis has prevented me from following
the details of the anatomy of the youngest Echinid to my
satisfaction, yet we may state positively that the essential
relations of the organs are not much altered during metamor-
phosis. If any mesentery exists at this stage (which is doubt-
ful) it must still occupy the same position as before—parallel
to the ambulacral surface, at the level of the water-pore ;
but since the total bulk of hydroccel and left body-cavity
combined is far greater than that of the right body-cavity
alone, it follows that the divisional line between the two cavi-
ties lies far on the aboral side of the equator of the young
Kchinid. Its approximate position is shown by a dotted line
in the diagram, fig. 835, which will be presently described in
connection with the skeleton.
In a previous paper (5) I described the position of the
basal plates in the Pluteus; though they do not form such a
regular longitudinal series as in Asterids, yet all (except
perhaps the madreporic plate) are formed over the right
body-cavity. Most of them possess spines, which when first
formed usually seem to end in three points, but when fully
formed are seen to be quadrangular and to terminate in four
points, not always of equal length; two of them are shown in
fig. 32.
Besides these plates a number of others are formed on the
left side, round the base of the “amnion,” into which their
spines project. They are most difficult to study in the Pluteus,
and I have not yet determined, in spite of much time spent
on them, their order of development; apparently a large
number of them are formed within a few hours of one another.
In a young post-larval stage their positions are more easily
THE METAMORPHOSIS OF ECHINODERMS. 79
determined, and I have therefore confined my figures to this
stage.
Their spines are of two different kinds, and are of great
assistance in identifying the different series of plates, the
general arrangement of which can be made out from figs. 34
and 35, and from the ventral view given by Lovén (11, pl. xvii,
fig. 149). On the aboral side of each primary tentacle lies a plate
with two quadrangular spines, such as we have seen on the basals.
In my latest stage (fig. 34) they curve round as if to embrace
thetentacle. Alternating with these, and usually rather nearer
the oral surface, are five interradial plates each bearing one
spine of quite a different pattern, with six longitudinal rods
instead of four (fig. 33). Théel (35, fig. 99) has given figures
of the development of these plates in Echinocyamus, which
agree with what I have observed in Hchinus, though rather
too diagrammatically regular. Several of the other plates,
the position of which need not be described, bear spines of the
same pattern (see figs. 84 and 35)—indeed those over the
primary tentacles are the only plates developed on the left
side which have spines of the quadrilateral pattern.
All these plates and spines have been carefully studied, both
in whole specimens and by means of maceration and dissection.
In my oldest larva, however, an accident prevented the use of the
latter method, and it is therefore possible that I have exaggerated
in fig. 384 the amount of curvature existing in the plates overlying
the tentacles; I do not think, however, that this is the case.
This (fig. 84) is the latest stage to which I have been able to
rear, from the egg, the young Echinus microtuberculatus.
The next stage (still of the same species) which I have been
able to obtain is very much older, and a considerable gap is
left between the two. Being unable to see the plates clearly
in the whole animal, I cut off the aboral portion and examined
it as a transparent object, the result of my observations being
shown in fig. 86: many of the spines are broken, and the
plates at the margin have been much injured in the process of
section, but still the figure shows much that is interesting,
The basal plates have already (5) been traced from the plates
80 HENRY BURY.
of the right side of the larva; but I may add here that in
figs. 11 and 12 of that paper the water-pore was drawn much
too large (a superficial hollow in the plate being mistaken for
it), and that the presence of two pedicellariz on the second
basal plate is much more common in the Pluteus than I then
supposed. Outside the ring of basals, and alternating with
them, we see the five oculars; each of them bears a pair
of quadrilateral spines, which are not found on any
other plates except the basals. When we compare
these plates with those just described in fig. 34, and take into
consideration the rarity of these quadrilateral spines, the
various facts known about the disappearance of the primary
tentacles, and the termination of the radial water-vessel of the
adult in the eye-spot, on the oral side of the ocular plate, we
can scarcely, I think, doubt that these oculars are identical
with the five plates which lie above the primary tentacles in
fig. 34. But these plates are developed on the left side, as are
the terminals of Asterids and Ophiurids, which also in the
adult embrace the terminations of the radial water-vessels; so
that we have, as it seems to me, new and important grounds
for accepting the homology, often suggested but never proved,
of the oculars of Echinids with the terminals of Ophiurids and
Asterids.
Another consideration follows from this identification of the
plates in figs. 34 and 36. In the younger stage the line of original
division between the right and left body-cavities (the mesentery
having probably disappeared already) lies somewhere between
the basal and ocular plates—probably in the position of the
dotted line in fig. 35, and nearly at the level of the water-pore.
In adults these two rows of plates fit into one another, but the
genital rachis encircles the intestine at the level of the basals
(through which the genital ducts pass) and of the water-pore ;
so that we have here somewhat better grounds than in Asterids
for believing that the genital rachis marks the line of division
between the right and left body-cavities.
The arrangement of the water-tube and the neighbouring
organs is almost exactly the same in Echinus microtuber-
THE METAMORPHOSIS OF ECHINODERMS. 81
culatus as in Dorocidaris (27), except that in the oldest speci-
men I have cut (diameter 3°5 mm.) the ‘‘ espace sous-madré-
porique” of Prouho does not extend down the side of the
water-tube.
In spite of the unsatisfactory histological condition of my
youngest radial specimens, and the gap which still exists
between them and the next stage obtained, the relations of
the parts in the Pluteus to those in the adult can be fairly
easily followed.
The pore or pores of the madreporic plate and the upper
end of the water-tube open in all stages into an ampulla
(figs. 830 and 39), which is continued down into a canal lying
alongside the water-tube, and enclosing the axial organ.
This is the ‘‘canal aquiferé annexe” of Prouho, and being
derived from the anterior enteroceel is apparently homologous
with the axial sinus of Asterids. (Fig. 39, “axial canal.’’)
Lying under the madreporic plate, on the aboral side of
the water-pore, is a closed vesicle into the floor of which pro-
jects a portion of the dorsal organ, which passes round the
water-tube into the axial sinus at a lower level than the section
drawn in fig. 39. This closed vesicle is Pronho’s “ espace
sous-madréporique,” and the portion of the dorsal organ pro-
jecting into it is his “ processus glandularis.” Its position is
exactly that of the “pulsating vesicle” of the Pluteus (for
what is on the right of the pore in the larva is on its aboral
side in the adult), and its obvious similarity to the dorsal sac
of Asterids justifies the conclusion already arrived at that
this sac is homologous with the “ pulsating vesicle” of the
Pluteus.
Fig. 39 very well illustrates the general relation of these
parts to one’another and to the intestine and ovary, though
the junction of ampulla and axial canal takes place at a higher
level, and the connection of the latter with the dorsal organ
(as already mentioned) at a lower level. The water-pores are
clearly adradial, being on the left of the ovary, which is inter-
radial. The water-tube in this figure appears to be on the
left side of this interradius, but at its oral end it opens into
VOL. 38, PART 1.—NEW SER. F
82 HENRY BURY.
the water-vascular ring in the same adradius as the water-
pores.
Biitschli (6) has already called attention to the fact that in
Lovén’s figure of a young Echinid (11, pl. xxi, fig. 170) the
flattened side of the dorso-central plate is directed towards
interradius E (compare fig. 36), and he infers from this that
the anus was originally in this position. I had observed the
same point in 18 88, before Butschli’s paper appeared, and am
able to give proofs of that which he could only conjecture.
As the anus does not break through till the young Echinus
microtuberculatus has attained a diameter of from 5 to 7
mm., it is no difficult matter to obtain the necessary material;
and by adding a few drops of chloral hydrate to the water
containing the animals, they can be stupefied in an extended
condition and easily examined.
Fig. 37 shows the position of the anus in one of the
youngest specimens in which it could be detected with absolute
certainty; it will be seen that it lies well within interradius
E. As the small plates of the perianal area (which sometimes
show a marked bilateral symmetry) increase in number, the
anus works gradually across into radius IV (fig. 38), and
thence into its adult position; but it is quite unnecessary to
illustrate the whole series of changes.
I have not succeeded in tracing the steps by which the long
and tortuous alimentary canal of the adult is evolved out of
the globular stomach and short intestine of the Pluteus. The
anus closes at the moment metamorphosis begins, and in my
youngest radial specimens I cannot even trace the intestine
with certainty. It is, however, interesting to note that in the
adult the cesophagus lies in interradius A, and from this
point the alimentary canal makes a complete circuit of the disc
till it again reaches this interradius. Then it turns back
(compare 14, pl. xiii, fig. 7) as far as interradius B, from which
it runs sharply back again to the anus in interradius D.
I am almost certain that in the youngest specimen of which
I have sections (diameter 1 mm.) this last loop (from inter-
radius D to interradius E and back again) does not exist,
THE METAMORPHOSIS OF ECHINODERMS. 83
and it seems to me not improbable that most of this coiling of
the intestine is secondary, and that the original condition was
that found in Crinoids—a simple curve round the disc, begin-
ning and ending in interradius A.
Lovén’s figure of Gonocidaris canaliculata (12, pl. iii,
fig. 10) is suggestive of this; but of course the matter can
only be settled by careful study of the development, for which
I have not been able to obtain the necessary material.
D. OPHIURIDs.
For a general account of the external changes which an
Ophiurid Pluteus undergoes before and during metamor-
phosis, I must refer to my former paper (5, p. 26) ; here I shall
only call attention to those features which will be referred to
in the second half of this paper, in connection with the phylo-
geny of the group.
Though not perhaps a very primitive group in themselves,
their Plutei offer us, in their external features at least, what
seems to me to be a much more accurate epitomeof phylogenetic
history than is met with in other Echinoderm larve. Unfortu-
nately, however, the difficulty of obtaining satisfactory sections
is so great that few of the internal changes can at present be
followed in detail. Iam full of confidence that better methods
will give all the facts we require, as all my material was used
up before I adopted the celloidin-in-paraffin method, which has
given me such satisfactory results elsewhere ; but meantime I
must content myself with inferences from studies of the exterior
and from our knowledge of other Echinoderm larve, which,
though attaining to great probability, are never so satisfactory
as direct observation.
The first stage in development which we need pause to con-
sider is that shown in 5, fig. 4. In it the hydroccel forms a
nearly complete ring round the cesophagus, while the posterior
part of the body still retains its primitive bilateral symmetry ;
the plane of the hydroccel is in fact about at right angles to
that of the mesentery.
I have already described and figured the changes of position
84 HENRY BURY.
which the calcareous plates of the Pluteus undergo at the time
of metamorphosis, showing that the plates developed over the
right body-cavity (radials and dorso-central) pass up on to the
dorsal surface of the larva, while those over the left body-cavity
(terminals) pass on tothe ventral side. At the same time the
madreporic plate is pushed forwards to the anterior end (finally
resting to the right of the middle line, see fig. 43), while the
mouth assumes a more decidedly ventral position, turning as
it does so towards the left (fig. 43).
All these changes in the skeletal plates are very easily
followed on the living Pluteus ; but a question of much more
importance to us now is, how far do the body-cavities follow
this movement—how far does the longitudinal mesentery
remain parallel to the lines of the radial and terminal
plates ?
It is on this point that my sections have most conspicuously
failed; but fortunately enough evidence remains to place the
matter (bearing in mind the analogy of Asterids and Echinids)
beyond any reasonable doubt. When the radial plates of the
dorsal side first begin to move across to the left, the edge of
the right body-cavity is still plainly visible in external views,
and certainly follows their movement as long as it can be traced
—that is, nearly to the stage shown in 5, fig. 5. Similarly, on
the ventral side the edge of the left body-cavity can be seen
passing, still parallel to the line of the terminal plates, over to
the right side (fig. 42). Between this and the first truly penta-
merous form, I have no observations worth recording ; but in
this stage (5, fig. 6) I find in sections traces of a mesentery
running round the edge of the stomach, still parallel to the
lines of the skeletal plates, though now at right angles to the
axis of the cesophagus (fig. 41). I cannot trace it all round
the body—indeed it appears to be in a very fragmentary
condition at this stage—but enough remains to make it
almost certain that we have here a remnant of the larval
longitudinal mesentery, which therefore in Ophiurids as in
other Echinoderms assumes in the pentamerous form a trans-
verse position.
THE METAMORPHOSIS OF ECHINODERMS. 85
If this is admitted, there cannot, I think, remain much
doubt that the left body-cavity, in the course of its movement,
encircles the wsophagus; though whether it ever forms a
mesentery in the interradius of the water-tube, must be left
for future investigation to decide.
Another point requiring further evidence is the behaviour of
the intestine. If the movement of the mesentery be admitted,
it is scarcely possible to doubt that the stomach moves with
it; and it would be at least a plausible conjecture that the
intestine, if it survived, would retain its primitive relation to
the stomach and body-cavities, and so assume a position trans-
verse to the axis of the adult esophagus. As a matter of fact
I believe in most larve the intestine and anus disappear almost
the moment metamorphosis sets in; but in a few larve I
have thought that I could trace the intestine, at the stage shown
in fig. 42, bending over to the right, exactly as we should
expect it todo. I have not, however, succeeded in obtaining
sections which prove this; and until I have done so, I do
not like to assert that it really occurs, though analogy renders
it extremely probable.
Up to the stage immediately preceding metamorphosis (fig.
40) the two body-cavities are practically equal in size; and it
would seem from fig. 41 that no great difference between them
exists even when the radial symmetry is fully acquired.
At this stage (5, fig. 6) the water-pore is nearly at the edge
of the disc; and from close by it starts (much later) the genital
rachis, which grows as a ring round the body (18, p. 138).
Subsequently, when the arms grow out, the water-pore moves
on to the ventral surface, while the radial portions of the
genital rachis remain dorsal to the arms, so that the whole
rachis assumes the form of an undulating cord, described by
Ludwig as an aboral vascular ring. Now I have no evidence
to offer except analogy; but I would suggest as possible, and
worthy of investigation, that this genital rachis may mark, as
it does in Crinoids, and I believe in Asterids and Echinids also,
the position of the larval mesentery—the original line of
separation between the two body-cavities, In this connection
86 HENRY BURY.
it is worth while to remember, though no great stress can be
laid upon it, that the cavities of the arms, bothin Crinoids and
Asterids, are primarily parts of the left body-cavity only, though
in both these forms (but not apparently in Ophiurids) the
right cavity secondarily extends into them.
Erratum.—lI take this opportunity of pointing out that the
calcareous plate marked over the “ anterior enterocel” in
fig. 4 of my former paper (5) is entirely due to a lithographer’s
error. A similar error has caused the omission of the madre-
porite in Bipinnaria (8, fig. 14).
E. CRINOIDS.
The development of Antedon rosacea, which alone among
Crinoids has been studied, is too well known to need any
description here; and though Seeliger’s (80) careful examina-
tion, while undoubtedly correcting some of my errors (4), con-
tains much debatable matter, yet fortunately few of the
points to which I wish here to call attention are open to
dispute.
(1) The plane of the hydrocel is not parallel to that of the
mesentery in the larva, but forms an angle with it. I may
have exaggerated this in my figure (4, fig. 59), but it is un-
questionably very marked (80, pl. xvi, fig. 67), and may be
compared with the somewhat similar, though by no means
identical arrangement in Asterina (15, woodcut iii, p. 156).
(2) The stalk, though connected with the preoral lobe of
the larva, does not include in the adult the whole of this lobe;
while it does contain in the larva other parts which do not
seem primarily to belong to this region. Thus the anterior
body-cavity, which extends into this region in all larve which
possess a preeoral lobe, is far removed from the stalk in the
Cystid stage of Antedon ; while several skeletal plates, and part
of the right body-cavity, are present in this region of the
larva. Now whatever may be said about the identification
of the terminal plate of the stalk with the dorso-central of other
Echinoderms, we have strong reasons for thinking that the
ancestral Crinoid (like many Cystidea) was sessile; and there-
THE METAMORPHOSIS OF ECHINODERMS. 87
fore it seems to me that the presence of stem-joints in the
larval Antedon indicates that this region is not a pure preoral
lobe, but a mixture of this with a structure (the stalk) belong-
ing to a much later (phylogenetic) date. The forward pro-
longation of the right body-cavity is due to the same preco-
cious development of the stalk; for it is impossible to compare
this, which arises so late, with the right anterior enterocel of
other Echinoderm larve, which is one of the first parts of the
ccelom to appear.
(3) The left body-cavity grows round the csophagus at the
time of metamorphosis, forming with the right body-cavity a
mesentery parallel to the plane of the hydrocel ring. The
two horns of the left body-cavity certainly both reach the
interradius of the water-tube, and appear to me to form a
mesentery supporting the latter; but this,in theface of Seeliger’s
researches (30, p. 292) must be admitted to be doubtful.
(4) In the free-swimming larva the right and left body-
cavities are approximately equal in size (Seeliger rightly points
out that in my diagrams I represented the right cavity too
small). In the Cystid, however, the left cavity is much smaller
than the right, and though, when the arms begin to grow out,
its relative size increases for a time, yet the subsequent exten-
sion of the right cavity into the arms apparently neutralises
this. The important point for us, however, is the fact that the
actual metamorphosis cannot in any way be said to be due to,
or even accompanied by, “ predominance of the left posterior
body-cavity ” (19, p. 434).
(5) The order of development of the first-formed pairs
of tube-feet in other Echinoderms is a little doubtful,
but apparently they are from the first (as the later pairs
certainly are) in centrifugal succession. In Antedon, how-
ever, the second pair is undoubtedly formed later than the
first pair of each ray; while the development of the later
tentacles in triplets (26, p. 177) is utterly unlike anything
observable elsewhere, and goes far to establish an absence of
homology between the arms of Crinoids and those of other
brachiate Echinoderms.
88 HENRY BURY.
(6) Perrier’s description (26) seems to leave little room for
doubt that the genital cords start at the level of the transverse
mesentery. They grow out, according to him (26, p. 202),
from the oral end of the dorsal organ, and pass along to the
arms in the septum which separates the oral and aboral arm-
cavities—a continuation (26, pl. ix, figs. 62 and 62) of the
transverse mesentery.
(7) In my former figures (4) 1 represented the stalk of the
larva as lying in radius V (compare fig. 14 of this paper). It
is extremely difficult, in the absence of radial plates, to deter-
mine its exact position, but I am strongly disposed now to
believe that its true position is adradial—close to radius V,
but actually in interradius A. There is some indication that
it undergoes a change of position during metamorphosis; but
further investigation of this point is required.
Part I].—PHYLOGENY.
Interesting as are the problems involved in the history of
the probable bilateral ancestor of Echinoderms, they have
very little to do with the subject of the present paper. The
relation of this ancestor to the Enteropneusta will indeed
be briefly discussed later on; but for the present we may
confine our attention to the first appearance of radial sym-
metry, and the changes represented in ontogeny by the meta-
morphosis.
On this point many opinions have been expressed in the last
few years, some of them of a highly speculative character; but
few attempts have been made to trace these changes in detail
from an embryological standpoint. The more general sketches
of phylogenetic possibilities, and expressions of opinion founded
on observation of post-larval and adult examples only, can,
where they call for any special remark, be more conveniently
dealt with in connection with those details of my views with
which they are immediately connected ; but those accounts
which endeavour to give a fuller explanation of the meta-
THE METAMORPHOSIS OF ECHINODERMS. 89
morphic changes must be briefly reviewed before my own
opinions are set forth.
Semon (82) assumes a bilaterally symmetrical ancestor,
which he calls “ Pentactea.” In it the hydrocel, which formed
a ring round the mouth, had five tentacles, and was connected
by the water-tube with the water-pore in the dorsal interradius.
A dorsal mesentery, embracing the water-tube, ran back from
near the mouth to the posterior end in the middle line; the
mouth and anus were ventral, and the animal was fixed at a
point somewhere on the dorsal surface. The representative
of this ancestor he professes to find in the young stages of all
Echinoderms (“ Pentactula”’ stage)—regarding the mesentery
of the water-tube as identical with the longitudinal mesentery
of the larva. He admits subsequent changes of position of mouth
and anus, but onlyin Crinoids does he recognise the parallelism of
the mesentery (or a part of it only, as he thinks) to the plane
of the hydrocel, and accounts for it briefly as due to a
secondary “ Drehung des Darmes.”
How utterly he has misunderstood the position of the larval
mesentery in Asterids and Kchinids will be evident without
further comment to any one who will compare his figures
(32, pl. vi, figs. 4 and 5) with the descriptions given in the
foregoing pages of this paper; while Ludwig’s observations on
Cucumaria (16 and 17) and my own re-examination of Synapta
(on which Semon’s views are founded) suggest that the
symmetry of adult Holothurians has not been quite so simply
derived from that of the larva as Semon would have us believe.
In fact, without entering further upon the details of his theory,
I think we may say that his fundamental assumption of the
retention of bilateral symmetry by the complete radiate form is
opposed to embryological evidence. To the further assumption
of the fixation of the common ancestor I shall return later.
Biitschli’s theory (6) is extremely ingenious and very
carefully reasoned ; but, while resting on no personal observa-
tion, is a little too prone to ignore, or set aside on purely
theoretical grounds, those statements of other observers which
do not conveniently fit into it. It is impossible here to review
90 HENRY BURY.
his paper as fully as the obvious care bestowed upon it deserves,
but the following is a brief summary of its main points:
The ancestor has a hydrocel with eight tentacles sur-
rounding the mouth (which is ventral) and two symmetrically
disposed body-cavities. It then becomes fixed by the tentacles
of the right side, three of which are thereby suppressed; and
after this the point of fixation shifts to the centre of the right
side, while the mouth (surrounded by the five remaining ten-
tacles) moves into the left side.
In this way he arrives at an arrangement of the body-cavities
and mesentery with relation to the alimentary canal which I
believe to be very nearly correct; but the steps by which this
position is reached are open to grave objections. In the first
place, there is no sort of evidence that more than five tentacles
ever existed. Biitschli begins with ten, but afterwards reduces
them to eight, with suppression of three, for the sole purpose
of explaining the remarkable bilateral symmetry observed by
Lovén in EKchinids. For the details of this explanation reference
must be made to the original paper; but as we have no sort of
evidence that this symmetry extends to other groups, I venture
to think that the assumptions made to explain it are wholly
unwarranted. Of fixation by the right side there is no more
embryological evidence than of the fixation by the dorsal side
assumed by Semon; but the whole assumption of universal
fixation is founded mainly on paleontological evidence (6, p. 137),
to which I shall return later.
In addition to these more important assumptions, there are
many other points unsupported by evidence (e. g. the pulling by
the cesophagus of a part of the right body-cavity into the
oral side—see p. 146; and the derivation of one of the five
permanent tentacles from the right side—see p. 157, note), as
well as others in which serious distortion of evidence occurs.
Thus he accepts Ludwig’s determination of the position of the
anus in Crinoids, in preference to mine (since proved by See-
liger to be correct), because it agrees with his theory
(6, p. 156), regardless of the evidence I produced in my last
paper to show that in other Echinoderms also the anus was
THE METAMORPHOSIS OF ECHINODERMS. 91
probably primarily in the same interradius as the water-pore.
Again, on purely theoretical grounds he assumes that the
closure of the water-vascular ring in ontogeny ought to take
place where we find it in Asterina; and he therefore boldly
denies (p. 157) the accuracy of the observations tabulated in
my last paper (5, fig. 28), except in the case of Ophiurids, in
which he assumes that the hydrocel has been turned com-
pletely round in ontogeny—its anterior end in the larva being
the original posterior end! I leave the facts in these cases to
speak for themselves ; but I cannot help expressing a regret
that Biitschli should have allowed his theories to carry him so
far without taking the smallest pains to find out for himself
where the truth lay.
MacBride’s hypothesis (19) is in many respects more nearly
in accordance with the facts of embryology than its predecessors,
and contains much that is suggestive ; but though it is perhaps
unfair to criticise it while only an abstract of it is before us,
yet it gives one the impression of being based too completely
upon the ontogeny of Asterina, the only form, apparently, in
which MacBride has personally followed the metamorphosis.
He assumes that the bilateral ancestor possessed two hydro-
coels (“‘ collar-cavities”’), of which one at least (the left) had
five tentacles. Whether there were five more on the right side
I cannot certainly gather, but at least they are not represented.
This bilateral form became fixed by the proral lobe, and then
the left hydroccel and left posterior enteroccel grew round the
cesophagus, the former embracing the base of the preoral lobe
in Echinozoa, but not in Crinoids. This encircling of the
cesophagus is regarded as brought about by “ the curious, and
as yet unexplained, peculiarity of Echinoderms, the predomi-
nance of the left side (left hydroccel and left posterior body-
cavity”). On these views I offer the following comments:
(1) Whatever may be thought of the existence at an early
period of a second (right) hydroccel (and to this question we
shall return later), it is improbable that it could have retained
enough importance to bear tentacles at the stage under con-
sideration, without leaving more trace of them in ontogeny.
92 HENRY BURY.
But if, on the other hand,as MacBride’s figure (fig. 2) indicates,
only the left hydroccel of the bilateral ancestor had tentacles,
then these tentacles, running down the left side, without any
special relation to the mouth, occupied an unparalleled and, to
my mind, most improbable position. Moreover, the position
assigned to them in this figure, though suiting fairly well the
larva of Asterina, is not at all in accordance with what we find
in Ophiurid Plutei, in which the hydroccl surrounds the ceso-
phagus at right angles to the dorsal mesentery, before
bilateral symmetry is lost. I shall endeavour to show later that
Ophiurids are more likely to be primitive in this respect than
Asterina.
(2) MacBride’s idea of the fixation of the ancestor, followed
by different changes in Echinozoa and Pelmatozoa respectively
(19, p. 434), will not suit paleontologists, but is for all that
more likely, in my opinion, to be right than those theories (such
as Semon’s, Biitschli’s, &c.) designed to satisfy the supposed
teachings of paleontology, to which we shall return. It must,
however, be borne in mind that it rests solely on the fact that
Asterina, as well as Antedon, becomes fixed by the preoral
lobe; no trace of such fixation has yet been found in any other
Echinozoan larva.
(3) We have seen that in post-larval stages of all Echino-
derms, except Holothurians, the mesentery of the water-tube
(formed, as MacBride rightly recognises, by the left body-cavity
only) is at right angles to the original longitudinal mesen-
tery of the bilateral stage, while the water-pore is almost at
the level of this longitudinal (now transverse) mesentery. I
am at a loss to understand how this condition can be brought
about by any amount of “ predominance of the left side.” No
doubt the greater this predominance the more nearly will this
condition be approached, but it can never beactually reached, and
the hypertrophy must be enormous before anything like it is
attained. But what is the evidence of this hypertrophy ?
No doubt it occurs early in Brachiolaria and Asterina, and
perhaps too in Echinid Plutei; but, on the other hand, metamor-
phosis seems to be accomplished in Ophiurids without any
THE METAMORPHOSIS OF ECHINODERMS. 93
marked change in the proportions of the two body-cavities ;
while in Crinoids it actually ends in the left cavity being
smaller than the right. On Holothurians I offer no opinion,
but I fail to see in them any support of MacBride’s views ;
while in Bipinnaria asterigera, lastly, in which the adult
relation of hydroceel and body-cavities is assumed long before
metamorphosis, I have shown that the preponderance of the left
cavity only arises when the arms are formed, and apparently asa
consequence of this formation—certainly not as a consequence
of the growth of the hydroccel and left body-cavity round
the cesophagus. Here, again, as it seems to me, MacBride
relies too much on the larva immediately under his notice.
Those who have approached the question of the origin of
Echinoderms from a_ paleontological standpoint, have almost
without exception derived all existing forms from the Cystidea.
Various genera are pressed into service as ancestral, but at some
period or other all the Kchinozoa are supposed to have passed
through a stage in which they are fixed by the aboral pole.
Of this there is not the slightest embryological evidence, for
even if we follow MacBride in regarding the fixation of
Asterina as an ancestral feature, that fixation is by the oral,
and not by the aboral surface, so that it does not in the least
satisfy the requirements of the palzontologists. Nevertheless,
almost all embryologists, apparently out of deference to paleeon-
tological conclusions, have thought it necessary to assume that
ontogeny is misleading, and that a period of fixation really did
take place, of which all traces have since disappeared.
Now this involves us in a question of fundamental import-
ance. If paleontologists have really proved beyond any
reasonable doubt that the Echinozoa are derived from fixed
ancestors, then ontogeny is misleading ; but if it is misleading
to such an extent as to obliterate all traces of a process of such
immense importance, I for my part do not see how we can
trust it in other particulars, and those who rely upon it for
indications of phylogenetic history had better re-consider their
position. (The fixation of Brachiolaria and Asterina obviously
94, HENRY BURY.
does not help us: if, with MacBride, we regard it as primitive,
we directly oppose the paleontological position; if it is
secondary, Asterids are in the same position as other Echinozoa
—they retain no trace of aboral fixation.) But after all, have
paleontologists so completely established their position as to
compel us to accept it? From the nature of the case no
details can be known of the internal anatomy of the Cystidea,
and consequently in connecting them with modern forms we
are obliged to rely solely on the general arrangement of the
skeleton, and on the position of the few apertures (mouth,
anus, &c.) which we can distinguish. The latter seldom help
us far, while the untrustworthiness of the former is proved by
the fact, so clearly emphasised by Neumayr (25, p. 497), that
in order to derive the Echinozoa directly from the Cystids at
present known, we are compelled to regard the resemblances of
the skeleton in Crinoids and Echinozoa as due to homoplasy,
not to homology. Neumayr thinks that the origin of Echinids
can be very clearly traced through the Cystid Cystocidaris
(25, p. 400) ; yet his description of this form does not give a
single really convincing item of homological resemblance,
while there is much that it is quite as easy to lay down to
homoplasy as the far more striking similarity of the basal
plates in Crinoids on the one hand, and Asterids and Echinids
on the other.
It is beyond the scope of the present paper to pursue this
subject further, but I submit that until paleontologists have
produced some far more striking intermediate forms between
fixed Cystids and free Kchinozoa than are at present forthcoming,
embryologists may be forgiven if they do not follow them.
But even if we deny, on embryological grounds, that the
Echinozoa ever had a stalk or disc of fixation on the aboral
surface, there remains the further question whether they may
not have been fixed, as MacBride supposes, by the preoral
lobe, which afterwards shifted—in them to the oral side, in
Pelmatozoa to the aboral pole. Since this actually occurs in
some Asterids, it cannot be said that this view is so violently
opposed to embryology as the one we have just been discussing ;
THE METAMORPHOSIS OF ECHINODERMS, 95
but here again the negative evidence ought, it seems to me,
to weigh very heavily with those who rely, as much as MacBride
does, upon ontogeny as a repetition of phylogeny. At present
the positive evidence is exceedingly weak—only in Asterids,
and not even in all of them, has this fixation been found, in
spite of most diligent search for it ; and so weak is the atavistic
tendency to recover this supposed phylogenetic character that
Amphiura squamata, though fixed to the body of the mother
in its young stages, is fixed by the posterior, not by the anterior
end. Here the fixation is clearly secondary, and until further
evidence is brought forward I am strongly disposed to regard
the fixation of Asterina as also secondary, and quite inde-
pendent of the fixation of Antedon. Fixation by the preoral
lobe is no uncommon thing, so that it may easily arise over and
over again ; and when we consider the apparent difficulties of
the transition from the bilateral to the pentamerous stage in
other larvee (as evidenced by the rapidity of the metamorphosis
and the frequent obliteration of the cesophagus), we can easily
see the advantages of such fixation, especially to a shallow-
water form exposed to wave action. ‘This, however, is pure
speculation ; what is really important is, I repeat, the strength
of the negative evidence, which to my mind is so great as to
make it unwise to assume the fixation of the ancestor so long
as any Other explanation is possible. That the phenomena
can be accounted for without this assumption I shall endeavour
to show in the following pages. Iam aware that the proof of
my views is still far from complete, and for that reason I shall
not attempt to follow out the details so far as some of my
predecessors have done; but the large number of speculative
suggestions as to the origin of Echinoderms which have
appeared in the last few years, almost all assuming original
fixation in some form or other, seemed to make it advisable
that I should attempt to show that such an assumption 1s
neither embryologically sound nor necessary as a basis for
phylogenetic speculation.
96 HENRY BURY.
The Bilateral Ancestor.
Into the most primitive stages of the bilateral ancestor I do
not propose to enter here; some few remarks on the subject
will be offered at the end of this paper, when I come to discuss
the relation of the Echinodermata with the Enteropneusta,
but for present purposes it will be enough to start with a stage
—of the existence of which I believe there is sufficient evidence—
in which the hydrocel already formed a ring round the
cesophagus, and five tentacles already laid the foundation of the
future pentamerous symmetry, while behind the cesophagus
the alimentary canal and body-cavities still retained the
primitive bilateral symmetry. Such a form is shown in figs.
44 and 45, and may be described as follows: —The mouth was
bent down on to the ventral surface, and probably opened into
an atrial cavity, though this is uncertain; the stomach was
globular or slightly elongated antero-posteriorly, and from its
posterior end the intestine ran forward, opening on the ventral
surface not far from the level of the anterior margin of the
stomach. Round the esophagus was the water-vascular ring,
with five tentacles or tube-feet, suitable to progression, but
capable of retraction within the atrial cavity (if that existed at
this stage). One tentacle was median and posterior, while in
the dorsal interradius, on the left side of it, was the water-tube
running back to the water-pore, which was situated over the
anterior end of the stomach, rather to the left of the middle
line. The left anterior enteroccel was probably already reduced
toasimple ampulla at the junction of water-tube and pore-
canal, such as we find in adult Echinoderms, though it probably
ran a little forward (see fig. 45) alongside and on the dorsal
side of the water-tube. The right anterior enterocel had
probably already disappeared. The two large body-cavities
were symmetrically disposed on the right and left sides of the
stomach, and there was certainly between them a longitudinal
dorsal mesentery, though how far this extended round to the
ventral surface is uncertain. In the middle line, just to the
right of the water-pore, I believe there was a “ dorsal sac,”
THE METAMORPHOSIS OF ECHINODERMS. 97
and under it a “dorsal organ” extending probably forwards
towards the water-vascular ring ; but though I have introduced
these into fig. 45, I shall, for the sake of clearness, omit them
almost entirely from my subsequent description, returning to
them when I deal with the relation to Enteropneusta.
The ancestral form just described is closely similar to Semon’s
** Pentactza,” from which it differs in not being fixed, and in
the body-cavities not extending forwards far enough to form a
mesentery enclosing the water-tube. Still more closely does it
resemble an Ophiurid Pluteus, just before metamorphosis,
deprived of its arms and its pelagic habits; indeed, almost the
only important points of difference lie in the facts that the
Pluteus has a pentamerously-arranged skeleton (which I shall
deal with immediately), but has not a very definite atrial
cavity, which, however, is not essential to the ancestor.
One of the first questions which meets us in trying to
reconstruct the history of Echinoderms is—what organ, or
group of organs, originated the pentamerous arrangement ¢
The Sarasins (28, p. 147) have given precedence on this point
to the longitudinal nerves and muscles; but I think most
embryologists will be inclined to follow more closely the
teachings of ontogeny, which seem to point to either the
hydroceel or the skeleton as the first to exhibit this symmetry.
Now in spite of the markedly metameric arrangement of the
skeleton in many larvee, there are very serious difficulties in
the way of making it the starting-point of the pentamerous
symmetry; indeed, all homologies of the skeleton between
Pelmatozoa and Echinozoa have been of late strenuously denied.
But without entering upon this very difficult question at
present, let us examine the primitive nature of the five pouches
of the hydroceel, and see to what results it will iead us; for,
after all, such assumptions must be judged more by the results
deducible from them than by the direct evidence in their
favour—provided always that ontogeny establishes a fair
prima facie case, which is not, I think, true of the Sarasins’
supposition.
vou. 38, pART 1.—NEW SER. Ga
98 HENRY BURY.
It is not my purpose in this paper to discuss how or why the
hydroccel came to encircle the cesophagus ; our present evidence
does not seem to me to be sufficient to allow of even a plausible
guess; but that it did assume this position before the general
bilateral symmetry was lost, there is a good deal of evidence to
show. In the first place, it actually does assume this position
in Ophiurids ; and secondly, in many larve its plane forms a
marked angle with that of the longitudinal mesentery—an
arrangement easily derivable from that of Ophiurids. This is
seen in Asterina (15, p. 156, fig. 3, taking the “antiambulacralen
Armanlagen” as parallel with the mesentery), and in Crinoids
(4, fig. 59). As we shall see in the next stage, this condition
may be considered as a derivative from that seen in Ophiurids,
approximating to a later arrangement; while it is not easy to
understand, if this or the later arrangement (with the planes
of hydroceel and mesentery parallel) is primitive, how the
Ophiurid position was ever arrived tt. In Holothurians, on
which Semon’s Pentactza is founded, this point is not so
clearly defined as he supposed, as my description of Synapta
will show; but as there is certainly nothing in this form
opposed to the view that the Ophiurid arrangement is primitive,
this need not detain us now; while the fact that no trace of this
arrangement is seen in Bipinnaria asterigera or in Echinid
Plutei is easily explained, when we consider that in these
larve the hydroceel ring is arranged in relation to the secondary,
not the primary, cesophagus. But all this will be clearer when
we have considered the next stage in ancestral history. The
same is true of several details in the positions assigned
to the water-tube, &c., but the situation of the water-pore is
clearly in accordance with what we find in most larve, while
the adradial position of the opening of the water-tube into the
water-vascular ring has been frequently commented on in the
foregoing pages. It is this position of the water-tube, together
with the probability of a symmetrical arrangement, which has
led me to assign a dorsal position to one interradius ; for though
it is tempting to assume a mechanical function for the posterior
unpaired tentacle, and perhaps for the number (five) of the
THE METAMORPHOSIS OF ECHINODERMS. 99
primary tentacles, it would be very unsafe to attach any im-
portance to such a speculation. That an atrial cavity was
present in the common ancestor at some stage or other is
rendered almost certain by its presence in such widely separated
groups as Crinoids, Echinids, and Holothurians ; and whether
it arose at the stage now under consideration or at a later one
is a question of no importance.
The only other point requiring justification is the limit
placed on the forward extension of the posterior body-cavities.
It has apparently escaped the notice of Semon and Bitschli
that these cavities rarely extend beyond the anterior margin of
the stomach ; even in Ophiurid Plutei (5, fig. 4) where they
extend further than elsewhere, they still lie posterior to the
water-pore. Asterid larve must, of course, be judged by those
early stages in which the anterior and posterior cavities are
separated on the left side, and they will then be found to agree
with other larvee in this respect.
It will be noticed that I have given this ancestor neither
preoral lobe nor point of fixation; a rudiment of the former
(which probably existed at an earlier epoch) may have been
present, and there may have been on it a sucker for temporary
fixation (though I refuse to admit that complete fixation can
have occurred without leaving stronger evidence behind) ; but
such assumptions, though leaving the main outline of my
hypothesis untouched, are wholly unnecessary, and to my mind
not justified by the evidence at present before us.
Transition to Radial Symmetry.
In all recent adult Echinoderms (with the exception of
Holothurians, to which we shall return later) the plane of the
water-vascular ring is at right angles to the axis of the ceso-
phagus and parallel to the longitudinal mesentery of the larva.
To arrive at this condition from that of our hypothetical
ancestor, it is only necessary to assume a movement of the
cesophagus, with the water-vascular ring, into the left side of
the animal. Of course I do not mean to assert dogmatically
that an actual migration, and that alone, of the cesophagus
100 HENRY BURY.
occurred, for it is quite possible that the result was produced
by a complicated system of hypertrophies and atrophies ; but
the predication of an actual migration gives us the simplest
process for descriptive purposes, and at the same time empha-
sises my objection to MacBride’s assumption that hypertrophy
of the left side has been the cause of the change of symmetry.
Though the water-vascular ring almost necessarily moved
with the cesophagus, which it surrounds, the water-pore, lying
over the anterior end of the stomach, was not involved in this
movement. The moment the cesophagus began to move it
came in contact with the margin of the left body-cavity, which
seems to have become invaginated to receive it, and by the
time the cesophagus reached the centre of the left side of the
stomach, to have surrounded it completely (see figs. 47 and 48),
possibly forming a mesentery to support the water-tube—but
to that we shall return. Figs. 46 and 47 will render the tran-
sitional stages sufficiently intelligible, while figs. 48 and 49
show the arrangement of the principal organs when the ceso-
phagus has reached its resting-place in the centre of what was
the left side of the stomach. The water-tube is elongated so
as to run over the surface of the stomach from the water-
vascular ring to the water-pore, and parallel to it as before
runs the anterior enteroccel (ampulla). Almost at the same
level as the water-pore the originally longitudinal mesentery
forms a sort of equatorial band round the stomach, and in this
plane lies the intestine, which has not changed its position
relative to the stomach and mesentery.
It will be noticed that in these diagrams (figs. 48 and 49) I
have assigned positions to the tentacles which do not quite
accord with the supposition of a simple movement of the
hydroceel into the left side. In the bilateral form the tentacles
were arranged symmetrically about a dorso-ventral plane, one
on the ventral side being median. If the movement into the
left side were as simple as I have so far represented it, this
plane of symmetry would now lie approximately along the line
X-Y in fig. 48 (its exact position depends on how we construct
fig. 44). But this, if we assume the water-tube still to open
THE METAMORPHOSIS OF ECHINODERMS. 101
into the water-vascular ring in the interradius through which
this plane passes, would give (as the construction of a simple
diagram will easily show) a very oblique course to the water-
tube between the water-vascular ring and the pore; and in
order to obtain the straight course for this tube, which it invari-
ably possesses, we must assume either that the tube has shifted
the position of its opening into the water-vascular ring, or that
the latter has been rotated. That the latter is the more probable
solution we shall see when we have examined the justification
of the main features of my hypothesis afforded by ontogeny.
If we may judge by external appearances, Ophiurid Plutei
repeat with most completeness the features of the supposed
ancestral migration of the cesophagus and water-vascular ring.
On the other hand, in Bipinnaria asterigera the hydrocel
lies from the first nearly in the middle of the left side of the
stomach, and a new esophagus is formed in the centre of it, so
that there is no migration of either organ. Between these two
extremes we have several very instructive intermediate steps.
In Crinoids there is no csophagus, but the atrial cavity
and water-vascular ring both migrate, so as to place the plane
of the latter more and more parallel to the ccelomic mesentery ;
but the nature of the process is much obscured by the early
change of position of the body-cavities and by the migration
being directed towards what appears to be the posterior pole.
In Echinids there is no migration of the cesophagus, but the
atrial cavity and hydroccel move over the left side of the
stomach, arriving fairly early at their final position. The forma-
tion of a new cesophagus is obviously secondary, but otherwise
the ancestral process seems to be fairly closely followed.
Brachiolaria (including Asterina) is still more interesting.
Here, as in Bipinnaria, the hydroccel is from the first nearly in
the centre of the left side (its position there being in my opinion
secondary ),anditconsequently undergoesnomigration. Butthe
larval cesophagus is retained intheadult, andat the time of meta-
morphosis is bent sharply into the middle of the hydroccel ring ;
at the same time it undergoes a complete change of position
102 HENRY BURY.
with relation to the longitudinal mesentery and water-tube,
which justifies us in asserting that here, too, there is an actual
migration over the left side of the stomach.
All these differences between the various larve are easily
intelligible as shortenings in ontogeny of the phylogenetic
migration—the shortest process of all being foundin Bipinnaria
asterigera, in which both hydroccel ring and permanent ceso-
phagus are produced in the positions which they will ultimately
occupy.
In close connection with this lies the point already alluded
to in connection with the bilateral ancestor—the angle which
the plane of the water-vascular ring forms with that of the
mesentery. If the facts above given are referred to, it will be
found that here too we have represented the steps by which the
ontogenetic process has been shortened—the two planes being
at right angles to one another in Ophiurids; inclined at a
lesser angle in Brachiolaria and Crinoids; and parallel from
the first in the Bipinnaria.
The facts just quoted with regard to the migration of the
cesophagus and water-vascular ring greatly strengthen the con-
clusion that the Ophiurid position of the latter—at right angles
to the mesentery—is the primitive one.
The encircling of the cesophagus by the left body-cavity has
been sufficiently emphasised in the early part of this paper;
but the frequent occurrence of a communication between the
right and left cavities makes it often difficult to say how far
this process is carried, and how far we are justified in asserting
that the mesentery of the water-tube (‘ oral mesentery,” let
us call it for brevity’s sake) is really bounded on both sides by
the left body-cavity. The following summary of the facts will
help us to a conclusion.
In Echinids there seems to be no fusion of the body-cavities
before metamorphosis ; but the dorsal and ventral horns of the
left cavity fuse completely, the oral mesentery being formed
later. Further investigation of these larve is, however, desirable,
In Brachiolaria it is fairly certain that the left body-cavity
bounds the oral mesentery (containing the axial sinus) on the
THE METAMORPHOSIS OF ECHINODERMS. 103
ventral side of the larva; for though the right and left cavities
are united, the point of union takes place so far to the right of
the intestine as to make it almost inconceivable that the right
cavity should have anything to do with this region. The
dorsal horn of the left body-cavity fuses with the axial sinus.
In Bipinnaria the early fusion of the two body-cavities
makes accurate determination of their limits impossible. The
dorsal horn of the left cavity behaves as in Brachiolaria.
In Crinoids, again, the ventral horn of the left cavity cannot
be traced with certainty up to the oral mesentery; but the
opposite side of this mesentery is unquestionably bounded by
the dorsal horn.
These facts, taken together, seem to me to establish almost
beyond doubt that the growth of the left cavity round the
cesophagus is complete (except for the possible intervention of
a mesentery between the two horns), and that the right cavity
has no connection with this region. Whether the oral mesentery
is primary or (as Echinids seem to indicate) secondary is a
question which need not detain us now.
That the water-pore did not move with the water-vascular
ring, but retained its original relation to the longitudinal (now
transverse) mesentery, the facts of ontogeny abundantly testify.
That it has since moved slightly either to oral or aboral side of
this position need not surprise us; but in all larve, even after
the establishment of pentamerous symmetry, it will be found
practically at the level of the mesentery, and I do not think
that even in adults it has ever migrated to any large extent.
The course of the intestine along the equatorial line marked
by the mesentery, and the opening of the anus in the inter-
radius of the water-pore, as shown in fig. 48, are best attested
by the Crinoids; but the former is well enough seen in
Echinid Plutei also, while in the same group, after meta-
morphosis, the intestine still coils right round the disc as far as
the above-mentioned interradius, though the anus does not
open there. Fig. 23 shows the intestine for the most part on
the right (aboral) side of the stomach in Bipinnaria, but the
mesentery is attached to its edge ; and the anus in this larva
104 HENRY BURY.
is in the same interradius as the water-pore (see fig. 24) before
metamorphosis. A further indication that this was the primi-
tive position of the anus is given by its relation to the calcare-
ous plates in Ophiurid Plutei. No doubt these plates (which
mark the radii) are precociously developed, but their situation,
when we compare this larva with Bipinnaria or Brachiolaria,
points pretty clearly to the conclusion just arrived at. On
the position of the anus in adult Asterids and Echinids, much
nearer the aboral pole than the water-pore, I shall have some
further remarks to offer later on.
Of course this question of the situation of the anus, though
interesting, has no vital bearing upon my hypothesis; it is
important, indeed, that the general course of the intestine
should be parallel to the plane of the water-vascular ring,
and that in early stages of ontogeny it should lie at the level
of the equatorial zone (mesentery); but the anus may be placed
in any interradius that ontogeny demands—that is simply a
question of the length of the intestine. In my diagrams I have
made it long, so as to bring it into the interradius of the
water-pore ;! but it would have been quite as easy to shorten
it into the interradius which it occupies in Asterids, or still
further into that in which it is found in adult Kchinids.
We saw that a straight course for the water-tube from water-
pore to water-vascular ring, which is demanded by the anatomy
of all known Echinoderms, was inconsistent with the very
simple movement of this ring into the left side which I had
postulated ; and we must now examine how the observed posi-
tion may have been brought about.
Seeliger (39, p. 261) takes up the somewhat remarkable
position that the water-pore is not in the same interradius in
all Echinoderms, giving as evidence the fact that it is in the
middle dorsal line in Holothurians (which is not strictly true),
but far removed from it (on the left side) in the larva of
Antedon. In making this statement, however, he seems to have
forgotten the fact that, before the pore appears, the body-cavi-
1 A slight alteration of my drawing has brought it into a radial position in
fig. 48,
THE METAMORPHOSIS OF ECHINODERMS. 105
ties have already shifted their position in Antedon, so that the
relation of the pore to the ‘* Medianebene ” of the larva as a
whole no longer has any morphological importance.
If we compare Antedon in the “Cystid” stage with an
KEchinid, we shall see that in both the water-pore may be stated
to lie in the same interradius as the cesophagus, alongside
which runs the mesentery holding the water-tube. From this
point in both cases the alimentary canal runs round the disc
till it again reaches this same interradius. It is true that only
in Antedon does the intestine end here, but remembering that
evidence which Echinids afford of the variability of the anal
interradius, this is of small importance; and it is a bold thing
to deny that the interradius of the water-pore is not homo-
logous in these two forms, yet Echinid Plutei have the water-
pore even more nearly in the “‘ Medianebene”’ than the Holo-
thurians on which Seeliger relies. A further argument might
be derived from the skeletal plates, but it is unnecessary to
pursue it; it is enough for my purpose to show, not the impos-
sibility of Seeliger’s assumption, but the extreme weakness of
the evidence. In the larve of all Echinozoa the water-pore
lies at the anterior end of the dorsal mesentery, though usually
somewhat to the left. In Bipinnaria the mesentery becomes
oblique (fig. 22), and the pore is consequently pushed to the
left of the ‘‘ Medianebene” ; in Antedon this mesentery is still
more oblique at the time the pore appears, and consequently
the latter is situated further still over on the left side—even,
indeed, on the ventral surface. This, at least, appears to me
to be a far easier explanation of the phenomena than Seeliger’s
supposition that the pore has changed its interradius.
A change of position of the union of the water-tube with
the water-vascular ring would be a good deal more difficult to
detect ; but in the entire absence of any evidence that it has
taken place, we are not justified in assuming it as the cause
of the straight course of the former, so long as any other
explanation is possible. That another cause is not only con-
ceivable, but actually supported by ontogeny, I shall now
endeavour to show.
106 HENRY BURY.
Turning to fig. 48, we see that a gradual rotation of the
water-vascular ring, as it moved into the left side, would
suffice to maintain the straight course of the water-tube,
without any change of interradius of either of its extremities
—the total angle of rotation being about 70°. Now compare
Ludwig’s diagram (15, p. 157, fig. v) of the changes by which
the “ambulacralen Armanlagen” (hydrocel pouches)
are brought into connection with the “antiambulacralen
Armanlagen” (formed over the left body-cavity, parallel
with the mesentery), and it will at once be evident that we
have here a twisting of the hydrocel through a considerable
angle (though not as much as 70°) in exactly the same direc-
tion (remembering that Ludwig gives a dorsal view and I a
ventral) as I have postulated ; the movement, in fact, may in
both cases be described as tending to unscrew the water-
vascular ring from the stomach.
Here, then, we have a possible explanation of a very remark-
able phenomenon. Of course so long as we are ignorant of the
meaning of the extraordinary variability in the point of closure
of the water-vascular ring noticeable in Echinoderm larve, we
cannot hope to explain all the peculiarities of development pre-
sented by Brachiolaria ; but the meaning just suggested for one
of the most striking of them fits in so completely with the needs
of my hypothesis that I cannot help attaching a good deal of
importance to it. In considering why this change is more
strikingly presented by Brachiolaria than by any other larva,
we must remember that this form is peculiar in that the
hydroccel early arrives at its position alongside the stomach,
while the cesophagus only joins it there at the time of meta-
morphosis. In Bipinnaria and in Echinids the hydroceel very
early assumes its final position, and a new cesophagus is
formed; so that the rotation of the hydroccel (if it is not
entirely omitted in ontogeny) probably takes place before the
development of the tentacles enables us to recognise it. In
Ophiurids the rotation ought to take place exactly as in the
ancestor; and I think when we come to consider the matter
carefully we are bound to admit that it does so, though the
THE METAMORPHOSIS OF ECHINODERMS. 107
details are a good deal masked by what appear to be secondary
and purely ontogenetic processes.
Hitherto, for simplicity of description, I have regarded the
stomach as fixed and the cesophagus as undergoing movement ;
but seeing that the mouth is always on the ground, to which
the animal adheres by its tentacles, it might have been more
accurate to regard this as the fixed point, and speak of a move-
ment of the stomach across the base of the cesophagus. One
of the first results of this movement is to throw the water-pore
and front end of the mesentery across to the right side, as seen
in fig. 46, and this finds its counterpart in ontogeny in the
position of the water-pore in a late Ophiurid Pluteus (fig. 43).
But a close comparison of these two figures will show us that
in the Pluteus some other changes have occurred as well. The
mouth was from the first ventral, and in order that the left
body-cavity may surround it, there must be a movement of
this cavity towards the ventral side. There are in fact two
movements, one tending to make the left side of the stomach
anterior, and the right side posterior (as in fig. 46); and the
other pushing the left side on to the ventral, and the right
side on to the dorsal surface. The former would lead to great
asymmetry, which the latter would to some extent counteract ;
and it is just possible that we have here the reason of this
second movement, though as we are as far as ever from the
reason of the first, this suggestion is not of much value.
Now if we consider carefully what the nature of this second
movement is, we shall see that it involves exactly that ‘ un-
screwing” of the stomach from the cesophagus which we have
already seen in Brachiolaria, and which we now see must also
take place in Ophiurid Plutei. An examination of the hydrocel
pouches in this larva points in the same direction. At first they
are arranged as a longitudinal series along the left side of the
cesophagus (fig. 40); then one by one they pass across the
dorsal surface of this organ, and encircle it; and finally, at the
time of metamorphosis the water-pore also moves forwards and
to the right (by a process already explained), and we reach the
rather curious result that that tentacular pouch, which was
108 HENRY BURY.
originally posterior, is now the most anterior of all. It would
be difficult and tedious to describe in full the changes of
position which the other pouches undergo, but in fig. 40 [
have placed numbers against them so that a comparison with
5, figs. 2—6, will show with which arm-rudiment each unites.
Much of this complicated process no doubt is due to ontogenetic
causes, and is unconnected with phylogeny, but the movement
by which the primarily anterior tentacle (see 5, figs. 2 and 4)
passes all round the cesophagus and finally is embraced by the
second terminal plate is so peculiar, and so exactly corresponds
in direction with the movement of the hydroceel in Asterina,
that it can hardly be without significance. In both larve it
will be noticed that the pouch which lies immediately anterior
to the water-tube subsequently unites with that terminal plate
which is the most posterior of the longitudinal series in the larve
(5, “ Terminal 5 ” in fig. 2).
The two movements spoken of above are combined in such a
manner in Ophiurid Plutei that at the end of metamorphosis
the original antero-posterior axis, still traceable with the help
of the arms of the Pluteus, passes distinctly to the left of the
water-pore (compare 9, figs. 5 and 6); this is due, as already
explained, to the first movement of the above description, while
the second movement is responsible for the fact that the former
left side of the stomach is at this stage (if we may trust the
external evidence of the calcareous plates) entirely on the
original ventral surface. In Crinoids, on the other hand, it
would seem that the first movement has little effect, while the
second carries even the pore far over to the left of the original
‘‘ Medianebene.” An intermediate condition between these
two is seen, as already indicated, in Bipinnaria (fig. 22).
As previously mentioned, the position ascribed to the dorsal sac
and organ in the bilateral ancestor will be discussed especially in
connection with the relation to the Enteropneusta; but it is worth
while here to show that, if that position be admitted, the move-
ments we have just been considering will bring these organs
exactly into the relation to surrounding structures which is
observable in adult Echinoderms. The dorsal sac has so far
THE METAMORPHOSIS OF ECHINODERMS. 109
only been found in the larve of two groups—Asterids (both
forms of larvee) and Echinids, but I shall assume for the present
that it has a phylogenetic value. Situated, as it is in these
larvee and in my hypothetical ancestor, just to the right of the
water-pore, it seems, like the latter, to have escaped the move-
ment into the left side, or perhaps I ought to say, has moved
with the pore and the anterior end of the stomach into an
equatorial position. A glance at my diagrams will show that
what was formerly on the right side of the water-pore must
now be on its aboral side; and this is precisely the position
of this sac in adult Asterids and Kchinids.
The axial organ was assumed to run forward along the dorsal
surface of the oesophagus, and it seems to have accompanied
the latter in its change of position relative to the stomach and
mesentery. As it lay to the right of the water-tube in the
bilateral stage, so now it still occupies the same relative position
when the animal is viewed from the aboral side with the inter-
radius of the water-pore directed away from the observer.
One end of the dorsal organ, however, was assumed to lie under
the dorsal sac; if it retained this position it must, in the
pentamerous stage, pass round at the aboral end of the water-
tube from the right side of the latter to the aboral side of the
water-pore, and this is precisely the course which, as
Ludwig has shown (18, p. 159) it follows in adult
Asterids. It seems to me that, apart from any possible
homologies with Enteropneusta, it is not an unimportant feature
of my hypothesis that it enables us to derive this very peculiar
and asymmetrical course from an originally symmetrical one ;
and I believe that, if ever the axial organ is discovered in the
bilateral stage of an Ophiurid Pluteus, it will be found to
occupy exactly the position ascribed to it in my bilateral
ancestor.
In fig. 45 I gave a forward extension to the anterior body-
cavity, in order that it, with the water-tube and dorsal organ,
might be brought down into the left side, and there by en-
veloping these two structures form the “axial sinus.” That
it might be made to do so is sufficiently obvious; but until
110 HENRY BURY.
more is known of the development of this sinus not much
importance can be attached to the suggestion. The fusion of
left anterior and posterior cavities in Asterids makes it rather
doubtful whether I was justified in asserting (5, p. 37) that the
axial sinus was a part of the former, though I see that MacBride
(19, p. 483) accepts my position. In Ophiurids, on the other
hand, the same authority states (18, p. 135) that the axial sinus
arises from the posterior cavity, quite distinct from the ampulla
(anterior cavity). In Echinids I confess I am not sufficiently
confident in my observations to assert a definite origin for this
sinus. Further investigation must be left to settle this point,
which fortunately is not very important to our present inquiry.
The nomenclature of all these parts is in a great state of
confusion ; but as long as we use the various synonyms merely
as names, and not as descriptions of position, not much harm
will accrue. Seeliger objects to the term “dorsal organ”
because this structure is apparently more ventral than dorsal
in Antedon; but what is to be said of the application of the
epithet “axial,” which he prefers, to Ophiurids and Asterids?
My hypothesis indicates that this organ may have been at one
time dorsal, but it certainly is not so in true Echinoderms, and
this being the case, both terms (axial and dorsal) seem to me
equally objectionable if held to be descriptive, and equally
unobjectionable if used merely as convenient names.
Further Development of Radial Symmetry.
The ancestral form at which we have now arrived would
probably, if found in a fossil state, be included in that very
heterogeneous group, the Cystidea, from the simpler forms of
which it differs chiefly in possessing only five tentacles. If,
however, I am right in supposing that it was not fixed, and that
these tentacles were used for locomotion, it must have been of
such extremely small size as to render its discovery as a fossil
very improbable ; for though, for the sake of clearness, I have
in my diagrams (figs. 45 and 46) made the tentacles more
slender than is probable, yet it is impossible to imagine an
animal of any large size supporting itself upon only five of such
THE METAMORPHOSIS OF ECHINODERMS. hit
organs. It was probably increase of size which led to the
further development of the ambulacral system, and this in its
turn led to the completion of that radial symmetry which is
among the most striking possessions of the Echinodermata.
The first addition to the ambulacral system probably took
the form of the production of five pairs of tentacles at the bases
of the original five, so that the total was raised to fifteen. The
next stage is not so easy to follow. In Crinoids five more pairs
are added between the previous pairs and the mouth, that is to
say, in centripetal order; and then, with a total of twenty-
five, there is a long pause. Many Cystidea never get beyond
this total, so that we may regard it as primitive, at any rate
for the Pelmatozoa.
As regards the Echinozoa, there is something of a pause
when the number twenty-five is reached in Ophiurids, but not,
I think, in other groups; and even here there is no evidence to
show in what order the pairs arise. In all later-formed tube-
feet the succession in Echinozoa is invariably centrifugal
(acropetal), and this seems to be the case even from the first in
Holothurians. In Crinoids, however, a totally distinct order
is observable. We are then driven to the conclusion that the
Pelmatozoa branched off at least as early as the stage with
twenty-five tentacles, and there is some evidence that the
common ancestor did not get beyond a total of fifteen.
At this point we may leave the Pelmatozoa, the origin of
which will be discussed later, and for the present direct our
attention chiefly to the Echinozoa, in which, so far as we can
see, the increase in number of tube-feet (tentacles) went on
pretty steadily, in acropetal order.
It is obvious that no great number of tentacles could start
from the water-vascular ring itself, nor would their concen-
tration there allow of any great increase in bulk in the body as
a whole, and this seems to have led, from a very early stage,
to the development of radial vessels, which may be described
as elongations of the bases of the five primary tentacles, from
which the paired tentacles sprang; and since these vessels
would have had no strength if they had grown out, like
112 HENRY BURY.
tentacles, free from the rest of the body, they were forced to
spread over the oral surface, which thus became divided up
into radial and interradial areas. This spreading of the
ambulacral area, which to a large extent seems to have
occurred independently in Echinozoa and Pelmatozoa, has
apparently been the cause of that “radial segmentation ” of
various organs which I shall now attempt, very briefly, to
follow.
It is probable—though we have no direct evidence on the
subject—that the concentration of a portion of the nervous
system into a ring round the mouth took place at an early
period, and that this ring supplied the tentacles. When the
water-vascular system spread over the surface of the body, the
nerve-bands would naturally accompany it, and thus we should
get a definitely radial arrangement of the nervous system. A
special development of the muscular system along the same
lines might be expected, and so it too would exhibit radial
symmetry.
The Generative Organs appear to have followed the new
(radial) symmetry at a fairly early period. What form they
assumed in the bilateral ancestor we are not in a position to
assert ; they may very possibly have consisted, as they do now
in Elasipoda, of a pair of glands opening by a median aperture
on the dorsal side, just behind the water-pore; then, when
radial symmetry was established, other pairs of glands (one
pair for each interradius) may have been developed, connected
together by the genital rachis ; but whether this segmentation
of the gonads took place in the common ancestor, and was
afterwards lost in Holothurians, or whether the latter branched
off from the parent stock before this was accomplished (and
therefore before the separation of Echinozoa and Pelmatozoa),
I am quite unable to determine. In any case the assumption
of a primary pore just behind the water-pore (or rather, being
median, behind the dorsal sac), enables us to understand why
it is, as I have endeavoured to show, that the genital rachis
marks the original line of division of the body-cavities—its
growth simply following the line of the mesentery, in which
THE METAMORPHOSIS OF ECHINODERMS. 113
the primary genital pore lay. It also agrees with the direc-
tion of the growth of the rachis round the disc in ontogeny.
Whether the Skeleton was one of the first structures to
assume radial symmetry, or whether this symmetry has been
independently acquired by Echinozoa and Pelmatozoa respec-
tively, is a question which is still too much sub judice for me to
attempt to decide it. It may freely be admitted that when once
the new symmetry was thoroughly impressed upon the body, any
set of organs might acquire it in independent groups (we shall
see directly that this has been the case) ; so that there is no
reason why homoplasy should not be responsible for all the
supposed fundamental homologies that P. H. Carpenter and
others tried to establish. But between this and asserting that
all the apparent resemblances are homoplastic, there is a very
great difference. I admit that we cannot rely upon the
skeleton alone to settle this question; but until we have
arrived at far more certainty than at present as to the rela-
tions of the various classes of Echinoderms, I do not think
we are justified in absolutely denying the possibility of homo-
logy between, for example, the dorso-central and basal plates
of Crinoids, and the similarly-named plates in Echinozoa. We
ought rather to return an open verdict and wait for fresh
evidence.
The utter unimportance of this question to my hypothesis
led me, as far as convenient, to omit all mention of the
skeletal plates in the first part of this paper; one or two bits
of fresh evidence, however, there introduced are worthy of con-
sideration. In the first place there is not the smallest
embryological ground for Neumayr’s statement (25, p. 498)
that the primitive arrangement of the plates in Echinids is in
rows of ten; indeed, the invariable development of all plates
in Echinoderm larve not directly connected with the ambu-
lacral system in groups of five, is a very striking coincidence if
the symmetrical arrangement has arisen out of primitive chaos,
at various times and in ancestors of various sizes, independently ;
while it is easily intelligible if the number five was established,
at any rate in a few plates, at that early stage when the
vol. 38, PART 1.—NEW SER. H
114 HENRY BURY.
ancestor was still extremely small and the tentacles few in
number.
Next I would point out that the correspondence of the
series of plates with the body-cavities indicates that their
symmetrical arrangement arose at a period when the distinc-
tion between these cavities was still very strongly impressed
upon the animal. In recent adult Echinoderms this relic of
bilateral symmetry is almost entirely lost; but in ontogeny,
as we have seen, it is often retained (with approximate
equality of the body-cavities) for some time after metamor-
phosis.
Lastly, my observations go far to prove that, whatever may
be true of other skeletal plates, we have in Echinids, Asterids,
and Ophiurids at least one set—the terminals—which are
homologous. They are developed over the left body-cavity,
and in all cases embrace the unpaired tentacles at the ends of
the radial canals, which may be the reason that in these
sroups, though not apparently in Holothurians, the ambula-
cral system has failed to extend itself over the region of the
right body-cavity. It must be admitted, however, that this is
also true of Crinoids, in which the terminal plates do not exist.
The organs hitherto considered may (though this is uncertain)
have assumed a radial arrangement before the separation from
the parent stem of any of the recognised classes ; but there are
a few cases of radial symmetry which must have been arrived
at independently by the groups in which they occur, This is
the case in the water-pores (five in number) of Rhizocrinus,
which seem to form a primitive feature in Crinoids (since
Antedon passes through such a stage), but are not found in any
other Echinoderms. Whether four of them were developed
(simultaneously (one of course being primitive), or in succession
as in Antedon), is not quite evident.
A similar case of the acquisition of radial symmetry by indi-
vidual organs is seen in the form of the stomach in Asterids
and Ophiurids. In Echinids, Holothurians, and Crinoids the
alimentary canal is a thin tube winding round the disc without
any trace of radial arrangement.
THE METAMORPHOSIS OF ECHINODERMS., LHS
We are strongly reminded by the above phenomena of the
varying degrees in which metameric segmentation has been
acquired in other animals. Indeed Bateson (8, p. 432) regards
the rays of Echinoderms as a successive, not a truly radial series.
This is not invariably true in ontogeny; and if the explanation
given above of the origin of radial symmetry in Echinoderms is
correct (though I am far from asserting that it is), it will be
seen that this symmetry was acquired by all organs except the
water-vascular system in the radial stage, and that therefore
the existence of pentamerism in the skeleton of bilateral larve
is precocious.
We have seen that, in the remote ancestors we have been
considering, there was probably but little difference in size
between the two body-cavities ; indeed, if the position of the
anus may be taken as an indication of the level of the mesen-
tery, the right body-cavity was, in many of the simpler Cystids,
much larger than the left, as in fact it is in the “ Cystid”’ stage
of Antedon. In Echinids and Asterids, however, the growth
of the ambulacral area (connected, as we have seen, with the
left cavity only) has led to an enormous preponderance of the
left over the right cavity. Hence the bulk of the alimentary
canal came to lie in the region of the left cavity, and it need
not surprise us if we find the mesentery forsaking its old posi-
tion and following where it is most needed. This seems to
have happened in Kchinids, in which the intestinal mesentery
undulates up and down in the region of the left cavity ;
while the old line of separation of the cavities is still marked
by the genital rachis, and, to a less extent, by the skeletal
plates.
In Asterids the mesentery probably retained its original
position for a longer period, the stomach being supported by
the septa; but with the outgrowth of the hepatic ceca into
the arms, it too forsook its former position, which the genital
rachis now alone marks.
The curious distribution of the two cavities in Ophiurids (as
indicated by the genital rachis) may perhaps be due to an early
116 HENRY BURY.
connection of the water-pore with one of the plates of the left
body-cavity (the so-called “ orals’”’). This pore was originally
situated between the two series of skeletal plates, as ontogeny
shows, and its connection with one or other of the plates adjoin-
ing it has apparently occurred independently in the different
classes; but in no case, to the best of my belief, has it ever
wandered far away from the original line of separa-
tion of the two body-cavities.
The freedom of the intestinal mesentery to move where it is
required has led in some cases to a portion of it traversing
part of the right body-cavity. In my hypothetical ancestor
the anus is placed in interradius A, at the level of the division
of the body-cavities, as we find it in the larva of Antedon. In
Asterids and young Echinids, however, we see that it has shifted
into interradius E,and into the region of the right body-cavity—
being aboral to the basal plates. This, however, may be due
not toa shortening but to a lengthening of the intestine,
which, in Kchinids at any rate (and in the larve of Asterids)
reaches interradius A, and then (in Echinids) turns back to
the anus in interradius E.
It is just possible that this position of the anus may be
primitive ; that at the very time that the mouth was working
its way into the left side, the anus may have been moving into
the right, but being (conceivably) prevented from reaching the
pole by the dorso-central plate, it turned a little to the side into
interradius E. In this case the position observed in Antedon must
be regarded as secondary, due to a shortening of the intestine
consequent upon the occupation of the aboral pole by the disc of
fixation ; and the “ aboral longitudinal mesentery,”’ found no-
where but in the larva of Antedon, and running from the aboral
pole in interradius E to the level of the transverse mesentery in
interradius A, very possibly marks the original course of the
terminal portion of the intestine. I put this suggestion for-
ward for what it is worth, knowing full well the weakness of
the evidence. The ontogeny of other Crinoids may show us
its true value, but the absence of the intestine in the larva of
Antedon deprives it of all importance in this connection.
THE METAMORPHOSIS OF ECHINODERMS. Ez
It would be altogether beyond the scope of the present paper
to try and follow further the separation of the different classes
of Echinoderms. The special features of the Pelmatozoa
and Holothurians will be briefly considered in tbe following
pages: but the exact period of separation of these and the
remaining classes seems to me to require much further evidence.
I will only add here, as following closely upon the point we
have just been discussing, a protest against Cuénot’s (7)
attempt to attach classificatory and phylogenetic value to
the persistence or closure of the anus in ontogeny ; it may or
may not possess this value, but in the present state of our
evidence it would seem almost as reasonable to attach import-
ance to the persistence of the larval cesophagus in Ophiurids,
some Asterids, and some Holothurians, or to the entire absence
of the intestine in the young stages of Antedon and Asterina,
as opposed to its invariable presence in all pelagic larve.
Origin of Pelmatozoa.
I have already given my reasons for believing that the
Echinozoa were never fixed by the aboral pole, and that the
utmost that embryological evidence will allow of is a fixation
of the bilateral ancestor by the przoral lobe; and even this
fixation, if it had been complete, and not a mere voluntary
process effected by means of a sucker, would, in my opinion,
have left more traces behind it than the study of embryology
has yet afforded us. The existence of such a sucker is certainly
possible, though the evidence for it is weak, and I do not
think the assumption of it necessary. But before pursuing
the subject further it is worth while to point out that whether
the idea of primitive fixation is accepted or rejected, it remains
certain that the changes of symmetry which we have been
considering in the foregoing pages are common both to
Echinozoa and Pelmatozoa ; and since the acceptance of this
idea, in whatever form, does not provide us with any explana-
tion of how these changes came about, my hypothesis—which,
indeed, only deals with the nature, not the cause, of these
changes—is obviously independent of it.
118 HENRY BURY.
Accepting for the present the supposition of a preoral
sucker in the bilateral ancestor, it would seem that it ought,
in the pentamerous form, to lie in the same interradius as
other organs (water-pore, water-tube, dorsal organ, &c.)
belonging to the same region; and here, I think, we meet
with the one serious objection to this view, which involves
the homology of the stalk by which Asterina is fixed with that
of Antedon. The latter, as we have seen, is either in radius
V, or, as I think, in interradius A, while the former, ex-
ternally at least, is in the next interradius (E). It is true that
it has its roots, so to speak, in interradius A, since it is there
that we find the remnant of the anterior body-cavity, which
before metamorphosis extended far into the przoral lobe; and
the whole stalk has undoubtedly a very oblique aspect in
Asterias rubens, though I have not satisfactorily traced
its internal relations. It is to be hoped that MacBride, in
his detailed account of Asterina, will give us some explana-
tion of this point, which at present seems to me opposed to
the homology he supports.
Thus the evidence of the existence of even a sucker in the
common ancestor is extremely weak, and it is worth while
to see whether the supposition that the Pelmatozoa became
fixed after the change of symmetry, instead of before it, is not
at least as probable.
Let us imagine that a sucker arose in the interradius of
the water-pore, somewhere between that pore and the mouth;
how or why it arose I cannot attempt to determine (expla-
nations of this kind are, indeed, very seldom possible), but
at least I see no a priori objection to the suggestion. The
selection of this particular radius may have been purely
a matter of chance, but the eccentric position of the mouth in
the Cystid larva of Antedon, as well as certain peculiarities
in the ambulacral fields—‘‘ hydrospires palmées ”’ of
Barrande (2)—of some Cystids suggest that perhaps this
radius may have been rather different from the rest in early
stages of pentamerism—possibly, even, it may have been (as it
still is morphologically) the anterior interradius, and loco-
THE METAMORPHOSIS OF ECHINODERMS. 119
motion may have been, as in the bilateral stage, in its direction.
However this may be, if fixation, beginning probably with a
mere sucker, afterwards assumed a more permanent character,
we have plenty of parallel cases to show the possibility (or even,
perhaps, probability) that the mouth would move away from
this point, and eventually reach the opposite pole. Here
again no certain cause can be assigned; we may suggest that
this movement was necessary to place the mouth in an advan-
tageous position for obtaining food ; but if the earlier position
was disadvantageous, why did fixation ever take place? All we
can say is that, if we may trust embryology, such a movement
is a very common sequel to fixation by a point near the mouth.
The exact position of this sucker in the ancestor of the
Pelmatozoa I cannot determine with any certainty. It may
have been anywhere on the oral surface, but we do not even
know what the extent of this surface was. In the later Cystid
stages of Antedon (26, pl. x, fig. 90) the mesentery is oblique
and the oral surface is fairly flat right up to the level of the
water-pore, and this is true also of young Ophiurids ; while in
some Cystids, though this surface is not so flat, an equatorial
line dividing the animal into equal oral and aboral halves
would lie far on the aboral side of the anus and water-pore, if
the identification of these apertures can be trusted. But
without going to such extreme cases as this, it is very easy to
understand that the hypothetical sucker may have occupied
almost any position between the transverse mesentery and the
mouth, though probably not within the atrial cavity. As its
subsequent migration to the aboral pole does not seem to have
affected the water-pore, it is probable that it lay nearly over
the centre of this interradius, and therefore to the right of the
pore, which, as we have seen, is adradial. But, while thus
avoiding both water-pore and water-tube, it may possibly have
involved some portion of the dorsal organ, which, as the diagrams
show, lay further to the right than either of these structures ;
or if it was very broad, and was situated at the level of the
mesentery, it might (though this is less probable) involve in
its movements the dorsal sac.
120 HENRY BURY.
In one of these two ways, it seems to me, we may find a
possible explanation of the very curious course taken by the
dorsal organ in Crinoids. So striking is the difference of
position of this organ in Echinozoa and Pelmatozoa, that Cuénot
denies the usually accepted homology. In combating this view
MacBride (18, p. 147) suggests that the dorsal organ of Crinoids
may be homologous with that portion of the “ovoid gland”
in Asterids and Ophiurids which lies on the aboral side of the
genital rachis. But this seems to me to imply misconception
of the relations of the parts involved. The remarkable point
about Crinoids is that the dorsal organ traverses the right
body-cavity, no part of it, so far as we know at present, lying
in the region of the left cavity. In Echinozoa, on the other
hand, almost its entire course is in the region of the left cavity,
the only portion which extends beyond this region being the
small aboral portion which ends in the dorsal sac in Asterids
and Kchinids, and lies under “ sinus b” in Amphiura (18,
fig. 2,e). There is not the smallest evidence that the dorsal
organ in any of the Echinozoa extends beyond the mesentery
into the region of the right body-cavity. This, so far as it
goes, furnishes an argument in Cuénot’s favour; but the
other objections to his view are so many and important, that
most embryologists will be loth to accept it, so long as any
other explanation is possible.
Now if, as I have supposed, the ancestor of the Pelmatozoa
was fixed by a point lying over the dorsal organ, and
this point afterwards migrated to the aboral pole, might
not the subjacent organ share in this migration, and after-
wards, taking the shortest course from this pole to the mesen-
tery, pass, as it does in Antedon, through the right body-cavity,
in the concavity of the alimentary canal ?
Of course this is only put forward as a suggestion, and
would require a great deal more evidence to prove it; but at
least it seems to me to be more satisfactory than Cuénot’s
denial of homology, which no amount of fresh evidence seems
likely to make wholly satisfactory, placing, as it does, a wider
gulf between the Echinozoa and Pelmatozoa than the many
THE METAMORPHOSIS OF ECHINODERMS, 121
similarities between the two groups would justify us in recog-
nising. With this exception the supposed migration of the
disc of fixation to the aboral pole would be for the most part
superficial in its effects, and the general arrangement of the
internal organs would not be altered by it.
If a dorso-central plate existed at the aboral pole of the
ancestor, I see no difficulty in supposing that the disc of fixa-
tion might come to lie external to it, the animal up to this
stage remaining sessile; and that when the stalk was formed
this plate was borne out on the end of it. But whether we
accept this skeletal homology or not, I do not see sufficient
erounds for adopting MacBride’s conclusion (19, p. 486) “ that
the abactinal poles of Asterina and Comatula are not
comparable with each other, and that all conclusions
based on the supposed homology of the dorso-central of
Echinids and Asterids, and that in Crinoids, are incorrect.”
The exact stage atwhich this fixation may be supposed to have
occurred is not, perhaps, a matter of great consequence. The
frequent occurrence in Cystids (some of which may almost
certainly be regarded as the earliest Pelmatozoa) of twenty-
five tentacles (five to each radius) suggests that this may have
been the number reached by the common ancestor of all the
Echinoderms ; but, as already mentioned, the absence of any
pause at this stage in most Echinozoa, as well as the apparently
anomalous order of development uf these twenty-five in Ante-
don, render this extremely doubtful, and make it perhaps
more probable that the separation of these two main divisions
of the Echinodermata took place at a still earlier period, though
whether at a stage with fifteen tentacles or with only five there
is no evidence to prove.
It remains for me to show that the hypothesis put forward
above is not inconsistent with the apparent teaching of embry-
ology that fixation took place by the preoral lobe. It seems
to me that we are apt to speak of this lobe as if it were a defi-
nite organ, instead of a region of the body possessing a
great number of parts; and that though Brachiolaria and the
larva of Antedon are both fixed, apparently, by the przoral
122 HENRY BURY.
lobe, we are not at present in possession of any facts to show
that the same part of the lobe is involved in both these cases;
so that even the proof of the homology of their position is
yet imperfect, while the proof that they had a common
origin, and are therefore completely homologous, is further
off still.
We have seen that the organs connected with the preoral
lobe of Echinoderm larve are situated in the radial stage, in
what I have called the anterior interradius; and it is evident
that a disc of fixation arising in this interradius might very
easily appear, in ontogeny, to be a part of the preoral lobe
itself. That some such confusion of two phylogenetically
distinct structures does occur in the larva of Antedon is very
strongly suggested by the fact, to which I have already drawn
attention, that the so-called przoral lobe of this larva contains,
at the time of fixation, certain structures (skeleton and con-
tinuation of the right body-cavity) which cannot well be re-
garded as primarily belonging to this lobe, but rather to the
stalk, which is itself apparently a secondary structure—the
earliest Pelmatozoa having been, as paleontology teaches,
sessile.
The position of the water-pore in the larva of Antedon also
accords very well with the view here advanced, though it is
not inexplicable on other hypotheses. It is difficult to illustrate
this point without undue multiplication of diagrams, but
perhaps fig. 50 may be of some service. I have assumed that
the disc of fixation arose in the interradius of the anus and
water-pore in such a stage as is shown in fig. 48, and then
moved round to the aboral pole. If, before this movement
had proceeded far, the stalk and the extension of the right
body-cavity into it were, in ontogeny, precociously developed,
something very like fig. 50 would be reached; compare this
with the larva of Antedon at the moment of fixation, and we
shall see a possible reason why the water-pore in this larva is
so far removed from that dorsal position which it assumes in
other Echinoderm larve.
Of course I am well aware that the above suggestions as to
THE METAMORPHOSIS OF ECHINODERMS. 1238
the origin of the Pelmatozoa are of an extremely speculative
character, and will require a great deal of evidence to support
them ; but a too rigid adherence to the apparent teachings of
the larva of Antedon is really open to equally strong objections.
In opposition to those who, like Seeliger, regard the ontogeny
of this larva as a safe guide to phylogeny, it cannot be too
strongly urged that at present we only know the development
of one Pelmatozoan larva, and that we have no reason for
regarding this as specially primitive ; on the contrary, the very
early loss of bilateral symmetry in the arrangement of the
body-cavities, as well as the entire absence, before fixation, of
either oesophagus or intestine, point most conclusively to its
being a much altered form.
Origin of Holothurians.
Until phenomena similar to those which I have described
in Synapta have been observed in other Holothurians, it
would be rash to attempt more than a cautious suggestion as to
the origin of this class; Even in Synapta my investigations
are unfortunately far from complete, but so far as they go
they appear to teach a fairly definite lesson.
In the light of our knowledge of other Echinoderms, we are
justified in regarding the asymmetrical movement of the atrial
aperture, and the formation of the mesentery of the water-tube
by the left body-cavity (a portion of which grows round the
cesophagus for this purpose), as indications that in the ancestor
of Holothurians also the movement of the esophagus into the
left side has taken place. But, on the other hand, although
we cannot determine the exact limits of the larval body-cavities
in adult Holothurians, we can certainly assert that the left
cavity is not symmetrically disposed about the cesophagus, and
that the mesentery of the water-pore is not, as in other
Echinoderms, at right angles to the mesentery of the stomach
and intestine (dorsal mesentery of the larva), but nearly in a
straight line with it. These facts, coupled with the observed
migration of the atrial aperture towards the anterior pole,
124 HENRY BURY.
suggest that the mouth, after first moving into the left side,
has undergone a secondary change of position, accompanied
of course by the water-vascular ring.
To some extent this is parallelled by the Spatangidea, and
(possibly) by Actinometra; but the case of the Holothurians
presents certain marked pecularities. In the first place the
secondary migration of the mouth has apparently been such as
exactly to retrace the line of the original movement—that is to
say, it has occurred in the direction of interradius A (compare
figs.47 and 48); though in this it does not differ very widely from
the Spatangidea, in which the secondary movement has been
towards radius I (see 14, pl. xiii, fig.8). Secondly,it has not been
a simple movement; for that, while it would reduce the length of
the mesentery of the water-tube, would still leave the same
angle between this and the mesentery of the stomach and intes-
tine which we have found in other Echinoderms. But in
Holothurians these two mesenteries are nearly in the same
straight line; and this can, I think, only be accounted for
by supposing that the torsion of the water-vascular ring
which we traced in the common ancestor has been reversed
and undone during the secondary movement, so that this
ring has returned very nearly to the position assumed for
it in the bilateral ancestor (fig. 45), in which the dorsal
mesentery may be said to lie in interradius A (the interradius
of the water-tube). One great difference, however, exists
between this secondary Holothurian ancestor and the ancestor
shown in fig. 45. In the latter the body-cavities do not run
forward on to the csophagus, but in the former, during the
time when, in common with other Echinoderms, its mouth was
on the left side, the left body-cavity learnt, so to speak, to
surround the «esophagus and form a mesentery for the
water-tube; and this peculiarity, once acquired, was not lost
during the secondary changes, but is still traceable in the larva
of Synapta; and it accords very well with this view that,
whereas in other forms (compare fig. 17) a dorsal as well as
a ventral horn of the left body-cavity is observable, in Synapta
the ventral horn alone is conspicuous; this, however, is a point
THE METAMORPHOSIS OF ECHINODERMS. 125
which cannot well be illustrated by figures in two dimensions,
but will be found by those who take the trouble to con-
struct models, to offer interesting evidence in favour of the
view here advanced. In fact, the only obstacle to this view
with which I am acquainted lies in the unusual position of
the water-pore on the right, instead of as usual on the left, of
interradius A (see fig. 14); but this is equally puzzling on any
other hypothesis, except Semon’s untenable one that the five
tentacles of Auricularia are radial; and indeed, as already
suggested, the difficulty may be more apparent than real,
being perhaps due to the precocious development of the ten-
tacles of radius I, while one of those belonging to radius V
only appears later (see 16, p. 183).
The further question of the exact stage at which the Holo-
thurians branched off from the parent stock, is not one on
which I care to express any very decided opinion. I would
point out, however, that it is difficult to conceive of any torsion
of the water-vascular ring occurring after the radial canals had
spread far over the disc and become united with parts of the
body-wall; and consequently I am inclined to the belief that
the separation of this class occurred very early, perhaps even
before that of the Pelmatozoa. The possibly primitive character
of the genital organs in the Elasipoda fits in very well with
this supposition ; while the fact that the ambulacral fields are
limited to the region of the left body-cavity in other Echino-
derms, but run to the extreme posterior end regardless of the
body-cavities in Holothurians, is, so far as it goes, opposed to
the derivation of the latter from any of the other groups of the
Kchinozoa.
Relation of Echinodermata to Enteropneusta.
Of the various features which have from time to time been
supposed to show affinities between the Echinodermata and
Enteropneusta, probably the least important, though one of the
first to attract attention, is the outward resemblance of certain
Echinoderm larve to Tornaria. No one who has seen the latter
and Auricularia alive can fail to be struck with their general
126 HENRY BURY.
similarity ; but it is difficult to regard Auricularia as a primi-
tive larval form, and even if we could, the details of the like-
ness are not sufficiently strong to prove a common origin.
Spengel ignores this resemblance, and suggests that Mor-
gan’s mention of Auricularia is a lapsus calami for Bipin-
naria,! though he does not himself attach any phylogenetic
importance to the resemblance of this larva to Tornaria. But
though these larve resemble one another in having the ciliated
band divided into two at the anterior pole, yet it is impossible
to regard this as a primitive feature in Bipinnaria, seeing that
no other Echinoderm larva normally possesses it; while the
ease with which it may be independently acquired is attested
by the fact that I observed precisely the same division of the
band into two, in one instance, in Auricularia.
Of far more importance is the presence in Echinoderms as
well as in Enteropneusta of at least one anterior body-cavity,
opening by a pore at its posterior end on the left side of the
body. My identification of this cavity in Echinoderm larve
(see 4 and 5) has met with a good deal of opposition, it being
by many regarded as a mere appendage to the hydroccel; but
the facts (1) that it always arises as early as, often earlier than,
the posterior cavities—generally earlier than the hydrocel ;
(2) that it is constant in position, arising and remaining
anterior to the stomach, with the pore at its posterior end;
(3) that it always has thin walls, while the hydroccel after the
first moment of its appearance has thick walls, seem to me to
go far towards refuting this view, and establishing the primi-
tive nature of the cavity in question. The homology of it
with the proboscis cavity of Balanoglossus may not be so well
established, but at least has too much plausibility to be lightly
set aside.
The existence of a second anterior cavity in many Echino-
derm larve is of less importance, since it seems to carry us
back to a far earlier period than the separation from a common
stock of Echinodermata and Enteropneusta. There is no clear
1 Morgan (22) has certainly made several statements about Auricularia
which are true, I believe, only of Bipinnaria,
THE METAMORPHOSIS OF ECHINODERMS. 127
evidence that the latter ever had a second anterior cavity,
though there are some grounds for believing it; but if they
had, its importance must have been subordinate before the
divergence of the two lines of descent, for we can hardly
suppose that the predominance of the left cavity has been in-
dependently acquired.
The same applies to the second pore discovered by Field,
and present in some Enteropneusta ; if it is really an ancestral
feature (which is not yet fully proved) it must have been lost
before the separation of the two groups, or we should not be
likely to find only one (the left) in the adult form of both.
Spengel’s suggestion, that the left collar-cavity of Tornaria
may be comparable with the hydrocel of Echinoderms, is
robbed of much of its value by his curious error in supposing
that Field and I have described in the latter a second (right)
hydrocel. There is, however, some plausibility in the
adoption of this comparision in the form which it assumes in
MacBride’s hands—a comparison of the true hydrocel (as
distinguished from the anterior enteroceel) with the collar-
cavity of Enteropneusta. The situation of the hydroccel in
young larve is certainly strongly suggestive of this; and the
obvious objection that the hydrocel is unpaired in most larve
is met by MacBride’s supposed discovery of a second hydroccel
in Asterina; and this writer even goes so far as to suggest
that in a pore leading directly from the hydroccel to the ex-
terior which he has found in one larva of Asterina, we have
the homologue of the collar-pore (20). As I have utterly
failed to find any trace of this second hydroccel in any of
the larvee I have examined, I may perhaps be forgiven if I
refuse, at present, to accept the evidence of such an obviously
secondary form as the larva of Asterina; and I would more-
over point out that one of MacBride’s own figures (19, fig. 4)
is out of accordance with his hypothesis, since the “ collar-
cavity” should be posterior to the anterior body-cavity, and
should not embrace its posterior end, though I must confess
that I am at a loss to understand why he ever drew his figure
in this form, as it is unnecessary for his hypothesis, and unsup-
128 HENRY BURY.
ported by evidence. Spengel’s idea evidently is that the whole
hydroceel ring is comparable to the two collar-cavities combined ;
and if there were any evidence of its two-fold origin, we might
readily adopt this view. It is just conceivable that the varia-
tion in the point of closure of the ring is in some way connected
with this, and that the hydrocel really does contain in itself a
right and a left element—its present development on the left
side alone being almost exactly paralleled by the development
of the right body-cavity in Auricularia from the asymmetrical
(left) hydro-enteroccel rudiment. But on this point I refrain
from offering an opinion.
As to MacBride’s “ collar-pore ” in Asterina, I would point
out the a priori improbability that the ancestral Echinoderm
ever possessed such a pore ; for if it did, why has the hydrocel
lost it own pore, and entered into a secondary connection with
that of the anterior enteroccel? Until further examples of its
occurrence have demonstrated a constant position for this pore,
it seems to me far more probable that we have in it simply
a case of multiplication of water-pores, such as has led in
Rhizocrinus to the presence of one in each interradius.
To the resemblances above mentioned, and some urged by
Morgan (22 and 23), I would suggest the addition of others,
which, if supported by further investigations, would go far to
bind the two groups together. The suggestions are not wholly
new, various scattered hints at them being found in the pages
of other writers; but the present paper offers, I think, far more
evidence than has hitherto been attempted.
At the base of the proboscis of Balanoglossus lies a*closed
vesicle ( pericardium,” ‘‘ Herzblase,’ “sac of proboscis
gland”), which is dorsal to the alimentary canal (notochord),
and, being in the middle line, has the water-pore on its left.
Underneath this “ pericardium,” and deriving its muscular coat
from it, lies the pulsating organ known as the “heart” it is
simply a lacunar space, as are the blood-vessels with which it
communicates, and may be regarded as a remnant of the em-
bryonic segmentation-cavity.
In intimate connection with these structures is the “ pro-
THE METAMORPHOSIS OF ECHINODERMS. 129
boscis gland.” It consists, according to Spengel, of a number
of folds of the epithelium of the proboscis cavity (anterior
body-cavity), and contains a number of cells with yellow
granules—apparently excretory; it is also supplied with
blood by a number of lacune.
Now I believe that we can recognise all these structures in
Echinoderms, though the metamorphic changes which the
larvee undergo greatly obscures their true position. In Asterid
and Echinid larve we find on the dorsal side, just over the
junction of stomach and esophagus, and to the right of the
water-pore, a closed vesicle of schizoceel origin. The floor of
this ‘‘ dorsal sac,”’ as I have called it, is raised up in Echinid
Plutei, and in these Jarve (and occasionally in Asterids) a
pulsation may be observed—not of the vesicle as a whole, but
apparently of its floor only.
Besides this, we have in all Echinoderms (except Holo-
thurians) a glandular organ known as the “ dorsal organ,”
‘‘axial organ,” “ovoid gland,” &c. The origin of this is
somewhat obscure. In Crinoids and apparently in Ophiurids
(18) it is at first a solid mass of cells; but in Asterids (Asterina)
MacBride describes it as “an ingrowth of the left posterior
celom into the septum separating the posterior coelomic cavities
from the axial sinus” (19, p. 433), that is to say, from the
anterior enterocel. I have not been able fully to satisfy myself
on this point either in Bipinnaria or Brachiolaria, but I would
point out that, owing to the fusion of the two cavities (anterior
and posterior), it must be almost impossible to determine with
certainty from which of them the organ in question is derived.
But whatever may be the case in the larva, its structure and
relations in the adult are very striking. In all forms it isa
much folded mass of apparently excretory cells, and in Asterids
and Kchinids, at any rate, if not in Ophiurids (Cuénot and
MacBride disagree on this point) it projects into the anterior
body-cavity (axial canal) in such a way that its folds may
be said to be involutions of this cavity.
It seems to me that we have a good prima facie case
in favour of the homology of the “ dorsal sac” and “ dorsal
VOL, 88, PART 1.—NEW SER. I
130 HENRY BURY.
organ” of Echinoderms with the “ pericardium” and “‘ pro-
boscis gland ” of Enteropneusta respectively.! It is true that
the observed position of the dorsal sac does not obviously
accord with this, but I have endeavoured to show in the fore-
going pages what its probable position was in the bilateral
ancestor, and that position accords very well with the homology
here suggested.
Our present knowledge of the blood-vascular system in
Echinoderms is too imperfect to allow of a detailed comparison
with that of Enteropneusta, though there is much in the con-
flicting evidence on the subject which is very suggestive. We
may say, however, without much fear of contradiction, that the
blood-vessels are simply lacunz, with no epithelial walls of
their own, and that these lacune penetrate all through the
complex structure of the “ dorsal organ ”—indeed in Holothu-
rians, where the latter is absent, the lacunz alone are left in
the place which it usually occupies (10).
Again, in Echinid Plutei I have shown that the observed
pulsation probably occurs in the raised mass of gelatinous
tissue which projects into the floor of the dorsal sac, which
gives this mass its only epithelial wall. Now this gelatinous
tissue, though not so represented in my drawings (special stain-
ing being required to demonstrate it) consists simply of a net-
work of protoplasmic threads, the interstices of which (filled
with a watery fluid) may be considered as parts of the
original segmentation cavity.
Without further discussion of the very obvious inferences to
be drawn from these facts, and the equally obvious gaps in the
evidence, I think I may claim to have established a case in
favour of the homology of the dorsal sac and dorsal organ of
Echinoderms with the pericardium and proboscis gland of
Enteropneusta which cannot be lightly set aside; and taking
these in connection with the other resemblances between the
1 The former is apparently suggested by Morgan (22, p. 442), though for
“ Auricularia’’ we must read “ Bipinnaria,” while the latter is suggested by
Koehler, on the ground of similarity of structure; but neither offer much
evidence,
THE METAMORPHOSIS OF ECHINODERMS. 181
two groups, we seem to have a chain of evidence of their con-
nection, which though not indeed conclusive—that, embryolo-
gical evidence alone can never be—is at least as strong as that
which binds together any two of the great subdivisions of the
Animal Kingdom.
15.
16.
LT.
LITERATURE.
. Acassiz, A.—‘ Embryology of the Starfish,” ‘Contrib. Nat. Hist. U.S.,’
vol. v, 1864.
. Barranpe, J.—‘Systéme Silurien du centre de la Bohéme,’ vol. vii,
Prague, 1887.
. Bateson, W.— Materials for the Study of Variation,’ London, 1894.
. Bory, H.—‘‘The Early Stages in the Development of Antedon
rosacea,” ‘ Phil. Trans. Roy. Soc.,’ London, 1888, vol. clxxix.
. Bury, H.—“Studies in the Embryology of Echinoderms,” ‘ Quart.
Journ. Mier. Sci.,’ April, 1889.
. Birscuut, O.—‘ Versuch der Ableitung des Echinoderms aus einer
bilateralen Urform,” ‘ Zeitsch. f. w. Zool.,’ vol. liii, suppl.
. Cuinor, L.—“ Etudes Morphologiques sur les Echinodermes,” ‘ Arch. de
Biologie,’ vol. xi, 1891.
. Frevp, G. N.—“ The Larva of Asterias vulgaris,” ‘Quart. Journ.
Mier. Sci.,’ vol. xxxiv, pt. 2, p. 105, 1894.
. Gottr, A.—“ Vergleichende Entwicklungsgeschichte der Comatula
mediterranea,” ‘Arch. f. Mikros, Anat.,’ vol. xii, p. 583, 1876.
. H&rovarp, E.—“ Recherches sur les Holothuries des Cétes de France,”
‘Arch. Zool. Exp. et Gén.,’ vol. vii, 2 ser., 1889.
. Lovin, S.—* Etudes sur les Echinoidées,” ‘Kéngl. Svenska Vetensk-
Akad. Handlungen,’ Bd. ii, No. 7, Stockholm, 1875.
. Loven, S.—‘ Echinologica,’ Stockholm, 1892.
. Lupwiec, H.—“ Beitriige zur Anatomie der Asteriden,” ‘ Morph. Stud.,’
vol. i, p. 150, Leipsic, 1877.
. Lupwic, H.—‘ Ueber den primaren Steinkanal der Crinoideen,” &c. -
‘Morph. Stud.,’ vol. ii, p. 34, Leipsic, 1880-82 (and ‘ Zeitsch. f. wiss.
Zool.,’ vol. xxxiv, p. 310, 1880).
Lupwie, H.—‘‘Entwicklungsgeschichte der Asterina gibbosa,”
‘Morph. Stud.,’ vol. ii, p. 3 (and ‘Zeitsch. f. wiss. Zool.,’ vol. xxxvii,
1882).
Lupwiec, H.— Zur Entwicklungsgeschichte der Holothurien,” ‘Sitz-
ungsberichte d. Kongl. Preuss.Akad. d. Wissenschaften,’ 3, Berlin, 1891,
pt. i, p. 179.
Lupwic, H.—‘‘ Zur Entwicklungsgeschichte der Holothurien,” ‘Sitz-
ungsberichte d. Kéngl. Preuss. Akad. d. Wissenschaften,’ 3, Berlin,
1891, pt. ii, p. 608,
132 HENRY BURY.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
MacBripz, E. W.—‘‘ The Development of the Genital Organs, &c., in
Amphiura squamata,” ‘ Quart. Journ. Micr. Sci.,’ vol. xxxiv, pt. ii,
p. 129.
MacBrinz, E. W.—‘‘ The Organogeny of Asterina gibbosa,” ‘Proc.
Roy. Soe.,’ vol. liv.
MacBraipz, E. W.—‘‘On Variations in the Larva of Asterina
gibbosa,” Philos. Soc., Cambridge, May, 1894.
Metscunikorr, E.—“ Studien iiber die Entwicklung der Echinodermen
und Nemertenen,” ‘ Mém. de |’Acad. Impér. de St. Petersb.,’ vol. xiv,
ser. 7, No. 8, 1869.
Moreay, T. H.—“ The Growth and Metamorphosis of Tornaria,” ‘ Journ.
Morph.,’ vol. v.
Morean, T. H.— The Development of Balanoglossus,” ‘ Journ. Morph.,’
vol. ix.
Miter, J.—“ Ueber die Larven und die Metamorphose der Kchino-
dermen,” 2te Abhandlung, ‘ Kéngl. Akad. d. Wissensch. zu Berlin,”
1848.
Neumayr, M.—*‘‘ Die Stamme des Thierreichs,” vol. i, Wien und Prag,
1889.
PERRIER, E.—‘ Mémoire sur |’organisation et le developpement de la
Comatule de la Mediterranée,’ Paris, 1886.
ProvHo, H.—‘‘ Recherches sur le Dorocidaris papillata,” ‘ Arch. d.
Zool. Exp. et Gén.,’ vol. v, 2nd ser., p. 213, 1888.
Sanastn, P. and F.—“ Ueber die Anatomie der Echinothuriden und die
Phylogenie der Echinodermen,” ‘ Ergebnisse Naturwissensch. Forsch-
ungen auf Ceylon,’ vol. i, pt. 3, Wiesbaden, 1888.
Sars, M.—‘ Beskrilvelser og Jagtagelser,’ &c., Bergen, 1835.
SEELIGER, O.—‘“ Studien zur Entwicklungsgeschichte der Crinoiden,”
‘Zool. Jahrb., Abth. f. Morph.,’ vol. vi.
Setenxa, E.—‘‘ Keimblatter und Organanlage der Hchiniden,” ‘ Zeit. f.
wiss Zool.,’ vol. xxxiii, p. 39, 1879.
Srmon, R.—“ Die Entwicklung derSynaptadigitata, und die Stammes-
geschichte der Echinodermen,” ‘ Jen. Zeitsch. f. Naturwiss.,’ vol. xxii,
new ser., 1884.
Semon, R.—“ Die Homologien innerhalb des Echinodermstammes,”
‘Morph. Jahrbuch,’ vol. xv, 1889.
SPENGEL, J. W.—‘ Die Enteropneusten des Golfes von Neapel und der
angrenzenden Meeresabschnitte,” ‘Faun. und Flor. des Golfes von
Neapel,’ No. 18.
Tuer, H.—*On the Development of Echinocyamus pusillus,”
‘Nova Acta Reg. Soc. Sc. Upsala,’ Ser, iii, 1892.
THE METAMORPHOSIS OF ECHINODERMS., tos
EXPLANATION OF PLATES 3—9,
Illustrating Mr. Henry Bury’s paper ‘‘ On the Metamorphosis
of Echinoderms.”
REFERENCE LETTERS.
Ant. B.C. Anterior body-cavity. 2.8.C. Right body-cavity. 2.B.C. Left
body-cavity. Dors. Dorsal. Vent. Ventral. 2. Right side. JZ. Left side.
W.V.R. Water-vascular ring. The numbers I—V mark the radii; the letters
A—E mark the interradii. [In Figs. 3—9 the numbers I—V refer to the
ciliated rings. ]
Fic. 1.—Auricularia, seen from left side, showing process of left body-
cavity running forwards to the hydrocel. x 75.
Fic. 2.—The same; older specimen, in which the ciliated band is beginning
to break up. x 75. (Hydroceel not quite correctly drawn).
Fic. 3.—Auricularia, from ventral side. The skeleton and “ nerve-bands”
are omitted, and the breaking up of the ciliated band is shown diagram-
matically. x 75.
Fic. 4.— Stage A” (transition of Auricularia to ‘‘ pupa”); from ventral
side. 75.
Fic, 5.—‘‘ Stage B”; from ventral side. x 75.
Fic. 6.—Diagram of the anterior pole of Auricularia (compare Fig. 3),
showing the breaking up of the ciliated band.
Fic. 7.—Diagram of the anterior pole in stage A.
Fic. 8.—Diagram of the anterior pole in stage B.
Fig. 9.—Diagram of the anterior pole in young “pupa.” The gradual
evolution of the ciliated rings can be followed with the help of the numbers
and letters (I, Il a, 4, c, &c.), the former referring to the ciliated rings of the
pupa,” and the latter to the parts of the ciliated band which form them.
The references are the same in all the figures (3 to 9).
Fie. 10.—Section of late Auricularia (between Figs. 3 and 4), showing
position of polian vesicle, and extension of “ventral horn” of left body-
cavity over to the right side. x 400.
_ Fies. 11, 12, and 13.—Sections (from a series) through a larva towards the
close of stage A, showing the extension of the “ventral horn” (‘part of
L.B.C.”) of the left body-cavity round the cesophagus to the dorsal surface.
Two other sections intervene between Figs. 11 and 12, and two between
Figs. 12 and 18. Only parts of the sections are shown in the last two. x
300.
Fic. 14.—Diagram of the water-vascular system of the “pupa” seen from
the dorsal (aboral) side.
134 HENRY BURY.
Fic. 15.—Longitudinal vertical section through “ pupa.” x 300.
Fic. 16.—Section through the posterior end of the stomach in stage A,
showing the extension of the right body-cavity on to the left side above
(dorsal to) the stomach. The arrow marks the approximate position of the
dorsal mesentery in preceding sections. Ectoderm and gelatinous tissue
omitted. x 540.
Fic. 17.—Lateral view (from left side) of a young larva of Asterias
glacialis (?), showing constriction of left enteroccl into anterior body-
cavity, hydroccel, and posterior body-cavity. Free-hand drawing. x 200
(approximately).
Fic. 18.—Ventral view of young “ Bipinnaria asterigera,” showing
the terminal plates on the left of the middle line. x 35.
Fie. 19.—Transverse section through Brachiolaria (Asterias rubens),
showing dorsal organ underlying dorsal sac. xX 180.
Fie. 20.—Transverse section through “ Bipinnaria asterigera,” show- —
ing depression in centre of hydroccl. The position of one of the terminal
plates is indicated (“terminal”), though the calcareous matter has been dis-
solved away. xX 220.
Fic. 21.—The same larva seen from the left side. The outlines of the
body-cavities (dotted lines) have been filled in from sections, and are only
approximate. From a decalcified specimen, somewhat shrunk. x 40.
Fie. 22.—Dorsal view of a larva at about the same age. The dorsal me-
sentery (not seen) runs just to the right of, and parallel to, the line of terminal
plates (compare Fig. 20). x 70.
Fic. 23.—Transverse section of a very old specimen of “ Bipinnaria
asterigera.” 55.
Fic. 24.—View from the left side of the same larva (oral side of adult),
reconstructed from sections. The interradial septum dividing the “‘axial sus”
from the left body-cavity is shown; the other four are omitted.
Fic. 25.—Section of the same larva, through two of the radial pouches of
the stomach. x 75.
Fic. 26.—Transverse section through an arm of young Lindia, showing the
hepatic ceca. xX 40.
Fic. 27.—Ventral view of young larva of Asterias glacialis (?), showing
the left body-cavity passing on the dorsal side of the intestine on to the right
side. x 100.
Fie. 28.—Transverse section through a larva of Asterias rubens at
about the same stage as the last. x 300.
Fic. 29.—Transverse section through a young pluteus of Echinus micro-
tuberculatus, showing the “dorsal sac.’ x 440.
Fic. 30.—Transverse section through a later pluteus of the same, showing
the raised floor of the “dorsal sac.’ x 900.
Fie. 31.—Longitudinal vertical section of the same. x 900.
~
THE METAMORPHOSIS OF ECHINODERMS. 135
Figs. 32a and 4.—Two spines with four points from an old pluteus of
Echinus microtuberculatus; a is the more typical form. xX 260.
Fie. 33.—Spine with six points from the same larva. x 260.
Fie. 34.—Very young Echinus microtuberculatus seen from the side.
x 180.
Fic. 35.—Diagrammatic view of the same, from a slightly different aspect,
to show the distribution of the two kinds of spines.
Fie. 36.—Aboral pole of an older specimen of the same, with a diameter of
about ‘75 mm. xX 75.
Fic. 37.—Aboral pole of the same, with a diameter of 7 mm. X 28.
Fic. 38.—The same, diameter 10 mm. In this stage the madreporic area
has spread all over the first basal plate. x 23.
Fic. 39.—Part of a section through a specimen of Echinus micro-
tuberculatus with a diameter of 3°5 mm., showing the relative positions of
the water-tube, &c. The ovary marks the centre of interradius A. x 90.
Fic. 40.—Ventral view of an Ophiurid pluteus (compare 5, fig. 2). The
water-tube (not seen from this side) enters the hydrocel between pouches I
and V. x 180.
Fic. 41.—Section of young Ophiurid just before its separation from the
remnant of the pluteus. x 500.
Fic. 42.—Ventral view of Ophiurid pluteus showing the anus pushed to the
right side. x 300.
Fie. 43.—The same, later stage. x 300.
Fic. 44.—Diagram of the bilateral ancestor, from the left side.
Fic. 45.—The same, dorsal view.
Fic, 46.—Transition to radial stage, dorsal view
Fic. 47.—The same, from the original left side. Atrium and tentacles
omitted.
Fie. 48.—Radial stage, from the former left (now oral) side. Atrium and
tentacles omitted as before. For the explanation of the line X—Y, see text.
Fic. 49.—The same, lateral view. Half the atrium, and two of the tentacles,
are supposed to have been removed.
Fic. 50.—Diagram of hypothetical ancestor of Pelmatozoa; the stalk is
greatly exaggerated (the original form being probably sessile) to show how a
precocious development of this organ would lead to the conditions seen in the
larva of Antedon.
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A CRITICISM OF THE CHELL-THEORY. 134
A Criticism of the Cell-Theory ; being an Answer
to Mr. Sedgwick’s Article on the Inadequacy
of the Cellular Theory of Development.
By
Gilbert C. Bourne, M.A., F.L.S.,
Fellow of New College, Oxford.
* Jedes Lebendige ist kein Einzelnes, sondern ein Mehrheit; selbst inso-
fern es uns als Individuum erscheint, bleibt es doch eine Versammlung von
lebendigen, selbstandigen Wesen, die der Idee, der Anlage nach gleich sind,
in der Erscheinung aber gleich oder ahnlich, ungleich oder unahnlich werden
kénnen. Diese Wesen sind theils urspriinglich schon verbunden, theils finden
und vereinigen sie sich. Sie entzweien sich und suchen sich wieder, und
bewirken so eine unendliche Production auf alle Weise und nach allen
Seiten.”—GortuHE (1807).
Mr. Avam Sezpewick has of late thrown himself with
considerable zeal into the part of a zoological iconoclast, and
has displayed an evident relish in battering the idols which,
he would fain make us believe, are turning away the minds of
men from the true faith, of which there are but few orthodox
exponents. Nor may we blame him for his fervour, for an old
faith always emerges purer, if not firmer, from the ordeal of
sharp antagonism. The idols in question are the develop-
mental law of von Baer and the cell-theory.
Seeing how important a thing it is that a science should be
guided by principles capable of being expressed in precise
language, it has been a matter of surprise to me that some
competent person has not taken up the challenges which
Mr. Sedgwick has thrown down. For, if his views are to
prevail, two of the fundamental principles of zoology, principles
which have hitherto directed and steadied the course of zoolo- |
gical speculation, are taken away from us; and unless some
138 GILBERT C. BOURNE.
better and more distinct principles are put in their place, the
course of speculation may be expected to be very erratic
indeed. It is not without serious misgivings as to my own
competence that I, in default of a better champion, take up
one of these challenges, and I propose to criticise Mr. Sedg-
wick’s recent article on the inadequacy of the cellular theory
of development, leaving for a future occasion the consideration
of his earlier article on von Baer’s law.
It is to be regretted that Mr. Sedgwick should, in putting
forward a view affecting one of the fundamental propositions
of biology, have chosen to adopt a controversial method, which
cannot but have the effect of weakening his case. And it is
still more a pity that he should be so unsparing in abuse of
his imaginary opponents, whilst he himself commits the very
fault. for which he so much blames them. For he lays, in the
front of his indictment, a charge of vagueness and unsub-
stantiality against the supporters of the cellular theory.
‘We are dealing,” he says, “ with a kind of phantom which
takes different forms in different men’s eyes. There is a want
of precision about the cell-phantom, as there is also about the
layer-phantom, which makes it very difficult to lay either of
them. Neither of these theories can be stated in a manner
satisfactory to every one. The result is that it is not easy to
bring either of them to book.”
I shall show, later on, that this charge of vagueness is not
altogether justified ; what I am at present concerned with is
to show that Mr. Sedgwick is as much open to the charge of
vagueness as the rest of the zoological world which he ceasti-
gates.
Read his article through as carefully as one may, one
cannot find any definite or precise statement of his own stand-
point, saving that he quotes passages from one of his earlier
works. The critic, therefore, must be content to infer
from the tenor of the whole article, and from particular
passages in it, as well as from his previous writings, what
Mr. Sedgwick does or does not believe with regard to the cell-
theory, and if he is misinterpreted, it is his own fault.
A CRITICISM OF THE CELL-THEORY. 139
It is probably a fair summary of his position to say that, for
the present, he limits his objections to the application of the
cell-theory to the process of growth during embryonic deve-
lopment; but that he scarcely conceals his preference for the
view that there are no such things as discrete cells in the
so-called multicellular organism. And as it is necessary, at
the outset, to have a perfectly clear idea of his meaning, I will
quote passages from the work to which he refers in his opening
paragraph, assuming that what he stated then he is prepared
to adhere to now, and that his last article is intended to
emphasise the views which he formerly propounded, and to
bring fresh evidence in support of them.
On p. 204 of the second part of his account of the deve-
lopment of the Cape species of Peripatus, he says :—“ It is
becoming more and more clear every day that the cells com-
posing animal tissues are not isolated units, but that they are
connected with one another. I need only refer to the connec-
tion known to exist between connective tissue cells, cartilage
cells, epithelial cells, &e. And not only may the cells of one
tissue be continuous with one another, but they may also be
continuous with the cells of other tissues. ... It is true
that the cells of blood and lymph and the ripe generative cells
are completely isolated. But the former, in their first stages
of growth, form part of the syncytium, as in all probability do
the latter also. This continuity, which for 4 priori reasons
we should expect, has hitherto been regarded as a fact of little
morphological importance and relegated to the category of
secondary features. The ovum, it is said, segments into
completely isolated cells, and the connection between them is
a secondary feature acquired late in development. It has
always been considered that the first stage in the evolution
of the Metazoa was a colonial Protozoon, i. e. a mass of perfectly
isolated unicellular organisms, derived by complete division
from a single cell. Now while I do not wish to exalt the facts
of the cleavage and early development of Peripatus to a posi-
tion of undue importance, or to maintain that of themselves
they are sufficient to destroy this conception of the origin and
140 GILBERT (0. BOURNE.
structure of a Metazoon, I think I am justified in pointing
out that, if they are found to be of general application, our
ideas on these subjects will have to undergo considerable
modification. The ancestral metazoon will no longer be
looked upon as a colonial protozoon, but rather as having
the nature of a multinucleated infusorian, with a mouth
leading into a central vacuolated tract of protoplasm. The
continuity between the various cells of the adult—the connec-
tions between the nerves and muscles and sensory epithelium,
recelve an adequate morphological explanation, being due to
a primitive continuity which has never been broken. In
short, if these facts are generally applicable, development can
no longer be looked upon as being essentially the formation of
a number of units from a single primitive unit, and the
co-ordination and modification of these units into a harmonious
whole. But it must rather be regarded as a multiplication of
nuclei and a specialisation of tracts and vacuoles in a con-
tinuous mass of vacuolated protoplasm.”
This is a temperate and lucid statement of a suggestion
which is still worthy of serious consideration, the more so
since it had been shown, but a short time previous, that
protoplasmic continuity between the tissue-cells of plants is of
very general occurrence, if not the rule. And, as a historical
fact, the continuity of protoplasm was a phenomenon familiar
to animal histologists long before it was proved for vegetable
tissues; indeed there were authors who, before Mr. Walter
Gardiner’s researches were published, were disposed to regard
protoplasmic continuity as a characteristic of animal organisa-
tion, discontinuity as a characteristic of vegetable organisa-
tion.
I have quoted at length because Mr. Sedgwick from being
temperate has become intemperate, and from being lucid he
has become obscure; so that, were I to deal only with his
latest utterances, I should be quite at a loss to know what his
maturer views might be.
What follows, then, may be taken to be a not unfair state-
ment of his position. That from the connection known to
A CRITICISM OF THE CELL-THEORY. 14]
exist between some cells composing adult tissues, there is an
antecedent probability that similar connections exist between
all cells composing all tissues ; and this probability is heightened
by observations made on the development of Peripatus, by the
fact that the so-called mesenchyme cells in Avian and Selachian
embryos are continuous, and not isolated, as was once supposed,
and by a study of the developing nerves of Elasmobranchs.
And that it follows from this that the morphological concept
of a cell, so far from being of primary, is altogether of secon-
dary importance, and that progress in the knowledge of
structure is impossible so long as men persistently regard
cells as the fundamental structural units on which the pheno-
mena manifested by organised beings depend. The true
method of enquiry must be a study of the growth, extension,
vacuolation and specialisation of the living substance—proto-
plasm.
It is in this sense that I propose to deal with Mr. Sedgwick’s
views, and he will pardon me if I have misinterpreted them.
At any rate, I have done my best to understand them.
I would wish to show, in the first place, that there is very
slender ground for the accusations which Mr. Sedgwick levels,
in an unsparing manner, against his zoological contemporaries.
He goes so far as to say that their eyes are blinded by theory
to the most patent facts, and that ‘‘ they are constrained by
this theory,’—the cell theory,—“ with which their minds are
saturated, not only to see things which do not exist, but
actually to figure them.” This is abuse and not argument ;
if Mr. Sedgwick were to remember the qualifying sentence in
his writings of 1886, “if they are of general application,” he
would recognise that there is little occasion for accusing
zoologists of perversely ignoring the views which he then
set forth.
For, in fact, the phenomena to which he draws our attention
have received their due meed of recognition from the time
that the cellular structure of tissues was first studied.
More recent researches have enlarged our knowledge of proto-
plasmic continuity, but it is still a phenomenon far from- being
142 GILBERT ©. BOURNE.
of such universal application as to constrain us to abandon
that very useful morphological concept—a cell.
For some years past the study of cells, of their ultimate
structure, of their chemical and physical properties, of pheno-
mena which accompany their growth and division, has been
carried on with a minuteness which a short time ago was
undreamt of. And attention has been directed, not only to
the cells composing adult tissues, but in the most marked
degree to the successive formation of cells from the primitive
unit, the oosperm, and to the fate which each subsequently
undergoes in the course of development. In place of the off-
hand statements of older embryologists, that the ovum divides
into two, four, eight, sixteen segments, and so forth, we have
the most accurate and minute accounts of the successive
formation of cells, of the place which each occupies in the
developing embryo, of its parentage and of its progeny, and
of the share taken by the last named in the building up of the
adult tissues. In short, we have a number of cell-lineages,
which show that in a number of animals, some of which are
widely separate from one another, the formation of cells from
the ovum follows courses which are either identical or so
closely similar that the differences excite our wonder far less
than the similarities. So minute are these investigations that
every karyokinetic figure has been followed in every cell, up
to a stage where their number becomes bewildering.
I refer, of course, to the remarkable series of observations
which were begun by Selenka, Arnold Lang, Hallez, Bloch-
mann, and others, and have been carried to the highest
perfection by von Wistinghausen, E. B. Wilson, Heymons,
and Lillie.
It would be impossible, in such an essay as this, to deal
adequately with the results obtained by these authors; and it
is unnecessary, since their works are within reach of everyone.
It is enough to say here that a perusal of them does not tend
to diminish the importance which we have been accustomed to
attribute to the cell in developmental processes.
Nothing can be more clear than the fact that, in Nereis or
A CRITICISM OF THE CELL-THEORY. 148
in Unio, there result from the division of the ovum separate
protoplasmic corpuscles, as distinct from one another as one
room in a house is distinct from another, each of which is not
only separate, but contains within itself definite, and probably
limited, qualities (at least at stages beyond eight or sixteen
cells), One might almost say that, after the earliest stages,
each blastomere has a definite task allotted to it, which it
faithfully and punctually performs, according to a prescribed
course. To each, it might be said in figurative language, is
given material, which it must place, not anywhere, but in one
particular part of the edifice.
In considering these very remarkable researches, it is not
sufficient, for the present purpose, to say that no connection
between the blastomeres was observed. Such connections
may have existed and have been overlooked; as the con-
nections, which undoubtedly exist, between plant cells were
for a long time overlooked. But, a priori, such connections
are improbable. For, as has been said, the qualities of each
blastomere are limited. ach is specialised before any form
changes become visible ; each plays one part, and one part
only in tissue formation. If their protoplasm were continuous,
being made so by uniting strands, then, as Mr. Sedgwick has
expressed it, the molecular constitution of any part would in
time spread through the whole mass. But the molecular
constitution of the blastomeres must be different, for their
manifestations are different, and we may possibly see, in this
case, some explanation, obscure though it may be, of the iso-
lation of the form elements from one another.
Further than this, there is objective proof that the cells
constituting the early embryos of these forms are separate.
They exhibit remarkable shiftings of position, which render
the existence of connecting strands of protoplasm highly im-
probable, and the migrations of some cells—e.g. those in
Nereis named c+: and d !*>* by Wilson—are of such an extent
that, if there were protoplasmic continuity, they would be
impossible.
It is no exaggeration to say that this is evidence which
144. GILBERT 0. BOURNE.
effectually disposes of the idea that a syncytial theory of
animal organisation is of general application.
It does more than this, it shows that there are not a few
instances in which cells possess a morphological and physiolo-
gical significance greater than was at one time supposed.
There are numerous other cases in which, at an early stage
of development, cells wander far from the position in which
they originated, and become placed so far from the parent
cells from which they sprung, that any idea of protoplasmic
continuity is impossible. As examples I may mention: the
outer layer cells of Cornacuspongiz and Silicispongiz, which, as
Maas has shown, go through remarkable migrations ; the me-
soblast of Callianira bialata, Beroe and Cydippe, as de-
scribed by Metschnikoff, whose statements are confirmed by
observations made (but unfortunately not published) by Mr.
Riches on Hormiphora plumosa; the lower endoderm
cells of Discocclis, Eurylepta, and Leptoplana, as described
by Lang, Hallez, and Selenka.
In short, the evidence is overwhelming, and it must be
taken to be very clearly established that there are numerous
cases in which there is not “a primitive continuity which has
never been broken.”
It is apparent, then, that morphologists have been amply
justified in refusing to recognise Mr. Sedgwick’s views as to
the syncytial nature of animals, and there is no justification
for the strong language which he uses towards them on
account of their refusal.
It is, on the other hand, quite possible that the frequency of
the occurrence of protoplasmic continuity between developing
tissue-cells may have been overlooked or ignored by a few
authors, and that those who have done so have been led into
the error of attributing too great and too fundamental im-
portance to the cell as an independent vital unit (Lebenseinheit).
But, in point of fact, I am unable to find, in the writings of
any reputable biologist, any statement to the effect that an
organism is composed of independent and isolated units. One
may, it is true, find passages here and there which, when
A CRITICISM OF THE CELL-THEORY. 145
removed from the context, might be made to bear such an
interpretation. I have questioned my pupils with regard to
such passages, and I find that they do in fact put such an
interpretation upon them. For instance, in Waller’s ‘ Intro-
duction to Human Physiology’ the following passage occurs
on page 2: “ The organism is a community; its individuals
are cells; groups of its individuals are organs.” Here we
have an example of the danger of the too free use of illustrative
language. In every illustration there lurks a fallacy. The
fallacy may not have been present to the mind of the author ;
but if the illustration alone is used, without a lucid explanation
of its meaning, the fallacy may be the one thing which im-
presses itself ou the minds of his readers. In this case there
is a fallacy in the analogy, so often made use of for purposes of
popular exposition, between an organism and acommunity. If
the analogy is used without the necessary reservations it leads
to confusion, for the reader is only too prone to transfer to
the organic unit the idea of the individual isolated man, who
is the social unit. The organic unit may in some cases be
individual and isolated, but in the great majority of instances
it has lost, wholly or partially, its individuality, and is not
isolated. It becomes a subordinate part of a higher individality,
which in its turn may be subordinate to an individuality of a
still higher order. This has been explained in the most lucid
and masterly manner by Hackel, in his ‘ Allgemeine Anatomie
der Organismen,’ published in 1866; and nobody who has
carefully studied that work can fail to have a clear under-
standing of the subject. Yet it is to Hackel that the doctrine
of a cell-republic is often attributed! Clearly by those persons
only who have not read his works. For he insists, over and
over again, upon a distinction (which since the researches of
Mr. Walter Gardiner no longer holds good) between the
organisation of plants and that of animals, namely, that the
special characteristic of plants lies in the preponderance of the
perfected and differentiated individuals of the first order—the
cells or plastids. ‘“ Der wesentliche tectologische Character
der Pflanzen liegt in der vorwiegenden Ausbildung und Differ-
VoL. 38, PART 1.—NEW SER. K
146 GILBERT C. BOURNE.
enzirung der Individuen erster Ordnung, der Plastiden” (op.
cit., p. 222). Of animals he says, on the contrary, “ Der
wesentliche tectologische character der Thiere liegt sowohl in
der verwickelteren Zusammensetzung der Thierleibes aus weit
differenzirten Individuen verschiedener Ordnung, als auch
besonders in der verschiedenartigsten Ausbildung der Indi-
viduen zweiter Ordnung, der Organe, welche viel mannich-
faltiger, als bei den Pflanzen und Protisten, differenzirt und
polymorph sind. Die Plastiden, die Individuen erster Ordnung,
sind bei Thieren allermeist Zellen, und zwar meistens Nackt-
zellen (ohne Membran) weniger Hautzellen (mit Membran).
Sehr haufig, und allgemein in den entwickelten Personen,
vereinigen sich bei den Thieren mehrere Nacktzellen zur
Bildung von Zellstécken (Nervefasern, Muskelfasern), was bei
den Pflanzen nur bei der Bildung der Milchsaftgefiasse und
der Spiralgefiisse geschie¢ht. Daher verliert bei den
Thieren stets wenigstens ein Theil. Zellen ihre in-
dividuelle Selbstindigkeit, wihrend sie dieselbe
in den Pflanzen meist behalten.”’
The last sentence, which I have put in italics, shows most
clearly that, as long ago as 1866, Hiickel did not regard the
animal organism as a community, whose individuals are cells ;
and it is the fact that he applied the term “ cell-republic” to
plants, intending thereby to emphasise the difference which
he believed to exist between vegetable and animal organisation.
So that, as a matter of history, whilst plants used to be
considered to be colonies of independent life units, animals
were not. A certain exchange of opinion seems to have taken
place more recently. Some few zoologists and animal physi-
ologists, borrowing from Hickel the term cell-republic, have
thoughtlessly applied it, with all its implications, to animal
organisation, whilst botanists, influenced by Mr. Walter Gar-
diner’s researches, have insisted more and more upon the
individuality of the plant as a whole, and the subordination of
its component parts, the cells. None the less, the facts of
cell fusion and cell communication have never been wholly
overlooked by zoologists, and recent years have brought to
A QRITICISM OF THE CELL-THEORY. 147
light facts, such as the continuity of cartilage cells, which were
unsuspected when Hickel wrote.
I am therefore far from being satisfied that the independent-
life-unit theory has had such a dominant influence as Mr.
Sedgwick would have us believe; and I am quite certain that
the picture which he draws of the teaching given to every
student of biology is a travesty of the truth.
Biology includes botany as well as zoology, and if we were
to allow (which I do not) that zoologists generally have
become as narrow in their conceptions of the processes of
development as Mr. Sedgwick says, it is quite certain that
botanists have not. And as all students of biology are—or if
they are not, they ought to be—put through a course of
elementary botany as well as of zoology (in many schools the
subjects are combined), grave blame must be imputed to those
teachers who have, in the later stages of their education,
warped the liberal conceptions which they must have formed
on the subject of organic growth and development. For I take
it that, after a study of Mucor, Vaucheria, and the Myxomy-
cetes, there is no student so dull but he will have imbibed
ideas respecting cell growth which impel him to ask the
question which as Mr. Sedgwick says it is so difficult to find
an answer to—“ What, after all, is a cell?” If, when he asks
this question, he is told that the cell is an isolated corpuscle
of protoplasm, the unit of vitality, and that there is “a most
fundamental distinction” between unicellular and multicellular
organisms, and so forth, the student may go on his way
rejoicing, for that he has at last been given a clear and tangible
statement; but none the less he will have been started on a
very wrong path. I have not a widespread experience of
zoological teaching, but I know, at least, that Professor
Lankester’s pupils are not started on that path. The truth is,
and, if I am not much mistaken, zoologists and botanists alike
have long been possessed of it, that there is no fundamental
but only a formal distinction between unicellular and multi-
cellular organisms; that the cell is a form concept founded
on a very wide basis of experience, whereby we can conveniently
148 GILBERT C. BOURNE.
interpret to our minds one of the most universal of organic
phenomena, viz. the splitting up of protoplasmic masses during
growth into a number of more or less distinct corpuscles.
It will not be out of place if I quote here a passage from
von Sach’s ‘ Vorlesungen uber Pflanzenphysiologie’ (English
edition, translated by H. Marshall Ward, 1887, p. 73). ‘To
many the cell is always an independent living being, which
sometimes exists for itself alone, and sometimes becomes
‘joined with others °—millions of its like, in order to form a
cell colony, or as Hackel has named it for the plant parti-
cularly, a cell republic. ‘To others again, to whom the author
of this book also belongs, cell-formation is a phenomenon very
general, it is true, in organic life, but still only of secondary
significance; at all events it is merely one of the numerous
expressions of the formative forces which reside in all matter,
in the highest degree, however, in organic substance.”
That this is a great limitation of the cell theory, both as
propounded by its authors and as held by many zoologists, is
not to be denied; and Mr. Sedgwick might well be content
if some such statement were made the established doctrine
as regards cells. It appears to me that some such limited
statement is necessary if we are to have any proposition
universally applicable to organic structure; but with this
reservation, that I cannot regard as of secondary significance
that which all experience shows to be the expression par
excellence of organic growth.
In admitting this much, a large part of Mr. Sedgwick’s
demand is conceded, for it is not to be denied that the cell
theory has been very differently and much more dogmatically
stated by quite recent authors.
We have, for instance, Dr. Oscar Hertwig’s recent work,
‘Die Zelle und die Gewebe.’ He begins dogmatically enough
by saying, ‘‘ Thiere und Pflanzen, so verschiedenartig in ihren
dausseren Erscheinung, stimmen in den Grundlagen ihres ana-
tomischen Aufbaues iiberein; denn beide sind aus gleich-
artigen, meist nur mikroskopisch wahrnehmbaren Elemen-
tareinheiten zusammengesetzt. ... Denn die Zellen, in
A QRITICISM OF THE CELL-THEORY. 149
welche der Anatom die pflanzlichen und thierischen Organis-
men zerlegt, sind die Triger der Lebensfunctionen, sie sind,
wie Virchow sich ausgedriickt hat die ‘Lebenseinheiten.’
Von diesem Gesichtspunkt aus betrachtet, erscheint der
Gesammtlebensprocess eines zusammengesetzten Organismus
nichts Anderes zu sein als das héchst verwickelte Resultat
der einzelnen Lebensprocesse seiner zahlreichen, verschieden
functionirenden Zellen.”” The whole book is written “von
diesem Gesichtspunkt aus,” and, admirable as it is, there is
reason to think that its value is somewhat impaired by the
excessive value attributed to the cell as an independent vital
unit.
In passing, I may remark that this passage of O. Hertwig’s
gives a very precise and definite statement of the cell theory,
as it is held now, bya very great authority; and a reference
to older works would have shown Mr. Sedgwick that, so stated,
it is practically the same as what its authors stated.!
For the original words of Schwann are these: “ The ele-
mentary parts of all tissues are formed of cells in an analo-
gous though very diversified manner, so that it may be asserted
that there is one universal principle of development for the
elementary parts of organisms, however different, and that
this principle is the formation of cells. ... .. In inferior
plants any given cell may be separated from the plant and can
grow alone. So that here are whole plants consisting of cells
which can be positively proved to have independent vitality.
Now, as all cells grow according to the same laws, and conse-
quently the cause of growth cannot in one case lie in the cell
and in another in the whole organism, and since it may be
further proved that some cells, which do not differ from the
rest in their mode of growth, are developed independently,
we must ascribe to all cells an independent vitality ; that is
such combinations uf molecules as occur in any single cell are
capable of setting free the power by which it is enabled to
take up fresh molecules. The cause of nutrition and growth
1 “Tam not concerned with what its authors held.”—Mr. Sedgwick, op. cit.,
p. 88.
150 GILBERT C. BOURNE.
resides, not in the organism as a whole, but in the separate
elementary parts, the cells.”
The definitions of Hertwig are a re-statement in other words
of the salient features of the theory of Schwann, and it is an
error to speak of an unsubstantial cell phantom. Nor is there
any unsubstantiality about the cellular theory of development,
which, I may remind my readers, originated with Remak,
The cellular theory of development, taking as its starting point
the conclusions of Schleiden and Schwann that all organisms
are cells or composed of an aggregate of cells, states that
every cell is formed by the division of a pre-existing cell, not
as Schwann had supposed, by differentiation within a structure-
less cytoblastema.! Hence Virchow’s well-known aphorism,
“ omnis cellula e cellula,”’ which, besides denying abiogenesis,
expresses the cellular theory of development as succintly as
possible.
It would have been a great advantage to his own argument,
and also to his critic, if Mr. Sedgwick had given the clear and
authoritative expositions of the cellular theory which lay ready
to hand, instead of confusing the issue by a whimsical account
of his experience of morphological teaching.
Let us now examine the cell-theory, as stated by Hertwig,
in the light of our present knowledge of animal and vegetable
structure.
It would not be a difficult task to demonstrate the general
truth of Virchow’s aphorism. Wherever there is a cell, it may
be shown to be the product, and generally the immediate
product, of a pre-existing cell. But it would seem that some
biologists have added an unwarrantable corollary to Virchow’s
generalisation, and would say, ‘‘ Nil nisi cellula e cellula,”
Now from a certain aspect this might be considered true;
everything depends on the question as to what is a cell?
Hertwig has pointed out, with much truth, that our pre-
sent conception of a cell is inseparably connected with our
conception of protoplasm. We are still very far from under-
1 Mr, Sedgwick appears to have leanings towards a cytoblastema, as I
shall show further on,
A ORITICISM OF THE CELL-THEORY. 151
standing the structure of protoplasm, and it might be said
that, if we know nothing of the component, it is useless to
make assertions about the compost; but it will at least be
useful to criticise the attempts which have been made.
Hertwig gives this definition, which is the same as that
originally given by Max Schulze. A cell is a corpuscle of
protoplasm in which is contained a specially organised con-
stituent, the nucleus. (Die Zelle ist ein kliimpchen von Proto-
plasma, das in seinen Innern einen besonders geformten
Bestandtheil, den Kern (Nucleus) einschliesst.) This at first
sight seems satisfactory enough, but the more one examines
it, the less satisfactory does it appear, in view of the different
kinds of organisms which are usually described as single cells.
If a corpuscle containing a nucleus is a cell, is a corpuscle
containing two or more nuclei also a cell? And still more, is
a large mass of protoplasm containing many nuclei to be
regarded as a cell? Such a mass, I mean, as Botrydium,
Caulerpa, or Codium, or even Pelomyxa. By many authors
these organisms are regarded as single multinucleate cells, but
I am far from being convinced that this is a right view of the
case.!
If there is one thing more than another which has come
into prominence as the result of recent research, both botani-
cal and zoological, it is the fundamental importance of the
nucleus to cell life. So many minute organisms, which at one
1 With regard to the argument which follows, I would remind my readers
that Hackel, thirty years ago, clearly expressed the view which I am now
urging (see his “ Allgemeine Anatomie den Organismen,” forming the first
part of the ‘ Generelle Morphologie,’ p. 296). ‘ Es muss hierbei ausdriicklich
errinert werden, dass wir unter eine Zelle nur einen Plasma-Klumpen mit
einem Kerne verstehen kénnen. Der hiaufig gebrauchte Ausdruck einer
‘mebrkernigen Zelle’ ist eine Contradictio in adjecto, da ja eben nur die
Einheit des Kerns die individuelle Einheit der Zelle als eines Elementar-
Organismus bedingt. Jeder Plasmaklumpen, der mehr als einen Kern
umschliesst, mége er nun von einer Membran umhiillt sein oder nicht, ist eine
Vielheit von Zellen, und wenn diese Vielheit eine bestimmte einheitliche
Form besitzt, so haben wir sie als Zellenstock zu dem Range eines Organes
erster Ordnung zu erheben.” ‘This view, however, has been controverted by
many authorities, as will appear further on,
152 GILBERT C. BOURNE.
time were believed to be non-nucleate, have since been shown
to contain nuclei, or at any rate nuclear matter, that we are
tolerably well justified in saying that the nucleus, or its
equivalent, is an essential constituent of the cell. At all
events we know that division of the nuclear substance, whe-
ther mitotic or amitotic, is all-important as a prelude to and
accompaniment of cell division. The experiments of Gruber
and Verworn show that if Ameebe are artificially divided, the
parts cut off will regenerate and lead an independent existence -
if they contain nuclear matter, but if they do not, they soon
perish. Fragmentation of the nucleus—by which is produced
a so-called multinucleate condition, often of considerable
duration—is a prelude to spore formation, i. e. to the
division of the cell into many parts. Mitotic division is highly
characteristic of division of the cell into two parts. It is very
difficult to draw distinctions, but it is worth consideration
whether the temporary multinucleate condition ending in
multiple fission, which is common in protozoa, has not a
different value to the permanently multinucleate condition of
some plants and animals, which are generally called unicellular.
In the one case (e. g. Podophrya, Thalassicolla, Actinospherium)
division or fragmentation of the nucleus leads, sooner or later,
to the separation of cells, each containing a fragment of the
original nucleus. In the Celoblastz (Siphonez, e. g. Caulerpa)
the repeated division of the nucleus is not followed by any
cell division, but the organism is throughout life a mass of
continuous undivided protoplasm. ‘The plant, as von Sachs
says, is of considerable size, develops roots, even leaf-forming
shoots, and in its protoplasm hundreds and thousands of cell
nuclei are contained, which with advancing growth are multi-
plied by division, and obtain a definite arrangement within the
protoplasm. And, as in the case in cellular plants, the nuclei
are specially aggregated at the growing points. The whole
behaviour is just that of a multicellular plant, but there are no
partition walls.
It is stretching the point very far to call this a single cell.
And, in fact, it is an inconsistency to do so, for where, by an
A CRITICISM OF THE CELL-THEORY. Lis
essentially similar process, a continuous sheet of protoplasm
containing many nuclei is formed as a tissue-constituent of a
multicellular animal or plant, we do not call the whole multi-
nuclear tract a single cell—we call it a syncytium, or take
some roundabout way of describing it. Such a case is the
formation of the endosperm in the embryo-sac of Phanero-
gams. By repeated mitotic division of the nucleus and growth
of the surrounding cytoplasm, a tract of continuous proto-
plasm is formed, containing many nuclei. At a later stage
partitions are formed and the mass is divided up into cells, but
for a period the endosperm has a structure which recalls that
of the Coeloblaste. Can we say that the condition in the
endosperm is to be regarded as multicellular because it is not
permanent, and that the condition in the Ceeloblastz is to be
regarded as unicellular because it is permanent? If this is
allowed the consequences are far-reaching, for it follows that
the multinuclear phase in Actinospherium and other Protozoa
is also multicellular, because not permanent.
Take, again, the case of the Mycetozoa. The plasmodium of
Badhamia or Fuligo is not unicellular, for it is formed by the
union of many cells: it is not called multicellular, because
there are no cell divisions: yet we draw, rightly enough, a
distinction between the plasmodium, where cell bodies fuse but
the nuclei do not unite, and the single cell resulting from con-
jugation, where the nuclei do unite,
A survey of the facts must lead to the conclusion that there
is an intermediate phase between the unicellular and the multi-
cellular condition, which is the multinucleate but non-cellular
condition,! and that there is no fundamental distinction
1 The term non-cellular does not exactly represent the condition which it
is intended to describe. Yet, if one adheres to existing nomenclature, it is
difficult to find a substitute. The term “cell,” though founded on an
erroneous conception, is so firmly established in biological language that it
would probably be impossible to eject it. Yet if one were to make general
use of the Greek equivalent xdri¢ (literally a little box), which has already
come into such favour as to have respectable claims on our attention, one
might adopt much more exact expressions. Thus the uninucleate Protozoa
might be said to exhibit a monocytial condition, multicellular organisms a
154 _ GILBERT C. BOURNE.
between Protozoa as unicellular, and Metazoa as multicellular
organisms, I should hardly have thought it worth while to
insist upon this had not Mr. Sedgwick written “that an
organism may consist of one cell or of several cells in associa-
tion with one another, We draw the most fundamental dis-
tinction between the two kinds of organism, and we divide the
animal kingdom into two great groups to receive them. Asa
proof of the importance which we attach to this feature of
organisation we assert that a man is nearer, morphologically,
to a tapeworm than a tapeworm is to a parameecium.”
Botanists, who have the great advantage of studying the
physiology concurrently with the morphology of their subject,
make no fundamental division into Protophyta and Metaphyta.
For them, unicellular plants, hypopolycytial plants, Fungi and
Algze are alike Thallophyta, and a passage from Goebel may
serve to illustrate the point of view which leads them to classify
together organisms which, from the point of view of “ inde-
pendent life units,” would appear widely separate. ‘‘ From
this initial stage ”—a single small cell—“ the process of de-
velopment may advance, yet still within the limits of a single
cell, and whilst the cell increases in size, often reaching
dimensions without parallel in the vegetable kingdom, either
the differentiation of the cell-contents or that of the external
form, as shown by the branching, may make most rapid pro-
gress, In other cases the growth of the cells is accompanied
by cell-division, the thallus becoming multicellular, and the
single cell producing, according to the nature of the plant,
a cell row, or a cellular filament, a cell surface or simple tissue
layer, or lastly a cell mass increasing in every direction,”
polycytial condition, and the so-called non-cellular condition of Cceloblaste
and Opalina might appropriately be called hypopolycytial, the preposition
§mo being used in a modifying sense, as expressing the intermediate stage
between one and many. The term syncytial, which is now used in a loose
sense, is strictly applicable to the early condition of the plasmodia of the
Myxomycetes, which are formed by the fusion of many units in a monocytial
condition, and are therefore different from organisms which exhibit a hypo-
polycytial condition. In later stages the nuclei of the plasmodia multiply by
division ; thus the hypopolycytial is added to the syncytial condition,
A CRITICISM -OF THE CELL-THEORY. 155
Although in this passage, which is descriptive of Thallophytes,
Goebel attaches too much importance, as I think, to the con-
tinuity of a vesicle as determining the unicellularity of a plant,
he shows clearly enough that he regards the growth and mode
of extension of the protoplasm, not its division into cells, as
the feature of fundamental importance.
There is the further property in plants that continuity
between the cells of highly organised multicellular plants has
been shown to be of very general, if not universal, occurrence,
And if complete separation were to be insisted upon as a
characteristic of a cell, any given Angiosperm, or other highly
organised plant, could no longer be considered as an aggregate
of life units, but rather as a conjunct mass of protoplasm,
imperfectly broken up into corpuscles, in each of which there
is a nucleus. It is but a step from the much-branched, multi-
nucleate Coeloblastz, which have no partitions, to the forma.
tion of incomplete partitions, breaking up the protoplasm into
small masses, which remain, however, linked with one another,
and so preserve an original continuity similar to that of the
Ceeloblastz, which has only apparently but never actually been
broken, ) :
So much has this idea impressed itself on the minds of some
observers, that Hofmeister suggested that the creeping motion
of the plasmodia of the Myxomycetes and their later transfor-
mation into fructification, is representative of the simplest type
of growth, even for more highly organised plants, This
opinion has been quoted with approval by von Sachs, who,
before even the continuity of the protoplasm of plant cells
was established, wrote that “fundamentally every plant, how-
ever highly organised, is a protoplasmic body, coherent in
itself, which, clothed without by a cell-wall and traversed
internally by innumerable partitions, grows; and it appears
that the more vigorously this formation of chambers and
walls proceeds with the nutrition of the protoplasm, the higher
also is the development attained by the total organisation.”
Expressed in this way, the phenomenon of cell-formation is
represented to us as being nothing more than a particular
156 GILBERT ©. BOURNE.
manifestation of growth, and Mr. Sedgwick may contend that
his views are thereby conceded, and that the ancestral meta-
zoon may, on this aspect, be considered as “‘ a multinucleate
infusorian with a mouth leading into a central vacuolated mass
of protoplasm.” There may be truth in the contention, yet
none the less we may hold fast to the concept of a cell, as I shall
attempt to show further on. And it may be observed in pass-
ing that Mr. Walter Gardiner, in describing and emphasising
the continuity of protoplasm in plants, expressly stated “ that
the presence of minute perforations of the cell-wall need not
lead to any modification of our general ideas as to the mechan-
ism of the cell,” a proposition which most reflective persons
will be cordially inclined to agree with. For this much is
certain, that the formation of cells is not merely the expression
of one out of many formative processes which reside in organic
matter, but is the formative process, par excellence, which
obtains both in animal and vegetable tissues.
Thus far I have endeavoured to show that the independent-
- life-unit theory has not held the minds of zoologists in an iron
bondage, much less the minds of biologists, for, when reference
is made to biologists, botanists must be taken into equal
account with zoologists.
It is, however, arguable that, whatever botanists have thought,
zoologists have not followed their example, but have publicly
maintained a complete adherence to the independent-life-unit
theory in its most limited form, whatever reservations they
may privately have made in their own minds.
But it may be doubted whether the argument holds good.
I have already shown that passages which seem to state most
dogmatically that cells are separate individuals, prove on
examination to be nothing more than illustrations ; and it is
to be remembered that ideas founded on botanical evidence
must always be reflected ou the minds of zoologists, and one
may certainly say that conceptions of animal structure have
of late years been considerably modified by the light thrown
upon organic structure in general by botanical investigation.
Some zoologists may possibly have given too little attention to
A ORITICISM OF THE CELIL-THEORY. 157
growth without division into cells, because there are not in
the animal kingdom any such striking instances of massive
growth without cell division as are exhibited by the Ceeloblastz,
especially if we leave out of consideration the Mycetozoa, as
belonging to the debateable territory between the two kingdoms.
Nevertheless, we have instances of growth and mitotic nuclear
division, unaccompanied by cell division, which are not ap-
parently a mere prelude to division. Take the single instance
of Opalinaranarum. Because this organism is microscopic,
and may be described, without offence to our sense of propor-
tion, as a corpuscle, it is invariably called unicellular. Yet in
essential features it resembles one of the Celoblaste. It
contains numerous nuclei, which divide mitotically, and their
division is an accompaniment of the growth of the mature
organism. Themultinucleate mature condition is of considerable
duration. In the reproductive process this multinucleate
corpuscle divides repeatedly, until a number of small offspring
are formed, each containing several, usually four or five, nuclei.
The minute product of fission then encysts, and it is remarkable
that either during or immediately after encystment the several
nuclei break up, and a single new nucleus is formed,—pre-
sumably it is constituted out of the chromatin of the several
nuclei. The form which emerges from the cyst grows, and
growth is accompanied by repeated mitotic division of the
nucleus till the mature condition is reached. The whole
history reminds one of that of a Mycetozoon, except that the
young do not fuse to form a plasmodium, but simply grow up;
in this respect Opalina resembles the Ceeloblaste, differing from
them, however, in the fact that the whole organism is concerned
in reproduction, not a special part. Although it has, as he
remarks, a distinct “‘ development,” Zeller, who first followed
its life history, has no doubt that Opalina is a single cell.
Now the multinucleate condition is far from uncommon in
the Protozoa, and it may almost be said to be the rule in the
Ciliata, if we regard macronucleus and micronucleus as two
separate nuclei. But putting aside this phenomenon, the
significance of which we do not yet clearly understand, there
158 GILBERT C. BOURNE.
are several Ciliata which have as many as one hundred nuclei,
e.g. Holophrya oblonga, Lagynus elongatus, and
Uroleptus roscovianus.’ I do not include as multinuclear
those forms in which, as in Trachelocerca phenicopterusor
Chenia teres, the chromatin is scattered throughout the proto-
plasm in the form of minute granules. Those Protozoa only may
be considered multinucleate in which there are several well-
defined aggregations of chromatin. And even if the Ciliata
above mentioned may not be considered truly multinucleate, but
to possess onlya fragmented nucleus, there can be no doubtabout
some Ameebe, e.g. Ameba quarta and others described by
Gruber.2. In the last-named the multinuclear state is con-
stant; as Gruber says, “es sich nicht etwa um vorubergehende
Entwicklungszustiinde handelt.”” He watched these Amebe
for a long period, expecting that the large number of nuclei
would at last find its explanation in reproduction by multiple
fission, but he was unable to observe any such culmination.
Dr. Gruber is a great authority, and he, equally with Zeller
and others, is quite positive that the multinuclear Protozoa
are truly unicellular. His reasons are, that closely allied
species are uninuclear, and that the protoplasmic body is con-
tinuous—contained in the case of Ciliata by a single cuticular
coat. But even he admits that the only reasonable interpreta-
tion of the multinuclear condition is that it is a prelude to
reproduction, that is, to cell division.*? It is, therefore, a con-
dition intermediate between the unicellular and the multi-
cellular condition, or, as I should like to call it, a hypopoly-
cytial condition, and nothing more need be affirmed of it.
Zeller is quite precise as to his reasons for regarding
Opalina as unicellular. “ Die kleinsten Thierchen aller be-
kannten Opalinen, so wie sie von Neuem sich zu entwickeln
beginnen, besitzen nur einen einfachen Kern und entsprechen
1 Maupas, “ Etudes des Infusoires ciliés,” ‘Arch, Zool. exper. et gen.’ (2),
i, 1883.
2 ¢ Zeit. fiir wiss. Zool.,’ xli, p. 186.
3 Aug. Gruber, “ Ueber vielkernige Protozoa,” ‘Biol. Centralblatt,’ iv,
p. 710.
A CRITICISM OF THE CHELL-THEORY. 159
unzweifelhaft, wie Engelmann schon fir die von ihm untersuchte
Art nachgeweisen hat, ‘ morphologisch vollstandig einer einzigen
Zelle.’ Aber auch mit der weiteren Entwicklung andert sich
daran nichts. Mag die Zellhaut zu einer aus vielen einzeln
zerlegbaren Bandern bestehenden muskuldses Hille werden
und mag der Kern in zwei Kerne zerfallen, wie in O. similis
und O. caudata, oder durch fortgestzte Theilungen eine
schliesslich sehr grosse Menge von Kernen aus sich hervorgehen
lassen, wie in O. ranarum, O. obtrigona und O. dimidiata,
die protoplasmische Korpersubstanz selbst zeigt
keine weitere Verinderung als die der Massen-
zunahme und blebt, wie auch Engelmann hervorhebt,
‘Zeitlebens eine einzige zusammenhingender Masse,
wie von eine einzigen Zelle.’” I have put the last passage
in italics, because it expresses most clearly why Zeller and other
authors regard multinucleate forms as unicellular, namely
because the protoplasm shows no other change than increase
in size, and because it remains, its life long, a single con-
tinuous mass. The same argument leads many to regard the
Ceeloblastz as unicellular. The continuity of the protoplasm,
then, is the test of unicellularity.
If anybody accepts this, he cannot escape from its logical
consequences. Not only are multinucleate Protozoa and
Ceeloblastz unicellular, but also the whole kingdom of plants,
for their protoplasm is continuous: the developing Peripatus
is unicellular, for its protoplasm is continuous ; the epithelial
cells of many animals, as Max Schulze, Pfitzner, Klein,
Paulicki, Th. Cohn, and others have shown, are united by fine
protoplasmic processes much as are the cells of plants, therefore
the epithelia are unicellular, for their protoplasm is continu-
ous. The same may be said for muscle cells (Werner and
Klecki), for connective tissue, for bone cells, for the developing
meseblast of Vertebrata (teste Sedgwick, Assheton, and
others), for the mesoblast (mesenchyme) of trochospheres and
Molluscan larve (see particularly von Erlanger), and for many
other tissues.
Thus the inevitable result of an argument which is meant
160 GILBERT C. BOURNE.
by those who use it to tighten the bonds of the cell-theory is
to loosen them altogether, and to hand us over unbound to
Mr. Sedgwick, who would fetter us once more with a new
doctrine, viz. there is no cell, all organisation is a specialisation
of tracts and vacuoles in a continuous mass of vacuolated pro-
toplasm.
We do not want to be bound, at least I donot, andif we are
to be free we must take refuge in some such lax but compre-
hensive statement as that of von Sachs, viz. that cell forma-
tion is a phenomenon very general in organic life ; but even if
we must regard it as only of secondary significance, it is the
characteristic expression of the formative forces which reside
in organic substance. |
Now this statement affirms the existence of cells, and it is
necessary to arrive at some understanding as to what is a‘cell ;
what properties are connoted by this term ?
It has become abundantly evident in the course of this
argument, that whatever other attributes may be affirmed of
the cell, the possession of a nucleus is one of the most im-
portant. It is impossible to disagree with Pfitzner when he
writes, ‘‘ Wenn wir aber den Kern tberall und zwar immer
und in allen Stadien als durchaus selbstiindiges Gebilde finden,
so ergiebt sich déraus dass er fur das Bestehen der Zelle
als solchen ein Organ von ‘wiel fundamentaler Bedeutung ist
als wir bisher geneigt werden anzunehmen.” ‘This is also the
view of O. Hertwig, and it is no new one, for Max Schulze
insisted upon it, and Hiackel wrote in 1866, “ Ein Plasmak-
lumpen ohne Kern ist keine Zelle mehr.”
But can we follow Pfitzner when he goes further and says,
“bei einer so ausserordentlich Konstanz in der ganze Reihe
der Thierformen, von den Protozoen bis zu dem Menschen,
kann ich nicht umhin auzunehmen dass iiberhaupt die ganze
Existenz eine Zelle als biologische Hinheit an das Vorhandsein
eines centralen Kérpers, von komplicirten inneren Bau,
gebunden ist, dass also die Chromatinstrukturen nicht etwas
sekundaren erworbenes, sondern die Grundbedingung vitaler
Existenz der Zelle darstellen. Und weiter folge ich hieraus
A ORITICISM OF THE CELL-THEORY. 161
das der als Karyokinese bezeichnete Vorgang nicht ein spe-
cielle Kerntheilungsmodus, sondern der Kerntheilungsmodus
kar’ éEoynv ist”?
I think not. Particles of chromatin scattered through the
protoplasm do not constitute a nucleus any more than a heap
of bricks constitutes a house. Under such a view, Ciliata like
Trachelocerca phenicopterus and Chenia teres would
not be cells, for they have no central nucleus of complex struc-
ture, nor have Oscillaria and Bacterium, in which chromatin
granules have been discovered. Though the case of Holos-
ticha scutellum, in which scattered nuclei (chromatin par-
ticles) unite and fuse to form a single central body or nucleus
previous to division, may help to clear our ideas, it is evident
that the demand for a central organised constituent is
more than the cell conception can bear, especially if the
demand carries with it a further demand for the universality
of mitotic division in nuclei.
In short, before we could accept Hertwig’s definition of
a cell, we should have to ask and answer the question, What
is a nucleus?
Here I may stop to ask whether it is worth while to discuss
the grounds of a definition which, when made, could not be
acceptable to the mind of everyone. An argument about
definitions would soon land one in the regions of scholasticism,
and I have no desire to enter into subtleties which would tax
the powers of a Duns Scotus. To give an answer which shall
be beyond all cavil to the question, What is a nucleus? would
be about as easy as to answer how many angels can dance on
the point of a needle.
The truth is that it is the attempt to frame short concise
definitions, applicable without exception to whole classes of
phenomena, which leads to trouble. The concepts of biology
may and should correspond with the phenomena we observe,
but they can very seldom be made into universal propositions.
There is no place in the science for definitions as exact and
universal as those of geometry. The qualities of a nucleus are
not to be defined like those of a point or a line. Such propo-
vou. 388, PART 1,—NEW SERIES. L
162 GILBERT C. BOURNE.
sitions as we may make are but resting-places for our minds
as we ascend the mazy scale of organisation. To attempt to
form definitions, to predicate the precise attributes of whole
classes of phenomena, is to run counter to the very genius of
the subject. For what do we mean by evolution if not that
life is labile, never resting, protean in its variety? And how
can we express this but in an incomplete way, contenting our-
selves with particulars, and trying to show that the stream,
though it flows in many tortuous channels, is one stream
nevertheless.
Cells and nuclei are protean in their variety, and since we
very rightly insist on objective study as a preliminary to the
understanding of them, it is not wonderful that they should
give rise to this concept in the mind of one man, and to that
concept in the mind of another man, and thus it is not sur-
prising that the theory of cells should be incapable of being
stated, as Mr. Sedgwick complains, ‘‘in so many words in a
manner satisfactory to everyoue.”
It is fairly obvious that Mr. Sedgwick’s quarrel with the
cell-theory began with the dissatisfaction which he felt when
he discovered that doctrines, which he believed to be of uni-
versal application, were in fact contradicted by several instances.
But he fell out of Scylla into Charybdis when he supposed
that he could reply to a universal affirmative by a universal
negative.
There is an old and respectable rule of logic that of two
contrary propositions both cannot be true and both may be
false, whilst of two subcontrary propositions both may be
true but both cannot be false. Had Mr. Sedgwick remem-
bered this, he would not have attempted to overthrow the
cell-theory by the statement of a contrary proposition of
equally universal import.
The cellular theory of development in the popular form in
which it is often presented may be briefly summed up some-
what as follows. The multicellular organism is a colony, con-
sisting of an aggregation of separate elementary parts, viz.
cells. The cells are independent life units, and the organism
A CRITICISM OF THE CELL-THEORY. 163
subsists in its parts and in the harmonious interaction of those
parts.
The falsity of this summary is evident when we consider
the known facts of vegetable organisation ; the development
of Peripatus; the union, by means of protoplasmic processes,
of epithelial, muscular, and connective-tissue cells; the evidence
lately adduced as to the continuity of the mesoblast in Elasmo-
branchs, Aves and Mammalia, and other well-known instances.
The absolute contrary, as expressed by Mr. Sedgwick, is
equally false, viz. that the metazoon is a continuous mass of
nucleated vascular protoplasm, subsisting in the unity of its
mass. For, as I have shown in the earlier part of this essay,
there are unequivocal instances of distinct isolated cells
occurring in the embryos of many Metazoa (Nereis, Unio,
Umbrella, Leptoplana). Moreover Iam convinced, by my own
studies on the histology of Ceelenterates, that, whilst there is
organic connection between many of the tissue-cells composing
these organisms, as was demonstrated long ago by the brothers
Hertwig, there are many other cells of which such continuity
cannot be affirmed.
To deal clearly with the cell-theory, or rather with the inde-
pendent-life-unit theory which has grown out of it, we must
split it up into as many separate propositions as it contains.
These are:
The multicellular organism is an aggregate of elementary
parts, viz. cells.
The elementary parts are independent life units.
The harmonious interaction of the independent life units
constitutes the organism.
Therefore the multicellular organism is a colony (cell-
republic according to Hickel).
It is not necessary to follow the theory further into the
cousequences which are deducible from these propositions,
e. g. that development consists in the separation of numerous
individual units from a single primary unit, the ovum. It is
obvious that the truth of the first proposition in no way
depends on the truth of those which follow, and that, in fact,
”
164 GILBERT ©. BOURNE.
the second proposition is an assumption which is made to
explain the first. We may make Mr. Sedgwick a present of
the last three, whilst we retain and value the first.
The essence of the whole question is this: are we justified
in considering the elementary parts of an organism to be
independent life units? Before we can answer this, we must
inquire why we do consider them to be independent life units?
The answer to this is probably to be found in the aphorism,
which commends itself to everybody, that reproduction is
discontinuous growth. From the observation that, in
unicellular organisms, division of the unit—the cell-corpuscle
—leads to the liberation of a new and independent unit, and
that in multicellular organisms it is the liberation of an
independent unit—the ovum—which constitutes reproduction,
it has become a settled conviction in men’s minds, that division
of a cell-corpuscle means the liberation of a new unit, that is,
the setting free of a new independent being. It is this con-
viction which has led to the belief that the units composing a
multicellular organism are in posse independent beings,
though in esse subordinate to the whole of which they form
a part. This was the argument of Schwann when he wrote
the passage which I have quoted on p. 149, and the argument
has been taken as conclusive.
But we know now that the power which Schwann and his
followers limited to cells is inherent in protoplasmic masses
not divided into cells. For instance, if the cell-membrane
of a Celoblastic alga is ruptured, portions of the exuded
protoplasm, provided they contain one or more nuclei, may
become, after a time, surrounded by a new cell-membrane,
grow, and form a new plant.
The experiments of Gruber show also, that portions of
Amecebe artifically separated may, provided that they contain
nuclear substance, recover from the operation, and lead an
independent existence.
May I ask, in parenthesis, whether there can be a better
illustration of the truth of the contention which I have en-
deavoured to establish above, that whilst a uninucleate cor-
A CRITICISM OF THE CELL-THEORY. 165
puscle of protoplasm is in esse as also in posse a unit of
independent vitality, a multinucleate corpuscle or mass of
protoplasm is in posse composed of separate cells (units of
independent vitality if one chooses to call them so) whilst still
in esse a single unit of independent vitality ?
To continue the subject. We now know also that division
into cells is not necessarily, though it sometimes may be,
division into units of independent vitality, but is often (may we
not say generally ?) incomplete separation into form elements
which may indeed, under certain conditions, be completely
separated, and exhibit an independent vitality (Begonia), but
under normal conditions participate in the vitality of the whole
plant or animal by means of their connections with their
fellows. Hence we must conclude, as it seems to me, that the
elementary parts of organisms are not independent life units
in esse. They may be soin posse in many cases, but as
differentiation and specialization progress they lose this power
also, and cannot, when separated from the whole of which they
form a part, exhibit independent activities.
This consideration leads to the apparent paradox, that the
higher the organisation the less conjunct and, at the same
time, the less independent are its parts ; the lower the organisa-
tion the more conjunct, but also the more independent are its
parts.
This is a puzzle which has, for years past, exercised the
minds of biologists. There is, I believe, but one solution of
the difficulty, and it is to be found in the physiological import
of cells.
But before we can enter into this question we must finally
satisfy ourselves, as far as circumstances allow, about the
morphological concept of a cell.
That the cell is a thing cognisable, and that it is not an
unreal figment, due to imperfect observation or to hopelessly
prejudiced interpretation of our observations, as Mr. Sees
would make us believe, I will try to show.
A cell is a “ body,” and therefore an external cause to winch
we attribute our sensations. I would submit that, without
166 GILBERT C. BOURNE.
prejudice to the metaphysical standpoint, we must conceive
that what is capable of giving rise in us to such very distinct
sensations, must have a real existence. I am referring now to
the component parts of the tissues of higher animals and plants,
and not to unicellular organisms.
If, then, the thing has existence, it must have attributes ;
we must be able to affirm something of it. What we have to
affirm is not the attributes of this cell or of that cell, but of
cells in general. We have to give expression to a morpho-
logical idea, in the sense in which Goethe used the word
morphological. Our concept of a cell must be an “ Allgemeines
bild,’ the generalised idea of a cell, derived from our ex-
perience of many kinds of cells. I have already shown, at
sufficient length, that we must now regard something of the
nature of a nucleus as an essential component of all cells, but
as the concept of a nucleus as a central organised body is not
applicable to all cells, I would widen Max Schulze’s definition
by saying that “a cell is a corpuscle of protoplasm, which
contains a specialised element, nuclein.”” This is a sufficiently
comprehensive statement of our “ Allgemeines bild,” though
I cannot pretend that it is not open to objection.
Cells, as thus defined, are not only of various kinds, but
they are variously compounded together. We may, by the
process of dichotomous division, classify them, according to
their relations to other cells, as discrete and concrescent.
By discrete cells, I] mean those whose protoplasm is not
in union with that of any other corpuscle.
By concrescent cells, I mean corpuscles whose protoplasm
is in union with that of other corpuscles.
Discrete celis may further be divided into:
Independent cells, living wholly apart from one another,
or separated by an appreciable interval of space, e.g. uni-
nucleate Protozoa, the mature ovum, leucocytes.
Coherent cells, which are in close apposition to others,
but not organically in union with them, e.g. the blastomeres of
many developing embryos.
Concrescent cells may also be further divided into :
A CRITICISM OF THE CELL-THEORY. 167
Continuous cells, whose protoplasm is fused but whose
nuclei are separate, e.g. Myxomycetes, Celoblaste,
Opalina.
Conjunct cells, those which having a protoplasmic body
of definite outline are united inter se by fine bonds of proto-
plasm, e.g. vegetable tissue cells, epithelial cells of many
animals; mesenchyme cells, &c.
Experience shows us that independent cells may, in process
of growth, give rise to coherent cells, continuous cells, con-
junct cells, or to all three together, and that coherent, con-
tinuous, or conjunct cells may, and in fact do, give rise to
independent cells. As thus stated, can there be a better
illustration of von Sachs’s principle that cell-formation is an
accompaniment of growth?
It will be observed that, in adhering to the present termi-
nology, I am obliged to classify organisms usually (though not
always) called unicellular as multicellular. I have tried to
escape from this necessity, but the limitations of language
compel me to it. I should be grateful for a better and more
logical definition.
The view of Mr. Sedgwick—if I do not misrepresent him—
is this, that there are no coherent cells; that all which I have
classified as continuous and conjunct cells are not cells, but
tracts of protoplasm; that the only cell, sensu stricto, is
the independent cell, and that morphologically and physio-
logically it is of no consequence.
I have already shown that there are cells which we must
regard as coherent. I cannot, for reasons which I will explain
directly, consider the independent cell of no consequence, and
the difference between us as to conjunct cells is simply this:
Are they to be regarded as one or many? I can, perhaps, best
express this difference by an illustration.
Is a house to be regarded as one room or composed of
separate rooms? A room isa certain portion of space enclosed
by walls, ceiling, and floor; but it is also in connection, by
means of the door, with other similar rooms. Is it, then, not
a separate room, but part of a larger room? Or if I shut the
168 GILBERT C. BOURNE.
door is it a room, and if I open the door is it no longer a
room? The subject might be argued with much ingenuity,
but the final answer is this—that “room” and “cell” are
terms which give expressions to certain states of our conscious-
ness, and for practical purposes they are very useful terms
indeed. Where distinct states of consciousness are called up,
of such a nature as to give rise to ideas of particularity, it
is a mere quibble to argue that the apparent parts are actually
merged in a whole. A cell is none the less a cell, in the sense
of a thing distinct in itself, because it is conjunct with its
fellow cell, than my room is the less a room because it has
one door opening into an adjoining room and another opening
into the passage.
Yet there is something more than a verbal quibble in Mr.
Sedgwick’s contention. He would have it that in the case of
mesenchyme it is incorrect to say that it is a number of stel-
late cells joined to one another by their processes. For him
the correct description is, ‘fa protoplasmic reticulum with
nuclei at the nodes.” Does he accept the logical consequences
of this, and say of the epithelial cells of the Salamander or of
unstriped muscle fibres that they are protoplasmic reticula
with nuclei at their nodes? And if so, how does he explain
the fact that, in the one case and in the other, the elements
when absolutely isolated by appropriate methods show a re-
markably constant and characteristic form’? Were they what
he describes, rupture of the internodes of the reticulum would
result in amorphous lumps of protoplasm, not in units of
characteristic form. It is the constancy of the various forms
of cells which convinces morphologists of their individuality
as form elements, and all the arguments which Mr. Sedgwick
or anybody else may choose to bring forward will not convince
the man who goes into a laboratory, makes a few maceration
preparations, and studies the results for himself.
Thus a tissue formed of conjunct cells is made up of many
and not of one, and as a form concept the cell holds its ground
and, pace Mr. Sedgwick, it will continue to hold its ground
against all comers.
A CRITICISM OF THE CELL-THEORY. 169
As a physiological concept it is hardly less useful, though
reflection may induce us to abandon the ‘ cell-republic”
theory, as, indeed, it has been tacitly abandoned by many.
I take it that the scheme of von Sachs very nearly expresses,
in general terms, the physiological importance of the cell.
An organism is a protoplasmic body, coherent in itself, which
grows, and as it grows it is divided by cleavage into innu-
merable corpuscles, and it appears that the more vigorously
this formatien of corpuscles proceeds with the nutrition of the
organism, the higher also is the development attained by the
total organisation. Nor does this statement stand in any con-
tradiction to the original theory of Schwann, from whom I
may quote two more passages: ‘‘ The elementary parts of all
tissues are formed of cells, in an analogous though very
diversified manner, so that it may be asserted that there is
one universal principle of development for the elementary
parts of organisms, however different, and that this principle
is the formation of cells.’ And again, he says of the relations
of cells to one another, ‘‘ Each cell is within certain limits an
individual, an independent whole. The vital phenomena of
one are repeated, entirely or in part, in all the rest. These
individuals, however, are not ranged side by side as a mere
aggregate, but so operate together in a manner unknown to
us, as to produce a harmonious whole.” It should be remem-
bered that Schwann regarded cells as so many separate vesi-
cles, and when allowance is made for this error, the second
part of the last passage must be allowed to have great signifi-
cance. The subordination of the parts to the harmonious
whole, leading to the loss of individuality of the parts, in
animal tissues, was insisted on by Hickel in his ‘ Generelle
Morphologie.’ The first of the two sentences which I have
quoted from Schwann is even more true to-day than when it
was written, for we have got rid of the cell-forming matrix,
the cytoblastema ; and I would wish to insist on this passage
as expressing in the clearest possible language the cell-theory
as we understand it to-day.
From this standpoint we can see, obscurely it may be, why
170 GILBERT ©. BOURNE.
cell-formation accompanies differentiation with growth of the
mass, and why specialisation is not possible in continuous
tracts of protoplasm. For, as Mr. Sedgwick himself admits,
in a continuous mass of protoplasm, changes of molecular
constitution in any one part would in time spread through
the whole, so that a differentiation of one part would in time
be impressed on all the other parts, and physiological division
of labour would be out of the question. The fact that in the
Protozoa there is differentiation within the limits of a single
corpuscle presents no greater difficulty than the fact that in
the epithelio-muscular cells of Ccelenterates, or the similar
cells in Nematodes, there is differentiation within the limits of
the cell.
Again, metabolism in a large mass is greatly facilitated by
its being broken up. As von Sachs says, “ It is very intelligible
that not only the solidity but also the shutting off of various
products of metabolism, the conduction of the sap from place
to place, and so forth, must attain greater perfection if the
whole substance of a plant is divided up by numerous transverse
and longitudinal partitions into cell chambers.” The same
thing applies, mutatis mutandis, to animals, and it is not
difficult to see that the difference between holozoic and holo-
phytic nutrition makes it impossible for the animal to grow
to a large mass without division into cells, whilst such growth
is possible in the case of plants which, like Codium and
Caulerpa, live in water, or like Botrydium in damp earth.
It is known that the spaces between epithelial cells which
are traversed by the connecting strands of protoplasm, and
were formerly supposed to be occupied by a cement substance,
“are in reality lymph spaces, and this gives us some insight into
the importance of the cell structure in animal organisation.
The formation of cells with spaces between admits of nutrient
fluid being brought to the very threshold of each constituent
corpuscle of the organism. (See on this subject Th. Cohn,
R. Heidenhain, Paulicki, Nicolas, Werner, and others.)
Whilst the necessities of cohesion, solidity, and transmission
of stimuli may explain the conjunct nature of so many tissue
A CRITICISM OF THE CELL-THEORY. 171
cells, recent researches on cell lineages may perhaps give us
a clue to the interpretation of the fact that blastomeres are
in sO many cases, no more than coherent. For it is noticeable
that wherever cell lineages, with marked isolation of the blasto-
meres, have been described, there is a decided tendency to the
precocious development of organs, or, at any rate, to the pre-
cocious isolation of the primordia (Anlage) of organs.
It seems probable that the discrete condition of the blasto-
meres is connected with the fact, to which I alluded in the
earlier part of this essay, that they are, from the very outset,
specialised. They have each a definite molecular constitution
different from the others, and, in figurative language, a
limited part to perform, which they could not perform to
advantage if they were conjunct with the other blastomeres
and shared in their different molecular constitution. But
this is a subject which I must leave for a future occasion when
I discuss the validity of von Baer’s law of development.
I have travelled in this essay over a great deal of ground,
and I have necessarily had to touch more lightly on many
topics than I should have wished. I hope that I may at
least have succeeded in presenting my arguments in a manner
which will make them clear to my readers, and that I have not
been too discursive. Starting from Mr. Sedgwick’s propositions
and accusations, I have tried to show what is or was the exact
extent and meaning of the cell-theory ; I have tried to examine
it and show how much was good and how much bad, and I
have finally been led to the conclusion—which is not quite
what I proposed to myself at the outset—that the cell concept
is a valuable expression of our experience of organic life, both
morphologically and physiologically, but that in higher or-
ganisms cells are much what von Sachs declares them to
be, not independent life units (Lebenseinzelheiten), but a
phenomenon so general as to be of the highest significance ;
they arethe constant and definite expression of the forma tive
forces which reside in so high a degree in organic matter.
Lest I should appear to have minimised the importance of
the cell too much, let me conclude by saying, that nothing
172 GILBERT C. BOURNE.
which has appeared above calls into question that great feature
of animal and plant development which most impresses the
biological student, viz. that organic growth is a cycle, beginning
in the single cell, and returning to the single cell again. And
therefore, in a limited sense, the cell is par excellence the
unit of life. Its growth takes various forms and shows many
complexities, but whatever the form, however great the com-
plexity, it is a progress from the state of an independent
corpuscle, through a state of many coherent, or continuous,
or conjunct, interdependent corpuscles, back again to the state
of a single independent corpuscle.
This was the great advance made by Remak on the theory
of Schwann, and summed up in Virchow’s aphorism, which I
believe to be universally true. For Schwann did not hold that
cells are the ultimate basis of life: he held that they are
formed, as a crystal is formed out of its mother liquor, from a
structureless matrix, the cytoblastema. To some such theory
Mr. Sedgwick wishes to take us back again, for his “‘ pale and
at first sparse reticulum ” bears a most suspicious resemblance
to the exploded cytoblastema. ‘“ The development of nerves,”
he says, ‘‘ is not an outgrowth from certain central cells, but
is a differentiation of a substance which was already in
position.” And earlier in his article, referring to the growth
and extension of the mesoblast between epiblast and hypoblast,
he says: ‘‘ What are the facts? The space between the layers
is never empty. It is always traversed by strands of a pale
tissue connecting the various layers, and the growth which
does take place between the layers is not a formation of cells
but of nuclei, which move away from their place of origin
and take up their position in this pale and at first sparse
reticulum.”
But surely nobody ever affirmed that the space between the
layers was empty except in the sense that it is devoid of
cellular structures. It is well known that it is filled with a
coagulable fluid, and it is worthy of remark that coagulable
fluids, treated with the reagents now most in use, frequently
form a reticulum of pale non-staining substance. I can speak
A CRITICISM OF THE CELL-THEORY. 1738
from experience, for not long since I was much puzzled by
such a reticulum, and had I been less cautious I should have
published, as a great morphological discovery, statements which
rested on a wholly insufficient basis of experience. The subject
requires further investigation, and the most that one can say
now is, that it is possible that Mr. Sedgwick, good observer as
he is, may have been mistaken. And he will pardon my
observing that the things which he states are not “ facts.”
They are his own inferences from his own individual observa-
tions, and will require very abundant confirmation before they
can take rank as what we agree to regard as “ facts.”” All the
“ facts ” we have at present, i. e. the accumulated observations
of hundreds of highly-trained and able observers, are funda-
mentally opposed to any such account of protoplasmic growth
apart from nuclear formation as Mr. Sedgwick gives us. But
there is another way of looking at it, namely, that he has only
overstated his case, and that the growth of the tissues in
question resembles the apparent creeping motion of the plas-
modia of the Myxomycetes. That this may be the case is
supported by a study of Mr. Assheton’s receut account of the
growth of the mesoblast and of the inner layer of the epiblast
in the embryo of the rabbit. It presents no theoretical diffi-
culties, but it should be remarked that Mr. Assheton figures
numerous nuclei at the very edge of the growing part of his
reticula, which is consonant with what we know of proto-
plasmic growth in other cases, but not with Mr. Sedgwick’s
account.
But if Mr. Sedgwick can prove that the reticulum is there
and that it grows and spreads far from the nuclei which sub-
sequently migrate into it, he must not suppose, as he is
apparently so ready to assume, that the inveterate prejudice
of morphologists will prevent their accepting his conclusions
because of their theoretical difficulties. If his case is proved,
it will be accepted, but he must prove it up to the hilt.
And if he does prove it, what then? It will be an isolated
case, of secondary significance: merely another addition to
our experience of the very various phenomena displayed in
174 GILBERT C. BOURNE.
organic growth. For thousands of instances point to the fact
that normal growth is effected in a very different way, by
mitotic division of the nucleus preceding and directing the
formation of a discrete or concrescent cell-corpuscle. The
recent researches of cytologists are too many, too good of
their kind, and too consistent to admit of any other conclusion.
DEC 1895
IRON COMPOUNDS IN ANIMAL AND VEGETABLE CELLS. 175
On the Distribution of Assimilated Iron Com-
pounds, other than Hemoglobin and Hematins,
in Animal and Vegetable Cells.
By
A. B. Macallum, M.B., Ph.D.,
Associate-Professor of Physiology, University of Toronto.
With Plates 10—12.
PAGE
J, PRELIMINARY REMARKS AND REFERENCES TO SePEecrAL LITERA-
TURE OF THE SUBJECT : ‘ ‘ ; pS
II. Metnops or Stopy . : Ray)
ILI]. GengRAL OBSERVATIONS ON THE DiaernUTTONs or ASSIMILATED
Tron In Hicuuy SPECIALISED ANIMAL AND VEGETABLE
CELLS : : » 205
In the Mnnteus of the Aaiinal Cell : é 5 DOE
In the Nucleus of the Vegetable Cell . ; opal
In the Cytoplasm of the Animal Cell ; . 214
In the Cytoplasm of the Vegetable Cell . : 227
TV. On tHe OccuURRENCE OF ASSIMILATED JRON IN SPECIAL Renu
oF LIFE ‘ ; : : ‘ 5
In Ascaris mayetae : : : ° » 229
In the Larve of Chironomus. F ; eo ol
In Protozoa f ; é - 9 - 2386
In Fungi. ; : - : : . 242
In Bacteria F : P ; . . 254
In the Cyanophyceze é 3 : : erp!
V. GENERAL REMARKS ; : ‘ : ; >, 208
VI. EXpLANATION OF FIGURES : . 5 ; 55 HA
I. PRELIMINARY REMARKS.
In 1891, in a communication to the Royal Society,! I described
a method by which the presence of iron in the chromatin of
1 “On the Demonstration of the Presence of Iron in Chromatin by Micro-
chemical Methods,” ‘ Proceedings Roy. Soce.,’ vol. J, p. 277.
VoL. 38, PART 2.—NEW SER. M
176 A. B. MACALLUM.
animal and vegetable cells may be demonstrated micro-chemi-
cally, and I referred to the results then obtained with it as
indicating, apparently, that iron is always a constituent element
of this substance. The interest which the subject had for me
led me to continue the investigation with improved methods of
research, and I am now consequently in a position to describe
a much more extensive series of observations in support of the
generalisation, then somewhat tentatively advanced, that iron
is a constant constituent of the nuclein substance proper.
From the commencement of the investigation I have been
fully aware of its difficulties, and I can, therefore, readily under-
stand that view of the subject which led Gilson to remark that
the solution of the question concerned is one “that seems to
require more than a single man’s activity.”’! The difficulties
encountered in the application of the micro-chemical method
are, however, very much less formidable than those met with
in the employment of the older. methods. Ihave pointed out, in
my first paper on this subject, how impossible it is to be certain
that the iron revealed by macro-chemical methods in isolated
quantities of nuclein is not present through absorption from some
other source, but due to a combination obtaining in unisolated
living chromatin, and I have indicated that the only way in
which the question could be settled definitely is by the employ-
ment of micro-chemical methods. I have shown in the suc-
ceeding pages of this paper that the acid alcohol upon which
Bunge relied to extract the iron of inorganic and albuminate
compounds from egg-yolk and other nuclein-holding substances,
and leave intact the organic (nucleinic) iron, does not perform
this function at all when the substance treated with it is in
mass, while it removes the iron of all three classes of compounds
from thin sections of tissues, if the time allowed for its action
be prolonged. We have, consequently, in a macro-chemical
investigation, no means whatever of distinguishing between
organic iron on the one hand and the iron of inorganic and
albuminate combinations on the other, and we are therefore
1 “On the Affinity of Nuclein for Iron and other Substances,” ‘ Report
British Association for the Advancement of Science,’ 1892, p. 778.
IRON COMPOUNDS IN ANIMAL AND VEGETABLE CELLS. 177
forced, more than ever, to depend on micro-chemical methods
to determine the relations of assimilated iron to the cell.
Objections may be urged against these methods also, based
chiefly on the facts that iron, free or combined, contaminates
everything, so to speak, and that what is shown to occur in
dead chromatin may not be present in the living compound ;
but these objections at once lose their force when the methods
are applied with all due care accompanied by such control
experiments as the conditions may suggest.
I have in my former communication made reference to the
investigations of Bunge and Zaleski upon iron-holding nucleins.
Since 1891 four other investigators have published observations
on the occurrence of iron in organic compounds.
Molisch! endeavoured to determine the relations of iron in
the vegetable cell by means of concentrated aqueous solutions
of potash. He found that when vegetable tissues were
immersed in this reagent for a day or longer, they gave a
reaction for iron not at all obtainable in the fresh tissues, and
he explained the result as due to the removal of the iron from
a firmly combined (‘ maskirt ” ) condition to that in which
it is readily detectable by ordinary reagents. The firmly com-
bined iron, as shown by this method, was sometimes in the
cell wall, sometimes in the cell contents, and sometimes again
in both. His results do not call for a fuller description than
this, since in a later publication? he has stated that his
solutions of potash were not free from iron, and he has con-
sequently withdrawn all the conclusions which he previously
based on the results obtained with this reagent.
Petit,? in investigating the occurrence of iron in barley,
employed Bunge’s method to separate the inorganic and
albuminate from the organic iron, using for that purpose a
1 per cent. solution of hydrochloric acid in absolute alcohol.
1 ¢Die Pflanze in ihren Beziehungen zum Hisen,’ Jena, 1892.
2 Bemerkung iiber den Nachweis von maskirterm Hisen,” ‘ Berichte der
deutschen bot. Gesell.,’ vol. xi, 1893, p. 73.
3 «Distribution et état du fer dans l’orge,” ‘Comptes Rendus,’ vol, exv,
p. 246, 1892,
178 A. B. MACALLUM.
The dried and finely pulverised barley was put, with the acid
alcohol, in a Soxhlet extraction apparatus and heat was applied
for six hours, during which time the reagent was renewed once,
but the second liquid extracted no iron. The result was the same
when the strength of the acid in the solution was 2°5 per cent.
From his experiments he concludes that nearly all the iron is
combined with nuclein (a l’état de nucléine) and exclusively
contained in the tegmen and embryo of the barley grain. Ina
second publication! he describes the separation of an iron-
holding nuclein from the malt-combs (touraillons) of barley,
free from sulphur and in which the iron amounted to 0195
per cent. The separation was made by extracting the pul-
verised matter with a 1 per cent. solution of potash at 60°C.
for some minutes, and filtering off under pressure the brown
liquid, which was then neutralised with dilute hydrochloric
acid. The precipitate formed was washed by decantation with
water, then with alcohol and ether, and finally dried over
sulphuric acid.
Gilson ® found iron in the nucleinic elements, not only when
ammonium sulphide, according to my method of using it, was
employed, but also after treatment with other reagents and in
nuclei which, without such treatment, gave no reaction for
iron with the ordinary methods of demonstration. He specially
mentions sulphuric acid and sulphurous anhydride as giving
the best results, although others, among which he includes
saline solutions, produce the same effects. He is, however,
inclined to regard the iron demonstrated in the nuclein as due
to a combination which is formed only after death, and similar
to that which dead nuclein effects with many other substances,
especially colouring matters. He showed that dead nuclein has
a very strong aflinity for iron compounds, the nuclei of freshly
extracted cells absorbing from a 0:05 per cent. solution of ferrous
sulphate more iron than could be demonstrated in them when
simply treated with sulphuric acid; and he maintains it is
extremely difficult to ascertain whether nuclein in a living
1 “Sur une nueléine végétale,” ‘Comptes Rendus,’ vol. exvi, p. 995, 1893,
2 Loe, cit,
[RON COMPOUNDS IN ANIMAL AND VEGETABLE CELLS. 179
condition contains iron, or contains it only after death, deriving
it by absorption from the blood or other surrounding fluids,
or even out of the reagents themselves, if these are not
absolutely free from iron. In his remarks upon my methods
he states that Bunge’s fluid, upon which I relied to extract the
iron of inorganic and albuminate combinations from sections
of tissues, does not take away the iron artificially combined
with dead nuclein even after six days.
Hammarsten! has isolated from the pancreas of the ox
an iron-holding nucleo-proteid containing 4°48 per cent. of
phosphorus.
II. Metuops or Srtupy.
In my first communication on the method of demonstrating
micro-chemically the occurence of ‘‘ masked” iron, the reagent
whose use I described was called, in a general way, ammonium
sulphide, This is a term that is properly applicable only to the
diammonium compound represented by the formula (NH,),S,
but it is sometimes given to solutions which contain either
ammonium hydrogen sulphide (NH,HS), or polysulphides of
ammonium, or to mixtures of diammonium sulphide and
ammonium hydrogen sulphide. At the time I was unable to
determine which of the two latter is the most effective as a
reagent in liberating the iron from the chromatin, since
either, when recently prepared, gave, with cellular elements
from the same piece of tissue, reactions in which differences in
intensity were not noticeable, and, while uncertain upon this
point, I felt justified in adopting the generic term “ ammonium
sulphide” to designate a reagent which might be held to
indicate either of the two compounds.
About two years ago I gave further attention to the question
whether one form of the reagent is more efficient than the other
in this respect, and the results of a series of experiments made
since then have led me to the conclusion that ammonium
1 “Zur Kenntniss der Nucleo-proteide,” ‘Zeit. fiir Physiol. Chemie,’ vol.
xix, 1894, p. 19.
180 A. B. MACALLUM.
hydrogen sulphide is more active than the diammonium salt,
and that none of the polysulphides of ammonium have any
action whatever on iron in its “ masked” form. These experi-
ments have been controlled by others made with these reagents
upon solutions of potassium ferrocyanide.!| Ammonium sul-
phide, when mixed with a solution of the latter salt and the mix-
ture kept at a temperature of 30—50°C. for one or more days,
will liberate the iron from its combination and precipitate it as
sulphide, the amount so liberated depending on the strengths
of the solutions forming the mixture, on the temperature and
on the time during which the reaction is allowed to goon. A
lower temperature will suffice when the time is prolonged.
By paying due attention to all the conditions, it is possible to
liberate, as sulphide, all the iron of such solutions. In this
ammonium hydrogen sulphide is more active than diammonium
sulphide, the amount of the sulphide formed being a measure
of the activity of either reagent.” These experiments have, in
all cases, given results which correspond with those obtained
with the two sulphides upon the chromatin of isolated cells,
but it was not possible in the latter case to estimate the effects
as definitely. I found that of two slide preparations of isolated
cells, one made with ammonium hydrogen sulphide, the other
with diammonium sulphide, the former as a rule gave the
1 T have not found any reference to the action of ammonium sulphide on
solutions of ferrocyanides in the literature of chemistry, although, on the pre-
sumption that some such reference exists, I made diligent search for it.
2 The results of one experiment upon this point may be mentioned. The
glass-stoppered cylinder a contained 10 c.c. of a 10 per cent. solution of
potassic ferrocyanide and 10 ¢.c. of ammonium hydrogen sulphide made from
an ammonia solution of 0°96 sp. gr., while to a similar cylinder 4, with like
quantities of the same solutions, 10 ¢.c. of dilute ammonia were added. At
the end of twenty-four hours’ stay in a warm oven with a temperature of
40° C., the precipitates were filtered off with iron-free filters, washed with
water containing hydrogen sulphide in solution, dissolved in dilute sulphuric
acid solutions, and, after care had been taken to reduce all the iron to the
ferrous condition, the amount of the metal in each case was estimated by
titration with a standardised permanganate solution. Results: the precipi-
tate in a contained 0:0113 grm. iron, while the iron of the precipitate in J
amounted to 0:0025 grm.
IRON COMPOUNDS IN ANIMAL AND VEGETABLE CELLS. 181
maximum reaction in about ten days, while the latter mani-
fested a reaction of moderate intensity at the end of that time,
which, with a longer stay in the warm oven, did not become
more marked. In the case of vegetable cells the reactions
were more quickly obtained and the differences in intensity
greater. This is illustrated in figs. 14,15, and 16, representing
preparations of cells of the ovary of Erythronium ameri-
canum, in which the reagents used had been made from dilute
solutions of ammonia (of 0°96 sp. gr.). Fig. 15 indicates the
depth of the reaction with ammonium hydrogen sulphide at
the end of twenty-four hours, the intensity attaining in
another cell in ninety hours the degree represented in fig. 16,
while in fig. 14 is shown how far the reaction had progressed
with diammonium sulphide in forty-eight hours. In the latter
case the reaction did not become more marked even on the
eighth day. Similar results were obtained in all the experi-
ments of this character, demonstrating that ammonium
hydrogen sulphide is more effective in liberating iron from
organic combinations than is the diammonium compound.
In the earlier stages of the investigation the reagent was
made from strong solutions of ammonia of sp. gr. 0°88; but
when thus prepared it deteriorates rapidly and becomes yellow
from the formation of polysulphides. Spoiled or unsuc-
cessful preparations were consequently frequently obtained.
Sometimes, also, difficulties were experienced in determining
whether, in the preparation of the reagent, the saturation of
the strong ammonia with sulphuretted hydrogen was complete.
For this reason, and also because dilute solutions of ammonium
hydrogen sulphide are less unpleasant in every way, I began
to use the latter, and found that it gives results not less decided
than those obtained with the stronger solutions. The dilute
solutions offer other advantages, for when made from pure
ammonia of 0°96 sp. gr., they retain their potency for three
weeks or longer, especially if kept in a bottle with a well-fitted
glass stopper, and in a cool place. The smaller the amount
of air in the bottle and the less frequently the stopper is
removed, the longer does the reagent retain its strength,
182 A. B. MACALLUM.
During the last two years the dilute reagent has, in conse-
quence of these facts, been exclusively employed.
The glycerine used was chemically pure.! It gave the best
results when diluted with an equal volume of distilled water.
In making the preparations, the cellular elements were teased
out on the slide in a drop of the dilute glycerine, and over this,
after thorough admixture with two drops of the dilute solution
of ammonium hydrogen sulphide, a cover-glass of 16—22 mm,
square was placed. The teasing-out process was done in each
case with a clean pair of goose-quill points. Every care was
taken to prevent the occurrence of impurities in the prepara-
tions. The excess of the glycerine and sulphide mixture is at
first uncovered, but if the slide be put in a warm oven with a
temperature of 60° C., the mixture rapidly concentrates and in
a few minutes is wholly under the cover-glass. When the
solution of ammonium hydrogen sulphide is deteriorated, a
deposit of sulphur forms at the edges of the cover-glass and the
mixture under the latter becomes yellow through the produc-
tion of polysulphides ofammonium. Such preparations never
yield anything of value. On the other hand, when the fluid
under the cover-glass remains colourless and free sulphur does
not form at the margins, the preparation, 1f kept at a tempera-
ture of 55—60° C. for a period of from two to fifteen days, is
almost always successful. Sometimes at the end of one, two,
or three days the mixture is further concentrated and has
receded from one edge of the cover-glass. This is remedied
1 Molisch (‘Die Pflanze in ihren Beziehungen zum Nisen,’ p. 107) states
that the glycerine of commerce—even the purest—contains traces of iron. I
have not found this to be the case with Price’s glycerine, quantities of which,
when mixed with ammonium hydrogen sulphide or diammonium sulphide,
gave not the slightest reaction or precipitate, even after two weeks, and
whenever portions of the stock supply used were evaporated at a low heat in
a platinum dish no appreciable residue was left, and not a trace of iron or lead
was detected. I found that in some samples of glycerine of other manufacture
the sulphide gave no immediate reaction, but at the end of a week, or later, a
small precipitate, composed partly of sulphide of iron, was at the bottom of
the test-tube. A similar precipitate was obtained in portions of the stock
supply of Price’s glycerine only when traces of an iron salt were added.
IRON COMPOUNDS IN ANIMAL AND VEGETABLE CELLS. 183
by placing at the dry side of the cover-glass a drop of a mix-
ture of one part of dilute glycerine and two of ammonium
hydrogen sulphide, the drop so placed running under the cover,
after which the preparation is replaced in the warm oven and
in the end usually proves successful. I have found that when
the isolated cellular elements are not very numerous and
uniformly distributed under the cover-glass, evaporation rarely
goes so far as to rendera resort to this remedy necessary ; but
when the tissues are only partially teased, and fragments tilt
or elevate the cover-glass, the mixture concentrates, the pre-
paration dries at one side, and the sulphide is largely converted
into polysulphide.
The solutions of ammonia used in the preparation of the
reagents were chemically pure, and in this respect, as well as
in the cleanliness of the slides and covers, I paid due regard to
the suspicion that there possibly exists a ferrous sulpho-hydrate
(FeS,H,), soluble to a certain extent in solutions of ammonium
hydrogen sulphide, the presence of which in the glycerine and
sulphide mixture of my preparations might, through its diffu-
sion into the nuclei and precipitation therein as ferrous sulphide
(FeS), give confusing results. That no such compound existed
in my reagents was shown repeatedly by allowing mixtures of
the sulphide and glycerine to stand for weeks, when all the
ammonium hydrogen sulphide was converted into the diammo-
nium salt, or into polysulphides of ammonium, in the presence
of which it would appear that the supposed existence of ferrous
sulpho-hydrate is impossible. In these experiments no iron was
found, nor did the mixtures in the end lose any of their trans-
parency,—a result which tells against the possibility of any such
iron compound existing in the mixtures employed upon teased-
out cells. ‘The cover-glasses and slides were cleaned in solu-
tions of hydrochloric acid to remove any adherent compounds
of iron, and afterwards passed through distilled water and
alcohol. The bottles in which the solutions of ammonium
hydrogen sulphide were kept were also, first of all, cleansed
in the same way.
Nothing was gained by making ‘‘stock”’ mixtures, in the
184 A. B. MACALLUM.
proper proportions, of glycerine and ammonium hydrogen
sulphide, for in such the reagent is more rapidly converted
into the non-active form than whenit is kept separate. Appa-
rently also in “stock” mixtures the polysulphides are very
rapidly formed, the fluids becoming deep yellow in twenty-four
hours or less, although the sulphide used may be nearly colour-
less. In summer the change of colour is rapid. That it is
due in part at least to the formation of polysulphides, appears
to follow from the fact that drops of the mixture, when
allowed to remain uncovered on the slide for a few minutes,
quickly become milky in appearance from the precipitation of
free sulphur. The mixtures retain a part of their strength
during the first two or three days, after which they become
useless.
The tissues which were teased out for treatment were always
hardened in alcohol wholly free from iron in solution. Latterly
I have employed for this purpose redistilled methylated spirit.
I have not used in this connection material fixed with any of
the mineral hardening reagents, since the latter frequently
contain iron, the presence of which in dying cells and tissues
might be held to contribute, under the influence of the harden-
ing reagent, to the formation of firm organic compounds of
iron. Some mineral reagents, moreover—as, for example,
corrosive sublimate and osmic acid—are difficult to remove
from the tissues upon which they have been allowed to act,
and their presence in preparations treated with ammonium
sulphide, which forms sulphides with these metals, gives
appearances obscuring, in a greater or less degree, the occur-
rence of iron compounds.
To facilitate the teasing-out I frequently used sections made
with a clean steel knife! covered with absolute alcohol, the
cells of such sections readily separating, and yielding sometimes
a number of free nuclei. In order to determine whether iron
in an inorganic or albuminate form is present, and to what
1 Jn my earlier paper (loc. cit.) I pointed out that the knife so used gives
no iron to the preparation. All my observations for the last two and a half
years have in no way called in question the correctness of this contention.
IRON COMPOUNDS IN ANIMAL AND VEGETABLE CELLS. 189
extent, it was my practice to allow the section to lie in the
glycerine and sulphide mixture for a few minutes before teasing
it out, the iron of these forms of combination giving an imme-
diate reaction on the penetration of the reagent. The removal
of all iron of this description is necessary, since its presence
may give confusing results in teased-out cells. For this
purpose I have used Bunge’s fluid, in which the sections were
kept for about an hour with the reagent at a temperature of
55° C., the subsequent treatment with alcohol and ammonium
hydrogen sulphide in all cases showing that the inorganic and
albuminate iron had been thereby removed.! Sections so
treated were teased out and mounted in the glycerine and
sulphide mixture in the usual way.
The disadvantages connected with the use of ammonium
hydrogen sulphide to demonstrate the presence of ‘ masked”
iron are that it effects, in the animal cell at least, structural
changes, that it is not successful on large nuclei or on nuclei
of large cells, and that it requires a great expenditure of time.
In regard to the structural changes it is obvious that, however
well hardened or well fixed cellular elements may be through
the action of alcohol, ammonium hydrogen sulphide or diam-
monium sulphide must, when heat is applied, sometimes alter,
to a greater or less degree, the structure of the cell, and
especially ofits nucleus. This is quite evident when we compare
such preparations with others in which the “ masked” iron has
been liberated by the use of sulphuric acid alcohol, and sub-
sequently treated with the sulphide. Figs. 23 and 24 illustrate
the differences obtained with the two methods, the former
representing liver-cells of Necturus lateralis treated for
ten days at 55° C. with the glycerine and sulphide mixture,
while the latter was drawn from a section of the same material
after it had been acted on by sulphuric acid alcohol for seven
hours at 85° C., and then with the glycerine and sulphide
mixture. The first difference to be noted between the
1 In regard to the capacity of Bunge’s fluid for extracting iron of all forms
of combination, see the description of the properties of hydrochloric acid
alcohol as given below.
186 A. B. MACCALLUM.
preparations represented is that of the iron reaction illustrated.
This is partly due to the fact that in one preparation the
ammonium hydrogen sulphide has not liberated all the iron of
the chromatin, but partly also to the fact that the reagent has
caused the delicate chromatin elements to become swollen,
thereby rendering the iron reaction more diffuse and less
marked. The effect on the cytoplasm is not less striking. It
is, however, chiefly with concentrated solutions of ammonium
hydrogen sulphide that preparations of animal nuclei exhibit
this phenomenon. Solutions of the reagent made from am-
monia of 0:96 specific gravity do not as readily produce this
change, and in many cases none at all may be shown. When
the reagent is fresh the reaction is quickly obtained, sometimes
in two or three days, and then no swelling of the nuclear net-
work occurs; but when it is not fresh, or when it gives an
odour of ammonia, the reaction is slowly obtained, and the
prolonged application necessary in order to bring out this
result, aided perhaps by the ammonia, causes a swelling of the
chromatic elements.
The slowness with which the reaction comes out is not
wholly a disadvantage, for by this means one may determine
whether the iron demonstrated is derived from other than
inorganic or albuminate compounds. With the exception of
hemoglobin, hematin, and the compound found in yolk-
spherules, the organic combinations in which the iron is
“masked” are affected very slowly by ammonium hydrogen
sulphide, and only when heat is applied ; whereas the reaction
comes out at once, or after a few minutes at the longest, and
without heat, in the case of inorganic and albuminate com-
pounds. The distinction between these and the ‘ masked”
compounds is, therefore, very marked. In one of the excep-
tions mentioned the distinction is not so clear, for when
ammonium hydrogen sulphide is added to the fresh yolk of
hen’s egg it gives a greenish reaction at once, but when the
yolk is hardened with alcohol or with heat the reagent gives
this result only after several days’ application at 50—60° C,
On the other hand, the yolk-spherules in Amphibia (Necturus
IRON COMPOUNDS IN ANIMAL AND VEGETABLE CELLS. 187
and Amblystoma), whether hardened or fresh, yield the
reaction in a few minutes. Such compounds are of too limited
a range of distribution to affect the value of the reagent in
making a distinction between the iron compounds. On
hemoglobin and myo-hzematin (myo-hzemoglobin) the reagent
has not the slightest action. I have kept mixtures of the
reagent with solutions of hemoglobin and myo-hemoglobin
for more than a year at a temperature of 55° C., and in no case
have I found that iron was liberated from these compounds as
sulphide. I have, moreover, mounted in the glycerine and
sulphide mixture on the slide finely powdered hemoglobin
which had been coagulated in alcohol, and applied heat to the
preparation for weeks without once obtaining the iron in an
inorganic form. When, therefore, in preparations of animal
tissues which have been hardened in alcohol one obtains with
the glycerine and sulphide method after a time an iron reac-
tion, it may reasonably be concluded that the iron so demon-
strated is not derived from hemoglobin in the tissues. One
may not, however, exclude hematin as a possible source of iron,
for although hemoglobin in all forms will not yield its iron to
ammonium hydrogen sulphide, the latter readily liberates the
iron of hematin, and from a solution of hematin in am-
moniated alcohol or in dilute ammonia, into which hydrogen
sulphide has been passed, part of the iron at ordinary tempera-
tures, but the whole at 50° C., is precipitated as ferrous sulphide,
in a few days.! Even in a solution of hematin in ammoniated
alcohol, if kept for several days at the temperature of the room,
1 The compound formed from the hematin in this process of liberating the
iron is neither hematoporphyrin nor bilirubin. With yellow nitric acid it gives
a play of colours in which violet, faint red, and yellow successively appear, the
mixture finally becoming colourless, and it yields an absorption spectrum like
that of bilirubin. It is insoluble in ether, and soluble in chloroform and hot
alcohol. The other properties of this compound are now under investigation.
It has one special claim to interest in that it is formed from hematin by a
method very much less drastic in its effects than those in which strong sul-
phuric acid or bromine in glacial acetic acid is used to form hematoporphyrin
or bilirubin (Nencki and Sieber, ‘Monatsh, fiir Chemie,’ vol. ix, p. 115,
1888),
188 A. B. MACALLUM.
a part of the iron of the hematin is precipitated as a greyish-
white hydroxide, which, if filtered off, gives at once with
ammonium sulphide the greenish-black sulphide reaction.
Very weak solutions of hydrochloric and other acids effect the
removal of the iron, and if solutions of hematin in alcohol are
kept for a week or more in contact with solutions of various
salts (potassium chlorate and sulphate and sodium chloride
and phosphate), decomposition of the hematin results, and
iron is liberated as an inorganic compound. In all these
respects hematin behaves like the ferrocyanides, while it differs
markedly from haemoglobin in the same points.
Experiments show, however, how little, if any, of the iron
demonstrated in animal cells is derived from hematin. Sec-
tions of the liver and other organs of Vertebrates, as well as of
vegetable tissues, were placed in alcoholic solutions of hematin
for twenty-four hours, then washed in alcohol for a few minutes,
and kept in a quantity of the glycerine and sulphide mixture
at a temperature of 35° C. for twenty-four hours. At the end
of the latter interval all the sections were blackened, and
under the microscope the nuclei were dark green from the
ferrous sulphide liberated from the hematin absorbed by the
chromatin. In order to get this result the sections do not
require to be teased out at all. The rapidity with which such
a strong reaction is obtained indicates that in ordinary teased-
out cells mounted on the slide in the glycerine and sulphide
mixture, the deep reactions obtained after several days or after
a week are due to a decomposition, not of hematin, but of
some other compound or compounds.
Ammonium hydrogen sulphide, then, may be regarded as a
reagent of very great value in the investigation of ‘‘ masked ”
compounds of iron, and it must constitute a final test for this
purpose, whenever the accuracy of the other reagents, used
also for determining the distribution of assimilated iron com-
pounds in cells, is called in question.
In June, 1891, Mr. R. R. Bensley, while carrying on under
my direction a research on the distribution of iron in the
ovary of Erythronium americanum, as demonstrated by
IRON COMPOUNDS IN ANIMAL AND VEGETABLE CELLS. 189
the employment of ammonium sulphide, succeeded in obtain-
ing some interesting results which necessitated control experi-
ments based on the removal of all traces of inorganic com-
pounds of iron from the tissues under investigation. for
this purpose Bunge’s fluid was used, and it was thought that
hardened specimens of the ovary, when subjected to its action
for a time, would not give, on the addition of ammonium
sulphide, any immediate reaction for iron, and that further
treatment with the latter reagent in a warm oven for several
days would show the presence of iron in the nuclei of their cells,
and possibly also in their cytoplasm. Much to our surprise,
however, the treatment of the ovary of Erythronium with a
quantity of Bunge’s fluid for two weeks at 20°C., and the
subsequent application of ammonium sulphide, resulted in the
production of a marked reaction for iron, which under the
microscope was found confined to the nuclei. I was at first
inclined to believe that the iron so shown was due to diffusion
into the nuclei of that present in an inorganic form in the
tissues, and this would appear to be Gilson’s view; but repeated
experiments have demonstrated the incorrectness of this
explanation, and that Bunge’s fluid liberates the iron of organic
compounds.! Experiments were also made on animal tissues
and similar results were obtained. The liberation of the iron
is to be attributed to the hydrochloric acid, the only active part
of the reagent. This conclusion suggested a number of
experiments, all based on the principle that whatever proper-
1 Gilson’s statement is difficult to interpret. He does not say whether he
applied the reagent to sections of tissues or to the latter in mass, and at what
temperature it was allowed to act. He appears to regard the iron absorbed
by dead nuclein as combined with the latter, and he remarks, in reference to
my statement that Bunge’s fluid removes all inorganic and albuminate iron
from sections after treatment with it for ten hours: “but I have observed
that Bunge’s liquid does not take away the iron artificially combined with
dead nuclein after six days.” I can explain his statement only on the sup-
position that he used the reagent on the tissues in mass, and that he thereby
obtained the same results that I did under similar circumstances; in other
words, the iron “artificially combined with dead nuclein” was in reality iron
liberated by Bunge’s fluid from its masked condition in the chromatin and re-
tained by the latter.
190 A. B. MACALLUM.
ties hydrochloric acid may have in this respect are possessed,
in a greater or less degree, by other mineral acids, whether in
dilute aqueous solutions or in alcohol, and the results were of
such a character as to induce me to employ these reagents on
all species of cells in which the distribution of iron had been
determined with ammonium hydrogen sulphide.
The more serviceable of these were found to be sulphuric
acid and nitric acid dissolved in alcohol of 95 per cent.
strength. The former was prepared by adding four volumes
of the strong acid to one hundred of alcohol, while the latter
contained three volumes of the acid (of 1:4 sp. gr.) in one
hundred of alcohol.
The chemicals used in the preparation of these reagents
were free from traces of iron, and care was taken to have all
bottles and vessels used to hold them also free from adherent
iron compounds. It was, of course, impossible to provide
against the iron in the glass, but I am not certain that the
reagents derived any from this source, even in infinitesimal
quantities. The alcohol used contained not a trace of iron,
During the last eighteen months re-distilled methylated spirit
was found to be in every way as serviceable as the pure ethyl
alcohol used earlier in the investigation.
The alcohol of these reagents largely prevents the occurrence
of digestive changes which the acids effect when, in aqueous
solutions, they are allowed to act on tissues for several days,
and especially at a slightly elevated temperature. Another
important function of the alcohol is to prevent a too rapid
extraction of the liberated iron, and thereby also its diffusion
from one part of the tissue into another, from nucleus to cell,
or from cell to nucleus. Acid alcohols dissolve iron salts
more readily than does alcohol alone, but less so than aqueous
solutions of the acids. For example, ferrous sulphate is
insoluble in absolute alcohol and in strong methylated spirit,
but it is soluble in these when they contain a small quantity
of sulphuric acid,—not, however, in any way as much so as in
distilled water, or in dilute aqueous solutions of sulphuric
acid. The smaller the proportion of the acid in the alcohol
IRON COMPOUNDS IN ANIMAL AND VEGETABLE CELLS. 191
the less readily does it dissolve the iron salts, and when used
upon tissues acid alcohols have a smaller capacity for extracting
the iron salts the longer the reagents are allowed to act, for
the liberation of the iron from its “ masked ” condition entails
the neutralisation of the acid, a very gradual process. Asa
result of this neutralisation the iron salts become less soluble
and would pass back into the tissues, but the danger of this
happening is minimised or altogether prevented by the
property which the chromatin has of retaining the iron that
is set free in itself by the acid alcohol. This is shown
specially in the case of nitric acid alcohol, for when sections
of vegetable tissues are allowed to lie for two weeks in a large
quantity of the reagent, the nuclei at the end of that time give
as intense a reaction for iron as they do at the end of two
days. The result is due to the fact that the tenacity with
which the chromatin holds the iron liberated in it counteracts
the extractive capacity of the reagent.
The results of the action of nitric and sulphuric acid alcohols
differ from those obtained with Bunge’s fluid! in one important
respect. The two former, whether they are used upon the
tissues in mass or on sections of the same, leave the iron, on the
whole, in the parts in which it is liberated ; but when sections
of tissues are treated with hydrochloric acid alcohol, the iron
is extracted as quickly as it is liberated, and consequently
such preparations on treatment with ammonium sulphide give
a feeble reaction for iron or none at all. This is most
distinctly seen when the temperature is raised, and if the
reagent is allowed to act for two or three days under these
conditions, no iron, organic or inorganic, is left in the prepara-
tions. When the tissues are in mass, on the other hand, the
quantity of acid that penetrates the preparations is largely
neutralised and extraction takes place very slowly, with the
result that teased-out portions of such tissues give a marked
reaction for iron, limited, as in preparations obtained with
1 Bunge’s fluid, or hydrochloric acid alcohol, consists of ninety volumes of
alcohol of 95 per cent. strength, and ten volumes of a 25 per cent. solution of
hydrochloric acid.
VoL. 38, PART 2.—NEW SER. N
192 A. B. MACALLUM.
the other acid alcohols, to the parts in which treatment
with the glycerine and sulphide mixture demonstrates its
occurrence.
In describing the properties of hydrochloric acid alcohol,
Bunge expressly states that while it extracts inorganic iron
it does not remove the iron from the nuclein (hzmatogen) of
egg-yolk.! Thisis not quite correct, for when hard-boiled yolk
is treated with ammonium sulphide it gives only a feeble reac-
tion for iron, even when kept for twenty-four hours at an
elevated temperature ; but when it has been acted on by a
quantity of Bunge’s fluid for a day at 30—35° C., the applica-
tion of ammonium sulphide, after all traces of the acid have
been removed with alcohol, gives an immediate and marked
reaction for iron. The iron under such conditions must be
in the form of chloride, and as an inorganic compound it
should be extracted by the reagent, if Bunge’s views con-
cerning the properties of the latter be correct, but this
happens only when the quantity of the yolk so treated is
very small, and then the whole of the iron is removed in a few
days, this fact demonstrating clearly that the reagent in its
action makes no distinction between inorganic and organic
iron. The latter is in its liberation from the ‘‘ masked ”’ con-
dition converted into the inorganic form, and it depends on the
quantity of yolk used whether or not the extraction may keep
pace with this conversion. If the quantity is large, the libera-
tion of the iron from its organic combination entails a diminu-
tion of the acidity of the reagent, and at length the extraction
of the liberated iron ceases. It commences again only when a
fresh quantity of the reagent is substituted for the exhausted
fluid.
The results of its action upon the iron-containing nucleo-
albumin of yolk are therefore practically similar to those which
it gives when applied to animal and vegetable tissues.
The fact that a considerable diminution of the acidity of
hydrochloric acid alcohol allows the liberated iron to be retained
1 «Ueber die Assimilation des Hisens,” ‘Zeit fiir Physiol. Chemie,’ vol.
ix, 1885, p. 49.
IRON COMPOUNDS IN ANIMAL AND VEGETABLE CELLS. 193
in its original position in the cell has led me to try the effects
of solutions in which the strength of the acid was less than
1 per cent.,! and they have been found, when used upon thin
sections of tissues, to give very successful preparations, permit-
ting the iron liberated to be demonstrated as fully as after the
employment of either sulphuric or nitric acid alcohol.
The time during which these reagents must be allowed to
act on a piece of tissue varies. I prefer to give general state-
ments on this point, because specific directions are impossible
in a case where the size of the object, the quantity of the
reagent, and the temperature constitute the conditions.
Bunge’s fluid extracts as readily as it liberates the iron in
thin sections of tissue, but when the latter is in mass the
reagent requires a length of time which may vary from a week
to two months, all depending on the size of the object and on
the temperature, which in summer may be that of the room
(20°—29° C.), but in the colder seasons that of the warm oven
(35° C.). Sulphuric acid alcohol acts more slowly, and con-
sequently requires a longer time for liberating the iron in
unsectioned objects, while in sections its action is complete in
from one to four days, this depending also on the temperature,
the most favourable being 35° C. A longer stay than is just
sufficient to liberate all the organic iron results in removing
from the sections some of the iron set free, the more being
extracted the longer the sections lie in the reagent. When
examples of the Protozoa and Protophyta were subjected to
1 These differences in extractive capacity exhibited by weak and strong
alcoholic solutions of hydrochloric acid have apparently not been noted by
Petit (loc. cit.), who used the diluted reagent in a Soxhlet apparatus to
remove the inorganic iron compounds from barley. As the boiling-point of
hydrochloric acid is higher than that of alcohol, it is obvious that little of the
former must pass from the 1 per cent. solution at the bottom of the flask as
vapour to condense above and act on the substance whose iron is to be ex-
tracted, while the alcohol is readily converted into vapour; in other words,
the reagent in the upper part of the apparatus must be much more dilute than
that in the flask below, and consequently its extractive power must be very
feeble. This method is, therefore, open to the objection that it does not
ensure the removal of inorganic iron compounds,
194 A. B. MACALLUM.
the action of the acid alcohol the full effect was obtained at
the end of twenty-four hours at the latest, when the tempera-
ture was 85°C. With nitric acid alcohol the liberation of
the organic iron was rapid, sections of vegetable tissue (Ery-
thronium and Iris) giving, after a stay of ten hours in the
reagent at 85°C., an intense reaction with the acid ferrocyanide
mixture. Ata lower temperature the result was less marked,
but the reaction was deeper than that obtained with sections
treated with sulphuric acid alcohol for the same length of
time and at the same temperature. The process of liberation
was usually completed in about thirty-six hours. So little
does nitric acid alcohol extract the iron it liberates that in
sections of the ovary of Erythronium americanum kept
for six weeks in it I found little diminution in that intensity
of the iron reaction which sections, placed in the same fluid at
the commencement with the others, gave at the end of two
days. With sections of animal tissue the intensity of the
reaction was less marked with the prolonged stay in the
reagent, which, after four or five days’ action, slightly alters
the cellular structures. When nitric acid alcohol is allowed
to act on a section for a longer time than is necessary to set
free all its organic iron, diffusion of the iron salts thus formed
is apt to occur, especially in vegetable preparations, the cyto-
plasm giving in such cases a reaction for iron.
That the iron demonstrated after the use of acid alcohols is
derived from organic compounds I have shown by numerous
experiments. I have found that when thin sections of animal
or vegetable tissue are covered with a large quantity of Bunge’s
fluid and kept for three days at 35° C. or higher, the teased-
out cells give no iron reaction when mounted with glycerine
and ammonium hydrogen sulphide on the slide, even after two
weeks and at 60°C. Furthermore, sections so treated with
Bunge’s fluid, when subsequently subjected to the action of
sulphuric acid alcohol or of nitric acid alcohol, yield no iron re-
action whatever. Bunge’s fluid, therefore, extracts the iron which
the prolonged application of ammonium hydrogen sulphide and
glycerine at an elevated temperature liberates and demon-
IRON COMPOUNDS IN ANIMAL AND VEGETABLE CELLS. 195
strates, and with this removal disappears the iron demonstrable
after treatment with either of the other acid alcohols. This
shows that the iron in such cases cannot be derived from the
reagent nor from the glass of the vessel used, and this is em-
phasised by the results of other experiments. I extracted with
Bunge’s fluid all the iron from a series of sections of an ovary
of Erythronium, and then subjected these to the action ofa
large quantity of sulphuric acid alcohol for twenty-four hours
at 85° C. These gave no iron reaction, while others did so
which had not been treated with Bunge’s fluid, and which were
put in the acid alcohol at the same time. That the absence of
an iron reaction was not due to a lack of absorptive capacity
on the part of the section, brought about by Bunge’s fluid,
was proved when such sections were allowed to stay in sulphuric
acid alcohol containing a little ferric salt in solution! for half
an hour. The reaction obtained was marked, and almost wholly
confined to the nuclei. These experiments were repeated
again and again with sections of animal and vegetable tissues,
and the results were always the same, proving that the iron
demonstrable after acid alcohol has been used on tissues is
derived from the latter, and not from the reagent or the vessel
used. These experiments indicate, however, how necessary it
is, in investigating the distribution of iron in tissues, that the
reagents should be absolutely free from iron, and that, in
sections of tissues containing iron in an inorganic or albuminate
form, there is danger, when either sulphuric acid alcohol or
nitric acid alcohol is used upon them, of its redistribution, and
especially of its deposition in those parts of the cell which
absorb various compounds readily. In order to guard against
this, I found it advisable to steep the sections in a quantity of
Bunge’s fluid for a time which varied with the temperature at
which the reagent was applied, as, for example, for one to
two hours at 50°—60° C., but for eight to ten hours at 35° C.
1 This solution was made in the following way. A quantity of sulphuric
acid alcohol was allowed to act on ferric oxide in powder for about a week,
when a portion passed into solution as a ferric salt. Of this solution 1 e.c.
was taken and added to 10 ¢.c. of pure sulphuric acid alcohol.
196 A. B. MACALLUM.
Bunge’s fluid extracts very little or no iron from sections when
the temperature is below 20°C., but at the higher temperatures
stated the extraction is complete at the end of the intervals
mentioned, and with a longer action more or less of the
“masked” iron is liberated and removed. When a tissue—
as, for example, that of the spleen in some animals—contains
an excess of iron in an inorganic form, the time of extraction
must be prolonged, and the extracting fluid large in quantity,
After the inorganic and albuminate iron has been thus removed
from a section—a result which may be demonstrated by
treatment of the preparation with ammonium sulphide,—it
may be subjected to the action of either of the two other acid
alcohols to liberate that portion of the “ masked” iron as yet
unaffected.
The acid alcohols do not readily attack and liberate the iron
of hemoglobin and hematin except at a high temperature.
Of this fact I have convinced myself by numerous experiments
on hemoglobin, whether prepared from alcohol material or
from that coagulated by heat. A quantity of it, in a powdered
form, put into a flask and covered with a quantity of Bunge’s
fluid, was heated for twenty minutes, and the fluid then, after
filtration through a filter free from iron, was neutralised and
treated with ammonium sulphide. The mixture gave no imme-
diate evidence of the presence of iron, but when the test-tube
containing it was put aside for twenty-four hours, a dark-green
sediment made its appearance, and this was shown to be sul-
phide of iron when it was separated on an iron-free filter and
treated with a quantity of an acid ferrocyanide mixture. This
iron was, in great part, derived from the hemoglobin and
hematin, as well as from organic combinations present in the
leucocytes and plasma, and but little had its source in the in-
organic and albuminate compounds of the same, a fact shown
by further experiments on the powder which had once been
acted upon by boiling Bunge’s fluid. The extract made with
a fresh quantity of the reagent gave, on neutralisation and on
the addition of ammonium sulphide, the same evidence of the
presence of iron that was obtained in the first experiment. A
IRON COMPOUNDS IN ANIMAL AND VEGETABLE CELLS. 197
third, fourth, and fifth extraction resulted in the same way.
When, on the other hand, a quantity of crystallised hemo-
globin was acted upon by the reagent for forty-eight hours at
35° C., the filtered fluid, tested for iron in the manner
described, gave a scarcely appreciable evidence of the presence
of the metal. The iron, therefore, which is found in animal
tissues after the use of Bunge’s fluid at either 35° or 50° C.
for short intervals cannot very well be supposed to be derived,
in any appreciable quantity, from the hemoglobin in them, and
as ammonium hydrogen sulphide does not affect the iron of
the pigment, yet reveals the iron of “ masked ” combinations of
an apparently less firm character, it follows that weak solutions
of hydrochloric acid at slightly raised temperatures must attack
such combinations more readily than it affects hemoglobin.
This was most clearly shown by results of experiments on
hemoglobin and chromatin with a quantity of Bunge’s fluid
for twenty-four hours at 35°C. When hemoglobin alone is
thus treated, neither the powder nor the extract gives any
appreciable indication of free iron, but the latter is readily
demonstrable in chromatin, or in mixtures of chromatin and
hemoglobin, after similar treatment. Since the iron in
hemoglobin is not affected to any perceptible degree by treat-
ment with the reagent for twenty-four hours at 35° C., one
may postulate that it is as little affected by treatment with
either of the other two acid alcohols at the same temperature,
and experiments with these have given results which bear out
this conclusion.
The substance chlorophyll, the relations of which to iron,
though generally recognised, have not been definitely deter-
mined, is, as is well known, an abundant constituent of the
cells in many vegetable forms, and, therefore, a brief discussion
of the possibility that this substance is the source of the iron
demonstrated in vegetable cells, is necessary.
Some of the more recent investigators of this substance have
made conflicting statements on the question of the presence of
iron in the molecule. Adolph Hansen! found it to contain
1 ‘Die Farbstoffe des Chlorophylls,’ Darmstadt, 1889, p. 58.
198 A. B. MACALLUM.
iron, while Emich, at the request of Molisch,' examined a
quantity of pure chlorophyll and found it free from iron.
Molisch also made observations on the subject, and determined
that, after every care was taken to prevent contamination with
iron salts through impure extracting fluids, the ash of chlorophyll
gave not the slightest reaction for the meta]. Gautier’ also
claims that it does not contain iron. Schunk,® on the other
hand, found ferric oxide in the ash of phylloxanthin, one of
the decomposition products of chlorophyll, even after that com-
pound had been treated with acids and after repeated solution
of it in ether.
The material from chlorophyll-holding organisms was, in all
cases, thoroughly freed from that substance before the disposi-
tion of the iron in it was examined. Chorophyll, however, has
not in any of my preparations yielded any evidence that it
contains iron, nor does its presence or absence at all affect the
question of the occurrence of iron in other compounds in the
cell. This is very distinctly shown when one compares the
results, obtained from experiments on vegetable cells holding
chlorophyll, with those determining the distribution of iron in
Fungi and in Monotropa uniflora and Corallorhiza mul-
tiflora, which are destitute of chlorophyll. In the two latter
the disposition of the assimilated iron is as it is in the chloro-
phyll-holding Phanerogamous plants, and consequently one
may dismiss the objection that the pigment constitutes the
source of the iron demonstrated by my methods in the nuclei
of vegetable cells. It may be proved also from Monotropa*
1 Op. cit., p. 87.
2 ‘Chemie Biologique,’ Paris, 1892, p. 20.
3 “Contributions to the Chemistry of Chlorophyll,” No. 4, ‘Proceedings
Roy. Soe.,’ vol. 1, 1891, p. 302.
4 The importance of Monotropa material for control purposes renders a
short description of the methods of preparation employed upon it necessary.
This plant, when hardened in alcohol, blackens more or less through the pro-
duction on the part of the dying cells of a dark greenish-blue pigment, but
it remains colourless when fixed in solutions of corrosive sublimate, a reagent
whose use is, for reasons already mentioned, objectionable when ammonium
sulphide is to be employed. To obtain material on which this reagent may be
IRON COMPOUNDS IN ANIMAL AND VEGETABLE CELLS. 199
that none of the iron found in the nuclei is derived from the
cytoplasm, for there is very little and often no cytoplasm in
the cells of the coats of the ovules in this plant, and yet the
nuclei of these give as intense a reaction as those of the ovary
of Erythronium, Iris, Hyacinthus, or any form in which
the cytoplasm is abundant.
In order to get the best results with the use of the acid
alcohols, I have found that the tissues must be well hardened.
If the tissues are fresh or imperfectly hardened, the application
of acid alcohols for a time sets free the organic iron, but the
structure of the cellular elements is more or less changed in
such cases by the acids—a change not at all found to occur
when the tissues have been carefully hardened. Strong alcohol
(90—95 per cent.) was used for this purpose, and it was found
to present, over the other hardening reagents, a number of
advantages. It can by redistillation be made free from iron,
and when it is of absolute strength it neither extracts any of
the iron compounds (hzmatins excepted) from tissues, nor
allows these to diffuse. There is the important point also, that
tissues fixed with it can be subjected to all the reactions for
iron, without incurring the risk of complications due to the
deposition of iron or other metallic salts, which occur when
other hardening reagents are used. In this way one may treat
pieces of a tissue with ammonium hydrogen sulphide and with
the acid alcohols, and thus allow the methodsto control each other,
When, on the other hand, it was not necessary to use ammo-
nium hydrogen sulphide on the tissues, other hardening reagents
were employed, but only such as did not, by their presence in
the tissues, interfere with or obscure the demonstration of the
iron. Saturated solutions of corrosive sublimate and } per
used advantageously, parts of the fresh plant are thrown into boiling distilled
water, and those which remain uncoloured at the end of ten minutes are
further hardened for several days in absolute alcohol. I have often treated
material so prepared with the warm glycerine and sulphide mixture for from
four to ten days, and then with an acid ferrocyanide solution converted the
ferrous sulphide demonstrated into Prussian blue. Such preparations are pro-
bably the most instructive obtainable in regard to the question of the relation
of iron to the vegetable cell.
200 A. B. MACALLUM.
cent. solutions of osmic acid were found serviceable, the latter
reagent also having been used in the combination known as
Flemming’s fluid.
The corrosive sublimate solution was allowed to act on the
preparations of tissue for about ten minutes, after which they
were washed for a few minutes in distilled water, and then in
50 per cent. alcohol. The hardening was completed with
alcohol of 70 and 90 per cent. strengths in the usual way.
When Flemming’s fluid was used the tissue was not allowed to
lie in it for more than half an hour, while for the osmic acid
solution not more than ten minutes were given, and the fixa-
tion was carried on further with alcohol of 50, 70, and 95 per
cent. strengths. Preparations, whether made with corrosive
sublimate or with osmic acid solutions, retain, even after
careful washing, traces of the metallic salt of the reagent used,
and the black or dark reaction which they give with ammo-
nium hydrogen sulphide, in consequence of the presence of
such metals, interferes with the proper demonstration of the
distribution of iron by that reagent. On this material the acid
alcohols only were used, and the preparations were subsequently
treated with the acid ferrocyanide mixture, the Prussian blue
reaction obtained not having been in the least affected by the
presence of minute quantities of the metallic salts of the har-
dening reagents. The latter were free from iron salts, a fact
of which I convinced myself by qualitative analyses.
To the use of all hardening reagents other than alcohol there
are objections. Those which contain an acid may assist in the
diffusion of iron salts in the tissues, and cause the deposition
of these in some other parts than those in which they originally
were held. Further, the acids of some of the reagents (e. g.
acetic acid in Flemming’s fluid) may liberate the organic iron,
which cannot in such a case be distinguished from the iron of
inorganic or albuminate combinations. For these reasons I
have used acid hardening reagents but occasionally, and then
the time allowed for their action was short, in order to reduce
to a minimum the risk of liberating organic iron, and of the
diffusion of iron salts through the tissues, Against corrosive
IRON COMPOUNDS IN ANIMAL AND VEGETABLE CELLS, 201
sublimate as a hardening reagent, which I have frequently
used, it may be urged that it possibly assists in the diffusion
through the tissues of the inorganic compounds of iron, and
that consequently the distribution of the latter, in preparations
thus hardened, may not correspond with that obtaining in the
fresh tissue. Where this is not under investigation it is a
matter of no importance, for treatment of sections of the tissue
with warm Bunge’s fluid for a few hours removes such com-
pounds, and the sections so treated may be subjected to the
action of the various reagents described to demonstrate the
organic iron; but when it is desired to study the distribution
of both classes of iron compounds in a tissue, the objection
urged would, if well founded, exclude corrosive sublimate as a
hardening reagent for this purpose. My experiments in rela-
tion to this were made on pieces of the same organ (liver and
kidney of guinea-pig and of Amblystoma) hardened with
alcohol alone, and with corrosive sublimate and alcohol, and I
have found, on comparing the distribution of iron in both series
of preparations, that though the possibility of the diffusion of
iron salts is not excluded when corrosive sublimate is used, yet
no appreciable evidence of it was manifestedin the preparations.
I have not, however, based my observations in any one case
alone upon material hardened in corrosive sublimate, but have
used material hardened in alcohol in all cases to control the
results obtained when that reagent was used.
When the iron was liberated by acid alcohol the whole of
it appeared as a ferric salt in some tissues, while in others a
very small portion of it also was set free as a ferrous com-
pound. The latter condition was illustrated in some of the
Protozoa. Insuch preparations all the iron set free is demon-
strated, after treatment with ammonium sulphide, as a ferrous
salt, and the preparations may then, on being acted upon with
a mixture of equal volumes of dilute solutions of hydrochloric
acid and potassic ferrocyanide, reveal all their liberated iron
as Prussian blue. The iron in the ferrous form is usually so
very minute in quantity, if present at all, that it may be
1 For an explanation of the preponderance of the ferric compound see p. 268.
202 A. B. MACALLUM.
neglected in the making of permanent preparations. In order
to prevent contamination of the sections with iron compounds
in the demonstration, the solutions of potassic ferrocyanide
were, on all the occasions used, not more than a week old,
although I found that those of longer standing, up to the end
of two months or so, when filtered carefully, gave preparations
which were free from any objectionable characters. The
strength employed was 1°5 per cent., and a volume of this
was mixed with an equal volume of hydrochloric acid of 0°5 per
cent. strength, when the mixture was required.
The sections, after removal from acid alcohol, were first
washed in pure alcohol, then in distilled water, after which
they were placed in the acid ferrocyanide mixture for not more
than five minutes. Again washed carefully in distilled water,
they were either dehydrated in alcohol, cleared in oil of cedar,
and mounted in benzole balsam, or, before they were put
through this course, stained with either safranin or eosin.
The staining reagents were of 1 per cent. strength in 30
per cent. alcohol, and the time allowed for the action of the
eosin was three minutes, while that for the action of safranin
was half an hour. The excess of the stain in either case was
removed with alcohol. The advantages given by the use of
these stains I have explained in the description of the con-
stituents of the nucleus. Very frequently I have found that
a preparation which illustrated, in a remarkable way, some
point in the distribution of iron in the cell, became useless
through a complete fading out of the blue. The causes of
this result are two: exposure of the preparation to the light
for a time, and the use of inferior oil of cedar, that is, impure
through the presence of minute quantities of water and other
matters. I found that when I used old oil of cedar to clear up
the sections and to remove all traces of alcohol, the prepara-
tions would keep their beauty unimpaired, if placed away from
the light in the slide box. In some way the preservation of the
blue colour depends on leaving a trace of the oil used in
clearing-up upon the section when the balsam is added, but
in this quantity allowed to remain there must be no alcohol or
IRON COMPOUNDS IN ANIMAL AND VEGETABLE CELLS. 2038
water. When oil of cloves or oil of lavender was used all the
preparations faded, for some reason at present unexplainable.
The presence of safranin or eosin in the preparation does not
influence, in any way, its chances of fading, but if the excess
of the stain has not been removed it is apt, while the balsam
is hardening, to diffuse, and thereby obscure the finer details
of the preparation. That it is not difficult to keep Prussian
blue preparations of animal and vegetable tissues, if carefully
made, is shown by the fact that I have had now for over two
years several hundred of such which retain unimpaired the
original intensity of the reaction.
I have always washed the sections with distilled water, before
putting them in the acid ferrocyanide mixture, because the
presence of acid alcohol, especially that containing nitric acid,
causes decomposition of the ferrocyanide and a deposition of
Prussian blue in parts of the preparation in which iron did not
occur originally. The acid ferrocyanide mixture itself decom-
poses after twenty minutes with the formation of Prussian
blue, but that this is not, even in an infinitesimal part, the
source of the blue that obtains in a section during the first
five minutes after the mixture is made, was shown by the com-
plete absence of a blue reaction in other sections of the same
tissue (e.g. cartilage, muscle, ovary of Erythronium) placed
in the mixture at the same time without having previously
been treated with an acid alcohol. The distribution of the
Prussian blue due to such a decomposition is quite different
from that which one finds in preparations treated with acid
alcohol, but in which this decomposition was avoided, for when
one leaves sections of animal or vegetable tissue in the acid
ferrocyanide mixture for two hours, the blue colour is uniformly
diffused through the section, not localised as it is when the
reaction is due to the iron of the tissue.
In the permanent preparations made to illustrate the distribu-
tion of iron, and on which no staining reagents were employed,
the parts revealed by the transparent blue are not as sharply
outlined as they would be if stained with hematoxylin for
example, owing to the cytoplasmic parts, over or under the
204. A. B. MACALLUM.
structures coloured blue, obscuring the latter. This may be
obviated, especially with high powers, by raising the Abbé
condenser to the level of the stage and removing altogether
its diaphragm, when the brilliancy of the light in the field of
the microscope enhances the blue due to the iron reaction,
while it renders more or less obscure the other details of the
preparation. It was only in this way that I was able to deter-
mine the occurrence of very minute traces of iron in the tissues
and, when the sections were stained with safranin, of bodies
which gave but a feeble Prussian blue reaction (figs. 45
and 46).
The sections of tissue were made, either by the free hand
with a polished steel knife, or by the paraffin or celloidin
methods. Care was taken that the knife should not yield a
trace of iron to the sections. When the paraffin method was
employed the surface of the cutting instrument was dry, but
with the other methods it was covered with absolute alcohol.
The transference of sections from one fluid to another was
done with goose-quill points or with glass needles.
I may not leave this part of the subject without a reference
to the potash method for the liberation of ‘“‘ masked” iron, as
described by Molisch, but afterwards determined by him to
be untrustworthy. I have studied the effect of concentrated
solutions of potassium hydrate upon vegetable tissues hardened
in alcohol, and have obtained, frequently, evidences of the
presence of iron in the cell wall, cytoplasm and nucleus, but
the amount thus indicated in the last was always very much
less than could be demonstrated by the other methods, while
the reagent so altered the nuclei that a determination of the
definite relations of the iron observed to the nuclear structures
was impossible. My observations have convinced me that a
very large part of the iron demonstrable after the use of this
reagent is derived from the latter, however pure it may appa-
rently be, and I am, therefore, upon this point in accord with
Molisch. One of the readiest ways of proving this is by
extracting all the iron from sections of vegetable tissues by
keeping them in a quantity of warm Bunge’s fluid for several
IRON COMPOUNDS IN ANIMAL AND VEGETABLE CELLS, 205
days and then transferring them to a quantity of a concen-
trated solution of potassium hydrate for an hour, after which
interval the sections give abundant evidence of the presence of
iron. That not a trace of iron is left in the sections by
Bunge’s fluid may be shown by incinerating some of the
sections so treated and examining micro-chemically the ash
for the presence of iron. Were one able to obtain the reagent
absolutely free from iron, its employment for this purpose,
limited as it must be through its drastic action on cellular
structures, would, however, still be open to objection on the
score that it dissolves and redistributes the iron of the
tissues.
III. GeneRAL OBSERVATIONS ON THE DISTRIBUTION OF
ASSIMILATED IRON IN HIGHLY SPECIALISED ANIMAL AND
VEGETABLE CELLS.
The greater part, and sometimes the whole, of the assimi-
lated iron in the cells of the higher forms of animal life is
held in the nucleus, in the chromatin of which it is chiefly
found. The chromatin fibrille, the chromatin granules, the
nodal points of the chromatin network, all exhibit, after the
employment of the methods described above, the clearest
evidence of the presence of iron. Though no definite com-
parison is possible, yet, judging by the depth of colour result-
ing from the Prussian blue reaction in a large number of
animal nuclei, one may say that the amount of iron thus
demonstrated appears to correspond in all cases with the
amount of chromatin present. This is probably best seen
after the use of sulphuric acid alcohol, followed by treatment
with an acid ferrocyanide solution, the sections thus prepared
being compared with others simply stained with a reagent like
Ehrlich’s hematoxylin so employed as to affect the chromatin
only. In this case the hematoxylin stain in the chromatin
is always found to correspond in intensity, in the object stained
and in the general distribution of the stain, with the blue re-
action obtained in the other sections. If, further, sections
206 A. B. MACALLUM.
illustrating the Prussian blue reaction, be stained also with
safranin, which, when carefully employed, affects only the
chromatin, it will be observed that all the elements coloured
by the safranin exhibit the blue reaction also, the combina-
tion of the red and the blue giving to the chromatin a colour
of a violet shade (figs. 46 and 48).
Tt is not, however, the chromatin alone in the animal nucleus
that possesses assimilated iron, for one sees in sections exhibit-
ing the Prussian blue reaction, but more readily in those which
have been also stained with safranin, that nucleolar elements
possess a light blue colour (figs. 45 and 48 a). Some diffi-
culty is experienced in observing this under ordinary condi-
tions, but this is overcome, when homogeneous immersion
apochromatic objectives are employed, by withdrawing the
diaphragm of the Abbé condenser, the great amount of light
thus transmitted causing all the blue parts to appear with
remarkable distinctness, and amongst these the nucleolar bodies
coloured light blue, while all the other elements are rendered
indistinct or invisible. When, however, safranin has also
been employed to stain such preparations, the chromatin
absorbs it but the nucleolar elements are absolutely unaffected
by it, and they thus stand out in marked contrast with the
other structures. Such nucleolar bodies take but a faint stain
with hematoxylin, a fact which, considered in connection with
the result of the employment of safranin, would seem to
demonstrate that they are not essentially formed of what the
cytologist comprehends under the term chromatin. The
number of these in a nucleus varies, and the shape and size of
each are not constant, while not unfrequently the central
portion appears free from iron, the outer or peripheral part,
coloured light blue, appearing as an envelope of greater or less
thickness for the uncoloured part (fig. 46, a and d). These
bodies 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. This is very distinctly seen in the safranin pre-
parations, the membrane in this case exhibiting a combination
IRON COMPOUNDS IN ANIMAL AND VEGETABLE CELLS. 207
of the blue and red reactions, and thus appearing in sharp con-
trast with the enclosed nucleolar body coloured light blue.
It is chiefly in the nuclei of the glandular cells that one finds
these nucleolar bodies, and they are most distinctly seen in
large nuclei, as, for example, those of hepatic and renal cells
and of the intestinal epithelium of Necturus lateralis.
They are very rarely seen in the nuclei of the muscle fibre
and in those of the cutaneous epithelium of the same animal,
while they are never present in those of leucocytes or lymph
cells, or in those of the red blood-corpuscles. In the search
for them in all these elements the greatest assistance is
obtained from the employment of eosin, which, in sections
exhibiting the Prussian blue reaction, gives these bodies an
ochre-red colour, while the parts showing a dark-blue reaction
are unstained by it (fig. 47). In the nucleus of the glandular
cell 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 fila-
ments and partly embraced by its substance, what appeared to
be the remains of sucha body. In later stages of mitosis
not the slightest evidence of this body or of its remains can
be observed.
Whether the iron which these bodies contain is that of a
small quantity of chromatin dissolved in them, I am unable to
say. The fact that they take sometimes a very feeble stain
with hematoxylin, seems to indicate that they may contain a
small amount of chromatin. The iron in them is held neither
more nor less firmly than in the typical chromatin elements,
since in hepatic nuclei containing them, prolonged treatment
with ammonium hydrogen sulphide in the warm oven does not
result in demonstrating any difference, except in the amount
of iron in the one and in the other. The substance which
holds the iron does not possess the slightest affinity for
safranin, but attracts eosin as no other cellular con-
stituent does, and in these properties, as well as in the
VOL. 38, PART 2,——NEW SERIES. O
208 A. B. MACALLUM.
very small amount of iron present, there would appear to be
distinctions which separate it from chromatin. My prepara-
tions were chiefly obtained from the organs of the fasting
animal, and as I did not succeed in my attempts at feeding
artificially some examples of Necturus that I had, it is not
possible for me to say whether the constitution of the nucleo-
lar bodies is always similar to, or ever different from that
described; but in preparations of the liver and other organs of
specimens of Amblystoma punctatum killed soon after
their capture, or after artificial feeding, the nucleolar bodies
appeared to present the characters noted in the cells of the
fasting animal, the smaller size of the elements in this case,
however, not allowing as clear a view of them as was desired.
In the nuclei of the liver-cells of Necturus, as illustrated
in preparations made after the manner described, I frequently
found a third element, whose significance is unknown to me.
It manifested itself by the red stain which eosin gave it, the
nucleolar bodies taking, in contrast, an ochre-red stain. It
had no constant shape or form, in some cases being of a fila-
mentous character, in others resembling a localised granular
deposit (fig. 47) ; and when the structures were filamentous,
several usually appeared in the same nucleus. The substance
forming them did not contain the slightest trace of iron, and
therefore appeared to have no relation to the nucleolar bodies
or to the chromatin. I have not in any other organ observed
similar structures.
The disposition of the iron-holding compound in the nuclei
of Amphibian ova deserves special mention. In the ovarian
ova, whose nuclei contain no peripheral nucleoli, the iron is
distributed as represented in fig. 36, the chromatin in this case
forming a fine reticulum, in the trabecule of which large
granules are found with lateral prolongations. The iron
demonstrated in this preparation was set free by sulphuric
acid alcohol, but a disposition of iron in the main like this may
be found in similar nuclei when the latter are, on removal
from the ova, broken into small pieces on the slide in the
glycerine and sulphide mixture, and, thus prepared and provided
IRON COMPOUNDS IN ANIMAL AND VEGETABLE CELLS. 209
with a cover-glass, kept for days ina warmoven. This method
must be resorted to in order to get the iron reaction, since other-
wise the large nuclei may be kept for a month in contact with
the reagent in the warm oven without resulting in demon-
strating, in the slightest, any iron reaction. In the peripheral
nucleoli, when they are present, the amount of the iron, as
indicated by the depth of the reaction, is great, but in the
remaining elements of such nuclei it is small. When such
preparations are examined with a strongly magnifying objective,
the chromatin network, as revealed by the iron reaction given,
is found to be less distinct, and instead of granules of iron-
holding substance arranged at definite positions along the
course of the fibrillz, as in ova much less developed, the iron
is now seen to be chiefly confined to beadlets, few in number,
sometimes regularly, sometimes irregularly, disposed on the
fibrille, which, in ammonium hydrogen sulphide preparations,
manifest but a feeble greenish tint. There is an inverse rela-
tion between the size of the nucleoli and the amount of the
chromatin in the network, and an examination of some nuclei in
which the formation of the peripheral nucleoli has commenced,
and of those in which the development of these bodies is much
more advanced, irresistibly suggests that the latter are derived
from the chromatin of the network. I have elsewhere! pointed
out that the solution of the substance of which these nucleoli
are composed and its diffusion from the nucleus into the cyto-
plasm of the ovum are connected with the formation of the
yolk-sperules in Amphibia. That a solution of the peripheral
nucleoli takes place has been noted by O. Schultze? and Born.
Schultze found that with the solution of the nucleoli (keim-
korperchen) the contents of the nucleus and the substance sur-
rounding the latter were affected in the same way by reagents
and staining fluids, and he believed that the dissolved sub-
1 «Transactions of the Canadian Institute,’ Toronto, vol. i, part 2.
2 “Untersuchungen tber die Reifung und Befruchtung des Amphibieneies,”
‘Zeit. fiir wiss. Zool.,’ vol xlv, p. 177.
$ “Die Struktur des Keimblaschens im Ovarialei von Triton taeniatus,”
‘Arch, fir Mikr, Anat,,’ vol. xliii, p. 1.
210 A. B. MACALLUM.
stance diffused from the nucleus of the ovum into the cell-
body. Born observed that the nucleoli are always placed as
closely as possible to the cell protoplasm, while the chromatin
in the development of the nucleus and ovum becomes so finely
divided in the karyoplasm that it is stainable with great diffi-
culty, and it is as difficult to demonstrate optically, a condition
which continues till the formation or deposition of the yolk-
grains (Dotterkérner) commences. In later stages the per-
sisting peripheral nucleoli lose their capacity for absorbing
colouring matters.
In support of these observations of Schultze and Born, I
may but add that the iron in the cytoplasm of the ovum makes
its appearance only after the solution of the peripheral nucleoli
commences. The substance forming the peripheral nucleoli
does not react with staining reagents as does the chromatin of
the nuclear network, and especially with the indigo-carmine
staining fluid of Shakespeare and Norris the resulting stains
of each are different, the chromatin of the network being
coloured red while the nucleoli are stained blue or green, the
latter colour obtaining also in the yolk-spherules of such pre-
parations. A further difference is noticeable in the effect that
ammonium hydrogen sulphide exercises when applied to these
structures for some time at an elevated temperature. In this
case the iron of the peripheral nucleoli reacts more readily
than that of the chromatin of the network, but less readily
than that of the yolk-spherules, which in the ova of Necturus
and Amblystoma give a green reaction in a few minutes
with the reagent. It would appear as if the iron compound
undergoes a change in its transference from the nucleus to the
cytoplasm.
The peripheral nucleoli appear to be formed at the nodal
points of the chromatin network, if one may judge from pre-
parations of which fig. 384 is an illustration; but there is a
possibility that these represent a pathological condition,
since they are not common in the ovary when, if they were
normal, they should be present in larger numbers. I have,
moreover, found that they were accompanied by examples of
IRON COMPOUNDS IN ANIMAL AND VEGETABLE CELLS. 211
another condition which I regard as pathological. In the
latter the nuclei were indistinct or disintegrated, their chro-
matin had disappeared, and the surrounding connective tissue,
with its blood-vessels and their red corpuscles even, gave in a
few minutes, with warm ammonium hydrogen sulphide, an iron
reaction, frequently so deep as to obscure largely the details,
while the tissues, a little further away from such examples, and
other ova under exactly the same conditions of treatment with
the reagent, gave nosuch reaction. It may possibly be that the
chromatin of such disintegrated ova furnished the iron observed
thus diffused in the connective tissue and blood-vessels.
In the nuclei of all the higher vegetable organisms the
assimilated iron compounds are, on the whole, distributed as
they are in the nuclei of the more highly developed animal
forms, a fact which may be demonstrated in any Phanero-
gamous plant, especially readily if its nuclei are large, as is
the casein Erythronium americanum. In many of the
preparations of the latter form the chromatin filaments were,
in the process of teasing-out, partially or almost wholly set
free from the nuclei containing them, and to the parts thus
set free, as well as to the remainder, the glycerine and sulphide
mixture always gave a distinct reaction for iron in a few days
(fig. 17). Mitotic figures in such preparations appeared very
sharply defined through the iron thus revealed in the chromatin
elements. In successful preparations made by this method
the reaction for iron is very marked, as much so as in those
made with sulphuric acid alcohol; and in this respect there
is a contrast between animal and vegetable nuclei, for in the
former the glycerine and sulphide mixture brings out, after
a longer application and less frequently, a reaction as intense
as that which may be obtained after treatment of the nuclei
with acid alcohol.
Of nucleoli and nucleolar bodies there are at least three
kinds. The reaction for iron given in one variety by the
glycerine and sulphide mixture was weak, and it was obtained
at the same time that it appeared in the chromatin network
or filaments. These are smaller, apparently, in the hardened
212 A. B. MACALLUM.
than they are in the living cell, for, as a rule, they only
partially occupy the cavity in which they lie (figs. 17 and 19).
I have in some cases isolated them from their nuclei in the
glycerine and sulphide mixture, and the greenish reaction
which they gave could, therefore, not have been due to the
iron of a compound which diffused from the chromatin
elements into them. When the sections were treated with
sulphuric acid alcohol and subsequently with the acid ferro-
cyanide mixture and the eosin solution, the result was usually
that of which fig. 42 is a representation, These nucleoli stain
intensely with eosin, which also colours very slightly the chro-
matin network, the blue of the latter thereby becoming violet,
but after being thoroughly washed in alcohol the bright blue
colour returns; while this treatment makes no difference in
the intensity of the stain in the nucleolar bodies. These
effects are most distinctly observed when the diaphragm of the
Abbé condenser is removed from the field, in which case it is
possible to see the most minute of the nucleolar elements,
a device that is necessary when the nuclei of ordinary paren-
chyma cells are under examination.
In the second class are those nucleolar elements which may
be found in the cells of the nucellus, and which are composed
of chromatin, since they give a deep iron reaction after the
employment of any of the methods of treatment for liberating
the element, and since, also, they stain in every respect like the
chromatin threads. They usually occupy cavities in the nuclei
like those which contain the eosinophilous nuclei last described.
I regard these as reserve masses of chromatin deposited in the
nuclei engaged in the formation of chromatin, which eventu-
ally is transferred to the cells of the endosperm. To this
subject I propose to refer again.
Nucleoli of the third class are to be found in the nuclei of
the embryo-sac (fig. 44, a and 6). They are not present in
the mitotic nucleus, but in the retrogressive stage 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, when the fila-
IRON COMPOUNDS IN ANIMAL AND VEGETABLE CELLS, 2138
ments became thinner and less rich in chromatin, is more
abundant. These nucleoli are eventually formed chiefly of
chromatin, and in stained preparations appear to contain nearly
all the chromatin of the nucleus. When mitosis again com-
mences 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 different from the eosino-
philous nucleoli in the animal cell, which appear to be unrepre-
sented in the vegetable cell.
Of an exceptional character are the nucleoli in Corallo-
rhiza multiflora and in Spirogyra. In these the greater
portion of the chromatin in each nucleus forms a single large
spherical element unconnected with the chromatin network,
which after prolonged treatment with the glycerine and sul-
phide mixture, gives a pronounced reaction for iron.
I have, on a few occasions only, in preparations illustrating
the iron reaction, seen the chromatin localised at points along
the course of the filament, and concluded that this was not due
to faulty methods of manipulation, for hematoxylin and other
dyes just as infrequently render such a distribution visible.
It was also, with the aid of the acid alcohols, found that in
the loops of the mitotic nucleus of the embryo-sac the chro-
matin is disposed under the membrane enclosing the filament,
in such a way as to make the latter appear as a tube of
chromatin.
In some of the elongated oval nuclei of the nucellus and
of the fibro-vascular bundle of the ovule, Mr. Bensley has
observed a point of some interest. This consists in the occur-
rence in the karyoplasm, amongst the trabecule of the
chromatin network in one end of such a nucleus, of an iron-
holding compound with all the characters of chromatin, and,
214 A. B. MACALLUM.
in some cases, in such abundance as to obscure the outlines of
the trabecule. He has found that in the fibro-vascular bundle
this end of the nucleus is directed toward the base of the
ovule, and is of the opinion, as a result of some investigation
of this subject, that the phenomenon in question is connected
with the processes of the formation of chromatin, which he
regards as taking place here.
The presence of assimilated iron, apart from its occurrence
in hemoglobin and hematin, is an exceptional feature in the
cytoplasm of the cells of the higher forms of animal life, but
the exceptional instances are themselves of a constant cha-
racter, and comprise, in addition to yolk-holding ova, the cells
of yolk-holding embryos, the hematoblasts of Vertebrates, and
the ferment-forming gland-cells of all descriptions.
The iron in the yolk of Amphibian ova is held in the yolk-
spherules, which manifest a strong affinity for dyes, and are
usually homogeneous in composition. These give with ammo-
nium hydrogen sulphide a dark green reaction, which makes
its appearance sometimes in a few minutes, but at the latest in
a few hours, when the preparation is kept warm. The reaction
is uniform throughout each spherule. The enclosing cyto-
plasm does not, before development of the ovum begins, con-
tain any assimilated iron; but in the developing embryo, with
the multiplication of the cells and the partition of the yolk,
the spherules gradually undergo solution, for they become
smaller in size, and then one obtains an iron reaction in the
cytoplasm of each cell. The solution of the yolk-spherules may
be studied also in preparations made with the carmine-indigo-
carmine fluid, the reagent giving, in the earliest stages of the
embryo, a green colour to the yolk-spherules, and a red stain
to the cytoplasm and nuclei; but in later stages the red colour
is rarely obtainable, and both cell and nucleus, the latter espe-
cially, are coloured blue-green or dark green. This result is
brought about by the solution of the yolk-chromatin in each
spherule and the diffusion of the dissolved substance through
the cytoplasm and nucleus of each spherule-holding cell, for
in those examples of larval Amblystomata which yield pre-
IRON COMPOUNDS IN ANIMAL AND VEGETABLE CELLS. 215
parations giving a dark green or blue-green colour in cell and
nucleus after treatment with the reagent mentioned, the cells
are found, after the prolonged application of the glycerine and
sulphide mixture, to exhibit an iron reaction in the cytoplasm
apart from the spherules, and a similar reaction diffused in the
karyoplasm independent of that manifested by the chromatin
network, the intensity of the reaction corresponding in each
case to the depth of the green stain given these elements by
indigo-carmine reagent. When in the advanced development
of the larval Amblystomata the yolk-spherules disappear
from the cytoplasm of the cells, the nuclei and all cells, except
those undergoing transformation into striated muscle, lose their
capacity for absorbing and retaining the indigo of the fluid of
Shakespeare and Norris. This indicates that the yolk chro-
matin is changed into some other compound, and the prolonged
application of the glycerine and sulphide mixture confirms this,
for the cytoplasm, except in secreting cells, the striated muscle-
fibre, and in the hematoblasts and red corpuscles, is destitute
of iron compounds, while the nuclei give, much more slowly,
and apparently with greater difficulty, a reaction for iron which
is, in contrast with what is observed in the earlier stages, con-
fined to the nuclear network and nucleoli. The iron-containing
substance is transferred to the nuclei, and with this transfer-
ence the iron becomes more firmly combined—a process the
very reverse apparently of that which is illustrated in the
formation of the yolk-spherules, for the iron compound of the
latter, though derived from the nucleus of the ovum, is less
firmly combined than that of nuclear chromatin giving origin
to it.
The yolk-spherules of the hen’s egg, as is well known, have
characters differing from those of Amphibian ova, but the most
marked difference consists in the distribution of the iron-con-
taining compound. The yolk-spherule in the ova of Ambly-
stoma and Necturus is homogeneous, and the iron compound
is uniformly distributed through it ; but in the hen’s egg ele-
ments of this character are to be found only in the constituents
of the “ white” yolk and in some of the “ yellow” spherules in
216 A. B. MACALLUM.
the most peripheral layers of the yolk, while in all the other
spherules the distinctive feature is the disposition of the iron
compound in a finely granular form. This cannot be deter-
mined with fresh yolk, for when treated with ammonium
hydrogen sulphide the greater part of it dissolves, and the solu-
tion becomes dark green owing to the formation of sulphide of
iron. Under the microscope no formed elements can be ob-
served in such a preparation, except those derived chiefly from
the ‘‘ white’’ region, and it is not possible to ascertain, under
these conditions, the relations of the iron-holding nuclein in
other parts of the yolk. Another difficulty experienced in
dealing with fresh uncoagulated yolk is that, when removed
from the egg the spherules disintegrate, the granular contents
escaping and obscuring more or less the characters of the other
elements. To avoid this the substance of the spherules must
be coagulated, and to accomplish this satisfactorily I placed
the eggs in boiling water for ten minutes. Thespherules were
thus fixed in polyhedral form, and, after these had lain in strong
alcohol for several days, it was an easy matter to determine the
distribution of the iron in them.
The results obtained were according to the variety of spherules
examined. In those known as “ white” the reaction for iron
was very distinctly obtained, but it was wholly confined to
their homogeneous spherical bodies. The reaction is, imme-
diately after the application of the glycerine and sulphide
mixture, light green, but this becomes deeper after a few days,
when the preparation is kept at a temperature of 60° C. The
homogeneous elements undoubtedly contain a quantity of
nuclein, for they resist the action of artificial gastric juice and
dissolve in weak alkalies, while they constitute the only part of
the “ white” spherules that possesses, like chromatin, the pro-
perty of absorbing and retaining colouring matters. This was
found to be the case specially when the spherules, coagulated
by heat, were further treated with Flemming’s chrom-osmio-
acetic mixture for twenty-four hours, then with alcohol, and
finally with a solution of safranin. When the excess of the
stain was extracted with alcohol and the spherules mounted in
IRON COMPOUNDS IN ANIMAL AND VEGETABLE CELLS. 217
balsam, it was always found that the spherical elements ex-
hibited an intense stain, while the remaining parts of the
spherules were absolutely uncoloured. I found it possible to
demonstrate this and the reaction for iron in the same pre-
paration. When the “ white” spherules, fixed with heat, were
kept in slightly warm sulphuric acid alcohol for twenty-four
hours, their spherical elements gave, on treatment with an acid
ferrocyanide solution, a Prussian blue reaction, and, when sub-
sequently stained with asafranin solution, became violet. These
results show how close is the relationship between the sub-
stance composing the spherical elements and chromatin.
A few of the spherical elements in the “ white ” spherules
are not of the character described, for in preparations made
with Flemming’s fluid one finds, now and then, a spherule in
which one or more large droplets of fat are demonstrated by
the intensely black reaction of the osmic acid. Apart from
the occurrence of these, there is comparatively little fat in
the “ white” spherules, a fact strikingly shown when a thin
section of the hard-boiled yolk, embracing portions of the
*‘ white” and “ yellow” zones, is submitted to the action of the
reagent for twenty-four hours, the ‘‘ white ” then exhibiting a
greyish appearance, while the “ yellow ” area is almost black.
The “‘ yellow” spherules are also richly supplied with the
iron-containing compound, but this is quite differently distri-
buted from what it is in the “ white” zone. The appearances
of these are subject to a great deal of variation. Some contain
only large round granules, in others the granules have a puncti-
form character, while in others again both kinds of elements
may be mingled with minute fat droplets. Owing to differ-
ences in the specific gravity of the constituents apparently,
the granules may be found, in some cases, to be gathered in
one portion of the spherule, the remainder of the contents
being occupied by a clear, non-granular substance of a firm
consistence, a character resulting from heat coagulation. Itis
in such spherules as these that one determines distinctly how the
iron compound is disposed, for, in those in which the granules
are uniformly distributed, it is sometimes exceedingly difficult
218 A. B. MACALLUM,
to decide whether the iron is contained in the granules or in
the extra-granular substance, so intimately are these usually
intermingled. The granules, whether of the large or of the
punctiform variety, always contain an iron compound, while
the substance in which they are shown is destitute of this
element. In demonstrating this fact the acid alcohols are of
the greatest service, the glycerine and sulphide mixture, owing
to the large size of the vast majority of the spherules, not
being as effective in liberating and demonstrating their iron,
but in the smallest spherules the complete reaction may be
obtained with the mixture in four or five days. In those
spherules which contain, as described, granular and non-
granular portions, the granules, closely aggregated as they
usually are, appear very prominent by reason of the reaction
for iron which they give with both methods of demonstration.
In some of the “yellow” spherules also, after treatment
with sulphurie acid alcohol, vesicles of different sizes were
observed, each of which appeared to be enveloped by an iron-
containing membrane-like structure. Their position near the
centre of the spherule often rendered the occurrence of iron
in the envelope obscure, owing to the light passing through so
many iron-holding granules above and below these vesicles.
What the latter contained it is not possible to say, for although
fat globules of a similar size can be demonstrated in some
spherules when these are subjected to the action of the chrom-
osmio-acetic mixture for twenty-four hours, it cannot be
demonstrated that the two classes of structures are connected
in any way.! The difficulty lies in the fact that in order to
show the occurrence of iron in the envelope, alcohol in some
form must be used, and by this the fat is largely, if not wholly,
removed ; while in those spherules treated only with osmic acid
solutions the black reaction of the globules prevents a demon-
stration by the Prussian blue reaction of any iron present.
1 In another paper (‘‘On the Absorption of Iron in the Animal Body,”
‘ Journ. of Physiol.,’ xvi, 1894, p. 268) I expressed the view that these vesicles
contain fat. After a more extended study of these elements than I was able
to make before that paper was published, I am doubtful of this interpretation
of their structure.
IRON COMPOUNDS IN ANIMAL AND VEGETABLE CELLS. 219
Apart from the question of the occurrence of fat in such
elements, there may be no doubt about the intimate associa-
tion of the iron-containing substance and the fat in the
spherules. Owing, however, to the size of the latter, as well
as to the density of the coagulated material in them, the osmic
acid used to demonstrate the fat penetrates but slowly, and
when, as usually happens, fat droplets stud the periphery of
the spherule, little or none of the reagent reaches its interior,
which then has only a straw-yellow colour. If, however, a
few spherules, coagulated with heat, are kept in a quantity of
Flemming’s fluid for twenty-four hours, the osmic acid pene-
trates the spherules in some cases and causes their granules
to become brownish-black, a fact which can be most distinctly
observed when the cover-glass is pressed down sufficiently to
disintegrate the spherules and set the granules free. If the
granules are large, the occurrence of fat in them is much less
readily demonstrated, possibly because the density of such
elements prevents penetration on the part of the osmic acid.
These granules are undoubtedly the source of the greater
part of the iron-holding nuclein isolated by Bunge from the
yolk,! since the “white” yolk is comparatively small in
amount. Miescher? regarded the nuclein, which he separated
from the yolk, as only in part localised in the homogeneous
spherical elements in the “white” portion, and he believed
that the greater part of it was derived from the granules in
the “‘ yellow ” spherules, and that none of it exists in a dissolved
form, a conclusion fully supported by the facts concerning the
localisation of the iron.
In describing the transference of the chromatin of the
spherules from the cytoplasm to the nucleus of each cell of the
larval Amblystoma, reference was made to an exception in
the case of developing muscle-fibre. In the cells undergoing
transformation into striated fibres, some of the chromatin dis-
solved in the cytoplasm finds its way into the nuclei as in other
1 « Ueber die Assimilation des Hisens,” ‘ Zeit. fiir Physiol. Chemie,’ vol. ix,
p. 49, 1885.
2 «Die Kerngebilde im Dotter des Hihnereis,” ‘ Hoppe-Seyler’s Med.-
Chem. Untersuchungen,’ 1871, p. 502.
220 A. B. MACALLUM.
cells generally, but the greater part appears to remain in the
cytoplasm of the developing fibre, and undergoes a transforma-
tion which is one of great interest in connection with the
origin of hemoglobin. In the cytoplasm of the muscle-cells
there is an abundance of yolk-spherules which, as in other
cells, gradually undergo solution, the dissolved substance
diffusing through the cytoplasm. When the striation makes
its appearance at one side of the now elongated cell, the dis-
solved substance passes into the striated area, for ammonium
hydrogen sulphide brings out an iron reaction in this part as
readily as in the undifferentiated cytoplasm and in the spherules,
but confined to the dim bands, the light bands giving no
evidence of the presence of the compound. In the fibre from
which the spherules have all but disappeared, and in which the
striated area embraces nearly the whole of its width, the reac-
tion with ammonium hydrogen sulphide is as distinct and as
marked as in the earlier stage, and this is true also of the fibre
in its final form. In this stage the iron is quickly liberated by
acid alcohols, as well as by ammonium hydrogen sulphide, and
its presence may be readily demonstrated by means of these
reagents up to the period when all traces of yolk disappear
from the cells of the larve. After this date the iron compound
becomes firmer, or, to speak more accurately, is less readily
attacked by acid alcohols or the sulphide reagent, and in the
muscle-fibre finally its presence may not be shown by these
methods. It is not that the iron is removed from the fibre,
but that the compound containing it is transformed, in red
muscles, into what is called myo-hematin by MacMunn, or
hemoglobin by Hoppe-Seyler and others. The latter com-
pound can, by means of the staining fluid of Shakespeare and
Norris, be clearly shown to be strictly confined to the dim
bands, which are given a grass-green colour distinctive of
hemoglobin, while the light bands and nuclei are coloured
red.!
1 T have pointed out the value of the reagent in this respect in my paper
entitled ‘‘ Studies on the Blood of Amphibia,” ‘Transactions of the Canadian
Institute,’ vol. i, 1898.
IRON COMPOUNDS IN ANIMAL AND VEGETABLE CELLS, 221
A similar conversion of a compound in which the iron is
easily attacked by ammonium hydrogen sulphide and by acid
alcohols into one from which the liberation of the iron is more
difficult, obtains in the dim bands of muscle-fibre in Inverte-
brates (Oniscus, Chironomus, Musca), but in this case the
transformation does not proceed as far as the production of
hemoglobin or myo-hematin, if one may judge from the
absence of pigment and from the fact that the liberation of
the iron, though difficult, is possible, while in the case of hemo-
globin the use of acid alcohols and of ammonium hydrogen
sulphide is ineffective for that purpose.!
In the development of the blood-corpuscles in the larve of
Amblystoma there is, as I have pointed out,? a conversion of
the chromatin of the hematoblast into hemoglobin, a change
that is analogous to that described above as occurring in muscle-
fibre. In hematoblasts, however, the chromatin so trans-
formed is not directly derived from that of the yolk-spherules,
as is the case in muscle-fibre, but from that compound after it
is transformed into nuclear chromatin. ‘This is very distinctly
seen in sections through the aortic arches of the larve, which
have been treated with acid alcohol to liberate the iron. In
the concave side of the arches are seen hematoblasts in all
stages of division, and in these one may, by the iron reaction,
differentiate between hzmatoblasts in which there is no cyto-
plasmic chromatin, and those in which the cytoplasm between
the chromatin loops of the mitotic figure contains dissolved
chromatin to an extent varying with the example of hemato-
blast noted. This cytoplasmic chromatin does not act in the
same way as ordinary nuclear chromatin does towards staining
reagents, as, for example, hematoxylin, eosin, and safranin,
1 The fact that ammonium hydrogen sulphide will liberate the iron from
hematin in solution, while it does not attack the iron in the compound called
myo-hematin by MacMunn, indicates that the latter cannot belong to the
hematin class. Its property in this respect shows that it is related to hamo-
globin. ‘lhe name given to it by MacMunn certainly appears to be a mis-
leading one.
Loc. cit.
yee A. B. MACALLUM.
although it has an affinity for them, and it persists with this
character for a long time after the stage of the hematoblast is
passed. I have found that in a large number of the fully-
formed red cells in the spleen of the larva of 35 mm. length
the disc contains a quantity of the modified chromatin, and
from this the iron is readily liberated, but in later stages both
the number of such corpuscles and the amount of iron in the
dise which may be liberated by acid alcohols gradually diminish
and disappear, the hemoglobin of the disc not yielding its iron
on the employment of such methods. The nuclear chromatin,
however, of all stages of the corpuscle, readily gives up its iron,
even when none can be set free in the disc.
It thus appears that the hemoglobin of the red corpuscles
and the analogous compound in muscle-fibre are formed in the
same way, the only difference obtaining between them existing
in the fact that the pigment of muscle-fibre does uot, in its
evolution in the developing ovum, comprehend a stage of
nuclear chromatin. The process by which they are formed is
a gradual one, and the position of the iron in the molecule
is apparently changed. The latter result may be partly ac-
counted for if we consider the composition of chromatin and
of hemoglobin. Chromatin is an iron-holding nucleo-albumin
in which the iron is attached to the nuclein, while in hemo-
globin the iron is held in the hematin molecule, and in the
transformations which result in the formation of hematin out
of nuclein, it is but natural to expect that the relations of the
iron to the molecule should change also.
In secreting cells, as, for example, those of the parotid,
Lieberkiinian, and pancreatic glands, a certain portion of the
cytoplasm gives evidence of the possession of “‘ masked” iron.
When the cells of the pancreas of an adult Amblystoma are,
after hardening in alcohol, subjected to the action of the
glycerine and sulphide mixture for six or seven days at a tem-
perature of 60° C.,in addition to the reaction for iron obtained
in the nucleus, one is found in the cytoplasm of the so-called
“outer zone,”’ in some cases almost as marked as in the nuclear
chromatin. The extent of the cytoplasm involved in the reac-
IRON COMPOUNDS IN ANIMAL AND VEGETABLE CELLS. 223
tion in all the specimens which I examined varied considerably,
whether according to the stage of secretory activity could not
be determined after the use of ammonium hydrogen sulphide,
for this reagent, in a day or two at an elevated temperature,
causes the zymogen granules to disappear; but in sections of
the pancreas from the same animal, after these had been acted
on by sulphuric acid alcohol, then with the acid ferrocyanide
solution and eosin, the iron-holding area in each cell was de-
monstrated by the resulting Prussian blue, while the zymogen
granules were given an intense red stain, and in this case it
was found that, apart from the granular zone, the cytoplasm
was uniformly blue. In other conditions of activity the iron-
holding area was increased or decreased in correspondence
with the decrease or increase in the extent of the granular
zone. In the exhausted condition of the gland-cell, that is, in
which there were but few granules, arranged in the “ border”
fashion near the lumen of the tubule, the whole of the cyto-
plasm exhibited the blue reaction, but the latter was less
marked than when it was confined to a narrow zone in the
neighbourhood of the nucleus. The relations of the extent of
the iron-holding area to the stage of secretory activity were
less easy to determine in the Lieberkiihnian and parotid glands,
for it is not possible to demonstrate the mucigen in the former
or the zymogen in the latter as prominently as the zymogen
granules may be in the pancreas, but in these the iron-holding
area appeared in all cases to correspond, in the main, with the
“ protoplasmic” or “outer” zone. In the “mucous” cells of
the submaxillary gland of the cat and dog only a narrow zone
of cytoplasm about the shrunken nucleus contains iron, but in
the large crescents of Gianuzzi in the cat the whole of the
cytoplasm is iron-holding. In the peptic tubules of Ambly-
stoma the cytoplasm in the outer half of each cell contains
iron, and this is also true of the chief cells in the cardiac por-
tion of the stomach in the dog and cat. In the parietal cells
in these animals the cytoplasm is absolutely free from iron.
The iron-holding zone in each chief cell appears to vary in
extent with the stage of secretion, but I am unable to speak
VOL. 38, PART 2.—NEW SER. PB
224 A. B. MACALLUM.
as definitely upon this as upon the relations, in this respect,
observed in the pancreatic cells of Amblystoma, for | have
not been successful in my efforts to obtain, from examples of
the latter animal, preparations of the gastric glands illustrating
marked variations in the stages of secretory activity, and have
had to rely upon those made from the cat and dog, in which
the chief cells are comparatively small and less favorable for
observation on this point.
It is only in the mucous glands of the skin of Amphibia, and
in the renal tubules of Vertebrates generally, that I find
exceptions to the rule that glandular secretion is associated
with the presence of an iron-holding cytoplasm. I have not
found any exceptions in Invertebrates to this generalisation,
but my observations have not been comprehensive enough on
this point, and I must speak with some reserve in regard to it.
In the Protozoa, as I will show further on, the presence of
assimilated iron in the cytoplasm seems to be a constant
feature, the iron not being confined to any part of the cell, but
uniformly distributed through it, and there is a probability
that this cytoplasmic iron-holding compound is also associated
with the secretion of ferments functioning in the digestion of
the ingested food. In the glands named above, which are
mentioned as exceptional instances, the absence of assimilated
iron from the cytoplasm may be explained on the ground that
the secretory process of a renal cell is widely different from
that of a pancreatic cell, the cytoplasm in the latter, but not
in the former, elaborating a portion of its own constituents to
furnish the secretion, whereas in the renal cell the process is
largely one of transference only. Ifthe explanation should hold
in all possible cases of exception, then it would follow that the
iron-holding compound is an important element in the elabora-
tion of the zymogens. I have elsewhere! pointed out the rela-
tions that obtain between the chromatin of the nucleus and of
the cytoplasm of the pancreatic cell, on the one hand, and the
formative process resulting in the production of zymogen on
1 “Contributions to the Morphology and Physiology of the Cell,” ‘Trans,
Canadian Institute,’ vol. i, part 2, p. 247, 1891.
IRON COMPOUNDS IN ANIMAL AND VEGETABLE CELLS. 225
the other; and so intimate did these relations appear that I
was led to apply the term prozymogen to the chromatin. I
have found, as a result of experiments on the active pancreas
of Amblystoma, that the zymogen granules under certain
conditions give an iron reaction. When the organ, hardened
in alcohol, is put in a quantity of Bunge’s fluid, and the pre-
paration kept at the temperature of the room (20° C.) for a
week, or when it is kept for two days in a quantity of sulphuric
acid alcohol, teased-out portions, after the removal of the acid
and on the addition of ammonium hydrogen sulphide, give pre-
parations of which that represented in fig. 38 is an illustration.
The zymogen granules give a greenish reaction, the colour
making them more prominent than the other elements in the
cells. The cytoplasm of the “ outer zone” gives but a feeble
iron reaction, and this appears only to a minor extent in the
nuclear elements, both results being caused by the lessened
action and feeble extractive capacity of the acid alcohols when
used on the tissue in mass. When the reagents are used for
longer periods than those specified the iron disappears from the
zymogen granules, while it becomes more strongly marked in
the nuclear elements and in the cytoplasm of the “ outer zone.”
Owing to the effect that ammonium hydrogen sulphide exer-
cises on the granules, causing them to dissolve or disintegrate,
an effect already referred to above, it is not possible to control
the results obtained with the acid alcohols by experiments with
this reagent, and one may, therefore, not regard the presence
of iron in the zymogen granules as conclusively demonstrated,
since it may be urged that the iron reaction which they gave
was due to the iron which diffused into them from that liber-
ated in the other cellular elements. When one remembers,
however, the fact that the zymogen is elaborated in a cyto-
plasm which is iron-holding and at its expense, the occurrence
of a faint reaction for iron in the granules after the use of
acid alcohols is best explained by the view that the zymogen
of the pancreas contains iron, and that its antecedent, the
prozymogen, is the iron-holding constituent in the cytoplasm
of the “ outer zone.”
226 A. B. MACALLUM.
In the rods and cones of the retina in Amblystoma and
Necturus an iron reaction was frequently obtained like that
represented in fig. 37. It was always feeble and confined to
the trabeculz, which stretched across the long axis of the rod,
or which formed the network in the cones. In some cases (as
in fig. 87, a) pigment-granules were observed attached to the
rods, probably derived from the cells of the tapetum nigrum, and
as the pigment probably contains iron, it is uncertain whether
the iron demonstrated in the rods and cones was not derived
by diffusion of some iron-holding substance from this source.
The eleidin granules in the stratum granulosum in the
human skin give, after treatment of sections of the epidermis
with sulphuric acid alcohol, a dark green reaction with ammo-
nium sulphide. I have not succeeded in obtaining a reaction
for iron in them when the containing cells, hardened in alcohol,
were simply subjected to the prolonged application of the gly-
cerine and sulphide mixture in the warm oven. Since the
chromatin of the nuclei in the underlying stratum mucosum
is, as elsewhere, iron-holding, while the nuclei in the stratum
granulosum are poor in chromatin, it is not improbable that
the iron, at least of that part of the latter which disappears
from the nuclei, is the source of the iron shown in the eleidin
granules. The homogeneous substance constituting the stratum
lucidum also gives a reaction for iron, which is diffuse and less
marked than in the granules of the underlying layer.
In my observations on preparations of the human thyroid
and of that of the dog, although it was easy to demonstrate the
presence of iron in the nuclear chromatin, and to a certain
extent in the cytoplasm of the cells lining the alveoli, I did
not succeed in finding any of it in the “colloid” matter.
Under certain conditions this substance absorbs staining
matters, and it also gives! the molybdate-pyrogallo] reaction
of Lilienfeld and Monti.” These facts suggest that the colloid
1 F, Gourlay, “The Proteids of the Thyroid and the Spleen,” ‘Journal of
Physiology,’ vol. xvi, p. 28, 1894.
2 “Die mikro-chemische Lokalization des Phosphors in den Geweben,”
‘Zeit. fiir Physiol. Chemie,’ vol. xvii, p. 410, 1893.
IRON COMPOUNDS IN ANIMAL AND VEGETABLE CELLS. 227
substance is allied to nuclein, and, according to Gourlay, the
nucleo-albumin which he isolated from the thyroid was derived
in large measure from the colloid matter which he, relying on
the reaction of Lilienfeld and Monti, found to contain phos-
phorus. If colloid matter is therefore a nucleo-albumin, its
freedom from iron renders it, in contrast with the chromatins,
a subject of special interest.
Assimilated iron is rarely found in the cytoplasm of the cells
of the higher vegetable organisms, and amongst the examples
illustrating its presence may be mentioned the cells of the
nucellus in the ovules of Erythronium americanum, and
those of the gluten layer in the wheat-grain. The cytoplasm of
the cells of the nucellus, when fertilisation has taken place, and
even before this occurs, gives, after treatment with sulphuric
acid alcohol, a distinct reaction for iron, which, however, in
respect to intensity, is not to be compared with that mani-
fested in the nuclei of the same cells. The iron in the cyto-
plasm in this case is not due to diffusion from the nuclei during
the course of treatment with the liberating reagent, for it is
also demonstrated in this situation in the glycerine and sul-
phide preparations. As the nuclei of the nucellus are much
richer in assimilated iron than those of other parts of the
ovule, except the embryo sac, it is possible that the cyto-
plasmic iron compound is intra vitam diffused from the
nuclei, and, further, as the cytoplasm of the embryo-sac of this
stage sometimes gives a diffuse reaction for iron after it has
been treated with acid alcohols, its presence here may be due
to a similar diffusion from the cells of the nucellus. I have
observed in certain preparations in which the nuclei of the
embryo-sac were in the stage of division, a large number of
iron-containing granules interspersed amongst the fibrils of the
achromatic spindles, and as in other preparations similar
granules were stained with hematoxylin, hke the chromatin
loops, it would appear as if the granules were formed of chro-
matin. The cytoplasm holding these granules gave no reaction
for iron.
The cytoplasm of the cells of the gluten, or so-called aleu-
228 A. B. MACALLUM.
rone layer (Kleberschicht) in the wheat-grain is richly supplied
with a “masked”? compound ofiron. In some cells it is chiefly
found in the large granules strewn through the cytoplasm ;
in others, again, apparently it is wholly contained in the latter;
while in certain instances, further, it was demonstrated only in
the extreme peripheral portions of the large granules. This
is most clearly shown in sections of the grain after they have
been treated with sulphuric acid alcohol for twenty-four hours
at a slightly raised temperature. When the individual cells of
other sections are treated with the glycerine and sulphide
mixture for several days the reaction for iron is readily ob-
tained in their cytoplasm, but its localisation, as observed after
the use of the other method, is thus less readily determined.
The “masked” compound apparently belongs to the class of
chromatins, for when sections are treated with the ordinary
staining reagents the cytoplasm stains deeply, especially with
safranin and hematoxylin, and the parts which are specially
affected are those which correspond with the iron-holding
structures in preparations treated with acid alcohols.
Haberlandt! has made experiments upon the question of the
site of origin of the diastase in the germinating rye-grain, and
these appear to show that the ferment is elaborated in the cells
of the gluten layer only. It is possible that the iron-contain-
ing compound in the cytoplasm of this layer is the zymogen or
prozymogen of the ferment.
IV.—On tHe OccuRRENCE oF ASSIMILATED IRON ComPouNDS
IN SPECIAL Forms.
What I have said in the foregoing pages with regard to the
presence of iron in the chromatin of higher forms of animal
and vegetable life is true also in regard to the types of lower
organisation in both kingdoms. In the investigation of the
less highly organised animal and vegetable forms, however,
1 “Die Kleberschicht des Grasendosperms als Diastase ausscheidendes
Drusengewebe,” ‘Berichte der deutschen botan. Gesellsch.,’ 1890, p. 40.
Abstract in ‘ Botan. Centralbl.,’ vol. xliii, p. 39.
IRON COMPOUNDS IN ANIMAL AND VEGETABLE CELLS. 229
some important variations were found in the disposition of
the iron-holding substance, and it was further determined
that in non-nucleated organisms the exceptional distribution
of the chromophilous substance is co-extensive with that of
the assimilated iron compounds observed. Such facts are
worthy of an extended description, and I now propose to
detail these and the more important observations allied to
them.
Ascaris.—In the species A. mystax the spermatozoids
and ova, both before and after fertilisation, manifest special
features in the distribution of the iron-containing substance.
When they are hardened in alcohol, the spermatozoids are
comparatively easily affected by the ammonium hydrogen
sulphide, the reagent, mixed with glycerine, giving in a couple
of days, under the usual conditions, a reaction for iron, which
usually is confined to the “nucleus,” a dense homogeneous
body (fig. 81) ; but in several instances the ‘“‘membrane” also
contained iron. The reaction in the latter varied in intensity,
and when most marked it revealed a structure in the “ menm-
brane ” like that represented in fig. 82. The iron compound
observed in such a case obtained only in the rodlets constitut-
ing the “membrane.” What the occurrence of assimilated
iron in this situation signifies I am unable to say, except that
it possibly represents an abnormal phase of a condition normal
to the spermatozoid after it has penetrated the ovum. When
the spermatozoid begins to penetrate the latter, its membrane
frequently manifests a weak reaction for iron (fig. 29), while
its cytoplasm does not give any evidence of the presence of
that element; but in the changes it undergoes after reaching
the interior, the “ nucleus”? becomes in part dissolved, and
the chromatin, as shown by the iron reaction, diffuses into
the cytoplasm and into the membrane, from which some of it
passes into the cytoplasm of the ovum immediately adjacent to
the spermatozoid. The membrane in this way becomes the
most prominent part of the spermatozoid. As the transforma-
tion proceeds, the membrane also dissolves, and the iron which
it contains appears to pass back again into the cytoplasm of
230 A. B. MACALLUM.
the spermatozoid, but what is held in the cytoplasm of the
ovum apparently is retained by the latter.
These observations on the diffusion of the iron-holding sub-
stance from the “ nucleus” of the spermatozoid into its cyto-
plasm coincide with those of van Beneden upon the changes
which take place in the spermatozoid of Ascaris megalo-
cephala after it penetrates the ovum. He found that the
protoplasm of the free spermatozoids' manifests no affinity
for staining compounds, while its capacity for absorbing and
retaining all colouring matters becomes remarkable immedi-
ately after it enters the ovum. As the “nucleus” at the
same time loses in part its affinity for stains, he came to the
conclusion that a part of the chromatic substance (chromatin)
of the “ nucleus’’ becomes dissolved in the cellular substance
(cytoplasm). O. Zacharias” has also pointed out that the proto-
plasm of the free spermatozoid, apart from its “ nucleus,” is
absolutely unstainable, but after it penetrates the ovum it at
once manifests an affinity for colouring matters. Kultschitzky,3
referring to the reactions with staining fluids, suggests that
possibly the “nucleus” gives off to the cytoplasm of the
spermatozoid a portion of its chromatin, or that, in other
words, not all of the chromatin of the ‘‘ nucleus” is employed
in the construction of the male pronucleus. I have found in
my preparations that the cytoplasm and “ membrane ” of the
spermatozoid which has penetrated the ovum, and, frequently
also, that portion of the cytoplasm of the ovum in the im-
mediate vicinity of the spermatozoid, have a slightly greater
affinity for colouring matters than the cytoplasm of the free
spermatozoid or of the unimpregnated ovum.
In many of his illustrations van Beneden represents that
part of the spermatozoid which I have called the “membrane ”
1 © Recherches sur a maturation de |’ceuf et la fécondation,” ‘ Archives de
Biologie,’ vol. iv, p. 265, 1883.
2 “Neue Untersuchungen iiber die Copulation der Geschlechtsprodukte
und den Befruchtungsvorginge bei Ascaris megalocephala,” ‘Arch,
fir Mikr, Anat.,’ vol. xxx, p. 111, 1887.
3 “Die Befruchtungsvorginge bei Ascaris megalocephala,” ‘Arch.
fiir Mikr, Anat.,’ vol, xxxi, p. 567, 1888,
IRON COMPOUNDS IN ANIMAL AND VEGETABLE CELLS. 231
as deeply stained, and in these one finds the existence of rod-
lets indicated, such as those to which I have referred above;
but these (les stries transversales de la queue) are more ap-
parent in the penetrating than in the free spermatozoid. I
observed only faint traces of such structures in the spermato-
zoid in the interior of the ovum, the rodlets apparently com-
mencing to disappear immediately impregnation is accomplished.
The chromatin of the nucleus of the ovum gives a deep
reaction for iron in whatever stage the nucleus may be found
(figs. 29 and 30). The chromatin also of the “ polar globules”
contains iron, and I made efforts to determine the ultimate
fate of this, but these were unsuccessful. It would appear,
however, as if the chromatin of the extruded elements were
dissolved eventually in the cytoplasm, for it is impossible to
find any traces of it after a time.
Chironomus.—Balbiani,! who was the first to call the
attention of cytologists to the structure of the nuclear elements
in the “salivary” glands of the larva of Chironomus, de-
scribed the nuclear filament as made up of a series of dim discs
or bands, each placed transversely, and separated from its
neighbour on either side by a band of clear substance, the
filament possessing, however, at certain points an annular
swelling, and terminating at its ends either in the polymor-
phous nucleolus or by an attachment to the nuclear membrane.
Leydig,” the next observer, found each dim stria to be made
up of a series of elements whose separation from each other
gives a composite character to the stria. The fine lines sepa-
rating the elements are, according to his observation, con-
tinued from one dim disc, through the light disc on either side
of it, to the adjacent dim disc. In this way a series of exceed-
ingly delicate longitudinal lines, in addition to the coarse trans-
verse ones described by Balbiani, make their appearance.
Leydig also believes that the substance forming the dim band
1 «* Sur la structure du noyau des cellules salivaires chez les larves de Chiro-
nomus,”’ ‘ Zool. Anzeiger,’ 1881, pp. 637 and 662,
2 Untersuchungen zur Anatomie und Histologie der Thiere,’ Bonn, 1883,
p. 90.
232 A. B. MACALLUM.
is situated immediately under the membrane. Korschelt’s
views on the structure of the filament are directly opposed
to those of Leydig and Balbiani. He regards the transverse
striation of the filament as due to a folding of the surface
membrane only, and explains the longitudinal striation ob-
served by Leydig as caused by the action of the reagents used.
In his opinion, also, the apparent differentiation of the fila-
ment is due to the differences in the reflected light.
So far as I know, no one has hitherto observed an arrange-
ment in the nuclear filament of Chironomus similar to that
described by Leydig, although Carnoy has found in the salivary
gland of a Nemocere larva that the dim disc is formed of a
series of longitudinally disposed rodlets, but he attributed the
delicate lines observed in the clear discs to folds in the mem-
brane of the filament.? The larve of the species of Chirono-
mus accessible to me offer preparations less favourable for
study than do those of the species C. plumosus studied by
Balbiani and Leydig, yet I have been able to determine, with
my methods for demonstrating the presence of assimilated iron,
the correctness of Leydig’s observations so faras they go. The
dim discs are of different thicknesses, the thickest appearing
to be five or six times the diameter of the narrowest. When
the salivary gland, after being hardened in alcohol, is kept
for several days in sulphuric acid alcohol, treatment with an
acid ferrocyanide solution gives all these dim bands a deep
blue reaction, the intensity of the reaction coming out very
markedly in the thicker bands. Under the highest magnifica-
tion of service in such a case (apochromatic immersion
15 mm. and compensation ocular 8, Zeiss), the bands of
medium thickness are resolved into a series of short rodlets
disposed parallel with the filament. If the filament has, in
the course of preparation, been isolated from the nucleus, one
may then determine that the rodlets forming one dim band
are connected by excessively delicate fibrils with the rodlets
1 Ueber die eigenthiimlichen Bildung in den Zellkernen der Speicheldriisen
von Chironomus plumosus,” ‘Zool. Anz.,’ vol. vii, pp. 189, 221, 241,
1884,
2 ¢ Biologie Cellulaire,’ p. 232.
IRON COMPOUNDS IN ANIMAL AND VEGETABLE CELLS. 2383
forming the two adjacent bands. The fibrils, or what are in
appearance such structures, have so little iron in them that
frequently in a large part of an isolated filament their blue
reaction may not be sufficiently deep to betray their presence,
but the chances of observing them may be increased by staining
such preparations carefully with safranin. Probably the ex-
pression fibril is not a correct one to apply to these appear-
ances, for they may be the optical sections of the partition
walls of compartments, the extreme ends of which would in
that case be formed by the dim bands. What appears to sup-
port the latter view is the fact that in some of the thickest dim
bands the Prussian blue reaction reveals the presence of a single
row of vesicles extending from one end of the band to the
other, the vesicles sometimes having an elongated form parallel
with the filament. It seemed to me that these were the
initial stages in the division of one dim band into two, that
the thinner bands represent those most recently formed, and
that, therefore, the vesicular mode of formation would result in
the production of a series of compartments the thin walls of
which, in the clear bands, would appear as fibrils. The struc-
tures observed are, however, so exceedingly minute that it is
impossible to determine definitely anything on this point.
The iron-holding substance in the filament is, therefore, dis-
posed in the rodlets of the dim band and in the fibril-like
elements connecting the rodlets of one dim dise with those of
its neighbours. The only exception to this statement may be
made in regard to the structure of the swellings which are
sometimes found on the course of a filament (fig.50). In this
case the dim discs are replaced by an iron-holding reticulum
disposed in the interior of the swollen portion of the filament.
A comparison of this portion with the adjacent portions
of the filament appears to indicate how the reticulum has
arisen and what its relations are. The iron-holding bands
on either side are less regular in their disposition than else-
where, and the fibril-like structures arising from them appear
to be directly connected with the iron-holding substance of the
reticulum referred to. The swollen portion of the filament
234, A. B. MACALLUM.
varies in its size and shape, but most frequently it has the
appearance represented in the figure.
I have never observed the annular swellings described by
Balbiani as present in the filament in C. plumosus, but I take
it that the swollen portions here described are the representa-
tives of such structures. Nor have I ever determined that
the filament ends by attachment to the nuclear membrane, or to
the ameehiform nucleolus, through which it may pass several
times in its course. The nucleolus varies not only in form
and size but also in composition. It may be homogeneous,
but more frequently the central portion contains vacuoles and
granules and stains more deeply with eosin or safranin, while
the peripheral non-granular portion may possess no staining
capacity whatever. In many preparations made from alcohol
material and stained with eosin, the nucleolar body alone is
stained, and this is particularly the case when the preparation
has been treated with acid alcohol and the acid ferrocyanide
mixture to demonstrate the iron present. The nucleolar sub-
stance, apart from its granules, contains iron, but the iron
present is very small in amount compared with that observed
in the filament, for, when the latter gives an intensely deep
blue reaction, the colour given the nucleolus is a very pale
blue, and when the nuclei are kept for a week mounted in the
glycerine and sulphide mixture in the warm oven, the isolated
nucleoli develop only a greenish colour, portions of the fila-
ments, on the other hand, giving in the same preparations a
marked dark-green reaction. Unlike the differences in staining
exhibited after treatment with eosin, the faint or light blue
reaction is uniform throughout the nucleolar substance.
The dim bands with the excessively fine fibrils in the filament
are formed of chromatin, as shown by treatment with the stain-
ing reagents, when the preparations have been properly
hardened. ‘There is, however, a difference between this chro-
matin and that of the ordinary animal cell in that while acid
methyl-green colours the former it leaves unaffected the
nucleoli and the swollen portions of the filament, which stain
deeply with hematoxylin and carmine.
IRON COMPOUNDS IN ANIMAL AND VEGETABLE CELLS, 235
Balbiani! concluded from such results that chromatin (sub-
stance chromatique) is present, not only in the dim discs, but
also in the annular swellings and the nucleoli. According to
Flemming,” safranin colours all these elements, but stains the
nucleoli very strongly. Flemming’s observation is correct only
for preparations made with the chrom-osmio-acetic reagent ;
but when the nuclei have been fixed with alcohol, or with
corrosive sublimate, treatment with acid alcohol for two or
three days affects the filament in such a way that its discs and
their excessively fine fibrils absorb and retain the safranin to a
very marked extent, while the nucleolus remains unstained,
and the swollen portions of the filament are faintly coloured.
It is possible to obtain in such preparations both the safranin
and the Prussian blue reactions, and then, with the exception
of the faint blue in the nucleoli, both effects are co-extensive
and of equal intensity. The marked difference between the
substance of the discs and that of the nucleoli is thus shown,
but it may be brought out in a more brilliant way by staining
Prussian blue preparations with eosin, which then affects the
nucleolus only.
The nucleolus thus resembles the similarly named structure
obtaining in the nuclei of Vertebrates, but it differs from this
in that it is amoeboid in form, and does not possess, in any case,
a chromatin envelope. The presence of granules and vacuoles,
moreover, appears to indicate that it is physically active, which
cannot be postulated of the vast majority of the nucleoli of
Vertebrate cells.
Whatever effects may be obtained by treating the nuclei
with various staining reagents, but one results in the cytoplasm
of the secreting portions of the salivary gland in Chironomus.
Acid methyl-green in the fresh preparations, and hematoxylin
and safranin in the hardened glands, demonstrate very clearly
that there is a stainable substance, in many respects like
chromatin, uniformly distributed through the cytoplasm ; that
it is chromatin would appear from the fact that the cytoplasm
} Loe. cit.
* *Zellsubstanz, Kern- und Zelltheilung,’ pp. 112, 113.
236 A. B. MACALLUM.
holds an assimilated iron compound, for if small fragments of
cells, hardened in alcohol, be subjected to the action of the
warm glycerine and sulphide mixture for a week or more, they
will manifest a dark-green reaction which, when the mixture is
washed away and replaced by an acid ferrocyanide solution, is
converted into that of Prussian blue. One may more readily
obtain the demonstration of the iron in these cells by allowing
sulphuric acid alcohol to act on the hardened gland for two
days, when the cytoplasm of the secreting cells and the sub-
stance of the thread (silk ?) in the lumen give evidence of the
presence of this element. Whether the iron thus demonstrated
in the substance of the thread belongs to the latter, or is
derived by diffusion from the cytoplasm of the secreting cells
during treatment with the acid alcohol, 1 am unable to say,
since my experiments made to determine this question, by the
use of the glycerine and sulphide mixture on isolated bits
of the threads, turned out to be failures.! The substance
forming the threads manifests a strong affinity for dyes, and
should it eventually be ascertained that the iron demonstrated
in it, after treatment with acid alcohol, is part of a “ masked ”
compound contained in it, the facts will then all indicate that
the iron-containing substance in the cytoplasm is the antece-
dent of at least a portion of the substance of the thread in the
lumen, and one will have then also a parallel of what was
pointed out as obtaining in the pancreas and other ferment-
secreting cells in Vertebrates.
Protozoa.—I have selected the genera Stentor, Epis-
tylis, Vorticella,and Param cecium for specially illustrating
the distribution of the assimilated iron in unicellular animals.
A very large number of other forms were used to confirm the
results which a study of the named organisms gave, but owing
1 Gilson (loc. cit.) has referred to the fact “that the silk of certain insects
seems to possess a stronger aflinity for this metal (iron) than nuclein itself.”
I have observed this peculiarity, but the iron absorbed is at once demonstrated
on the application of any form of ammonium sulphide, a fact which shows that
the iron so revealed does not enter into a “ masked ” condition, and ought not
to be confused with that of “ masked” compounds.
IRON COMPOUNDS IN ANIMAL AND VEGETABLE CELLS. 237
to the difficulty experienced in getting examples of such forms
in the numbers required, it was impossible to make a fully
satisfactory, systematic investigation of their iron-holding
character. On the other hand, examples of the genera named
could be obtained at all times in abundance, and I regard the
opportunities thus presented as compensating in some measure
for the limited range of genera studied.
One of the difficulties encountered in attempting to study
the distribution of iron-compounds in Protoza is the fact that
many of the motile forms, and some also of those which are
sessile or attached, have in their cytoplasm inorganic com-
pounds of iron, in great part, if not wholly, derived from the
food matters ingested, and when such organisms, after being
hardened in alcohol, are treated with the glycerine and sulphide
mixture, they give at once a deep reaction for iron which, in
many cases, obscures other details in the cytoplasm and
nucleus. When, moreover, attempts are made with acid
alcohols, and especially Bunge’s fluid, to remove the inorganic
iron, the conditions under which the experiments are made
enable the reagent to liberate the “‘ masked ” iron at the same
time, in which case the liberated portion becomes indistinguish-
able from that present previously in an inorganic form. To
avoid such difficulties it is necessary to select forms in which
the amount of inorganic iron is small or infinitesimal, and by
determining the amount of the reaction obtained during the
first ten minutes after the application of the glycerine and
sulphide mixture, one may thus prevent confusion arising from
the study of results obtained by the more prolonged applica-
tion of the reagent. Such forms may be found in the genera
above named, and one may, by attention to the character of the
medium of the organisms, without any difficulty secure such
examples as offer the most favourable conditions for investi-
gating the distribution in them of the assimilated iron. The
specimens of Hpistylis, for example, which were used by me
for this purpose, were obtained from a colonial form attached
to the sides and limbs of the common crayfish, and their cyto-
plasm gave no immediate reaction for iron. Examples of
238 A. B. MACALLUM.
Stentor and Paramecium, in sufficiently large numbers,
and all but completely free from inorganic iron compounds,
were readily obtained. The cytoplasm in Vorticella, on the
other hand, usually contains such compounds, but these
are very often in the form of granules situated in vacuoles, or
at the periphery of the same, a disposition of the compounds
which gives every facility for studying the distribution of the
assimilated iron.
In the examples of Epistylis there were, as stated, no in-
organic compounds of iron, at least none were demonstrable
in the glycerine and sulphide mixture within the first hour
after the application of the reagent, but on the third and fourth
day both cytoplasm and nucleus gave a marked reaction for
iron. The latter was, of course, most prominent in the nucleus,
in which was revealed, by the dark-green colour, in some exam-
ples a granular structure, in others a fibrillar arrangement.
The reaction of the cytoplasm was a diffuse one, with here and
there large granules in which it had developed more markedly.
The membrane and stalk were, in these cases, free from iron.
All these points were more readily observed in preparations
treated with sulphuric acid alcohol or with Bunge’s fluid for
twenty-four hours (fig. 28).
In Vorticella a similar distribution of the assimilated iron
was observed in both cytoplasm and nucleus, and a diffuse
reaction for iron was also obtained in the central or axial por-
tion of the stalk, after the preparation had been kept in the
warm glycerine and sulphide mixture for several days. The
reactions are represented in fig. 27, drawn from a preparation
which contained inorganic iron compounds disposed in vacuoles.
In this the central portion of the stalk is shown to be continued
into a funnel-shaped organ at the base, which also contains
“masked” iron. Iwas unable to determine how this organ
was connected with the cytoplasm. I found no difficulty in
obtaining the complete reaction in all the parts at the end of
a five days’ application of the warm glycerine and sulphide
reagent.
Examples of Stentor polymorphus, free from inorganic
IRON COMPOUNDS IN ANIMAL AND VEGETABLE CELLS. 239
iron compounds, were, after being hardened in alcohol and
after treatment with ammonium sulphide, isolated from those
more or less impregnated with iron salts, the large size of the
organisms enabling one to do this readily. One of such, after
treatment for fourteen days with the warm glycerine and sul-
phide mixture, is represented in fig. 25. In this no distinct
reaction was obtained during the first two days, definitely
showing that no inorganic iron was present. In the interior
of the spherical elements constituting the nucleus there
appeared eventually a diffuse iron reaction, as well as one
localised in granules, and the cytoplasm gave a diffuse reaction
like that given by the cytoplasm in Epistylis and Vorticella.
I do not think that in this case the reaction had developed to
the fullest extent of which it was capable, for I found other
examples in which the nuclear and cytoplasmic elements gave
amore intense one; but it is usually difficult in such large
cells to obtain the best effects of the réagent, since in two
weeks’ time it is apt to undergo decomposition, when the
development of the iron reaction ceases. In order to ascer-
tain how abundant the assimilated iron is, 1 employed acid
alcohols to liberate it, and, after the removal of the acid,
treated the preparation with ammonium sulphide. Sul-
phuric acid alcohol is the best reagent for the purpose, since
with it there is less iron diffused from the parts in which it is
liberated; but, in order to get the most exact results, the
examples of Stentor used should be free from inorganic iron
compounds, a point of which one may be certain by putting the
hardened examples in ammonium sulphide for a few minutes,
when, if they pass this test, they may be washed in alcohol to
remove all traces of the reagent and placed in the acid alcohol
for one or two days. I have represented in fig. 26 an example
of S. polymorphus, in the wall of the funnel-shaped ceso-
phagus of which was found the only inorganic iron compound
present, and in this, after it had been treated as described, the
ribbon-like nucleus appeared intensely greenish-black, while the
cytoplasm gave a deeper reaction than was obtained in any
specimen simply by prolonged treatment of it with the warm
VOL. 38, PART 2.—NEW SER. Q
240 A. B. MACALLUM.
glycerine and sulphide mixture. In examples absolutely free
from inorganic iron compounds the reaction in the cytoplasm
and nucleus was as marked as that represented in the figure.
The method is, of course, open to the objection that it may
permit a diffusion of the liberated iron from the nucleus to the
cytoplasm, but that the latter contains assimilated iron is
shown by prolonged treatment with the warm glycerine and
sulphide reagent.
In examples of different species of Paramcecium, the
cytoplasm, which gave no reaction for inorganic iron, mani-
fested with the warm glycerine and sulphide reagent after ten
days a reaction as distinct as that obtained under similar
conditions in the cytoplasm of Stentor, Vorticella, and
Epistylis. These organisms were the only ones in which
the micro-nucleus was revealed by the iron reaction, and the
latter appeared to me to develop more slowly than that in the
macro-nucleus; but the explanation for this may be that the
large quantity of chromatin in the latter renders a reaction
of any degree of intensity obtaining in it much more promi-
nent than a reaction of a similar intensity would appear in
the micro-nucleus. In both the reaction was almost wholly
confined to the granules and fibrillar elements.
All the forms of Protozoa studied illustrated the fact so
prominently indicated in the organisms referred to above, that
an assimilated compound of iron is a constant element in
their cytoplasm. It is probable that this compound belongs
to the chromatin class, for the cytoplasm in Protozoan
organisms generally stains much more readily, and holds the
dyes more tenaciously, than the cytoplasm in higher organisms
does. In support of this may be urged other facts. I
pointed out, when dealing with the relations of assimilated
iron compounds to the ferment-forming cells in Vertebrates,
that the substance which elaborates the ferment, or out of
which it is prepared, contains iron and acts towards staining
reagents like chromatin. Digestion in Protozoa is, in all
probability, effected by ferments derived, as in higher forms,
from the cytoplasm, and it is only reasonable to suppose that
IRON COMPOUNDS IN ANIMAL AND VEGETABLE CELLS, 241
the antecedent of the ferments is, in this class also, an iron-
holding chromatin.!
Euglena viridis is a form whose position, whether as a
vegetable or as an animal organism, has not by any means
been definitely determined, but the distribution of assimilated
iron in its interior appears to indicate that if it does not be-
long to the animal kingdom, its physiological processes pos-
sibly resemble those of the Protozoan cell, and it is for this
reason that I deal with it in this place. Examples of this
organism free from inorganic compounds of iron may be ob-
tained readily, and when hardened in alcohol, they may be
subjected to the action of the glycerine and sulphide mixture
for twenty-four hours, without manifesting a reaction for iron,
but when the application is extended for three days or longer,
a reaction for iron is obtained in the nucleus and cytoplasm.
The chromatin network is usually so affected by the reagent
that its nodal points only manifest the reaction, while the
nucleolus exhibits a less intense dark-green colour. The
cytoplasmic trabecule separating the “ amylaceous ” corpuscles
from each other develop a dark-green reaction, which is found
to be most intense at the nodal points. All these features are
more clearly seen in specimens which have been hardened in
alcohol, then treated for two days with sulphuric acid alcohol,
and finally, after being acted on with the acid ferrocyanide
mixture to produce the Prussian blue reaction, mounted in
balsam (fig. 49). In these preparations the iron revealed in
the cytoplasm is most abundant in its nodal points, which, with
the reticulum of the nucleus, are thereby rendered most pro-
minent. The nucleolus, separated from the other elements
by a clear zone, in which the light blue observed is derived
from the nuclear elements above and below the focal plane,
gives a less intense reaction than one of the much smaller
nodal points of the nuclear network. If the preparation has
also been stained with eosin the nucleolus alone appears to be
1 The ferment or ferments, according to M. Greenwood (‘Journal of Physio-
logy,’ vol. viii, 1887, p. 263), pass into the fluid surrounding the ingested
matter,
242 A. B. MACALLUM.
markedly affected by it, exhibiting an ochre-red colour so
characteristic of the nucleoli in the hepatic cells of Necturus
after similar treatment. Safranin leaves the nucleolus un-
affected, but colours deeply the chromatin network and the
iron-holding portions of the cytoplasm. When, however, the
organism has been hardened in picric acid, the nucleolus
exhibits no affinity for eosin, while it colours as deeply as the
chromatin network does with hematoxylin and picro-carmine.
From this it would appear as if the nucleolus were intermediate
in composition between the nucleolus of higher animal cells
and the chromatin of the nuclear reticulum.
The occurrence of assimilated iron in the cytoplasm of
Euglena viridis, if it is not chemically associated with the
chlorophyll present, appears to indicate that the organism is
closely related to the Protozoa, in common with which it has
other characters.! If the view, that the assimilated iron in
the cytoplasm of Protozoa is part of the antecedents of the
zymogenic compounds of these organisms, is correct, it would
explain the phenomenon in Euglena in which the presence of
a short digestive “‘ tract”’ also postulates, to a certain extent,
the occurrence of processes of nutrition belonging to the animal
type.
Fuugi.—The presence of nuclei has not yet been demon-
strated in a large number of the Fungi, nor has the occurrence
of a substance similar to the chromatin of other organisms
been determined with any degree of certainty, except in a few
forms ; and, therefore, the question of the occurrence and dis-
tribution of assimilated compounds of iron in the cells of this
class is not quite as easy of solution as that dealing with the
1 G. Klebs, who has given special attention to the Euglenacee (‘“ Organiza-
tion einiger Flagellaten-Gruppen und ihre Beziehungen zu Algen und Infu-
sorien,” ‘ Untersuch. aus dem Bot. Inst. zu Tiibingen,’ 1881-85), is of the
opinion that this group should be classed amongst the Protozoa. Khawkine
(‘Recherches biologiques sur l’Astasia ocellata, ns., et ’Huglena
viridis. Seconde Partie, L’Euglena viridis.’’ ‘Ann. des Sciences Nat.,
Zoologie,’ Serie 7, vol. i, 1886, p. 319) came to the conclusion, as a result
of experiments, that Euglena takes in organic compounds in the dark, but
in daylight assimilates only inorganic compounds.
IRON COMPOUNDS IN ANIMAL AND VEGETABLE CELLS. 243
presence of these compounds in higher organisms. I have,
however, endeavoured to solve it by the investigation of a few
widely different forms, and the results now to be described
show the presence of “ masked” iron compounds similar to
those found in all the higher organisms. These forms com-
prise: Saccharomyces cerevisix, 8. Ludwigii, Hy-
phelia terrestris Fries,a leucosporous Agaricine, Cystopus
candidus, and Aspergillus glaucus.
The question of the occurrence of a nucleus in Saccha-
romyces bears upon that relating to the presence of iron-
containing chromatin-like substances in this genus ; and, con-
sequently, it is necessary to give an account of the various
observations that have been made on this subject.
The earlier botanists, Nageli! and Schleiden,? claimed that
they had found a nucleus in the yeast-cell, and the later
observers, Schmitz,> Strasburger,* Zalewski,® and Zacharias,é
have maintained that it exists, while Zimmerman’ speaks
reservedly on the question. Raum’ found in yeast-cells which
had been fixed on the cover-glass by heat or by solutions of
corrosive sublimate, and stained, first with warm methylene-
blue and then with bismarck brown, black spherical granules,
varying in number from one to fifteen, in a more or less
brown-tinted protoplasm. These were usually arranged in the
1 «Zeit. fiir wiss. Botanik,’ vol. i, p. 45. Reference in Raum’s paper.
> «Grundziige der wiss. Botanik,’ 1849, p. 207. Referred to by Raum.
3 “ Untersuchungen iiber den Zellkern der Thallophyten,” ‘Sitzungsber.
der Niederrhein, Gesell. fiir Natur- und Heilkunde zu Bonn,’ Sitzung. am
4 Aug., 1879.
4 «Das Botanische Practicum,’ p. 339, 1887.
5 “On Spore Formation in Yeast Cells,” ‘Transactions of the Scientific
Academy of Cracow’ (Polish), 1886. Abstract in ‘Bot. Centralbl.,’ vol.
ZXVe po L.
6 « Beitrage zur Kenntniss des Zellkerns und der Sexualzellen,” ‘ Bot.
Zeitung,’ 1887, Nos. 18-24.
7 “Die Morphologie und Physiologie der Pflanzenzelle,” Breslau, 1887,
p. 25. Ihave not had access to this publication, and my attention was first
called to it by a reference made by Raum.
8 “ Zur Morphologie und Physiologie der Sprosspilze,” ‘ Zeit. fiir Hygiene,’
L891, vol.x, p. 1.
244 A. B. MACALLUM.
form of a circle or of a segment of a circle at either pole of the
oval cell, and there was no relation between their size and that
of the cell containing them, although they appeared to have
some connection with the budding process, since he observed
them undergoing transference to the protoplasm of the bud.
What the nature of these granules is Raum does not say, but
the results of his experiments would seem to indicate that they
are not formed of nuclein, for on submitting the yeast-cells to
digestion with an artificial gastric fluid at a temperature of
40° C. for one or two days, and afterwards on washing with
ether and alcohol, every trace of the granules had vanished.
Nuclein is undoubtedly present in yeast-cells, and Raum
prepared some of it from this source, which he mounted in
egg-albumen on a cover-glass, and stained, first with methylene
blue and afterwards with bismarck brown, when he found that
the nuclein particles took a brownish stain while the albumen
appeared light yellow, a reaction in marked contrast with that
obtained in the granules of the hardened yeast-cells after the
employment of the same staining methods. Raum appears to
be doubtful concerning the existence of anything resembling a
nucleus in the yeast-cell.
The more recent observers who claim to have found a
nucleus in the yeast-cell are Moller aud Janssens. The
former! found in older yeast-cells a spherical corpuscle which
he regards as a nucleus, but without a membrane or nucleolus.
This changes its shape readily, and therefore its position in
the cell varies. Owing to this property, it is capable of
assuming a thread-like form when budding occurs, a portion
of it being thus enabled to pass into the protoplasm of the
bud through the narrow tube which connects the mother and
daughter elements. The part in the latter eventually breaks
off, and both portions become spherical. Janssens,? who used
1 “Ueber den Zellkern und die Sporen der Hefe,” ‘Centralbl. fiir Bakt.
und Parasitenkunde,’ vol. xii, 1892, p. 537; also “ Weitere Mittheilungen
iiber den Zellkern und die Sprosse der Hefe,” ibid., 1893, vol. xiv, p. 358.
2 « Beitrige zu der Frage iitber den Kern der Hefezelle,” ‘Centralbl. fiir
Bakt. und Parasitenkunde,’ vol. xiii, 1893, p. 639.
IRON COMPOUNDS IN ANIMAL AND VEGETABLE CELLS. 245
in his investigations the species 8S. cerevisie, 8. Ludwigii,
and S. Pastorianus, states that he found in the two former a
nucleus provided with a membrane and a nucleolus, the latter
spherical and homogeneous and of a diameter one third that of
the nucleus. The remaining portion of each cell is occupied
by a cytoplasmic network with fine meshes, whose nodal points
readily absorb colouring matters, and, in the opinion of
Janssens, constitute the granules of Raum. He claims to
. have observed mitotic stages of the nucleus, which obtain when
budding commences and when spore formation occurs.
Two observers only, Briicke! and Krasser,? have denied the
existence of a nucleus in the yeast-cell. Krasser in his later
publication asserts that the body described by Mdller as a
nucleus is not such an organ, and he found, after employing
Moller’s methods on beer yeast-cells, that the latter possessed
no body like the one described by that observer. He further
observed that the bodies described by Moller as nuclei, after
being submitted to digestion with artificial gastric juice, gave
no evidence of the presence of nuclein. The occurrence of the
latter substance in yeast-cells, which is readily demonstrable
in a macro-chemical way, Krasser attempted to show micro-
chemically, and, after many failures, succeeded in finding it in
a few specimens in the form of granules at the side of the body
regarded by Moller as a nucleus.
I have followed the methods of hardening and staining
adopted by Moller, for the purpose of ascertaining the nature
of the body considered by him to be a nucleus, and have
compared the results thus obtained with those found in yeast-
cells after hardening the latter in saturated solutions of cor-
rosive sublimate and staining them with hematoxylin and
eosin. I have also used Flemming’s fluid for hardening, and
stained preparations so made with safranin. Moller’s methods
certainly do reveal, now and then, a structure like that which
1 “Die Hlementarorganismen,” ‘Sitzungsber. der K. Akad. d. Wiss. zu
Wien, Math.-Nat. Classe,’ 1861, vol. xliv, Abth. 2.
2 “Ueber das angebliche Vorkommen eines Zellkerns in den Hefezellen,”
‘Oesterreich. Bot. Zeits.,’ 1885, No. 11; also “ Ueber den Zellkern der
Hefe,” ibid., 18938, p. 14.
246 A. B. MACALLUM.
he took to be a nucleus, but this body, when hardened with
corrosive sublimate, stains with eosin but not with hema-
toxylin, while after fixation with Flemming’s fluid it appears
to have no particular affinity for any dye. On the other
hand, in S. Ludwigii, as it usually develops in the sap of the
iron-wood tree (Ostrya virginica), there is in the great
majority of cells a corpuscle which corresponds with the
“nucleus” of Méller. This structure is round, homogeneous,
and in diameter sometimes more, sometimes less, than half
the length of the shorter axis of the cell, in the centre of
which it is usually placed, and after being hardened with
corrosive sublimate it exhibits a special affinity for eosin,
but none for hematoxylin, while it acts like the cytoplasm
towards safranin. In preparations made with Flemming’s
fluid the results were practically the same, and therefore not
indicating on the part of the body in question the possession
of a substance in all points like chromatin.
A substance like chromatin appears to be distributed
through the cytoplasm. In S. cerevisiz, after being hard-
ened with corrosive sublimate, the cytoplasm takes, when
treated with hematoxylin (Delafield’s and Ehrlich’s), a blue-
violet tinge. With favourable illumination and apochromatic
objectives, the stain is found to be localised in the trabeculee
of the cytoplasmic network, and, where the vesicular character
of the cytoplasm appears pronounced, all the cytoplasm, except
the contents of the vesicles, is coloured. In some of the cells
granules were observed with a stain slightly deeper than that
of the cytoplasm, and similar elements were found in cells
hardened with Flemming’s fluid and stained with safranin.
These, possibly, are those described by Raum. In 8. Ludwigii
the cell is usually very much larger, and the structure and
staining reactions are, therefore, much more distinct. In this
form, when hardened with corrosive sublimate and stained
with hematoxylin, the vesicular structure of the cytoplasm
comes out quite markedly through its blue-violet stain, which
also is found now and then to characterise prominently gran-
ules in the cytoplasm between the vesicles. The granules of
IRON COMPOUNDS IN ANIMAL AND VEGETABLE CELLS. 247
Raum are, however, much more common elements than these,
and are to all appearances quite different structures, as is
apparent in ordinary cover-glass preparations made after
Raum’s methods. The larger examples of the granules of
Raum seem to be less abundant in corrosive sublimate prepa-
rations stained with hematoxylin and eosin.
From these results I am inclined to regard the existence of
a nucleus in the yeast-cell, in its usual condition, as extremely
doubtful, and, on the other hand, to support Krasser’s con-
tention that nuclein is disseminated through the cytoplasm.
Whether, in other stages, as, for example, those in which spore
formation occurs, there is a nucleus I cannot say, but there
appears in the ordinary stages of the organism to be nothing
which may be looked upon as a specialised chromatin-holding
structure.
These conclusions are, on the whole, confirmed by the
results of experiments made to determine the distribution
of assimilated compounds of iron in these organisms. When
specimens of S. cerevisiz, hardened in alcohol, are subjected
to the action of the glycerine and sulphide mixture at a tem-
perature of 60° C. for several days, their cytoplasm acquires a
greenish tint. Sometimes, however, the latter reaction may
not appear except in a few granules scattered through the
cytoplasm (fig. 4). On account of the small size of the cells
and of the alteration produced in them by the reagent, one
cannot definitely determine whether the granules correspond
to those described by Raum. When the cells have been sub-
jected to the action of sulphuric acid alcohol, the subsequent
application of an acid ferrocyanide solution gives their cyto-
plasm a faint blue colour, which is more distinct and deeper
when the light transmitted passes through several cells in
succession. Blue granules are sometimes observed in such
preparations.
It is in specimens of S. Ludwigii that one obtains the
clearest evidence of the occurrence of an assimilated iron
compound. In these, after being hardened in alcohol, the
glycerine and sulphide mixture eventually gives results like
248 A. B. MACATLLUM.
those represented in fig. 5. The differences observed appear
to depend on the cytoplasmic structure in the specimen ex-
amined. When there are a few large vesicles in the cell, the
iron-holding substance seems to be, in great part, at their peri-
pheries. This disposition also obtains in the buds. The
remaining portion of the cytoplasm in each element is very
slightly coloured greenish, but whether that is due to ferrous
sulphide is uncertain. When, on the other hand, the cells are
markedly vesiculated, the glycerine and sulphide mixture gives
the cytoplasm between the vesicles a distinct reaction for iron,
In the majority of such cells there are one or more large
spherical elements, which, in the glycerine and sulphide mix-
ture, after the third or fourth day appear dark green, much
more so than does the surrounding cytoplasm. They are
homogeneous, manifesting a uniform reaction throughout their
substance, and their position is, if not in the centre of the
cell, at least in that neighbourhood ; but smaller granules of
the same character may be more remotely situated. From
their position, size, and shape, they would appear to be the
bodies which, in preparations made with corrosive sublimate,
hematoxylin, and eosin, stain exclusively with the latter
reagent. In cells which are treated with acid alcohol, then
with an acid ferrocyanide solution, and finally, after being
stained with eosin, mounted in balsam, similar bodies are
given a violet tint, while the cytoplasm is coloured bluish, the
violet being undoubtedly due to a combination of the Prussian
blue colour with the eosin stain. As the granules of Raum
are not specially selected by eosin, it would appear that the
iron-containing body observed does not belong to that class.
It is thus seen that in 8S. cerevisiz the assimilated iron is,
like the substance which absorbs hematoxylin, distributed
through the cytoplasm and sometimes also in the latter in the
form of granules, but in S. Ludwigii it may be chiefly found
at the periphery of each large vesicle when only a few vesicles
are present, while in those cells in which the whole of the
cytoplasm is vesiculated, the latter gives a uniform reaction
for iron corresponding in its depth with that given by hema-
IRON COMPOUNDS IN ANIMAL AND VEGETABLE CELLS. 249
toxylin. Further, there is a substance which constitutes cor-
puscles of a nucleolar character in cells of this form, which
stains with eosin and gives a marked reaction for iron, but
differing from the chromatin substance in remaining unstained
after treatment with hematoxylin. There is no nucleus,
although such an organ may occur in other stages, especially
in 8. Ludwigii.!
When the mycelial threads and hyphe of Hyphelia ter-
restris, Fries, are hardened in alcohol and stained with hema-
toxylin, the cytoplasm generally is coloured, but it is specially
affected by the stain in the terminal portions of the hyphe on
which the elements of fructification are developing. One can
find also, in such preparations, deeply-stained granules scattered
in the cytoplasm of the hyphe, and at times also a vesicular
cavity and a membrane enclosing these granules, which then
simulate nucleoli. Sometimes such structures strongly re-
semble nuclei, and mitotic conditions are suggested by the
presence of pairs of rows of deeply-coloured granules placed
opposite, and at avery short distance from, each other. In
the fully-formed fructification these vesicular cavities and their
deeply-stained granules may be most readily seen. Whether
such structures are nuclei in the proper sense of the term it is
difficult to say, but if they are, they contain only a small por-
tion of what may be considered as the chromatin, which is
diffused in the cytoplasm of the mycelial threads in the younger
stages, but appears to be transferred to the hyphe when the
fructification of the latter commences. When the latter stage
is fully attained the mycelia and lower portions of the hyphz
are found to have little or no cytoplasm and to stain very
feebly, a result quite different from that obtained in the fructifi-
cation.
The distribution of the “ masked ” iron in this form is found
to coincide very closely with the distribution of the stainable
substance. In the simplest form of the hypha, the glycerine
Ludwig (‘Lehrbuch der niederen Kryptogamen,’ 1892, p. 201) appears
to regard S. Ludwigii as merely a stage in the development of Endomyces
Magnuusii.
250 A. B. MACALLUM.
and sulphide mixture gives in twenty-four hours a reaction
like that represented in fig. 13 a, while in the slightly more
developed structure the reaction is deeper with large dark-
green granules (fig. 13 6). A similar result is obtained in
the hyphe which terminate in two, three, or more pear-shaped
outgrowths (fig. 12). In the hyphe below the fructification
the cytoplasm is of a vesicular character, the walls of the
vesicles being formed of an iron-holding substance, and as the
terminal element develops, the vesicular character becomes
less marked and the iron reaction less distinct, so that, finally,
no iron may be found in this part of the filament. At the
same time the granules in the fructification become more
numerous, larger, and manifest a deep reaction for iron
(fig 11). These granules are then found to be situated in
small vesicles very much like the vesicles which, in hema-
toxylin preparations, resemble nuclei. The granules revealed
by the iron reaction are the same as those indicated by the
hematoxylin stain. This is also true of the granules in the
younger hyphe. The cytoplasm of the mycelial threads is, at
this stage, free from ‘‘masked’’ compounds of iron, but in the
earliest stages the mycelial threads give at once, on the applica-
tion of the glycerine and sulphide mixture, a slight reaction for
iron, which, however, becomes deeper at the end of twenty-four
hours if heat be applied, this indicating the presence of
“masked” iron. Granules in the cytoplasm along the course
of the threads give a marked reaction for the metal like that
manifested in the hyphe. It is probable that the absence of
iron in the later stages of the threads may be due to the
transference of the iron-holding compound to the hyphe.
The question concerning the occurrence of nuclei in the
Hymenomycetes has been dealt with by Strasburger,’ Rosen-
vinge,? and Wager.? The two former describe them as obtain-
1 ‘Das Botanische Practicum,’ pp. 380] and 4338, 1887.
2 «Sur les noyaux des hyménomycétes,” ‘ Annales des Sciences Nat., Bot.,’
1886, Serie 7, vol. ili, p. 75.
3. “On the Nuclei of the Hymenomycetes,” ‘Annals of Botany,’ 1892,
vol. vi, p. 146.
IRON COMPOUNDS IN ANIMAL AND VEGETABLE CELLS. 251
ing in the hyphe, in the basidia, and in the spores of the
various species, in the form of small elements which are
brought into view only when alcoholic material is acted on by
very dilute solutions of hematoxylin. Their number in a
hypha varies, but in each basidium there is at first only one,
which, when the sterigmata are being formed, divides, the
daughter nuclei undergoing division also, sometimes a
second time, each of the four or eight thus resulting passing
through the tubes of the sterigmata into the spores at the
end of the latter. When the spores are mature they thus
contain, according to the species, one or two very minute
nuclei, while the basidia at this stage contain none. Wager
also found nuclei in the basidia, but maintains that the spores
do not contain any until after the formation of the thick spore-
membrane.
It is an easy matter to demonstrate in the hyphe and some-
times in the basidia and in the mature spores of leucosporous
Hymenomycetes,! the structures regarded by Strasburger and
Rosenvinge as nuclei, but, as was the case in Hy phelia ter-
restris, such elements contain only a small portion of the chro-
mophilous substance, for when preparations are made, as recom-
mended by Strasburger, with very dilute solutions of hema-
toxylin, the cytoplasm also stains though not quite so deeply
as the minute nuclei, especially in young hyphe. This and
other staining reactions indicate that chromatin is dissolved in
the cytoplasm, a conclusion borne out by the results of experi-
ments with the glycerine and sulphide mixture and with acid
alcohols, in which case the hyphal elements of a very young
stage of growth give a reaction for iron diffused throughout
the cytoplasm, but when the spores are formed the hyphal cells
and their shrunken nuclei rarely give a reaction for iron. At
this stage also, in sections of the lamelle, a reaction for iron is
obtained in the hymenium and in the spores, while the hyphal
elements of the “trama’’ appear free from the metal. If the
spores and the basidia are teased out and mounted in the
The pigment in the spores of the other divisions of the Hymenomycetes
greatly obscure the reaction obtained with the glycerine and sulphide mixture.
252 A. B. MACALLUM.
glycerine and sulphide mixture, the application of heat to the
preparation for a week will bring out appearances in the
isolated elements like those represented in fig. 10. The most
prominent feature in these is that the cytoplasm in both classes
of structures contains “masked” iron. When the bodies
regarded by Strasburger and Rosenvinge as nuclei were
observed, they manifested a slightly deeper reaction for iron
than the cytoplasm generally, but no structure was detected
in them and they appeared as large granules rather than nuclei.
The most marked reaction for iron was obtained in the spores
in which a cytoplasmic reticulum was thus demonstrated.
When, however, the spores are provided with a thick mem-
brane, a reaction with the glycerine and sulphide mixture does
not appear, but is obtained after the use of acid alcohols. As
a rule, the reaction is uniform throughout the cytoplasm of
the basidia. There are, however, constituents of the hymenium
occasionally observed in which no iron was found. They
possessed no sterigmata or spores, and from their association
with the basidia I was inclined to regard them as paraphyses,
but from the comparative scarcity of such elements free from,
or poor in iron, they can scarcely be looked upon as belonging
necessarily to that class, which in stained preparations is
abundantly represented. The subhymenial cells also give a
faint reaction for iron.
It thus appears that in the leucosporous Hymenomycetes the
cytoplasm of the hyphe in the early stages of the fungus con-
tains iron, which is also present in the minute “‘ nuclei,” and
that in later stages this cytoplasm gives a faint reaction or
none at all for iron, while the cytoplasm of the basidia and
spores contains enough “masked” iron to give a marked
reaction. This distribution of the iron corresponds with the
distribution of the stainable substance, and it may, therefore,
be fairly concluded that the chromatin is here also iron-
holding.
In my earlier communication reference was made to the
occurrence of an iron-containing substance in the gonidia of
Cystopus candidus, and I stated that the iron compound
IRON COMPOUNDS IN ANIMAL AND VEGETABLE CELLS. 253
was found to be localised in spherical elements of 16m dia-
meter, corresponding to the nuclei of the zoogonidia. I have,
since that date, investigated the cytological character of this
organism, and have found that though there are, as Fisch,!
Wager,’ and others have observed, nuclei in the mycelia and
in the gonidia, the whole of the protoplasm, except in the
mature gonidia, is chromophilous, that is, it contains chro-
matin. ‘The nuclei are, indeed, of the more regular form in
the mature gonidia, but in the mycelia amongst the cells of
the host (Capsella bursa-pastoris) they are chiefly, if not
wholly, small masses of chromatin, like those forming the
‘“nucleoli” in the abjointing gonidia. I have not succeeded
in finding the mitotic phase either in the mycelia or in the
developing gonidia, although I have carefully looked for such
in a large number of preparations.
The disposition of the assimilated iron corresponds closely
with the distribution of the chromophilous substance in this
form. The cytoplasm of the haustoria and of the mycelia
gave a marked reaction for iron in all the methods of demon-
stration.2 The mycelial membrane gave no evidence of the
presence of the element. The small masses of chromatin were
found to be rich in organic iron. In the terminal enlarged,
sometimes club-shaped, sometimes truncated, portion of each
hypha the iron was found to be in a localised as well as in a
diffuse form. The “nucleoli”? gave abundant evidence of its
presence, these structures thus appearing in marked contrast
with the remaining portions of the nucleus, which contain
relatively less iron than the surrounding cytoplasm in this
stage. In the subsequent development of the abjointed gonidia,
the nuclei appear to take up from the cytoplasm all, or nearly
1 “Ueber das Verhalten der Zellkernein fusionirenden Pilzzellen,” ‘ Ver-
sammlung deutscher Naturforscher und Aerzte in Strassburg,’ 1885. This
paper I have not seen, and the only references to it that I can find are those
made by Wager and Dangeard (‘Comptes Rendus,’ cxi, 1890, p. 882).
“ Observations on the Structure of Cystopus candidus,” ‘Rep. Brit.
Ass. for the Adv. of Science,’ 1892, p. 777.
3 The material was hardened in alcohol, which was renewed until every
trace of chlorophyll was removed from the tissues.
254 A. B. MACALLUM.
all, of the iron-holding substance, and with this the character
of the nuclei seems to change. The “nucleoli,” first of
all, are converted into fine granules distributed through the
nuclear cavity, and, finally, in the mature gonidia the nuclei
appear, in the glycerine and sulphide preparations, to be
simply more or less homogeneous masses of iron-holding sub-
stance, while the cytoplasm does not contain a trace of the
metal (fig. 6 /).
In Aspergillus glaucus the cytoplasm of the young
mycelia and the gonidiophores, especially their globular ends,
absorbs staining matters readily, but it contains also, scat-
tered through it, granules of a nucleolar character, which,
in very dilute solutions of hematoxylin, applied for twenty-
four hours or more, stain deeply. The cytoplasm of the
sterigmata and of the immature gonidia is similarly affected.
In the mature gonidia hematoxylin selects large granules
which are distributed through the cytoplasm. In what appear
to be old mycelial threads, the cytoplasm is stained with diffi-
culty, while the membrane may be deeply coloured. These
results correspond in the main with those obtained in regard
to the ‘‘ masked” iron present. When the warm glycerine
and sulphide mixture is applied for about a week, the cyto-
plasm of young mycelia gives a diffuse reaction for iron, while
a deeper one appears in the large granules referred to as
affected by hematoxylin. In the cytoplasm and granules
of the gonidiophores a relatively deeper reaction makes its
appearance, and a marked one is obtained in the sterigmata.
In the immature gonidia the reaction is diffuse, a special one
at the same time obtaining in granules collected or scattered
in the cytoplasm. In mature gonidia the granules are larger,
and give a deeper reaction for iron, the cytoplasm otherwise
showing no trace of its presence (fig. 7). The same results
are obtained, but more readily, when sulphuric acid alcohol
has been employed to liberate the iron present.
Bacteria.—The question of the occurrence in bacteria
of a substance like the chromatin of more highly developed
organisms has been investigated to a certain extent by
IRON COMPOUNDS IN ANIMAL AND VEGETABLE CELLS. 250
Ernst,! Babes,? Wahrlich,? Biitschli,* Trambusti and Galeotti.’
Ernst found in a large number of species of bacteria granules
which stain with hematoxylin and other dyes, while the sur-
rounding protoplasm is coloured faintly or not at all. These,
which on account of their direct transformation into spores he
termed sporogenous, undergo in their earlier stages solution in
artificial gastric juice, but in the more advanced condition
resist digestion. From Babes’ observations, which agree in
the main with those of Ernst, it would appear that the granules
which absorb and retain colouring matters and take part in
spore formation, also stand in some relation to the division of
the bacterial cell. According to Wahrlich, the protoplasm is
formed of two constituents at least, a ground substance of
reticular structure resembling linin, and one forming granules
distributed in this reticulum, and, owing to its capacity for
absorbing and retaining dyes, regarded by him as chromatin.
In Bacillus pseudoanthracis the small granules which
appear before the spores are formed are constituted of chromatin,
and from them is derived the main portion of each spore, while
the plastin serves apparently for the construction of the spore
membrane. Biitschli found in species of Beggiatoa, Chro-
matium, in Spirochete serpens, Spirillum undula,
Bacterium lineola, andinsome Cyanophycee, a faintly stain-
able peripheral portion, and a central body, readily stainable,
in which a honey-comb structure (Wabenbau) was distinctly
1 “Ueber den Bacillus xerosis und seine Sporenbildung,” ‘Zeit. fur
Hygiene,’ vol, iv, p. 25, 1888; also “ Ueber Kern- und Sporenbildung in
Bacterien,’’ ibid., vol. v, p. 428, 1889.
2 “Ueber isolirte, farbbare Antheile von Bakterien,” ibid., vol. v, p. 173,
1889.
3 * Bacteriological Studies.’ Reprinted from ‘ Scripta Botanica,’ vol. iii,
St. Petersburg, 1890-91. I have not seen this work, and the representation
of Wahrlich’s observations and views is taken from ‘ Bot. Central.,’ vol. xlix,
1892.
4 «Ueber den Bau der Bakterien und verwandter Organismen,’ Leipzig,
1890.
> «Neuer Beitrag zum Studium der inneren Struktur der Bakterien,”
‘Centralbl. fir Bakt. und Parasitenkunde,’ vol. xi, p. 717, 1892.
vol. 38, PART 2,—NEW SER. R
256 A. B. MACALLUM.
seen. The central body is, in Bitschli’s opinion, a nucleus.
In or on this organ were observed granules which became red
after treatment with hematoxylin, and were identified with
the granules described by Ernst. Trambusti and Galeotti
found in one stage of a very large bacillus isolated from
drinking water, that the whole of the protoplasm stained uni-
formly and deeply with safranin, while in a later stage of the
same the stainable substance was converted into granules, dis-
posed at the periphery and arranged in the form of a garland
of oval outline. The granules eventually fused to form a
homogeneous garland out of which arose from three to four
elliptical rings, at first connected by their ends, but afterwards
independent of each other, and in this condition became free.
These changes the observers regard as analogous to those of
mitosis in the cells of more highly specialised organisms.
Schottelius! and Ilkewicz® have described structures in the
bacterial cell which they regard as nuclei, and Sjobring ®
claims to have found many of the phenomena of mitosis, as it
obtains in the cells of higher organisms, exemplified in bacteria.
The results of these observers appear to me to have been due
to defective methods of technique.
I find that in Bacillus subtilis, B. anthracis, B. mega-
therium, B. tuberculosis, and in the root bacillus, there are
granules like those described by Ernst and Babes, and which
stain with hematoxylin, and in B. pseudosubtilis (?), in
which there is only one granule to each rodlet, each granule is
developed into a spore, the remaining protoplasm at the same
time losing all its affinity for colouring matters. The struc-
tures observed are the same whether alcohol, corrosive subli-
mate, or heat has been employed for their fixation.
Ernst found, as already stated, that the granules, except in
the later stages, undergo solution in artificial gastric juice.
1 «Beobachtung Kernartiger Kérper im Innern von Spaltpilzen,”
‘Centralbl. fiir Bakt. und Parasitenkunde,’ vol. iv, 1888, p. 705.
2 « Ueber die Kerne der Milzbrandsporen,”’ ibid., vol. xv, p. 261, 1894.
* “Ueber Kerne und Theilungen bei den Bakterien,” ibid., vol. xi, p. 65,
1893.
IRON COMPOUNDS IN ANIMAL AND VEGETABLE CELLS. 257
This would seem to indicate that they are not constituted of
typical chromatin.! I have endeavoured to determine whether
they contain iron in a “ masked” form; but the results of my
experiments, except in the case of B. megatherium, have
not been decided enough to permit a general conclusion on
this point. The organisms are very small, and their size
would postulate the occurrence of a very small amount of iron
in them, and even in the larger spores. When, therefore, a
cover-glass preparation of B. megatherium is treated with
sulphuric acid alcohol for twenty-four hours, it is not surpris-
ing that the subsequent treatment with an acid ferrocyanide
solution should give but a very faint blue reaction. When the
granules referred to were under observation they manifested
themselves by a blue colour slightly deeper than that apparent
in the rest of the protoplasm of the organism. In B. subtilis
the granules are the only parts of the bacillus which appear to
contain iron, the reaction for which is very faint. I have in
none of these forms obtained a reaction with the glycerine and
sulphide mixture distinct enough to permit certainty of opinion
in regard to this. Sulphate of iron, when present in very
minute quantities in preparations, appears less distinct than the
same amount of iron when revealed by the Prussian blue
reaction, and on this account the apparent absence of the
sulphide reaction determines nothing. In some preparations
of B. pseudosubtilis the largest granules and the spores
gave, after treatment with acid alcohols, a blue reaction with
the acid ferrocyanide mixture. The root bacillus gave fre-
quently a diffuse and faint blue reaction under the same condi-
tions.
It is obvious that these organisms are too minute to furnish
results which would allow the question, whether they contain
“masked ” iron, and how it is distributed, to be definitely and
decisively answered, and I had to employ other forms, of such
a size that no difficulty would be experienced in this respect.
1 Vandevelde (“ Studien zum Chemie des Bacillus subtilis,” ‘Zeit. fiir
Physiol. Chemie,’ vol. vill, p. 867, 1884) states that he has isolated nuclein
from B. subtilis.
258 A. B. MACALLUM.
The most readily accessible form was Beggiatoa alba. This
organism, as is well known, manifests itself in five different
conditions: long threads composed of cells of varying lengths,
shorter filaments also formed of cells usually free and motile,
spirillum-like elements, comma-shaped, two-celled, swarming
bodies, and simple “cocci.” Cover-glass preparations of all
these forms, fixed first with heat and subsequently with
alcohol, were subjected for about two weeks to the action of
the glycerine and sulphide mixture at 60°C., while like prepa-
rations were treated with sulphuric acid alcohol for about two
hours at a temperature of 30°C. The results of both methods
agreed. In the long threads the abundance of sulphur granules
causes the cytoplasm to have a reticular, or more properly
speaking, a vesicular appearance, brought out very prominently
when the glycerine and sulphide mixture has dissolved out the
sulphur and at the same time given the cytoplasm a greenish
colour, developing into a faint blue on treatment with an acid
ferrocyanide mixture. At times the greenish or the blue re-
action appears most prominently in some of the nodal points
of the “network,” but this is doubtless due to the fact that
more of the cytoplasm is condensed at such points. The
shorter free, motile filaments, which contain, as a rule, very
many fewer sulphur granules, have a more homogeneous cyto-
plasm, and in these the reaction for “ masked” iron obtained
was a diffuse one. A similar result was obtained in the
examples of the spirillum form. In the comma-shaped forms
the reaction obtained was, as a rule, slightly deeper, and
it frequently appeared most markedly in the central portions
of each of the two cells. In some examples a granule in this
central mass gave a marked reaction for iron. I did not suc-
ceed in determining the relations of the iron in the “ cocci.”
So far as these results go they correspond with those ob-
tained when cover-glass preparations of Beggiatoa alba are
stained with hematoxylin, which colours diffusely the cytoplasm
in all the forms, but rarely reveals the existence of special
chromatin elements. I have been unable to determine, except
in a few comma-shaped elements, the occurrence of the denser
IRON COMPOUNDS IN ANIMAL AND VEGETABLE CELLS. 259
central portion described by Biitschli, and I am inclined to re-
gard the structure observed in the exceptional cases as due to
shrinkage caused by the method of preparation. In some of
the comma-shaped elements the hematoxylin stain demon-
strates granules like those which were observed to manifest an
iron reaction in the glycerine and sulphide preparations, The
use of Loffler’s solution of methylene blue, followed by that of
a saturated solution of bismarck brown, as recommended by
Ernst, stains similar granules in the “ comma” elements, and
in a few of the spirilla only ; but it is doubtful if these may be
classed with the “sporogenous”’ granules of other bacteria
revealed in the same way. I have not found that there are any
granules in the spirilla which contain ‘ masked” iron, although
there is the possibility that spirilla, containing granules, were
not present in the preparations made with the glycerine and
sulphide mixture or with acid alcohol.
The diffusion of the ‘‘ masked” iron throughout the cyto-
plasm of Beggiatoa corresponds, on the whole, with what was
observed in the other bacteria, but the interpretation of
the results in the latter has an element of obscurity in it, It
is evident that the iron-holding compound is not, as a rule,
localised in granules or in special structures; and although
the distribution of this compound, in Beggiatoa alba at least,
corresponds with that of the substance which stains with
hematoxylin and other dyes, it is uncertain whether the two
compounds are identical. It is possible that experiments with
some of the larger forms, as, e. g., Beggiatoa mirabilis or
Crenothrix Kihniana, may result in determining a solu-
tion of the question. Unfortunately I had no opportunity of
studying the distribution of iron in such large forms.
I did, however, obtain a few preparations of a form which is
possibly allied to Crenothrix, and whose size (2°8—3:2 x
6'4—8 yw) rendered it favourable for such observations as I
had an opportunity of making, This organism grew on the
surface of some sewage water collected in the fall of 1898, in
which also myriads of examples of Euglena viridis throve.
It multiplied by fission. Some of them exhibited rounded
260 A. B. MACALLUM.
ends, while others had an oval shape, but the majority were
cylindrical with flat end-surfaces. Several cover-glass prepara-
tions of the organisms, fixed by heat and subsequently placed
in alcohol, were made, but no cultivations were attempted,
since before its value for the purpose of this investigation
was ascertained, the original culture fluid had been thrown
away.
One of the cover preparations was subjected to the prolonged
action of the glycerine and sulphide mixture, but, as sometimes
happened in other cases, no result was obtained. The other
two were placed in sulphuric acid alcohol for about eight hours
at a temperature of about 25° C., and then treated in the usual
way with the acid ferrocyanide mixture. One of the prepara-
tions was also stained with eosin, and both were, after being
washed in water, dehydrated with alcohol and mounted in
balsam. Examples of the organism exhibiting the Prussian
blue reaction and the eosin stain are represented in fig. 53.
The eosin reveals a large central body, sometimes of irregular
shape, and always lying free in a cavity in the markedly reticular
cytoplasm. The body in question contains no iron, but in other
respects resembles the large body present in Saccharomyces
Ludwigii. Theiron demonstrated appears to be ina granular
form distributed in the trabeculz of the cytoplasm, though
sometimes a very large granule, richly supplied with iron, was
found adjacent to, or in contact with, the large central body
destitute of iron.
As inorganic iron is a constituent of the sheath and other
parts in species of Crenothrix and allied forms (C. Kiihni-
ana, Leptothrix ochracea and Cladothrix dichotoma),
it is possible that all of the iron observed in the form described,
and whose relationship to Crenothrix has been suggested,
was not derived from a ‘‘ masked” compound. The amount
of inorganic iron must, however, have been very little, for, in
the cover preparations subjected to the prolonged action of
the glycerine and sulphide mixture, but a few of the forms
gave an immediate reaction for iron. The chief difficulty lies
in the fact that through the failure of the last-mentioned
IRON COMPOUNDS IN ANIMAL AND VEGETABLE CELLS. 261
method of liberating the “ masked ” iron in this organism, it is
uncertain whether the iron demonstrated after the use of
sulphuric acid alcohol had the distribution it obtained in the
living organism, or in the cytoplasm before it was treated
with acid alcohol. Apart from these matters it seems to me
quite certain that the results indicate the presence of iron in
a “masked ” form in this organism.
Cyanophycez.—These organisms, which are generally
regarded as closely related to bacteria, offer, on account of
their much larger size, fewer and less formidable difficulties
to an investigation of the morphological and micro-chemical
characters of their cells, and I have, therefore, endeavoured to
give a careful attention to the question of the presence of
assimilated iron in them. The determination of the relations
of the iron compounds in these organisms has entailed also an
investigation of the morphology of their cells, and I have, in
consequence, obtained a very large number of results, the
description of which is beyond the scope of the present paper.
These, and a fuller account of the literature of the subject, I
propose to detail on a future occasion, and I now deal with
the ascertained facts relating to the iron compounds and, in
so far as morphological characters are associated with these,
with the structure of the cells themselves.
The literature on the subject of the Cyanophycez has grown
considerably in the last ten years, but as it is only within the
last six that improved technical methods have been employed
in the investigation of their structure, a short sketch of the
more important publications, which have appeared in the latter
period, will suffice for present purposes.
Zacharias found that the cell is constituted of a coloured
peripheral part, and an uncoloured central portion of a reticu-
lated or granular structure. In the central portion he observed
two substances, one exhibiting the characters of a plastin, the
other, which he termed the “central substance” (Central-
substanz), varying in amount in the different cells, and re-
sembling nuclein in its chemical reactions. In the central
portion he found granules destitute of nuclein, and related in
262 A. B. MACALLUM.
many of their characters to the nucleoli of highly developed
vegetable organisms.! He considers that the central portion
differs very greatly from a nucleus, but whether it performs
the functions of the latter he is not prepared to say.
Biitschli,? whose observations on the structure of bacteria
have been already referred to, found one type of structure
prevail in both these and the Cyanophycee. ‘The cytoplasm
is, according to his view, formed of a faintly stainable peri-
pheral zone, and of a denser, deeply stainable central portion
which, in the living Cyanophycee, is always uncoloured. Both
parts are vesiculated. He found that hematoxylin colours
the cytoplasm blue, while it gives a red stain to granules
situated in the central portion, and, in the nodal points of the
vesiculated structures, more especially of those of the peripheral
zone. These disappear after subjecting the cells to the action
of artificial gastric juice, but he nevertheless regards them as
chromatin elements, and he looks upon the central portion
as a nucleus. Besides these granules, he found in certain
Oscillariz, in the extreme peripheral portions of the cell, and
especially adjacent to the transverse cell walls, others which
did not stain with hematoxylin, but which exhibited a strong
affinity for eosin.
Deinega® could formulate no conclusion in regard to the
presence or absence of a nucleus in these organisms, and also
in regard to the nature of the granules, although he is disposed
to regard the latter as formed of an isomer of starch. These,
of which he found but one species, stain specially with picro-
carmine, and dissolve in weak hydrochloric solutions (0°3 per
cent.).
Passing over the observations of Zukal,* who appears to
1 “Ueber die Zellen der Cyanophyceen,”’ ‘ Botanische Zeitung,’ 1890,
Nos. 1—5.
2 Op. cit.
3 « Der gegenwartige Zustand unserer Kenntnisse iiber den Zellinhalt der
Phycochromaceen,” ‘ Bulletin de la Soc. impér. des Naturalistes de Moscow,’
année 1891, p. 431.
4 “Ueber den Zellinhalt der Schizophyten,” ‘Sitzungsber. der K. Acad.
der Wiss. Wien,’ 1892, Math.-Nat. Classe, vol. ci, p. 301.
IRON COMPOUNDS IN ANIMAL AND VEGETABLE CELLS. 263
regard all the granules as nuclei, the next investigator of this
subject is Hieronymus,! who found in these cells a thin hya-
line membrane externally, a chromatophore, and a central
body consisting of a single much-wound fibril, comprehending
in its turns all the granules in the cell. The granules he looks
upon as crystals belonging to the regular system, and composed
of a substance ‘ cyanophycin,”’ which, though not identical
with nuclein in its reactions, he regards as related to the
chromatin and pyrenin of highly specialised vegetable cells.
The central body is, in his opinion, an “ open nucleus.”
According to Palla? the cells in the Cyanophycez consist of
a chromatophore with a vesiculated structure, of a central
homogeneous body, and of granules of different composition
always outside the latter. The central body is affected, like a
nucleus, by staining reagents. In preparations fixed with
corrosive sublimate and stained with Bohmer’s hematoxylin
the granules adjacent to, or in contact with, the central body
are stained reddish-violet, while those scattered in the chroma-
tophore are coloured blue. He finds that those which thus
become blue dissolve in dilute solutions of hydrochloric: acid
(0°3 per cent.) and do not stain intra vitam when treated
with solutions of methylene blue. The substance constituting
these, and which he calls ‘‘ cyanophycin,” he regards as the
first assimilation product of the activity of the chromatophore.
Those which stain reddish-violet with hematoxylin are com-
posed of a viscid substance, are not soluble in dilute acids, and
in the living cell manifest a strong affinity for methylene blue,
To such structures he has applied the name ‘‘ mucous sphe-
rules,” first given them by Schmitz. They correspond with
the granules which, in Bitschli’s preparations, stained red
with hematoxylin, but, in opposition to the views of that
observer, Palla regards it as extremely doubtful if they contain
any compound comparable to chromatin.
1 « Beitrage zur Morphologie und Biologie der Algen,” Cohn’s ‘ Beitrage
zur Biologie der Pflanzen,’ vol. v, 1893, p. 461.
2 “Beitrag zur Kenntniss des Baues des Cyanophyceen-Protoplasts,”’
Pringsheim’s ‘ Jahrbiicher fiir wiss. Bot.,’ 18938, vol, xxv, p. 511.
264 A. B. MACALLUM.
From all this it may be gathered that nuclei, in the strict
sense of the term, are not present in the cells of the Cyano-
phyceze, and that if any structure performs the functions of
such an organ, it must be the colourless central body. In
regard, however, to the composition, the position, and the
number of varieties of the granules, there is less of concord-
ance. All the observations quoted would appear to indicate
that a typical chromatin substance is absent. If a ‘‘ masked ”
iron compound is present in these organisms, with what part
of the cell is it associated ?
The forms which I used, in endeavouring to determine an
answer to this question, were: Oscillaria Froelichii, Oscil-
laria princeps, Oscillaria sp., Tolypothrix sp., Scy-
tonema sp., Microcoleus terrestris, Cylindrosper-
mum majus, Anabena (Spherozyga) oscillarioides,
and Nostoec commune. The fixative reagents used were
alcohol, corrosive sublimate, the stronger Flemming’s fluid, and
saturated solutions of picric acid ; while the staining fluids
employed were hematoxylin (Khrlich’s and Delafield’s), alum
cochineal, picro-carmine, safranin, and eosin. In determining
the presence of iron compounds, material hardened by alcohol
only was used.
The results of my experiments, so far as they affect the
question of the relations of iron to the cytoplasm of these cells,
may be summarised as follows:
1. The cytoplasm consists of a dense central portion and of
‘a vesiculated peripheral zone, the former staining with hema-
‘toxylin, alum cochineal, and safranin more deeply than the
latter when it is free from granules or vesicles, but when
vesicles are present they stain deeply, while the remainder
of the central portion acquires a faint colour only slightly
more marked than that of the peripheral portion. ‘The size of
these vesicles of the central portion varies from that, in which
they appear as scarcely larger than granules, to that observed
in Tolypothrix sp., in which they measured in diameter a
third of that of the cell. The stainable substance of these
forms a thick membrane enclosing an apparently inert sub-
-
IRON COMPOUNDS IN ANIMAL AND VEGETABLE CELLS. 265
stance, and when subjected to the action of artificial gastric
juice for two or three days it lessens slightly in volume, but
its presence is quite as readily demonstrable then as it was
previously. In this case the central portion of the cell also
diminishes in volume slightly, the diminution entailing a
shrinkage of the peripheral portion away from the original
limits of the cell. Digestion does not affect the capacity,
on the part of the central substance or of the membrane of
the vesicles referred to, of absorbing staining matters, but on
subsequently treating such preparations with a solution of
potassium hydrate of 0-1 per cent. strength for twenty-four
hours, the vesicles disappear and the central body, now some-
what swollen, has lost its capacity for fixing colouring matters
in itself. Evidently there is here a substance which has the
characters of nuclein. This is confirmed by the results of
experiments to determine the presence of ‘ masked” iron.
The central body always gives, with the glycerine and sul-
phide mixture, in an interval of from two or three days to
two weeks in length, depending apparently on the size of the
cell, a diffuse greenish reaction which is changed to light blue
on the addition of a drop of an acid ferrocyanide solution.
When granules and vesicles stainable with hematoxylin are
present, they also give a reaction for iron, but it does not
always manifest the same intensity. The iron in them is most
readily demonstrated after they have been treated with sul-
phuric acid alcohol (fig. 51).
2. In the peripheral portions of the cytoplasm, in well-
nourished forms only, are granules not so readily stainable
with hematoxylin, but which are intensely coloured with picro-
carmine. These are dissolved out of the fresh cell with dilute
hydrochloric ac id, and even in preparations thoroughly hard-
ened in alcohol they are but slightly less soluble in the same
reagent. In Oscillariz they are placed in a row at each end
of the cell and adjacent to the transverse walls, but in Micro-
coleus terrestris and Cylindrospermum majus they are
disposed in all the peripheral portions of the cytoplasm. In
the spores of the latter some of them appear as if embedded
”
266 A. B. MACALLUM.
in the central body. These are the ‘‘cyanophycin” granules
of Palla and such as Biitschli found in Oscillariz to be un-
affected by hematoxylin but markedly stained by eosin. They
may give a reaction for iron, but not always one of the same
intensity, for in Oscillariz it was very slight, and in one pre-
paration of Microcoleus terrestris none was obtained,
while in preparations from the same specimen of fresh material
made a few days later than the other, the reaction was quite
distinct. In two preparations of Scytonema sp. the granules
gave no reaction, aresult which I attribute to a deterioration
of the solution of the sulphide reagent then used. In Cylin-
drospermum majus these granules give an intense reaction
for iron (fig. 8). Theiron is not less firmly combined in the
substance of these granules than it is in the chromatin, for
in the last mentioned species the glycerine and sulphide mix-
ture brought out its complete reaction only after an applica-
tion of ten days or more. Within twenty-four hours after the
addition of the mixture, they gave, in all the species in which
they were iron-holding, a slight greenish reaction. I have
not succeeded in demonstrating the presence of iron in them
after the use of sulphuric acid alcohol, and the explanation
for this is that the latter reagent liberates, but at the same
time wholly extracts the iron in these granules, the substance
of which, unlike chromatin, is incapable of retaining it.
8. Beyond the fact that the “ cyanophycin” granules may
contain iron, there is nothing to show a relationship, chemical
or physiological, between them and the vesicles. From their
situation the “cyanophycin” granules would, as Palla sug-
gested, appear to be the assimilation product of the activity of
the chromatophore, while the chromatin vesicles and granules
might be regarded as due to processes of elaboration on the part
of the central body. In Cylindrospermum majus, which
grows on soft mud, the former are usually extremely abundant,
but in twenty-four hours after placing the thallus in water, the
granules diminish very much in number, and on the third day
they may be wholly absent in very many of the filaments.
Central vesicles, on the other hand, are in this form extremely
IRON COMPOUNDS IN ANIMAL AND VEGETABLE CELLS. 267
few in number, and the conditions which greatly influence the
number of the “cyanophycin” granules have apparently no
effect upon them. In Oscillaria Froelichii a filament may
contain large numbers of both elements, another may contain
“ eyanophycin” granules only, while a third may be free from
the latter but contain a large number of vesicles, and all in
the same preparation. In Cylindrospermum majus the
“cyanophycin” granules of the spore diminish somewhat in
number and volume during the formation of the episporium,
and in the spore which is undergoing its initial division their
number is very greatly reduced, the central body appearing at
the same time increased in volume.
4, In the heterocysts of Nostoc commune, Cylindro-
spermum majus, and Scytonemasp., picro-carmine demon-
strates the presence of “‘ cyanophycin”’ substance in a button-
shaped body at one or both ends of the cell, according as the
heterocyst is terminal or intercalary. A strand of ‘ cyano-
phycin” connects this body with the contents of the neigh-
bouring cell. In the heterocyst the ‘‘ cyanophycin” body is
quite unconnected with the homogeneous cytoplasm which occu-
pies the remainder of the cavity, and stains but faintly with
hematoxylin and not at all with picro-carmine. When sub-
jected to the prolonged action of the glycerine and sulphide
mixture the “cyanophycin,” both of the button and of the
strand, gave a deep reaction for iron, and a feebler reaction
was obtained in the cytoplasm (fig. 8).
It thus appears that in the Cyanophycee there is a substance,
containing ‘ masked ”’ iron, in many respects like the chromatin
of more highly organized cells, and that the ‘ cyanophycin,”
a compound of undetermined nature, may, in some forms at
least, also give evidence of the presence of the element in a
firmly combined condition.
1 This connection has already been described by Hansgirg, ‘ Physiologische
und Alyologische Studien,’ Prague, 1887, pp. 125, 126. The description is
quoted in full by Deinega.
268 A. B. MACALLUM.
GENERAL REMARKS.
The facts described in the preceding pages appear to indi-
cate that a substance, in which iron is firmly held, is a constant
constituent of the nucleus, animal and vegetable, of the cyto-
plasm of non-nucleated organisms and those possessed of
apparently rudimentary nuclei, and that, further, a similar
iron-containing substance obtains in the cytoplasm of ferment-
forming cells. This substance, to which cytologists apply the
term chromatin, cannot, on theoretical grounds, be regarded as
constant in its molecular structure, even in the same organism,
and its most marked characteristic, apart from the iron in its com-
position, is the occurrence in it of nuclein or nucleinic acid.
Beyond the fact that the iron is firmly held, it is difficult
to say how it is disposed in the molecular structure of the
nuclein or nucleinic acid. It is, possibly, united directly to
the carbon of the latter. The acid alcohols liberate it as a
ferric salt, but this fact cannot be held to indicate that it is
combined in the nuclein or nucleinic acid in a ferric state,
since from solutions of potassium ferrocyanide, in which the
iron is contained in a ferrous state, acids liberate the iron in a
ferric condition,! as evidenced by the formation of ferric ferro-
cyanide or Prussian blue.
It is also difficult to say whether there is, in the way in
which the iron is held in the animal cell, anything different
from that obtaining in the vegetable organism. I have, as
a rule, found it easier, in the case of the vegetable cell than in
that of the animal cell, to liberate the iron with ammonium
hydrogen sulphide; but upon this no conclusion may be
founded, since the same reagent liberates the iron of free
hematin readily, while it does not affect the iron of hematin
in hemoglobin, and it is possible that in the animal cell the
1 The iron immediately on liberation may be in the ferrous state, but it
quickly assumes the ferric form. Similarly, the iron liberated in the chro-
matin may at first be a ferrous compound which, with the continued action of
the liberating reagent and under the conditions obtaining in the hardened
tissues, may further undergo a conversion into a ferric salt.
IRON COMPOUNDS IN ANIMAL AND VEGETABLE CELLS. 269
proteid molecules attached to the iron-containing nuclein or
nucleinic acid may more greatly affect the activity of the
reagent than those of the vegetable cell are capable of doing.
Since, on the other hand, hemoglobin, which, as I have
pointed out, is derived, in Amblystoma, from chromatin,
occurs in a large number of animal forms, but is present
in no vegetable organism, it would appear to follow that
the iron is combined in animal chromatin in a way unlike
that in which it is held in the vegetable cell.!
The apparently universal occurrence of such iron com-
pounds renders intelligible the fate of the iron salts absorbed
by plants from the soil, and of the iron compounds found
by Raulin®? and Molisch? to be necessary for the growth of
Aspergillus niger. Chromatin, to the formation of which
the iron absorbed contributes, is, as the results of cytological
investigations show, a substance of primary importance to the
cell, and a diminution in, or a cessation of, the supply of
iron to the vegetable organism, which produces the condition
known as chlorosis, instead of affecting only the formation of
its chlorophyll, as generally supposed, strikes at its very life.
The conditions known as anemia and chlorosis in the
higher Vertebrates have been hitherto explained as caused
by a diminished production of hzmoglobin directly from
organic or inorganic iron compounds absorbed by the intes-
tine from the food matters; but they must now be referred
to a deficient supply of the primary iron-containing com-
1 Compounds which appear to resemble, somewhat remotely, the hematins
of animal organisms have been found in Palmella cruenta (Phipson, “Sur
la matiére colorante du Palmella cruenta,”’ ‘Comptes Rendus,’ vol.
Ixxxix, p. 316, 1879), and in Aspergillus niger (Linossier, “Sur une
hématine végétale ; l’aspergilline, pigment des spores de l’Aspergillus
niger,” ‘Comptes Rendus,’ vol. exii, p. 489, 1891). The colouring matter
of the latter is, as I have found, held in the membrane, but not in crypto-
plasm of the spore, and it would, therefore, appear to be simply a degeneration
product.
2 « Btudes chimiques sur la végétation,” ‘ Annales des Sc. Nat.,’ Bot., Série
5, vol. xi, 1869, p. 93.
3 Op. cit., pp. 97—117.
270 A. B. MACALLUM.
pound, chromatin, not only in the hematoblasts, but in all the
cells of the body. The consequently lessened proliferation of
cell and tissue would explain the hypoplasia of the imper-
fectly developed vascular system observed by Virchow! in
chlorotic human subjects.
Accepting this explanation of the nature of chlorosis, one may
infer that this condition is not limited to animal organisms
in which hemoglobin is found, although its occurrence in
others may be difficult to detect because of the total absence
of this pigment. From this point of view animal chlorosis
is fundamentally similar to the chlorosis of the vegetable
kingdom.
The oxygen-carrying property of hemoglobin and of hema-
tin is generally attributed to the iron present in these, because
when hematin is deprived of its iron, the resulting compound,
whether hematoporphyrin or bilirubin, manifests no affinity
for oxygen. The proof may not be quite conclusive, for we
cannot be certain that either compound represents the un-
changed remainder of the hzmatin less its iron, but assuming
that it is correct, it follows, as I have pointed out in my pre-
vious communication, that the antecedent of hemoglobin,
chromatin, has the capacity of absorbing and retaining oxygen,
and that one may attribute the processes grouped under the
term “ vital,” to an alternation of the conditions of oxidation
and reduction in the iron-holding nuclear constituent. This
hypothesis, reasonable as it now appears to me to be, I do
not regard as free from difficulties, since in vegetable cells the
two processes of respiration and assimilation, involving two
activities of different natures, so far as the oxygen is concerned,
appear to postulate the existence of two different iron com-
pounds in the same nucleus.” There are no facts to indicate
1 “ Ueber die Chlorose und die damit zusammenhingenden Anomalien im
Gefassapparate, insbesondere tiber Endocarditis puerperalis,”’ ‘ Vortrag.,’
Berlin, 1872.
? On the relations of the vegetable nucleus to the processes of assimilation,
see Strasburger, ‘Ueber Kern- und Zelltheilung im Pflanzenreiche,’ 1888,
pp. 194—204.
IRON COMPOUNDS IN ANIMAL AND VEGETABLE CELLS. 271
the occurrence of such, and it is scarcely possible to explain
away the objection without advancing some hypotheses regard-
ing the action of the sulphur and the phosphorus in the
nuclein. I propose to detail these on another occasion.
EXPLANATION OF PLATES 10—12,
Illustrating Dr. A. B. Macallum’s paper “On the Distribution
of Assimilated Iron Compounds, other than Hemoglobin
and Hematins, in Animal and Vegetable Cells,”
EXPLANATION OF FIGURES.
Note.—In the preparation of all the figures Abbé’s camera lucida was em-
ployed when the size of the objects represented permitted its use, and all
except 25, 26, 35, and 36 are illustrated as they were seen with an apocbro-
matic immersion objective (Zeiss 8 mm., 2 mm., or 15 mm.). The excep-
tions are represented as they appeared under a Zeiss D. Figs. 1—40 show
the distribution of assimilated iron as it was demonstrated by the dark green
colour of ferrous sulphide, but in Figs. 41—53 the disposition of iron com-
pounds of this kind is indicated by the colour of the Prussian blue reaction.
Fic. 1.—A nucleus and a cell from the testicle of Necturus lateralis.
Alcohol, the glycerine and sulphide mixture eleven days. x 620. This and
the two succeeding illustrations were drawn from the very first preparations
made with this reagent.
Fie. 2.—Testicular elements of another example of N. lateralis. Alcohol,
the glycerine and sulphide mixture eleven days. x 620.
Fic. 3.—a, a leucocyte, 4, a red corpuscle, of N. lateralis. Alcohol, the
elycerine and sulphide mixture six days. x 500.
Fie. 4.—Two yeast-cells, Saccharomyces cerevisiz. Alcohol, the
glycerine and sulphide mixture ten days. x 1500,
Fic. 5.—Four yeast-cells, Saccharomyces Ludwigii. Alcohol, the
glycerine and sulphide mixture four days. x 1640.
Fie. 6.—The developing and fully-formed spores of Cystopus candidus.
a, b, c, d, e, alcohol, sulphuric acid alcohol two days, ammonium hydrogen
sulphide in glycerine. x 750. /, alcohol, the glycerine and sulphide mixture
ten days. x 680.
VOL. 388, PART 2.—NEW SER. s
O72 A. B. MACALLUM.
Fic. 7.—Spores of Aspergillus glaucus, a, in the unripe, 4, in the ripe
condition. Alcohol, the glycerine and sulphide mixture three days. x 1640.
Fic. 8.—Cells, heterocyst (4.), and spore (sp.) of Cylindrospermum
majus. Alcohol, the glycerine and sulphide mixture fourteen days. x 1640.
Fic. 9.—Three cells of a filament of Microcoleus terrestris. Alcohol,
the glycerine and sulphide mixture four days. x 2000.
Fic. 10.—a. Spores (immature), 4, c, and d, basidia of a leucosporous
Hymenomycete. d. Basidium with sterigmata and one attached spore.
Sterile (?) element in &. Alcohol, the glycerine and sulphide mixture eight
days. xX 820.
Fies. 11—13.—Portions of hyphe of Hyphelia terrestris Fries,
illustrating the development of the fructification, 13 a and representing the
simplest form. Alcohol, the glycerine and sulphide mixture three days.
x 820.
Fries. 14—18.—From the ovary of a specimen of Erythronium ameri-
canum hardened in alcohol. Fig. 14 illustrates the effect produced by diam-
monium sulphide and glycerine in two days; Figs. 15, 17, and 18 represent
that produced by ammonium hydrogen sulphide and glycerine in the same
time; and in Fig. 16 is shown how intense the reaction appeared after treat-
ment for four days with the same reagent. x 1240.
Fies. 19—22.—From the ovary of a specimen of Erythronium ameri-
canum hardened in alcohol. Sections treated for thirty hours with sulphuric
acid alcohol, and mounted in a mixture of glycerine and ammonium hydrogen
sulphide. xX 1240.
Fic. 23.—Four hepatic cells from a specimen of Necturus lateralis.
Alcohol, the glycerine and sulphide mixture eight days. x 620.
Fic. 24.—Two hepatic cells from the same animal, illustrating the distri-
bution of the iron and the nuclear structure after they were treated with sul-
phuric acid alcohol for twenty-four hours, and mounted in a mixture of
elycerine and ammonium hydrogen sulphide. x 620.
Fic. 25.—An example of Stentor polymorphus. Alcohol, the glycerine
and sulphide mixture two weeks. X 305.
Fic. 26.—An example of Stentor polymorphus. Alcohol, Bunge’s
fluid thirty-seven hours, ammonium hydrogen sulphide and glycerine. x 305.
Fic. 27.—Examples of Vorticella sp. Alcohol, the glycerine and
sulphide mixture seven days. x 600.
Fic. 28.—An example of Epistylis sp. Alcohol, Bunge’s fluid twenty-
four hours, glycerine and ammonium hydrogen sulphide. x 600.
Fic. 29.—An ovum of Ascaris mystax, fixed during impregnation. Only
a portion of the ovum is represented. Alcohol, the glycerine and sulphide
mixture eight days. x 820.
IRON COMPOUNDS IN ANIMAL AND VEGETABLE CELLS. 273
Fie. 30.—An impregnated ovum of Ascaris mystax, showing the division
of its nucleus (~.) and the condition of the spermatozoid (sp.). Alcohol, the
glycerine and sulphide mixture ten days. x 750.
Fies. 31 and 32.—Spermatozoids of Ascaris mystax. Alcohol, the
glycerine and sulphide mixture nine days. 31, x 820; 32, x 1640.
Fics. 88—36.—Ovarian ova of the lake-lizard, Necturus lateralis,
illustrating differences in the distribution of the “masked” iron. In 35 is
shown the iron-containing peripheral nucleoli, and @ represents a more highly
magnified (= x 1240) portion of the nuclear structure. In 386 is seen an
earlier stage with a, a portion of its nuclear network, more highly magnified
(x 1240). Alcohol, sulphuric acid alcohol thirty-six hours, glycerine and
ammonium hydrogen sulphide. x 305.
Fic. 37.—Retinal rods and cones from a larva of Amblystoma. Alcohol,
whole of retina in Bunge’s fluid two days, glycerine and ammonium hydrogen
sulphide. x 620.
Fic. 88.—Cells from the pancreas of a larva of Amblystoma. Alcohol,
Bunge’s fluid (on the whole of the organ) two days, glycerine and ammonium
hydrogen sulphide. x 620.
Fig. 39.—A portion of a section of the human epidermis, illustrating the
occurrence of “‘masked”’ (?) iron in the granules (eleidin) of the stratum
granulosum and in the stratum lucidum. Alcohol, sulphuric acid alcohol two
days, glycerine and ammonium hydrogen sulphide. x 620.
Fic. 40.—Strands of fibrils from the muscle fibre of a larva of Ambly-
stoma. Alcohol, sulphuric acid alcohol two days, glycerine and ammonium
hydrogen sulphide. x 750.
Figs. 41 @ and 6.—From the ovary of a specimen of Erythronium ame-
ricanum; 4 represents an isolated nucleus. Alcohol, sulphuric acid alcohol
thirty hours, acid ferrocyanide mixture, balsam. x 1240.
Fie. 42.—A cell from a section of the ovary of the same specimen, with the
iron demonstrated as in last case, but the preparation, before being mounted
in balsam, was stained with eosin. x 1240.
Fies. 43 and 44.@ and 4.—Nuclei of the embryo sac of a specimen of E.
americanum. Alcohol, sulphuric acid alcohol thirty-six hours, acid ferro-
cyanide mixture, balsam. x 620.
Fies. 45 a and 6.—Nuclei from the liver of a specimen of Necturus
lateralis. z. Nucleoli. Alcohol, sulphuric acid alcohol thirty-six hours,
acid ferrocyanide mixture, balsam. x 1240.
Fics. 46 a—d.—Hepatic nuclei treated as in foregoing case, also stained
with safranin to illustrate the differences between the chromatin network and
the nucleoli in regard to the effect of this reagent. x 1240,
Fic. 47.—Two hepatic nuclei treated as in the preparation illustrated by
274 A. B. MACALLUM.
Fig. 45, but also stained with eosin, which deeply colours the nucleoli and
non-iron-containing constituents (see text). x 1640.
Fie. 48.—Nuclei of the epithelial cells of the intestinal mucosa of Nec-
turus lateralis. Alcohol, sulphuric acid alcohol thirty-six hours, acid ferro-
cyanide mixture, balsam. The preparation from which 4 was drawn was, before
being mounted in balsam, stained with safranin. x 1240.
Figs. 49a and 4.—Two examples of Euglena viridis. Alcohol, sul-
phuric acid alcohol thirty-six hours, acid ferrocyanide mixture, balsam,
x 820.
Fie, 50.—A portion of a nuclear filament from the “salivary” gland of a
larva of Chironomus. Alcohol, sulphuric acid alcohol thirty-six hours,
acid ferrocyanide mixture, balsam. x 1640.
Fics. 51a, 6, ce, d.—Cells and portions of filaments of Oscillaria
Froelichii; 4 and d represent the isolated cells as seen through their trans-
verse walls. The “‘cyanophycin”’ granules are coloured red. Alcohol, sul-
phuric acid alcohol three hours, acid ferrocyanide mixture, picro-carmine,
balsam. xX 1640.
Fie. 52.—A portion of a filament of Microcoleus terrestris. Alcohol,
sulphuric acid alcohol three hours, acid ferrocyanide mixture, balsam. x
1500.
Fic. 53.—Examples of an organism obtained from sewage water (see text).
Alcohol, sulphuric acid alcohol eight hours, acid ferrocyanide mixture, balsam.
x 2000.
CHANGES IN REPRODUCTIVE CELLS OF ELASMOBRANCHS, 275
On the Structural Changes in the Reproductive
Cells during the Spermatogenesis of Elasmo-
branchs.
(From the Huxley Research Laboratory, R. Coll. Sci. Lond.)
By
J. E. Ss. Moore, A.R.C.S.
With Plates 13—16, and figs. in text.
“ Plagiostomorum spermatosomata, que magnitudine et peculiari quadam
forma in primis ad evolutionis studium apta sunt, hac ratione iam plurium
observationes in se contraxerunt.”—LAVALETTE St. GEORGE.
1. Durine the development of a metazoan embryo, after the
differentiation of the generative cells from those of the general
somatic “anlage,” the reproductive elements pursue a course
of evolution peculiar tothemselves. Instead of attaining to the
high specialisation, decay, and final dissolution, characteristic
of somatic tissues, their variation is of much less amplitude,
but cyclical, and returns at length to the production of elements
similar to those in which both series started.
In animals, the course of such a reproductive cycle appears at
first sight to be differentiated into two distinct periods of activity,
the one extending from the earliest embryonic development of
the generative elements to the commencement of the proper
spermato- or ovo-genesis, the other beginning with the spermato-
or ovo-genesis and ending with the formation of the mature
reproductive cells.
In reality, however, the transition from the first of these
periods to the second is much more expressive of changes
276 J. E. S. MOORE.
incident to the surrounding parts, than any alteration in the
structure of the generative cells themselves.
In fact, no definite change occurs in them till late in the
second period, when ,the advent of the so-called “ reduction
process” produces a sharp and definite alteration in the
morphological value of all the elements affected. So character-
istic is this change, that the time of its commencement can be
used as a point of reckoning in all those reproductive cycles in
which it is apparent.
For the sake of clearness, that part of the reproductive cycle
in Elasmobranchs which comes before the proper spermato- or
ovo-genesis will be called the primary or embryonic period,
while the cellular generations of spermato- or ovo-genesis before
and after the numerical reduction of the chromosomes will be
distinguished as those of the first and second spermato-
or ovo-genetic series.
It is my purpose in the present paper to give a detailed ex-
position of the changes witnessed in the generative cells during
the reproductive cycle of Elasmobranchs, after the completion
of the embryonic period in the male, i.e. during the proper
spermatogenesis in the fish, and then to draw such conclusions
as may seem legitimate from a comparison with other forms.
I. Technical.
2. The materials with which the present investigation was
carried out were obtained while I occupied the British Asso-
ciation Table in the Naples Laboratory during the months
of October, 1893,to July of the following year. They consist of a
large number of Elasmobranch testes, including those of
Scyllium canicula, Scyllium catulus, Pristiurus,
Torpedo, Raja macrorhynchus, and Raja maculata,
The testes were cut up into small pieces about the size of half
a cubic centimetre, and fixed in various ways. I obtained the
most successful preparations after the use of Flemming’s strong
solution, Hermann’s fluid, osmic acid in various strengths, and
corrosive sublimate, both with and without acetic acid. Valu-
able comparative material was also obtained by treating the
CHANGES IN REPRODUCTIVE CELLS OF ELASMOBRANCHS. Pata
testes with glacial acetic acid,and washing quickly in water, by
tearing up the fresh material in acetic carmine, by fixing in
a 2 or 8 per cent. solution of formic aldehyde, by the use of
Carnoy’s fluid, and last but not least, by a formic acid method
which I hit on quite by accident. This consists in placing
small fragments of the living testis in a 50 per cent. solution
of formic acid for a few seconds, and then transferring directly
to 50 per cent. alcohol, after which they are treated for
sectioning in paraffin, in the usual way. By this means the
chromosomes were in some cases rendered admirably distinct,
but the fixation, so far as I have yet tried it, renders the
material difficult to stain.
Although the cells of these different kinds of fish examined
necessarily differ somewhat inter se, the differences do not
appear to be of any morphological importance, and the following
description, although taken more especially from those of
Scyllium canicula, is, so far as I am aware, applicable to
them all.
II. The Resting Cells in the First Spermatogenetic
Period.
8. The successive generations of the smaller and smaller
cells which gradually fill up the testicular ampulle during the
first spermatogenetic period are all alike, and I find no essential
difference between their structure and that of the single em-
bryonic elements (fig. 1) budded off into the stroma from which
they gradually arise.
The chromatin is dispersed as a coarse reticulum throughout
the interior of the nucleus, and is not in the least specially
related to its surface; in fact it is really denser towards the
interior than at the periphery, and contains a large oval or
flask-shaped nucleolus, generally attached to the nuclear wall .
(figs. 2, 11, 12, n.).
The chromatic threadwork itself is composed of innumerable
staining granules, embedded in a scaffolding of clearer sub-
stance (linin) (fig. 12), and the unoccupied nuclear space is
filled up with a thin nuclear sap.
278 J. E. S. MOORE.
4. The cytoplasm presents the usual fine reticulation in its
substance (figs. 1, 11), and the nucleus is placed excentrically
within its mass, so that there is more cell body on the one side
than on the other.
The whole reticulation of the cytoplasm is disposed radially
towards a point just outside that part, of the nuclear wall which
faces the larger mass of the cytoplasm (fig. 2, 7.), and the point
itself is occupied by two small centrosomes (c.), which can be
stained bright red by treatment with fuchsin and orange G.
There is hardly any archoplasmic substance round the cen-
trosomes, and they, together with their cytoplasmic radiation,
which extends quite out to the periphery of the cell,’ constitute
a good example of what I have elsewhere called? a simple
sphere.
There are one or two small chromatic bodies in the cyto-
plasm (fig. 2, b. c.).
Ill. The Divisions of the Cells of the First Spermato-
genetic Period.
5. Just as the successive generations of resting cells in the
first spermatogenetic period are all alike, so also are the
divisional metamorphoses by which they are produced.
At the commencement of mitosis the nuclei become swollen,
their smooth round contours appearing as if turgid with an
excess of intra-nuclear sap (fig. 13), and at the same time the
chromatic framework shortens up into a lesser number of
stouter threads. But I have not seen any indication of early
1 Dr. Heidenhain, in his paper published in the ‘Arch. fiir Mikr, Anat.,’
Bd. xliii, p. 496, 1894, is anxious to claim priority over me in the discovery of
radii extending from the sphere in leucocytes to the periphery of the cell. The
words I used in a former paper were these : “a delicate radiation spreads from
the whole sphere to the periphery of the cell” (‘Quart. Journ. Mikr. Anat.
Sci.,’ vol. xxxiv, p. 188); and my meaning would have been equally well ex-
pressed had I used the word towards” instead of “to” the periphery. I have
therefore no claim at all in the matter, and it seems to me to have about as
much importance as the conclusions which Dr, Heidenhain has drawn from it.
2 “On the Morphological Value of the Attraction-sphere,” ‘Science Pro-
gress,’ vol. ii, No. 10.
CHANGES IN REPRODUCTIVE CELLS OF ELASMOBRANCHS, 279
splitting in this threadwork or the granules which com-
pose it, like that given by Brauer! in the corresponding stage
of the spermatogenesis of Ascaris. The individual granules
(microsomes) become completely massed together into axial
cores, from which delicate filamentous radii of linin (figs. 12,
13, /.) spread in all directions, and the continuation of this me-
tamorphosis in the chromatin results eventually in the for-
mation of one or two long chromatin threads coiled round the
inner surface of the nuclear membrane (figs. 14, 15), the nu-
cleolus lying a little more within.
These threads, as soon as they are found, break up appa-
rently simultaneously into twenty-four bent rods, which form
the twenty-four chromatic elements characteristic of the
divisions of the first spermatogenetic period ; and at the same
time the nucleolus, becoming smaller and smaller, breaks up
and disappears.
6. Concomitantly with the above intra-nuclear changes, the
centrosomes, originally occupying the focus of the cytoplasmic
radiations, separate from one another and pass successively
through the positions represented in ¢., figs. 11, 12, 13, 14.
Owing to the absence of the archoplasmic constituent of the
sphere, there is in these cells no real archoplasmic spindle
formation (as in the spermatocytes of Salamandra described
by Hermann’). Each centrosome, with its crown of radiations,
simply travels away from the other, until, at the period of the
nuclear evolution reached in the last paragraph, they lie on
opposite sides of the nucleus, with almost the whole diameter
of the cell between them (cf. c., figs. 12, 13, 14).
7. As soon as the cytoplasmic conditions just described have
been attained, the nuclear wall becomes irregular and dis-
appears, while the chromosomes, collecting under the con-
traction of their connecting linin filaments, form a long oval
mass stretched across the nuclear sap between the centrosomes
(figs. 15, 16, 17).
1 «Zur Kenntniss der Spermatogenese von Ascaris megalocephala,”
‘Arch. fiir mikr. Anat.,’ Bd. xlii, pp. 153—208, figs. 3—5, Taf. xi.
9°
2 «Arch. fiir. mikr. Anat.,’? Bd. xxxvii.
280 Jc Hs 8; MOORE:
At first the chromosomes are attached to the centrosomes by
a few faint protoplasmic strands, which are apparently of
cytoplasmic origin ; but as time goes on the chromatin assumes
a more and more equatorial position, and the linin filaments,
being left stretched towards the centrosomes, help to form the
central portion of an achromatic spindle figure, the equatorial
moiety of which is nuclear, while its extreme ends appear to
be cytoplasmic (figs. 17, 18, 19).
8. A portion of the astral radiation round the centrosomes
becomes connected with the outer ends of the chromatic rods,
clothing the inner achromatic spindle with a sheath of cyto-
plasmic fibres (m., fig. 19), structurally equivalent to Her-
mann’s *‘ outer mantle.”
9. The chromosomes to which these fibres are attached
assume the form of short bent rods, and lie (fig. 19) at all
angles on the equatorial plane, being by no means specially
related to the surface of the spindle figure, and in surface view
they consequently present the appearance of a somewhat
irregular chromatic disc (fig. 21).
10. The achromatic spindle would thus appear to have a
dual origin, its superficial portion and extreme ends originating
in the cytoplasm, while its greater internal and equatorial
mass arises from the nucleus—a state of things approximately
coinciding with Flemming’s! views respecting its complex
origin in Amphibia, as opposed to the general acceptation of
its wholly cytoplasmic nature among plants.
11. When the equatorial plate is fully formed, the chromo-
somes, after becoming extremely broad and flat, split longi-
tudinally down the middle, each into two daughter-threads,
which gradually separate from one another towards the
spindle-poles (figs. 19, 20, 22, 23). During their transit
those daughter-elements, which were at first internal, work
outwards to the surface of the spindle in such a way that by
the time they are halfway from the equator to the poles, the
chromosomes of each daughter-nucleus have assumed the
1 “Neue Beitrage zur Kenntniss der Zelle,” ii Theil., ‘Arch. fiir mikr,
Anat.,’ Bd, xxix, p. 389.
CHANGES IN REPRODUCTIVE CELLS OF ELASMOBRANCHS. 281
well-known open ring-form of the diastral figure represented
in fig. 24.
12. In consequence of this outward motion of the inner
chromosomes, the spindle (now intra-zonal) fibres with which
they are connected become drawn out from their original
axial position, and form a central fibrous tube (fig. 24, 7.8.)
enclosed by (i) the remains of the nuclear sap, (ii) the external
spindle (intra-zonal) fibres (0. s.), and (iii) some linin fila-
ments left stretched between the separate chromosomes, which
last, in my opinion, represent the true! “ Verbindungsfaden.”
The “ Verbindungsfaden”? and the intra-zonal fibres now
become indistinguishably fused, while the differentiation of
the achromatic spindle into an inner and an outer sheath,
becoming more distinct, gives to that structure the appear-
ance of two concentric tubes, one of which (0. s., fig. 24) is
stretched directly between the outer edge of the chromatic
rings, the other (@. s., fig. 24), passing internally through
them, to its termination in the centrosomes. The unstained
fluid which separates the outer from the inner of these
sheaths is that which previously filled the interspaces- be-
tween the younger spindle-fibres, and it was once the
parental nuclear sap. It contains a few irregular chromatic
particles (fig. 24, b. c.), which appear to have been left as
débris of the previous chromosome formation, and which
sooner or later pass (fig. 26, 6. ¢.) into the cytoplasm of
the cell.
While the above changes are in progress the centrosomes
become gradually surrounded by a dusky zone (figs. 20, 26, 27),
which is caused by the shortening up and coalescence of the
cytoplasmic fibres between them and the chromosomes, 7. e. by
those described above (§ 8) as structurally equivalent to
Hermann’s mantle.
13. The chromosomes in the two daughter-rings (fig. 24)
are at first quite distinct from one another, although lying
closely side by side, but as time goes on they fuse together,
1 See Ishikawa, “Studies of Reproductive Elements; Noctiluca,” ‘Journ.
Sci. Col. Imp. Univers. Tokio,’ vol. vi, p. 322.
282 J. HS. MOORE:
until the chromatin eventually forms two solid chromatic rings,
one in each daughter-cell (figs. 25, 26).
14. About this time the outer spindle-fibres begin to
spread so widely in the equatorial plane (fig. 24) that they
actually come in contact with the membrane of the cell, and at
each of these rather angular connections there appear slight
thickenings of the fibres (07.), which stain, and thus constitute
an interesting stepping-stone between the true cell-plate and
Flemming’s! intermediate bodies.
The chromatic rings now gradually lose their original con-
nection with the outer spindle-fibres, which begin to bulge out,
and pass round them to the poles (fig. 27’) ; the chromatic rings
are thus left in a surrounding vacuole, but the core of fibres
(0.5s.,%. s., fig. 27 a) still passes through them to the poles. The
bulging out of the spindle-fibres round the nucleus increases,
and is accompanied by a corresponding collapse of the same
in the division plane of the daughter-cells (figs. 25, 26, 28).
This brings the outer and the inner spindle-tube together in the
division plane (figs. 26, 27, 28), and the whole spindle figure at
last acquires the appearance of a sharply differentiated fusiform
body between the daughter-cells (fig. 27 a). The terminal por-
tions of this body (the remains of the spindle-fibres) as they pass
round the daughter-nuclei (fig. 27 a, n.g.), are at first distinctly
seen, but they become shortly indistinguishable from the sur-
face of the vacuole and are consequently lost; but the conical
extremities of the fusiform equatorial portion which remains are
still prolonged as delicate protoplasmic filaments, which extend
towards the nuclei in each daughter-cell (figs. 29, 80). These
thread-like prolongations are the remains of that inner core of
spindle-fibres above described (§ 12) as passing through the
daughter-rings. In apolar view (fig. 27, /.) they perforate the
daughter-nuclei very much to one side, and the little orifice (0.)
is all that is left of the originally wide passage through the
chromatin.
15. The closing up of the chromatic rings commences on
the equatorial side, and is produced by the formation of a
1 Flemming, loc. cit.
CHANGES IN REPRODUCTIVE CELLS OF ELASMOBRANCHS. 283
thin chromatic floor. In consequence of this, the spheres
(fig. 27, a. b.) appear to occupy the hollow of a little nuclear
cup, and by the continuance of this filling-up process the
remnant of the inner spindle-core is pushed gradually to one
side, and eventually out of the nucleus altogether, but it con-
tinues to pass round the nucleus (in a more or less deep furrow)
(fig. 27 a, n.g.) towards the spheres.
16. The course of the terminal spindle-filaments becomes
generally coincident with the surface of the vacuole about
each nucleus, and they consequently take a curved course from
each end of the fusiform remains of the original spindle-figure
(between the cells) to the spheres in the polar faces of the
nuclei. So that the whole arrangement. of the daughter-nuclei
and spindle-fibres at this time bears (see fig. 27 a) a curious
resemblance to the figures seen in the divisions of the micro-
nucleus! during the conjugation of many infusoria.
17. The chromatin in the daughter-nuclei now blows up
once more into a foam, and eventually completely fills the
vacuoles originally surrounding them (figs. 28, 29, 30) while a
nucleolus appears in the reticulum of each, generally at the
base of the shallow depression (mg., fig. 29), which persists as
the remains of the nuclear cup described above (§ 15). This
depression, together with the spheres, is gradually rotated
somewhat to the equatorial side (as in fig. 29), and the chromatic
granules existing in the cytoplasm, becoming fewer in number
and larger in size, assume the characters of the chromatic
bodies described by Hermann in the spermatogenesis of Mam-
malia (fig. 30, 0. ¢.).
18. The cells are now practically at rest once more, but
the fusiform spindle remnant, with its equatorial band of
intermediate bodies (fig. 30, 6. 7.), continues long after the
daughter-cells have come to rest, and eventually degenerates
and vanishes in the equatorial plane.
Mitoses of the above description are carried out with hardly
any variation in their details, through all the cellular divisions
' See Maupas, “‘ Le Rajeunissement Karyogamique chez les Ciliés,” ‘Arch,
de Zool.,’ exp. tm. vii, 1889 (pl. ix, figs. 14—20).
284 J. E. S. MOORE.
of the first spermatogenetic period, and in the course of this
the features which appear to be of primary comparative im-
portance may be summarised as follows :
I. The existence in the resting cells of a large round nucle-
olus lying near the nuclear periphery.
II. The evolution during the prophasis of division, of twenty-
four bent chromosomes, which shorten up, and split longitu-
dinally in half to form the same number of chromosomes,
twenty-four, in each daughter-cell.
III. The existence of an extra-nuclear attraction sphere,
which, during this period of the spermatogenesis, is practically
destitute of archoplasm, being surrounded by a simple cyto-
plasmic radiation like that observed in many forms of tissue
cells.
IV. The consequent non-formation of an archoplasmic spindle
figure, and the dual origin of this latter structure, partly from
the simple cytoplasmic radiation, partly from the intra-nuclear
substance.
V. The differentiation of the spindle during the dyastral
figure into an outer and an inner fibrous sheath, which, after
the escape of the parental nuclear sap, collapse and coalesce,
forming a delicate connecting thread between the attraction
spheres of both daughter-cells.
VI. The formation of extra-nuclear chromatic bodies from
the débris of the nuclear chromatin.
IV. The Rest of Transformation (Synaptic Phase)
between the First and Second Spermatogenetic
Periods.
19. As I have already pointed out, the transition from the
first into the second spermatogenetic period is completed in
the cells during the rest which follows the last division of the
first, and when the elements in the ampullee are seven or eight
rows deep (fig. 34). Such cells, although at first retaining the
characteristics of those of previous generations, gradually acquire
new ones, but so gradually that itis some time before we realise
the profound nature of the changes wrought, and that, while yet
CHANGES IN REPRODUCTIVE CELLS OF ELASMOBRANGHS. 285
apparently at rest, the cells have passed completely over from
the first into the second spermatogenetic period. The com-
mencement of this metamorphosis is marked by an increasing
fineness of the reticulum in the nuclei, which continues to
increase until cells with nuclear elements like that represented
in fig. 835 are seen, and about the same time there appears a
curious secondary nucleolus surrounded by a vacuole (fig. 31 n’),
which, so far as I can ascertain, is in these fishes diagnostic of
the change. After a while the nuclear threadwork again
grows coarser and thicker, displaying at the same time a
peculiar tendency to contract to one side of the nucleus, leaving
a great clear space (fig. 39) across which stretch numerous
linin filaments. The contraction is not so marked when the
cells have been preserved with osmic acid, nor on the outside
of sections which have been preserved with Flemming’s fluid,
where the osmium has acted directly upon the cells. I have,
however, seen it in elements of Torpedo which were simply
immersed in dilute glycerine ; and whether it exists in nature
or not, the cells display at this period, and at no other, a
remarkable tendency to have their chromatin contracted, in
consequence of some internal change which renders these
nuclear figures diagnostic of the particular period in question.
Similar figures have been obtained at corresponding periods in
the spermatogenesis of Amphibia, Mammals, Nematodes,!
and various Arthropods,’ and I do not think it probable that
the contraction in many of these cases has anything to do with
the reagents used.
20. In the cytoplasm the conversion from the first to the
second spermatogenetic period is marked by a gradual increase
in the small dark zone about the centrosomes, until it eventu-
ally attains the dimensions of a veritable spermatic ‘‘ Neben-
kern” or archoplasm (figs. 85, 36,37), and from what has been
said (§ 12) it follows that this body is here of an entirely
cytoplasmic origin. The archoplasm, with its contained cen-
1 Brauer, loc. cit. (pl. ix, figs. 12—18).
2 See Toyama, “On the Spermatogenesis of the Silkworm,” ‘ Bull. Agric.
Coll. Imp. University Tokio,’ vol. ii, No. 3, 1894, pl. iv, figs. 25, 26.
286 J. He So MOORE:
trosomes, is at first closely applied to that part of the nuclear
wall within which the curious lopsided condensation of the
chromatin goes on.
21. The fine-meshed, tightly-coiled condition of the chromatin
persists some time, but it gradually resolves itself into a coarse
chromatic network on the nuclear periphery (figs. 37, 38).
The strands of this network are sharply polarised towards the
position occupied by the archoplasm and the centrosomes.
The large oval nucleolus present in the resting cells of the
first spermatogenetic period becomes now somewhat modified,
both in position and character. Instead of being disposed
casually along the nuclear circumference, it takes a position,
generally, but not always, in line with the long axis of the
archoplasm (fig. 37, ”.). Along this line there is still to be seen
the secondary nucleolus (fig. 36, 37, m.') surrounded with a
vacuole, which I described in § 19.
These two peculiar forms of nucleoli are always to be found
after the transition from the first into the second spermato-
genetic period, and throughout all the generations of the
latter.
22. The archoplasm, which at first lies closely applied to
the nuclear wall, during the early stages of the conversion
of the first into the second spermatogenetic period, migrates
away, quite into the cell body, while the two centrosomes
which it contains, moving faster in the same direction, appear
shortly on its exterior surface just beneath the membrane of
the cell (fig. 37).
V. The Divisions of the Second Spermatogenetic
Period.
23. The advent of the first division in the second spermato-
genetic period is characterised by the strong polarisation of
the chromatin, represented in fig. 37. The chromatic strands
are seen on close examination to be composed of a thick core
of innumerable microsomes, which, collecting together into
groups, give to the strands their curious monilated appearance,
CHANGES IN REPRODUCTIVE CELLS OF ELASMOBRANCHS. 287
also described by Hermanu! in the prophasis of the great
heterotype division of the spermatogenesis in Salamandra.
These monilations in Elasmobranchs, however, do not consist
of one large microsome, as Hermann’s figure would lead one
to expect, but are each formed by a group of numerous chro-
matic granules,and these are embedded in a scaffolding of linin.
Delicate connecting filaments of this substance spread from
the monilations on the threads in all directions. The polarised
threadwork is disposed throughout the nucleus in long parallel
loops (figs. 37, 38), the free ends of which, if they exist,
are difficult to discern. After a time the threads begin to
show longitudinal splitting (figs. 38, 40), and the double ropes
thus formed, dividing into equal segments, eventually give rise
to twelve thick loops which (fig. 42) form the twelve ring
chromosomes (fig. 43) typical of the divisions of the second
spermatogenetic period.
24. There are thus, after the rest of transformation, only half
as many chromosomes, 1. e. separate chromatic masses, as there
were before, and the halving of their number, being brought
about while the nuclei are still at rest, is to that extent
comparable to what is now known to go forward during the
maturation of the reproductive elements of plants. I there-
fore propose the term Synaptic phase® to denote the period at
which this most important change appears in the morphological
character of reproductive cells.
25. Concomitantly with the formation of the twelve ring
chromosomes, the centrosomes (figs. 40, 41, c.) begin to separate,
and their greatly enlarged archoplasmic envelope (Nebenkern)
(fig. 41,a@.) is drawn out between them into a little archoplasmic
spindle (fig. 42), strictly comparable to that described by Her-
mann during the division of the spermatocytes of Salamandra.
As in the divisions of the previous spermatogenetic period,
the separation of the centrosomes occurs with great rapidity,
the archoplasm being drawn asunder into two parts (figs. 43
and 44), although it sometimes presents the appearance of a
? Loc. cit.
* Gr, cvvarrw, to fuse together.
vou. 38, PART 2,—-NEW SER. T
288 J. #, S; MOORE.
fine achromatic line stretched round the nuclear membrane.
My preparations indicate both these methods of procedure.
The protoplasmic contents of the cell become radially dis-
posed, not directly to the centrosomes, as in the divisions of
the previous spermatogenetic period, but towards the outer
surface of the daughter-archoplasms (fig. 43, 7.), and it conse-
quently follows that the sphere of the first period is structurally
less complex than that of the second. In cells which possess
an archoplasm, any radiation in the cytoplasm external to this
structure has not generally been considered a part of the
attraction sphere; neverthelesss, such external radiations are
obviously similar to those directly related to the centrosomes
in the cells of the first spermatogenetic period, where they
would certainly be regarded as a portion of the sphere. To
save confusion, therefore, I shall speak here, as I have done
elsewhere, of spheres which possess an archoplasm, as compound,
and those which do not, as simple, and thus avoid the neces-
sity of determining whether any particular set of radiations
should or should not be regarded as constituents of the
sphere.
26. The ring chromosomes, which, when fully formed,
become dispersed over the nuclear periphery, like those of the
first spermatogenetic period, are in like manner connected to
one another by numerous filamentous strands of linin (fig.
43, 1.).
The nuclear membrane eventually becomes irregular, and,
giving way at various points, leaves the chromosomes to collect,
by the contraction of these connecting filaments, into a long
oval mass stretched across the nuclear sap between the centro-
somes (fig. 44). The nuclear sap is traversed from the first by
numerous fine strands, putting the chromosomes into connection
with the outer cytoplasmic network, and which are in all pro-
bability part of the latter, dragged inwards from without after
the disruption of the nuclear wall. The central chromatic
mass 1s somewhat stretched, and more firmly attached to
the old nuclear surface in the direction of the spheres, appear-
ing as if slung between the centrosomes (fig. 44).
CHANGES IN REPRODUCTIVE CELLS OF ELASMOBRANCHS. 289
As time goes on, the chromosomes assume a more and more
equatorial position; but their linin filaments remain stretched
out towards the centrosomes, and form the greater portion of
an achromatic spindle, the equatorial part of which is con-
sequently nuclear.
27. The astral radiations which surround the centrosomes
become connected with the chromosomes in such a way as to
clothe the achromatic spindle with a fibrous sheath, structu-
rally equivalent to that described by Hermann (ante, § 8),
while even at this early period in the formation of the spindle
figure, the centrosomes are sometimes divided at the poles
(figs. 45, 46, c.).
28. The exact form of the chromosomes, when they appear
in the monaster of this first heterotype of the second spermato-
genetic series, varies a good deal from cell to cell; but in the
majority of cases the loops are at first bent up upon them-
selves, in the manner represented in figs. 45, 45’. The rod-
like bodies thus produced at first stand stiffly out from the
surface of the spindle (fig. 45’), but after a time they flatten
down in the manner represented in fig. 45. In consequence
of this, the two limbs of the loop appear in profile to have the
form of two Greek Q’s place, side by side, and the outer
surface of the bends being greatly thickened, the original open-
ing of the loop is reduced between them to the merest slit
(fig. 45, s.). These thickenings on the outer curves of the Q's
would appear to correspond with the thickenings on one side
of the heterotype loop of Salamandra, but in Elasmo-
branchs they developed equally on both limbs. I was conse-
quently interested to find that in the great heterotype division
of the spermatogenesis of newts, these thickenings sometimes
occur on one, sometimes on both limbs of the elongated loops.
From the drawings given by Hermann, Flemming, and vom
Rath, who deal with this form of chromosome in the salaman-
der, it would appear that the loops are often intentionally repre-
sented with the plane of their openings at right angles to the
surface of the spindle, that is, with one limb on and the other
off the spindle. However this may be, it is certainly rarely if
990 J. E. S. MOORE.
ever the case in either Elasmobranchs or newts, in both of
which the loops lie flat, with both limbs on the spindle surface.
As the loops lengthen out towards the poles, the outward
bends are gradually reduced, but they never disappear, and at
the time the chromosomes divide (fig. 47) they separate from
one another in such a way that the original openings of the
loops are clearly seen, The separation into daughter-elements
is effected by a transverse splitting of the loop across the central
thickening, at right angles to the then long axis of each
chromosome (fig. 47), as is usual in Heterotype metores.
After their separation, the daughter-chromosomes form super-
ficial chromatic rings (fig. 48—51), as did those in the divi-
sions of the first spermatogenetic period (see § 13), and the
spindle-fibres in like manner become differentiated into two
concentric tubes, separated from one another by the nuclear
sap. This differentiation of the spindle into fibrous tubes is
carried further than in the divisions of the first spermatogenetic
period, the whole structure appearing to be composed of two
completely closed cylinders of fibres, one (fig. 49, 0. s.) stretched
directly between the outer edge of the chromatic rings, and
the other (¢.s.) passing internally through them to the centro-
somes. The unstained fluid which separates the outer from
the inner of these sheaths, is that which previously filled
the interspaces between the fibres of the younger spindle, and
it was once the parental nuclear sap. It contains irregular
chromatic granules (0. ¢., fig. 50) which appear to have been
left as débris of the chromosome formation, and which sooner
or later pass into the cytoplasm of the cell (0.c., fig. 54).
29. While the above changes are in progress, the centro-
somes become surrounded by the dusky zone, created by the
shortening up and coalescence of the cytoplasmic fibres between
them and the chromosomes (figs. 48, 49, 50 a).
30. The chromosomes of each daughter-nucleus are at this
time quite separate and distinct, although lying closely side by
side; but as time goes on they begin to fuse together, so that
the chromatin eventually forms two solid chromatic rings, one
in each daughter-cell (figs. 51, 52, 53).
CHANGES IN REPRODUCTIVE CELLS OF ELASMOBRANCHS. 291
31. About this time the intra-zonal fibrils spread out, until
those from either pole meet at the circumference of the cell
(fig. 49), and at these somewhat angular connections there ap-
pear beaded thickenings in the threads, which (fig. 50, 0.2.)
stain and thus form an interesting stepping-stone between
Flemming’s intermediate bodies and a true cell-plate.
32. At the same time the chromatic rings gradually lose their
original connection with the outer spindle-fibres (0.s., fig. 52),
which begin to bulge out and to pass round the nuclei towards
the poles (fig. 53). The chromatic rings are thus cast loose
in a surrounding fluid vacuole, but the inner core of fibres
(fig. 52, 7. s.) continues to pass through them.
The bulging out of the intra-zonal fibres round the nuclei is
marked by a collapse at the point of their original distension
at the equator (figs. 51, 52, 53), which brings the outer and
the inner sheath together in the median plane (figs. 52, 53).
Thus the central portion of the residual spindle figure presents
the appearance of a sharply differentiated fusiform body be-
tween the cells (figs. 53, 54, 55). Across its middle there is a
chromatic band, produced by the fusion of the intermediate
bodies (figs. 63, 54, 55, 56, 57, b.i.).
While contemplating the changes I have just described, it is
impossible to avoid the impression that the rupture of the outer
spindle-sheath and the consequent outflowing of the enclosed
fluid to form the nuclear vacuoles (figs. 54, 55, 56, n. v.), are
the primary causes by which the expanded equatorial spindle-
figure is made to collapse, and that it may also have a direct
mechanical connection with the formation of the primary con-
striction between the daughter-cells.
33. As in the divisions of the first spermatic period, the spheres
(fig. 37, a) begin now to travel over the surface of the nucleus,
generally along a groove (n. g., fig. 54) like that described in
§ 15, towards its equatorial face.!
34. The expanded central portion of the spindle remnant
now lies between the daughter-cells (which are otherwise quite
1 *Arch. fiir mikr, Anat.,’ Bd. xhii, p. 423; cf. M. Haidenhain, op. cit.,
Taf. xxv (figs. 14, 21, 22, 23).
292 J. E. 8S. MOORE.
separate from one another), and inserts a conical termination
into both (figs. 53, 54); but the delicate filaments into which
these terminations are prolonged, after the translocation of the
spheres from the polar to the equatorial surface of the recon-
structed nuclei, disappear, the last function of the remains
of the outer and inner spindle-tube being to form an open
connection with an equatorial chromatic band (the inter-
mediate bodies) between the daughter-cells (0. 7., figs. 56, 57,
58, 59).
35. The spheres, during their passage from the polar to the
equatorial nuclear faces, pass in a more pronounced manner
through a similar metamorphosis to that described in
§ 22, and which, when rightly understood, appears to be
of the most profoundly interesting nature. When the archo-
plasm (a, figs. 58, 59) has reached some point halfway between
the pole and the equatorial nuclear side, it begins to move
away from the nucleus, while the centrosomes, travelling faster
in the same direction, pass from the centre to the surface of its
mass (figs. 58, 59, c.). From this point (c.) there grows out a
fine protoplasmic thread (fig. 59, f.), extending to the cell
periphery. The cell membrane is indented slightly where the
thread approaches it (fig. 59), but the thread itself is pro-
longed beyond it as a fine protoplasmic process, comparable toa
short flagellum (figs. 59—64, f.). By the time this structure has
been formed the archoplasms of each daughter-cell are more or
less facing each other, with the tubular remains of the spindle
stretched between (figs. 59 and 61).
36. When the cells come perfectly to rest, there appears on
each side of the archoplasm, or in its immediate vicinity, a
marked condensation of the cytoplasmic substance (fig. 62, x.),
which, in the absence of the attraction sphere, might readily
be taken for an enlarged representative of that body ; and as
this mass is of some importance in understanding the process
of conversion of the next generation of cells into the sperma-
tozoa, I shall speak of it as the Nebenkern.
37. Before the prophasis of the next division, the remains of
the spindle become no longer visible between the cells, and
CHANGES IN REPRODUCTIVE CELLS OF ELASMOBRANCHS. 293
the rudimentary flagellum is withdrawn, but the centrosomes
remain immediately beneath the membrane of the cell.
VI. The Last or Second Heterotype Phase of the
Second Spermatogenetic Period.
38. The last division in the second spermatogenetic period
is a heterotype, like the first, but the elements are scarcely
more than half the size. The number of the chromosomes is
again twelve, and, like those of the earlier divisions, they
become eventually grouped together as a globular mass in the
centre of the nuclear sap (figs. 66, 67, and 68).
When the spindle has been formed, the ring chromosomes are
not altogether on its surface, and, owing to their small size, a
polar view of the monastral figure often presents the curious
appearance represented in fig. 70, on account of the upper
and lower edges being in focus at the same time. The chro-
matic loops eventually split transversely, like those of the
previous division, the daughter V’s travelling to the pole in the
manner represented in figs. 69, 71, and 72.
Thus the last division in the spermatogenesis of these fishes
is, as I pointed out so early as in 1893,! a perfectly normal
affair, each mother-chromosome splitting into two daughter-
elements, so that the two cells produced contain each the same
number (twelve) of residual chromosomes. Consequently,
like the last division in Mammalia, it presents nothing in
common with the “ Reductionstheilung”’ described by Hert-
wig, and upon the assumed universality of which so much of
Weismann’s latest theory of heredity is built.
39. The process of return to rest of the daughter-cells (sper-
matids) is in all respects essentially the same as that in the
previous generation. The distension of the outer spindle-sheath
(fig. 73) and the formation of intermediate bodies (0. 7.) (cell
plate) is perhaps more marked, the inner tube (¢.s.) appearing
consequently more isolated and alone. But there is the same
detachment of the chromatin from the fibres of the outer sheath
1 «On the Germinal Blastema of Cartilaginous Fishes,’ ‘Anat. Anz.,’
Bad. ix, p. 547.
294 Jo Bea S. MOORE:
(figs. 75 and 76), the same formation of a nuclear vacuole in
each daughter-cell (fig. 76, v.v.), the same gradually diminish-
ing connecting spindle filament between the nuclei (figs. 76
and 77), and lastly, the same formation of nuclear grooves
(fig. 77, m.g.) along which the spheres travel to the equatorial
side. Further, when the spheres have reached some point
halfway between the polar and equatorial nuclear faces, the
archoplasm leaves the nuclear wall. The centrosomes (fig.
79, c.) pass on to the outer archoplasmic surface, and from this
there passes a fine protoplasmic strand (fig. 80, f.) to the cell
periphery, and the cell membrane is indented where this
_ strand perforates it as the whiplash-like spermatozoon tail. It
thus becomes evident that the metamorphoses described in
§ § 22, 35 are nothing more nor less than abortive attempts
at tail-formation, and it consequently follows that the
synaptic phase in these fishes marks the assumption
by the cells, during spermatogenesis, of a flagellate
condition.
40. Besides the nucleus, there appears a dusky condensation
of the cytoplasm (#., figs. 80—83), which at first sight gives the
cells the appearance of possessing more than one attraction
sphere, and is obviously similar to the nebenkern of the
preceding generation (of § 36). This body when it first appears
is closer to the nucleus than the archoplasm (fig. 80), and
in the latter there is seen at the point of origin of the
flagellum a clear round vesicle (fig. 82, a. v.), which enlarges
and eventually moves, with its archoplasmic surroundings and
the centrosomes, into close apposition with the nucleus
(fig. 83, a. v.).
41, The nuclear chromatin rises up into a shallow collar
round the base of the archoplasmic vesicle, while the rest
of the chromatic substance, contracting from the nuclear
membrane, becomes condensed into a flask-shaped mass below
the collar (figs. 84 and 85). This contraction increases rapidly
while the collar, elongating, spreads into the nuclear membrane
at the base of the archoplasmic vesicle, to form a small chro-
matic flange round the neck of a_ bottle-like structure
CHANGES IN REPRODUCTIVE CELLS OF FLASMOBRANCHS. 295
(figs. 85, 86, 87), the body of which is filled with nuclear
chromatin, and the neck of which is stopped with the archo-
plasmic vesicle (fig. 85, a.v.). Beyond the chromatic flange
the nuclear membrane encases the whole, much in the same
way as the basket-work used to protect an Italian wine-flask,
the nuclear sap between it and the chromatin representing the
glass.
42. The base of the intra-cellular part of the flagellum, with
the centrosomes, now lies between the archoplasmic vesicle
and the chromatic flange, but the point of attachment of the
flagellum moves round the surface of the nucleus, the archo-
plasmic substance penetrating the nuclear membrane, and
resting with the base of the flagellum on the chromatin, in a
funnel-shaped mass (fig. 85, a.). The centrosomes are no
longer visible, being either lost or becoming indistinguishable
among the rest of the chromatic substance at the base of the
tail. The translocation of the point of attachment of the
flagellum continues (figs. 86, 87) until it finally comes to
rest at the side of the nucleus opposite to the neck and the
archoplasmic vesicle where it started (fig. 88). The ‘ neben-
kern” is implicated in this motion, and its substance is
eventually mixed up with that of the true archoplasm, both
structures forming a distinctly differentiated protoplasmic mass
extending along the intra-cellular part of the flagellum, from
its base in the nuclear chromatin to its exit through the nuclear
wall (figs. 85—89, v.a.). The whole of this mass (composed
of the “nebenkern’”’ and the archoplasm, together with the
intra-cellular part of the flagellum) eventually forms the long
Mittelstiick of the mature spermatozoon (figs. 90, m.). The
origin of the Mittelstiick in these fishes will thus be seen to
coincide with what I have related respecting this structure
in Mammalia, and probably with Hermann’s description of its
formation in Salamandra together with what occurs in a
number of Invertebrate spermatogeneses.
43, At the oppositeend of the nucleus the archoplasmic yoeiele
(a. v., figs. 88, 89, 90) becomes first flattened, and then elongates
out, together with the nuclear chromatin, forming. a definite
296 J. E. S.. MOORE.
cephalic point to the spermatazoon head. The nuclear jacket
(figs. 88, 89, 90, .v.), formed by the sap separating the nuclear
chromatin from the nuclear wall, continues well marked even
at maturity, and the swelling on the cell membrane, where
the flagellum originally passed out, remains (fig. 90, md.) as
the little bead at the hinder end of the Mittelstiick.
The spermatogenesis is now practically complete, and the
facts of the second spermatogenetic period which appear to be
of primary comparative importance are:
I. The transformation of the cells of the first spermatogenetic
period into those of the second, which I have termed the
Synapsis, is accomplished while the celis are in complete
repose, and is marked by a peculiar evolution in the chromatin
with the formation of peculiar nucleoli (which are repeatedly
characteristic of the succeeding cellular generations) and by the
formation of an archoplasmic constituent round the centro-
somes.
II. The evolution during the prophases of the first and
second divisions of the second spermatogenetic period of
twelve ring chromosomes, which split transversely to form the
same number, twelve, in each daughter-cell.
III. The differentiation of the spindle during the diastral
figure into an outer and an inner fibrous sheath, which coalesce,
forming a delicate connecting thread between the attraction
spheres of both daughter-cells.
IV. The existence during the synapsis of a peculiar evolu-
tion among the constituents of the attraction sphere, whereby
the centrosomes are brought to its exterior surface, beneath
the membrane of the cell.
V. The repetition of the process in a more pronounced
manner, after the first heterotype division in the second sperma-
togenetic period, so that a short flagellum is protruded from
the centrosomes through the membrane of the cell.
VI. The origin of the long whiplash tail of the spermato-
zoon in a similar manner, after a corresponding metamor-
phosis of the sphere during the formation of the final cellular
generation.
CHANGES IN REPRODUCTIVE CELLS OF ELASMOBRANCHS. 297
44, The whole course of the spermatogenesis may now be
diagrammatically represented as follows: In Diagram I the
rings under a represent a succession of resting cells in the first
spermatogenetic period, while the signs of division m! (24),
n® (24), &e., stand for the successive divisions by which they
are produced. The number 24 represents the constant number
of chromosomes in each.
The cone under represents the synaptic change, while
under y are represented (by black dots) the cellular generations
of the second spermatogenetic period, up to the formation of the
final spermatozoa.
a p Y
we2------ ~—_—
77 (24) No(2) 73 (24) 1(2) 2 PE
Ree o + ——o+ —o + =u @—- : Eebee
Ds NS liste a oe
ea
Diagram J, illustrating the course of Elasmobranch spermatogenesis. (a)
First spermatogenetic period. (6) Synapsis. (y) Second spermato-
genetic period. 7, (24), &c., number of chromosomes in each division of
first period, where x represents an indefinite number of previous divi-
sions. 1 (12), &c., same in second.
VII. Comparative.
45. As the majority of the operations performed by living
protoplasm are inexplicable on any structural arrangement in
the parts of cells which has hitherto been observed, it is
obvious that the structural relationships which, if known, would
render the actions of protoplasm self-explanatory, lie some-
where below the present range of vision, and it consequently
follows that the theoretical explanations of this or that property
which protoplasm exhibits are, at bottom, nothing more nor
less than hypothetical forecasts of the ultimate structure on
which this or that manifestation of vitality depends. The
probability of any forecast being true is proportionate to the
298 J. E. S. MOORE.
capacity which its premises exhibit of being logically worked
up into harmony with what has been actually observed. Thus,
according to Weismann,! the phenomena of heredity depend
ultimately on the existence of innumerable little unities in the
“germplasm,” or “ ids,”’ and these are in reality the hypothe-
tical doers of everything that is done. ‘They are capable of
influencing the protoplasm which surrounds them in different
ways, and by coming into action successively during develop-
ment, they produce the structural differentiation of a complex
form. Representatives of all the different kinds of “ ids,”
actual or potential, which exist in any given animal or plant,
are continually being locked up for future use in every ovum
or spermatozoon formed, and in consequence of the indefinite
multiplication of the “ids,” which must occur after every act
of fertilisation, it appears, according to Weismann, a logical
necessity from the premises of his theory, that the reproductive
cells, before fertilisation, must each get rid of half their here-
ditary substance (i.e. “ must each get rid of half their nuclear
rods”’). ‘This is supposed to be accomplished by there being
two kinds of division among cells. In the first of these (the
ordinary somatic division) the chromosomes split in half, there
being consequently the same number in each daughter-cell,
and this method of division has consequently been termed
“ Kquationstheilung,” to distinguish it from the second or
“ Reductionstheilung,” which is apparently introduced only
during the final stages of the development of the reproductive
elements, and is brought about by half of the entire number of
chromosomes formed during a mitosis passing unsplit into one
daughter-cell, and half into the other.
The value of these hypothetical speculations touching the
nature of the phenomena immediately antecedent to fertilisa-
tion, appeared to be enormously enhanced by O. Hertwig’s
description of a process answering to the Reductionstheilung
in the final stage of the spermatogenesis of Ascaris, because
if this process should turn out to be universal, as at one time
seemed probable, it would give to the “id” theory an actual
>
1 «The Germplasm,’ English trans.
CHANGES IN REPRODUCTIVE CELLS OF ELASMOBRANCHS, 299
demonstration in fact. Unfortunately, however, for the Re-
ductionstheilung, as well as for the enormous superstructure
which Weismann has lately piled upon it, O. Hertwig’s obser-
vations have been shown by Brauer to be quite erroneous,
there being in the spermatogenesis of this animal no such
thing as a division in which alternate chromosomes pass
unsplit to daughter-cells. So also, during the Elasmobranch
spermatogenesis with which we have been dealing (the
course of which will be found summarised in these pages at
the end of each spermatogenetic period), there is nothing com-
parable with the ‘‘ Reductionstheilung ” of Hertwig, which is
made such an integral part of Weismann’s last theory of
heredity. It is true that there is a numerical halving of the
chromosomes, between the first and second spermatogenetic
periods, but this is brought about in the synapsis which
separates the one period from the other, and has nothing to do
with division at all.
It is so necessary to be quite clear about this, that I have
subpended a few lines of Weismann’s treatise in which his
conceptions of the ‘‘ Reductionstheilung ” are given in full.
On page 11 of the English translation of the ‘Germplasm,’
its author, after speaking of the necessity of a “ Reductions-
theilung,” and as though the universality of its occurrence was
an established fact, goes on to say :—“ The hypothesis of the
Reductionstheilung has been thoroughly substantiated by sub-
sequent observations—in fact, it has even been proved that in
many cases this reduction occurs exactly as I had foretold and
represented in a diagrammatic figure; that is to say, by the
non-occurrence of the longitudinal division of the chromo-
somes, which occurs in ordinary nuclear division, and by the
distribution of these in the daughter-nuclei. This holds good
for the ovum as well as for the sperm-cell in animals, and as
far as is known,in plants also. The germ-cell must in
all cases by division get rid of half its nuclear rods.” Again on
page 236 :—‘‘ We now know that this reduction in the number
of the ids, by one half,is of general occurrence, and is effected
by means of the nuclear divisions which accompany cell divi-
300 J; i. 8+ MDORE;
sion. The divisions which result in the formation of the polar
bodies perform the function of the Reductionstheilung as re-
gards the ovum, and the final divisions of the sperm mother-
cells have this function in the case of the spermatozoa. In
both cases the Reductionstheilung does not consist in the
idants (chromosomes) becoming split longitudinally, and in
their resulting halves being distributed equally amongst the
two daughter-nuclei, as in ordinary nuclear division, but in one
half of the entire number of rods passing into one daughter-
nucleus, and the other half into the other.”
46. The absence in the spermatogenesis of Elasmobranchs of
any Reductionstheilung is thus of peculiar interest, because the
fundamental way in which Weismann has used this conception
of a Reductionstheilung as a basis on which to build up his
supposed explanation of heredity, renders it evident that any
widespread collapse in the alleged universal existence of this
process, either among animals or plants, will in the long run
bring down the whole speculative superstructure with it.
47. Now with respect to plants, the two highest living
authorities, Strasburger! and Guignard,? have already dissented
from Weismann’s views; and the former, in his address to the
Biological Section of the British Association meeting at Oxford
last year, expressed his opinion of the Reductionstheilung as
follows :—‘‘ There is no such thing among plants as nuclear
division resulting in the reduction of one-half of the chromo-
somes. Such a conception involves the assumption that the
entire, not longitudinally-split, chromosomes of the mother-
nucleus become separated into two groups, each of which
goes to form a daughter-nucleus.” So we may take it that the
Weismannistic conception of the “ Reductionstheilung,” “so
far as is known in plants,” fails.
48. With respect to animals, Boveri,’ in his “ Befructung,” as
far back as 1890, after postulating the chromatin as the
1 © Ans. Bot.,’ vol. vili, 1894.
2 « Anns. d. Sc. Nat.,’ Bot., 1891.
3 «Hrgebnisse der Anat. und Entwicklungsgeschichte,”’ Bd. i, 1891,
pp. 458, 459.
CHANGES IN REPRODUCTIVE CELLS OF ELASMOBRANCHS. 3801
hereditary substance, argues, like Weismann, for the necessity
of some sort of chromatic reduction, before the maturation of
sexual cells; but he comes also to the conclusion that the
reduction processes hitherto described are numerical reductions
of the chromosomes, and not quantitative with respect to their
substance. He draws a sharp distinction between “id”? re-
duction and chromosome reduction, the latter of which he
apparently disregards, but he seems to entertain the idea that
the former, by the numerical reduction of the chromosomes,
may in reality be carried out. He shows further that the
numerical reduction of the chromosomes in ovogeneses, like
that of Echinus and Pterotrachea for example, repre-
sented in the diagram which I have borrowed (Diagram II),
and which will become intelligible on reference to my § 44, is
not brought about by any of the divisions in the first ovogenetic
period, a, and up to the formation of the first ovocyte after the
rest (3, i.e. the ovum before the extrusion of the polar bodies,
but that there are only half as many chromosomes in the first
ovocyte when it emerges from the rest, (3, in the polar body-
spindle as there were before, and this number is retained after
the polar bodies are extruded in the ovum. ‘Therefore the
numerical reduction is not brought about by any division of
the ovogenesis, but occurs during the synaptic rest, 3, and
before the prophasis of the first “ Richtungspindel.”’
a p Y
; ‘ LS SSS
Ag)»
4) No) 1(¥) 4
Diagram II, representing course of typical ovogenesis (after Boveri).
Reference letters same as in I.
Finally, he shows that the so-called reduction processes
hitherto described are irreconcilable among themselves, and
302 J. 5. So MOORE:
concludes with the following characteristic phrase :—‘‘ Durch
die vorstehenden Erorterungen, glaube ich gezeigt zu haben
dass zwar gewisse Vorginge bescrieben worden sind, die
vielleicht mit der Chromosomenreduction in Zusammenhang
stehen, dass uns aber eine wirkliche Einsicht in diesen
Vorgang bisjetzt fehlt. Ks bleibt weitere Forschung vorbe-
halten, dieses Dunkel aufzuhellen.”
49. In 1893 I published! a preliminary account of some
investigations concerning the course of the spermatogenesis of
mammals, which I summarised in these words: “‘ There is in
the Rat (i)? a period of indifferent cell formation, terminated
by a mitosis with sixteen chromosomes, both in the primary
and daughter-nuclei ; (ii) a period of growth (or rest) during
which the sixteen chromosomes are converted into eight, and
terminated by a division in which the daughter-nuclei
spermatids still retain the number eight; (iii) a period in
which the spermatids are converted into spermatozoa.” If
a B y
On ery tpl se cg, _—_
1(46) 1(8)
itttese rector <a
~~
Diagram III, showing course of Mammalian spermatogenesis. Reference
letters same as in I.
we now construct a diagram of the first and the second
spermatogenetic periods in Mammalia (as in Diagram III),
and place it side by side with that of the Elasmobranchs given
' “ Mammalian Spermatogenesis,” ‘Anat. Anz.,’ Bd. viii, 1893, pp. 6883—
688.
2 Dr. Toyama, who apparently writes under the wing of Professor Ischi-
kawa, speaks of my results respecting these phenomena in mammals as being
“very improbable,” but as he produces no evidence relevant to the subject, I
fail to see the force of such a criticism, and have therefore refrained from
applying it to several portions of his own treatise, more especially as very
little trouble with any native mammal would have enabled him to see whether
the ‘ improbable” was true,
CHANGES IN REPRODUCTIVE CELLS OF ELASMOBRANCHS. 3038
in § 44, it will be seen that the former differs from the
latter only at the beginning and the end. These differences
are produced by the shortening up of the generations of the
first spermatogenetic period in mammals, (a) into what is
practically a kinetic budding, so that there is only one distinct
homotype division with sixteen chromosomes (I, 16) before
the synapsis ; (6) (equal growing cells) in which the chromatic
individuals are reduced or fused together into eight. The
process of transformation and the succeeding heterotype cor-
respond exactly with that of the Elasmobranchs. But the
daughter-elements produced do not, as in Elasmobranchs,
divide again. They are converted directly into spermatozoa,
and it thus appears that one of the two generations of ciliated
cells, present after the heterotype in Elasmobranchs, in some
mammals is unrepresented.
50. Brauer,! as I have said, in his admirable account of the
spermatogenesis of Ascaris, published in 1891, also denies
the existence of the “ Reductionstheilung ” described by O.
Hertwig, both in the uni- and bivalent form of this curious
worm. There is a period of cell multiplication, equivalent to
the first spermatogenetic period, with two or four chromo-
somes, as the case may be, and in the divisions of which, as
Professor Brauer has recently informed me, the chromosomes
split longitudinally, like those in ordinary divisions. Then a
period of rest, equivalent to the rest of transformation, in
which the number of the chromosomes is halved, followed by
divisions of a totally different character, in which there
appears to be precocious splitting of the chromatic elements
and rapid separation of daughter-cells,? without the nuclei
1 Loe. cit.
? It is probable that all the cases of the so-called “ Reductionstheilung ” are
in reality referable to a process of precocious splitting among the chromosomes,
whereby the elements for several daughter-cells are produced at once, and are
then distributed, either by successive divisions without rest, or by multipolar
spindle formation. An admirable example of the latter method is afforded by
Farmer’s description of the spore formation in Pallavicinia decipiens.
In these plants the number of the chromosomes in the sporophyte genera-
tion is always eight, but as soon as the spore mother-cells are formed, the eight
VOL. 38, PART 2,—NEW SER. U
304 J. E. S. MOORE.
returning into rest. The closeness of the similarity of the
spermatic reduction described by Brauer with those detailed
above is perhaps best seen when presented in the same
schematic form.
51. There are thus several well-established cases of spermato-
genesis in which the reduction process described by Weis-
mann is departed from. Besides Boveri’s account, it is appa-
rent from Brauer’s! figures of the ovogenesis of Branchipus,
published in 1889, that the twenty-four chromosomes of the
cells of the first ovogenetic series are reduced to twelve, during
the synapsis, before the commencement of the second, while
each of these twelve chromosomes splits twice at the beginning
of the first “‘ Richtungspindel.” The quadripartite chromo-
somes thus formed, divide and redivide in the two subsequent
mitoses, without any intervening rest, so that there are twelve
single chromosomes left finally in the ovum.
52. There are thus several well-established cases of both
spermato- and ovo-genesis in which the reduction process
described by Weismann is departed from, not only in the
absence of the ‘‘ Reductions ”—as opposed to the ‘‘ Equations-
theilung,”’—but also in the fact that the halving of the number
of the chromosomes takes place in resting nuclei, one or more
generations before the formation of the final sexual cells—from
all of which it will have become sufficiently apparent that
the Reductionstheilung of Weismann is universal neither among
animals nor plants, and although an attempt may possibly be
made to foist the theoretical burden which it carries on to the
“ synapsis”’ instead, there are cogent reasons for believing
that the advocates of such a process will simply travel further,
and in the end fare worse. It is obvious that the objections
chromosomes have, during the previous rest stage, been numerically reduced
to four. These four chromosomes now split up, first into eight, and then into
sixteen, and all these residual chromosomes are distributed by a quadripolar
spindle figure in groups of four, amongst four spores, and this final number of
the chromosomes persists through all the succeeding divisions of the gameto-
phyte generation. (‘Annals of Bot.,’ vol. viii, pp. 35—51, 1894.)
1 “ Uber das Hi von Branchipus Grubii,” ‘ Abhandl. d. preuss. Ak. d. Wiss.,’
Berlin, 1892.
CHANGES IN REPRODUCTIVE CELLS OF ELASMOBRANCHS. 3805
which have been raised, by botanists, against the numerical
halving of the chromosomes in the resting reproductive cells
of plants having anything in common with the Reductions-
theilung, can be used with equal weight in the case of the
synaptic phenomena in the animals I have just described. And
there is yet another and much more formidable obstacle to
such a view, namely the possibility, if not probability, of both
the synapsis among animals and the analogous processes
in plants being interpretable on common and quite different
grounds.
53. It will have been seen that throughout the whole course
of the evolution by which the halving of the number of the
chromosomes in the above animals is produced, there exists at
least a superficial similarity to that accompanying the forma-
tion of the spore mother-cells and embryo-sacs in plants;
and Strasburger,! in the address to which I have referred
above, has already put forward, in a more or less provisional
way, the ingenious suggestion that the halving of the number
of the chromosomes in the reproductive cycles of living
organisms may be interpretable on phylogenetic and not on
physiological grounds. This attempt, however, to bring the
whole of the phenomena into line is sadly hampered, thanks
to the influence of the “ Reductionstheilung ” on investigation,
by the insufficiency of recorded observation on the zoological
side. I am enabled now, however, with these new facts
relating to the reproductive cycles of Elasmobranchs, to draw
Strasburger’s comparison between animals and plants much
closer, and to show that the phylogenetic interpretation of the
numerical halving of the chromosomes of both is probably
true.
54. It will be seen on reference to § 19 of the descrip-
tive part of this paper that the prophasis of the heterotype
division following the synapsis in Elasmobranchs is _pre-
ceded by a peculiar readiness of the chromatin to contract into
forms like those represented in fig. 39, which is charac-
teristic of this particular phase in the spermato- and ovo-
1 Loe. cit,
306 J. E. S. MOORE.
genesis in a great variety of animal forms. Now, exactly simi-
lar figures are obtained before the division of the pollen
mother-cells, during the formation of the so-called “ para-
nucleus” in plants; but considerable diversity of opinion
exists on the botanical side as to the real or artificial nature of
the paranucleus and its associated contraction figure, 1. e.
whether the whole appearance is not in reality more a ‘‘Gerin-
nung’s Erscheinung” than a reality. However this may be,
I do not believe that the one-sided nuclear figures seen at a
corresponding period in the spermato- and ovo-genesis of
animals, and with which most histologists must be quite
familiar, are artifacts at all; and whether the contraction
really exists in plants or not, it has been generally conceded
by the botanists I have interrogated on the subject, that it is
especially related to the time in question, while Professor
Farmer tells me that such shrunken nuclear figures are practi-
cally diagnostic of the synapsis in certain liverworts of Ceylon,
and so there can be little doubt that there exists, at any rate at
this period, a peculiarly sensitive condition of the chromatin,
common to both animal and plants.
55. In Elasmobranchs, Mammals, Amphibia, and probably
many other animals, the division which immediately follows
the synapsis is, as will be seen from §§ 23—387, different
from all those preceding it. The chromosomes as_ they
emerge from the reticulum of rest, being no longer longitudi-
nally-split rods, but closed loops or rings, the divisions thus fall
under the category of Fleming’s heterotype. In animals the
exact form and placing of these closed loops differs a good deal
in different forms, but they all agree in this, that the loops
split finally in the equatorial plane. In Elasmobranchs,
Amphibia, and many other forms, the loops at first become bent
up in the equatorial region of the spindle, so that when seen
in profile they present the appearance of two Greek omegas
placed side by side, the ends of which unite towards the poles
(Diagram IV, 1,4, c). The outer curves and the closed ends
of these figures are much thickened, and consequently the space
between the two enclosed omegas is reduced to a mere slit.
CHANGES IN REPRODUCTIVE CELLS OF ELASMOBRANCHS. 307
Viewed from above, such chromosomes present the appearance
represented in Diagram IV, 1, c, and when the chromosome
divides, the polar extremities lengthen out, while a transverse
split appears across the equatorial thickening, and the daughter
V’s, gradually separating, present a curious fourfold appear-
ance represented in d, e.
Diagram IV, representing division of heterotype chromosomes. (1) In
Hlasmobranchs. (2) In Phanerogams (according to Guignard and
Strasburger). (3) In Phanerogams (after Farmer). a, 0, ¢, d, e,
corresponding stages in division.
56. In phanerogams the division which succeeds the long rest
after the formation of the spore mother-cells, and which in
general superficial characters corresponds to the synaptic
phase (cf. § 19) among animals, differs, like its zoological
counterpart, entirely in the arrangement of the chromatin
from all the previous mitoses of the reproductive cycle: But
the manner in which the daughter-chromosomes separate and
go apart is, according to Strasburger and Guignard, quite
different from what obtains in the corresponding animal cells.
. According to these authors, the chromosomes, after arising as
stout, longitudinally-split rods (Diagram LV, 2, 0) are attached
308 J. BS,’ MOORE:
at one end to the spindle surface, the two halves gradually
separating in the manner represented at 2,d,e. Quite recently,
however, Farmer showed! that in the division of the pollen
mother-cells of Lilium candidum this description of the
origin of the daughter-chromosomes by no means fits the
facts. After arising as long closed loops of microsomes, the
chromosomes assume the rod form previously known ; but the
apparently longitudinal splitting extends only part of the way
towards the outer end. When they have become flattened
out on the spindle surface—sometimes before, and always as
soon as the transverse fission is apparent (Diagram IV, 3, d.)—
there is seen another longitudinal split, which converts the
chromosomes into a closed loop, exactly comparable to those
of the animals I have just described (1V, 1), the four masses
into which the equatorial thickenings are divided at the time
of separation being very marked (IV, 3, d, ¢.).
57. Professor Farmer has kindly given me photographs of
these details, which I have copied in Diagram IV, 3. It
therefore appears extremely probable that the chromosomes
described by Guignard and Strasburger are, in reality, like
those of Lilium candidum, closed loops bent upon them-
selves, but that the great shortening up and thickening they
undergo leads here, as it often does in animals, to the internal
opening being difficult to see.
58. The outcome of all this is that the reproductive cycles of
animals and plants correspond, not only in the number of the
chromosomes typical of the somatic cells of any species being
halved, but also in the successive and complex phases by which
their numerical reduction is brought about, as well as in the
type of modification which the post-synaptic cellular genera-
tions may undergo.
59. Now, with repect to the nature of these post-synaptic
generations, it is made obvious by the fact that in Klasmobranchs
there are two, while in mammals there appears to be only one,
that their number is of no physiological importance in the
! «Ueber Kerntheilung in Lilium-Antheren in Bezug auf die Centrosomen-
Frage,” ‘Flora. Bot. Zeitung,’ 1895, Heft 1.
CHANGES IN REPRODUCTIVE CELLS OF ELASMOBRANOHS. 309
formation of the mature sexual cells. They appear rather
to constitute a sort of vanishing quantity, the existence of
which becomes intelligible only on the supposition that they
represent a phylogenetically decreasing succession of post-
synaptic generations.
The flagellum which I found in the first cellular genera-
tion after synapsis in Elasmobranchs appears to me to indi-
cate more clearly than anything I know, that the cells,
before and after the synaptic phase, are morphologically dis-
tinct. If the spermatogeneses of Mammalia were the only ones
we knew, the tail developed in the generation following the
synapsis might legitimately have been regarded as a purely
physiological structure acquired simply to suit the exigencies
of the case. But the fact that in Elasmobranchs the com-
plex initial phases of tail formation (ef. § 35) are gone through
in the first as well as the second post-synaptic generation,
is to me quite unintelligible, unless these flagella represent
similar structures once possessed by the representatives of the
cells’ remote ancestry. This view is greatly strengthened by
the complete analogy of structure which exists between the
post-synaptic generations of Elasmobranchs and some of the
simplest forms of sexually reproductive cells. It is well known
that in many Algz, reproduction can be carried on by means
of fusion between two flagellate gametes, and quite recently
Strasburger has discovered! that the flagella of such gametes
arise from the kinoplasm, a structure which there is every
reason to believe is the vegetable homologue of the archoplasm.
Moreover, among these organisms there exist species which
exhibit every gradation between those in which both gametes
are alike and flagellate, and others in which there is a true
tailed spermatazoon, and a tail-less ovum.
60. It would appear thus that if the foregoing comparisons
are just, the existence of cellular generations with vestigial
flagella, after the synapsis and before the spermatids have
been formed, indicates that the synaptic phase marks a period
in the reproductive cycle at which the cells return to a flagel-
1 *Histolg, Beitr.,’ 1892, Heft iv.
310 J. E. S. MOORE.
late condition, with only half as many chromosomes as they
had before.
61. It is conceivable that this capacity of periodically
altering their chromatic valency, which the cells of both
animals and plants possess, and which is accompanied in some
by incipient tail formation, may have arisen in either of two
ways, Viz. :
It is conceivable that sexual reproduction may have begun
before the periodic alteration in chromatic valency was evolved
at all; but that, owing to the constant doubling of the number
of the chromosomes after every act of fertilisation, a reduction
in their number became physiologically necessary ; or it is con-
ceivable that the periodic variation in chromatic valency was
evolved first, and that after its introduction, sexual conjugation,
with all its attendant advantages, became physiologically
possible. I donot know of the existence of any evidence which
is decisively in favour of one view or the other. This much,
however, is certain: on the one hand, variations in the number
of the chromosomes, of a capricious and indeterminate character,
are known to exist to-day, as in the case of the cellular struc-
tures accessory to the reproduction of many plants ; while, on
the other hand, variations which are neither capricious nor
indeterminate exist, as we have seen, at certain times and
places among the elements of complex animal and vegetable
forms ;—and I think most people will probably be disposed to
agree in regarding this orderly variation as the expression of
adaptive selection, which has worked towards some physiological
end in the past, and the disorderly variation, if I may use the
term, as the remains of something approaching a primitive
chaos, on which adaptive selection has not yet acted.
I regard the above speculations, however, of relatively small
importance beside the long series of structural homologies
which I have established before, during, and after the synaptic
phase in the reproductive cycles of both animals and plants,
because this close correspondence, among a host of complex
structural details, renders it improbable in the extreme that
the two series of phenomena can have been independently
CHANGES IN REPRODUCTIVE CELLS OF ELASMOBRANCHS. 811
evolved ; and whatever the synapsis may eventually turn out
to be, it is evidently a cellular metamorphosis of a profoundly
fundamental character, which would appear to have been
acquired before the animal and vegetable ancestry went apart,
and to have existed ever since.
In conclusion, I would express my sincere thanks to Prof.
Howse for much help, and to the Royal College of Science
for granting me the Marshall Scholarship for the completion
of this investigation.
DESCRIPTION OF PLATES 138—16,
Illustrating Mr. J. E. S. Moore’s paper, “On the Structural
Changes in the Reproductive Cells during the Spermato-
genesis of Elasmobranchs.”
REFERENCE LETTERS.
a. Archoplasm. 6.c. Chromatic body. c¢. Centrosome. c.f, Foot-cells.
ch. Chromosomes. c.s. Seminiferous cells. 4.7. Flemming’s intermediate
bodies. fr. Fragmented portions of foot-cells. 2. Linin filaments. m.
Mittelstiick, and portion of flagellum contained in it. %. Nucleolus. x.y.
Nuclear groove. z.v. Nuclear sap-spaces. i.s. Inner spindle-sheath. 0..
Outer spindle-sheath. 7. Cytoplasmic radiations round the sphere. f. Fla-
gellum. z. Indeterminate body at the base of the flagellum.
The figures were drawn with Zeiss’? hom. immer., 2 mm., 140 ap., and
oculars 12, 18, except Figs. 1, 3—10.
Cells of the first spermatogenetic period.
Fic. 1.—Single primitive cells from which the contents of the ampulle are
formed,
Fic. 2.—Single seminiferous cell, of the first spermatogenetic period.
Fig. 3.—Early relative position of seminiferous and foot-cells.
Fie.4.— Do. do. with foot-cell in division.
Fie. 5.— Do. do. later.
Fig. 6.—Fragmentation of foot-cells.
312 J. E. S. MOORE.
Fic. 7.—Migration of foot-cells.
Fic. 8.—Arrangement of the contents of an ampulla, before migration of the
foot-cells.
Fie. 9.—Arrangement of the contents of an ampulla, with fragmentation
products.
Fic. 10.—Division of foot-cells.
Fic. 11.—Karly stages of division in the first spermatogenetic period.
Fies. 12—20.—Successively later stages of division in the first spermato-
genetic period.
Fic. 21.—Polar view of monastral spindle-figure.
Fie. 22.—Details of the division of the chromosomes.
Fies. 23—26.—Later stages of division.
Fic. 27.—Final details of the spindle, before separation of the daughter-
cells.
Fic. 28.—Final details of the spindle, before separation of the daughter-
cells.
Fic. 29.—Reconstruction of the nucleus in its surrounding vacuole.
Fic. 30.—Reconstruction of the nucleus in its surrounding vacuole.
The synapsis and the divisions of the first spermatogenetic
period.
Fie. 34.—Characters of the ampulle at the time of the synapsis.
Fic. 35.—Seminiferous cell in stage of transformation.
Fies. 36—41.—Successive stages of chromosome formation.
Fie. 42.—Separation of the centrosomes, and initial spindle formation.
Fic. 43.—Superficial distribution of the chromosomes when fully formed,
with further divarication of the centrosomes.
Fic. 44.—First stage of spindle formation.
Fies. 45—52.—Successively later stages of the same.
Fic. 53.—Seminiferous daughter-cells in later stage, with remains of
spindle figure between them.
Fies. 54—56.—Successively later stages of the same.
Fic. 57.—First stage in the reconstruction of the daughter-nuclei, with
residual spindle filaments attached to sphere.
Fie. 58.—Later stages of the same, showing translation of, and meta-
morphosis in the spheres.
Fic. 59.—The same.
Final division and structure of the spermatozoa.
Fie. 60.—Daughter-cells of first heterotype division, with vestigial fla-
gellum.
Fie. 61.—Daughter-cells of first heterotype division, with vestigial fla-
gellum.
CHANGES IN REPRODUCTIVE CELLS OF ELASMOBRANCHS. 313
Fig. 62.—Daughter-cells of first heterotype division, with vestigial fla-
gellum, showing cystoplasmic condensation about the sphere.
Fic. 63.—Daughter-cells of first heterotype division, with vestigial fla-
gellum, showing cytoplasmic condensation about the sphere.
Fig. 64.—Daughter-cells of first heterotype division, with vestigial fla -
gellum, showing cytoplasmic condensation about the sphere.
Fic. 65.—Initial stage of final division.
Fie. 66.—The same later.
Fie. 67.—The same later, showing divarication of the centrosomes at the
cell periphery.
Fie. 68.—Complete formation of chromosomes of the final heterotype.
Fie. 69.—Monastral spindle figure.
Fig. 70.—Monastral spindle figure, polar view.
Fies. 71—78.—Successively later stages of final heterotype division.
Fic. 79.—Daughter-cells of the final heterotype, showing translation of the
spheres, and initial stages of tail formation.
Fics, 80—81.—Successively later stages of the same.
Fie, 82.—Cells showing the origin of the archoplasmic vesicle, and peculiar
form of nucleolus.
Fic. 83.—Cells showing the origin of the archoplasmic vesicle, and peculiar
form of nucleolus ; showing elongation of the intra-cellular part of the flagel-
lum, and the attachment of the archoplasmic vesicle to the nuclear wall.
Fic. §4.—Cells showing the origin of the archoplasmic vesicle and peculiar
form of nucleolus; showing elongation of the intra-cellular part of the flagel-
lum, and the attachment of the archoplasmic vesicle to the nuclear wall.
Fie. 85.—Cells showing the origin of the archoplasmic vesicle and peculiar
form of nucleolus ; showing elongation of the intra-cellular part of the flagel-
lum, and the attachment of the archoplasmic vesicle to the nuclear wall;
showing position of the archoplasm, and re-formation of ring chromosomes,
Fics. 86—87.—Two views of the tail during its passage away from the
archoplasmic vesicle.
Fies. 88—90.—Successive stages in the elongation of the spermatozoids.
Fie. 91.—Optical section of spindle in first heterotype division, showing
outer and inner spindle tubes, o. s., 7. s.
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ON FECUNDATION, MATURATION, AND FERTILISATION. 315
Notes on the Fecundation of the Egg of Sphe-
rechinus granularis, and on the Matura-
tion and Fertilisation of the Egg of Phallusia
mammillata.
By
M. D. Hill, B.A.Oxon.,
Assistant to the Professor of Zoology in the Owens College, Manchester.
With Plate 17.
Ir is somewhat remarkable that since the late Professor Fol’s
(4) account of the “ Quadrille des Centres” in the Echinoderm
egg, up till very recently, no further work had been published
either to confirm or refute Fol’s original statements. It is
therefore with all the more satisfaction that zoologists will
welcome the conjoint paper of Messrs. Wilson and Matthews
(10), being, as it is, a record of so much careful and accurate
work. To the student of cytology, however, the theoretical
importance of a clear insight into the complicated phenomena
accompanying the maturation and fertilisation of ova is so
great, that all evidence gained from independent work, with
- the help of reliable optical instruments and the most fitting
reagents, necessarily becomes of some interest. I propose,
therefore, to give in this paper an account of a portion of the
work done by me whilst holding the British Association’s table
in the Marine Zoological Station at Naples. I have, however,
to regret that circumstances forced me to leave Naples before
I had completed my observations, especially on the maturation
of the sexual cells in Phallusia mammillata. In all cases
where a doubt still exists as to the real facts, I have been careful
to state so expressly. My thanks are due to the British
316 M. D. HILL.
Association for kindly allowing me the use of their table, and
to the officials of the station, by whom I was treated with all
possible friendliness.
The form I chose to work upon was Spherechinus granu-
laris, which is exceedingly abundant at Naples, and could always
be had in any number. Mr. Wilson studied Toxopneustes
variegatus, while Mr. Matthews divided his attention between
Asterias Forbesii andArbacia punctulata. With regard
to Wilson’s interesting observations on the polarity of the egg
and the axial relations of the two pronuclei, I am not in a
position either to corroborate or refute his statements, my
attention having been almost entirely directed to the behaviour
of the centrosomes. I will therefore pass over the first part
of his paper, which has to do with the living egg, without
further comment, The result of his and Mr. Matthews’ study
of series of sections of the eggs of the different forms they
worked upon are summed up in the following words :—“ After
the formation of the second polar body the egg archoplasm soon
disappears, and no egg centrum, or egg archoplasm (‘ ovocentre’
as opposed to ‘spermcentre’) can be discovered at any subse-
quent period. There is nothing like a quadrille to be seen save
in doubly fertilised eggs (‘Toxopneustes). The archoplasm of the
first cleavage-amphiaster is developed entirely from, or under
the influence of the sperm archoplasm (‘spermocentre’ of Fol),
and this is derived not from the apex of the spermatozoon, but
from its base, undoubtedly from the middle piece (Toxopneustes
Arbacia). . . . There is no centrum save as an artefact.”
With regard to all but the last sentence, my results are
practically the same as Wilson’s. In as far, therefore, as my work
agrees with his, I shall deal very shortly with the facts, dwelling
in greater detail on those points where our results differ.
Method.—At once after the fertilisation of a great number
of ova certain quantities were preserved, in a mixture of
corrosive sublimate and acetic acid, at intervals of about five
minutes, until the first cleavage-plane made its appearance.
This usually took place about one and a half hours after fertilisa-
tion, but the time varied greatly with the temperature of the
ON FECUNDATION, MATURATION, AND FERTILISATION. 317
surroundings. After being hardened in alcohol, the eggs were
embedded in paraffin, cut into sections, and stained with
Heidenhain’s iron-hematoxylin.
Fig. 1 is a drawing of a section of an unfertilised ovum,
killed immediately after leaving the parent’s body. The polar
bodies are given off before the egg leaves the mother, but the
nucleus has not yet taken up a central position. At this
stage there is no sign of an astrosphere or of a centrosome.
The next stage is taken (fig. 2) shortly after the entrance of
the spermatozoon, which may be at any point on the surface,
and is not affected by the position of the female pronucleus.
The tail of the spermatozoon has dropped off, and the head
and middle piece have turned completely round, so that the
latter is nearest the centre of the egg. That this rotation
takes place, as Wilson is the first to point out for Echinoderm
eggs, I had convinced myself long before seeing his paper.
The process is exactly parallel to that described by Fick (8) as
occurring in the egg of the axolotl. For avery short time
the middle piece persists as a small faintly-stained body attached
to the sperm head. It soon becomes separated, and is con-
verted into the astrosphere. At first the rays are very short,
and all start from a central point, but gradually they lengthen
out, and a finely granulated central mass makes its appear-
ance, in the midst of which lies a single centrosome. As the
astrosphere grows the granular central mass at first becomes
reticulate, and in this condition the division of the astrosphere
takes place, Fig. 5, but finally the network disappears, leaving
a clear homogeneous central mass. The centrosome is of
extreme minuteness. Figs. 2, 38, and 4 show the above stages.
The sperm head consists of a mass of chromatin enclosed in
a loose membrane. At first cone-shaped, it gradually becomes
more irregular in contour, till it appears as a roundish or oval
lump, and the membrane so closely approximated as to become
indistinguishable. As regards the so-called ‘fusion’ I have
nothing to add to Wilson’s account. The astrosphere divides
into two about the same time as the sperm head comes iuto
contact with the egg-nucleus, which has by this time taken
318 M. D. HILL.
up a central position. The two products of division both
resemble the original astrosphere at the stage drawn in fig. 3.
There is a finely reticular central mass in each, but in no single
instance was I able to see a centrosome. Although I find a
similar absence at this stage in Phallusia, still I believe that
further examination is all that is necessary to prove that the
centrosome exists at this stage also. The two astrospheres
gradually travel to opposite poles of the ovum, as Wilson has
already described. Shortly after taking up their positions
there, they exhibit each a clear inside space! (thegranular central
mass having disappeared), in the middle of which are two clearly
distinct centrosomes (fig. 6). It is probably this stage that
Fol interpreted as being the one in which the two halves of the
egg- and sperm-centrosomes respectively are about to fuse, one
sperm half with one egg half, to form two single composite
centrosomes (cf. Fol, fig. 9). From his drawings, however,
even when it is taken into consideration that they are but
rough woodcuts, one is tempted to doubt whether Fol ever
really saw a centrosome at all. It is quite certain, as Wilson
points out, that the clear area round the nucleus in which
the “ quadrille ” is drawn as taking place is an artefact. Ihave
obtained like results after using certain reagents, especially
with the eggs of sea-urchins which had been kept some time
in the tanks previously. With regard to the decrease in the
length of the rays (mentioned by Wilson) during the Amphiaster
(two-starred) stage, I cannot confirm his results. On the con-
trary, in some instances they are larger than at any other
time, reaching nearly to the periphery of the ovum, and the
“stratum corneum” is extremely well marked.
Further than the Amphiaster (two-starred) stage of the first
segmentation nucleus I have not investigated. The conclusions
which I think may be drawn from these facts I shall reserve
till later, in the general summary at the end of the paper,
1 By the word “space” I do not mean a hollow cavity in which the
centrosome is somehow suspended, but a portion of the protoplasm which is
so homogeneous as to give the impression, even when looked at under high
powers of the microscope, of being an empty space.
ON FECUNDATION, MATURATION, AND FERTILISATION. 319
Phallusia mammillata.
I studied the maturation and fertilisation of the ova of this
Ascidian, partly with a view to comparing the origin of and
the rdle played by the centrosomes with what I had found to
be the case in the egg of Spherechinus, but more especially to
compare the case of Phallusia with that of Styelopsis described
fairly recently by Julin (5). The method employed was pre-
cisely similar to the one already given for Sphzerechinus,
A mixture of 90 to 95 per cent. saturated solution of corrosive
sublimate with 10 to 5 per cent. glacial acetic was found in
both cases to be the best preservative when followed by Hei-
denhain’s iron hematoxylin stain.
Here again I took no account of the changes to be observed
in the living egg. These were sufficiently described by Stras-
burger (7) so long ago as 1875.
The ova were examined by means of sections in the ovary,
oviduct, unfertilised after leaving the parent’s body, and after
fecundation. Unfortunately with regard to the development
of the ova I can give no details. I never succeeded in getting
satisfactory preparations of the nuclear figures of the ovogones
whilein the germinative zone. (I use Boveri’s (1) nomencla-
ture in his well-known diagram of the sexual cells of Ascaris.)
I can therefore give no such details as described by Julin
for Styelopsis. Although I obtained preparations showing
karyokinetic division in the ovaries of very young Ascidians,
yet the cells themselves were so small that counting the
chromosomes was impossible. I found precisely the same
difficulty with the testes.
Transverse sections of the ovary show ovogones in various
stages in the zone of growth. The nucleus is relatively of
enormous size, is vesicular, and contains a large circular
“nucleolus” of chromatin (fig. 8). As the egg passes into
the branches of the oviduct, the nucleus begins to get smaller,
and lessens so quickly in size that it becomes hardly one
sixth of its original diameter. The nucleolus disappears, the
chromatin is more regularly dispersed in the form of a long
vol, 38, PART 2,—NEW SER. x
320. M. D. HILL.
thread, and then is split into eight chromosomes. The details
I have not been able to follow (fig. 9).
Formation of the first polar body.—The polar bodies
are given off shortly after the ova are shed into the sea water,
irrespective of whether they are fertilised or not. The
nucleus loses its membrane, a spindle is formed, and the
eight chromosomes are arranged in the equatorial plane. As
to what follows I do not wish to lay down any absolute facts.
The polar spindles are so excessively small, and the chromo-
somes lie so close to one another, that accurate observation is
a matter of extreme difficulty. From the study of a large
series of sections, however, I am convinced that the réle played by
the chromosomes is very different from what has been described
for Ascaris, and on which so many theoretical speculations
have been based. The eight chromosomes of the first polar
spindle lengthen out, become dumbbell-shaped, and finally
divide in the middle (fig. 10). Eight chromosomes pass into
the first polar body, which also divides karyokinetically into
two, each having eight chromosomes. (I have never counted
more than six or seven chromosomes in the products of
division of the first polar body, but I think it may be taken for
granted that there must be eight [fig. 11].) The chromo-
somes left in the first polar spindle again divide in the same
manner (?), and about eight—certainly more than four—chro-
mosomes pass into the second polar body (I have counted six
distinctly), and eight (?) remain. To exactly determine the
number of chromosomes left in the female pronucleus after
the formation of the second polar body, and before it passes
into the resting condition, is a matter of great difficulty, as the
time between the two phases is very short (fig. 12). I have
counted four, six, seven, eight, and nine in different instances.
This discrepancy is partly due to the great tendency the
chromosomes have to lump themselves together into one mass,
so that the female pronucleus resembles the nucleus of the
ovogone in having a large “nucleolus” of chromatin. This
is broken up into a network as soon as the nucleus develops
a membrane and passes into the resting condition. It then
ON FECUNDATION, MATURATION, AND FERTILISATION. 3821
withdraws somewhat from the periphery of the egg, though
still maintaining an excentric position. It should here be
remarked that throughout the whole process of maturation
there is no sign of a centrosome or archoplasmic sphere.
As is shown, however, in fig. 9 c, at either end of the spindle
is a deeply-stained body, which may be called a pseudocentro-
some. ‘This is nothing more than the point where the slightly
stained spindle-threads meet. Although there is no astro-
sphere, the protoplasm at the ends of the spindle is distiactly
modified, the reticular structure giving way to a more granu-
lated condition.
Fertilisation.—The spermatozoa consist of the usual
three pieces—head, middle piece, and tail. When ripe they are
very active, piercing through the two layers of test and follicle
cells in a very short space of time. The egg puts out a “ cone
d’attraction,’ which embraces the head of the spermatozoa.
The tail drops off, and the head rotates very rapidly. In fig. 13
the stage is drawn where the head has rotated 90°. At the
end of the middle piece is a deeply-staining body, which may
be the centrosome. Already the rays of the astrosphere are
apparent. The “cone d’attraction” subsides as soon as the
sperm-head has made its way into the ovum. The head itself
broadens and grows rapidly, until it reaches about twice its
original size. It then suddenly splits into two. At first regular,
these pieces gradually take on a beaded irregular shape, and
subsequently break up into small irregularly-shaped chromo-
somes, usually about eight or nine in number (figs. 14,15, and
16). These chromosomes are only transitory structures, at
least as far as outward appearances go, for very shortly after-
wards the male pronucleus passes completely into the resting
condition. Meanwhile the astrosphere has grown considerably,
and passed through the same phases of formation as described
in the case of Spherechinus. The rays are remarkable for
their length and thickness, the whole structure being much
coarser than the astrosphere of Spherechinus. The centro-
some is also very large and distinct, and soon after the centre
of the astrosphere has become finely granular, divides into
qe M. D. HILL.
two, although the corresponding division of the astrosphere
itself does not take place till somewhat later. As Boveri (2) has
described and figured for Ciona intestinalis, the astrosphere
lengthens out,— the rays contracting somewhat,—becomes dumb-
bell-shaped, and finally constricted off in the middle into two
separate spheres. In Phallusia, however, the pronuclei fuse
somewhat differently, as will be seen by comparing fig. 18 with
fig. 29 in Boveri’s paper (2). The two astrospheres and also
the two nuclei are at this stage very difficult to stain, and this
may account for the fact that from this until the Amphiaster
stage there was no trace of a centrosome to be found. The
interior of the astropheres show,a reticular structure, which is
maintained till the Amphiaster stage, when the centroscmes
become visible again, and the network gives place to a clear
“space,” or at most a slightly reticular central mass (figs,
17 and 18).
Tn the first cleavage-spindle I have counted from thirteen
to sixteen chromosomes, and on theoretical grounds it is neces-
sary to assume that the latter is the correct number, which
would give eight derived from the female, and eight from the
male pronucleus respectively. I do not wish, however, to lay
too much stress on the exactitude of these numbers, though I
believe they are approximately correct (fig. 19).
With regard to the conditions of the chromatin in the
development of the spermatozoa, it was only possible to sub-
stantiate the fact that one spermatocyte I.gives rise to two
spermatocytes II, and these again each divide into two, form-
ing in all four spermatids. Further, the two nuclear divisions
take place without any intermediate resting phase. Beyond
this, however, nothing could be definitely ascertained as to the
number of the chromosomes in any stage of development,
owing to the extreme minuteness of the cells themselves.
It may be interesting to note that the ova of certain
specimens, which had been kept in the aquarium tanks for a
long time, were found to be infested with long rod-like bodies
seemingly of a bacillic nature. Although these ova were
mixed with ripe sperm, no single fertilisation ever took place,
ON FECUNDATION, MATURATION, AND FERTILISATION. 323
That they were not, however, quite destitute of vitality is
shown by the fact that polar-spindles were formed, although of
an apparently pathological nature (fig. 20).
For the structures needing high powers of the microscope,
Zeiss’s apochromatic 2:0 mm., apert. 1°30, homogen. immers.
was used with compensating ocular No. 8, and an Abbé
condenser.
SUMMARY AND CONCLUSION.
The above results may be shortly summarised as follows:
(1) InSpherechinus granularis and Phallusia mam-
millata there is no egg astrosphere or egg centrosome. Both
these structures are brought into the ovum by the spermatozoon,
and they give rise by division to all the subsequent astrospheres
and centrosomes throughout ontogeny. There is, consequently,
no such thing as a “ quadrille.”’
(2) In both forms the sperm head rotates through 180°, and
the astrophere and centrosome are elaborated out of or under
the influence of the middle piece.
(3) In Phallusia the nucleus of the ovocyte I-contains eight
chromosomes irregularly dispersed throughout its substance.
(4) In the two succeeding nuclear divisions these eight
chromosomes divide into sixteen each time, eight passing out
into the first and eight into the second polar body. There is
consequently no equalling or “reducing division” at this
period.
(5) The sperm head breaks off into eight chromosomes, and
sixteen are found in the first segmentation spindle.
The origin and fate both of astrospheres and centrosomes in
Spherechinus and Phallusia certainly tend to support Boveri’s
generalisation, that the ovum when ready for fertilisation
possesses two out of the three essentials for cell division, viz.
cytoplasm and nucleoplasm, but is without the third, or
centrosome; while, on the other hand, the ripe fspermatozoon
possess nucleoplasm and centrosome, but little or no cytoplasm.
It is therefore only when the union of the male and female cells
takes place that the requisite conditions for development are
324. M. D. HILL.
fulfilled. Where careful observations have been made this
supposition has almost always been found to hold good, e.g. in
the case of Rhynchelmis (Vejdoffsky, 8), Ascaris (Boveri, 2),
Axolotl (Fick, 8), and Styelopsis (Julin, 5); while only in
one instance has it been incontestably denied, viz. by Wheeler
(9) for Myzostomum. When, however, it is borne in mind
that nuclear and cell-division can take place without the
presence of an astrosphere or centrosome, the supposed im-
portance of the latter as an organ of division is greatly lessened.
It is, moreover, extremely difficult to offer any explanation as
to why the first cleavage spindle should have two astrospheres
and two or four centrosomes, while the polar spindles
(Phallusia, Ascaris, Sagitta, Ciona, &c.) may have none.
Again, the relation between centrosome and astrosphere is
very obscure; are the rays produced by the action of the
centrosome, or vice versd? The former alternative seems to
the most probable in the case of Phallusia, where, as is shown
in fig. 18, while the spermatozoon is quite at the surface of the
ovum the posterior half of the middle piece has become a
centrosome, and already acted on the cytoplasm of the egg to
produce a radial appearance. In spermatozoa simply killed
and stained there is no sign of a centrosome in the middle
piece, which points to a direct metamorphosis of part of the
middle piece into a centrosome as soon as the spermatozoon
penetrates into the ovum.
Neither in Spherechinus nor Phallusia is there any evidence
to point to the centrosome as being artefact. In the former it
appears to be all that remains of the middle-piece, and is
brought to light by the latter’s disintegration into granules
and final disappearance.
Finally, with regard to a more important matter, viz. the
relations of the chromatin substance during the maturation
of the ovum, Phallusia agrees more with the vertebrates
than the invertebrates. I cannot go into the subject, however,
in any great detail, because I have been unable to trace the
history of the eight chromosomes in the nucleus of ovocyte I.
Still, there are one or two points worth noting. In the first
Ww
ON FECUNDATION, MATURATION, AND FERTILISATION. 320
place, Phallusia agrees with all the other forms in the ovocyte I,
containing half the number of chromosomes typical for the
given species. Riickert (6) puts this first and foremost among
the few ascertained facts that we have at present in this com-
plicated subject. He writes: —‘‘ Alle genauere Untersuchungen
der letzen Jahre stimmen darin ueberein, dass schon vor der
ersten Reifungstheilung Chromatin-portionem auftreten deren
Zahl die Hilfte betriigt von der Normalzahl der Chromosomen
der betreffenden Species. ... Am klarsten liegt der Rei-
fungsvorgang, wenn sie aus vier deutlich geschiedenen Unter-
abtheilungen, Stabchen oder Kugeln, bestehen ( Vierergruppen).
In diesem typischen Fall geht die Reifung bekanntlich folgen-
dermassen vor sich: Durch zwei ohne Ruhephase auf einander
folgende mitotische Reifungstheilungen werden die Vierer-
gruppen in der Weise gevierteilt dass in jede Enkelzelle
(Spermatiden, Reifes Ei) von jeder Gruppe ein einziges
Stabchen als Chromosoma gelangt, womit die schon durch
die Zahl der Vierergruppen vorbereitete Reduktion definitiv
vollzogen wird.”
It is obvious that if these “ Vierergruppen” are to be looked
for in Phallusia, it is not in the nucleus of the ovocyte I-that
they are contained, but in the three polar bodies and the female
pronucleus. That is to say, there are eight ‘‘ Vierergruppen,”’
as each of the eight chromosomes divides twice, making thirty-
two pieces in all. Julin, on the other hand, looks upon the
eight chromosomes in Styelopsis as themselves forming two
“‘ Vierergruppen;” and the subsequent formation of the polar
bodies, albeit on these he confesses to have made few observa-
tions, bears out this view. At the first polar division four
chromosomes pass out, and at the second two, leaving two
quarters of the two original chromosomes, which had divided
twice precociously to give rise to two ‘‘ Vierergruppen.” The
spermatozoon brings into the ovum two quarters likewise, so
that the first segmentation nucleus possesses four chromosomes,
or double the number contained in the nucleus of the ovocyte I.
In fact, as regards the number of chromosomes, Styelopsis
exactly resembles Ascaris bivaleus. In Phallusia each of
»
326 M. D;) Hid:
the original eight chromosomes is a single complete structure ;
and if the process were like what occurs in Styelopsis, the
nucleus of the ovocyte I.would contain thirty-two chromo-
somes. Owing to its extremely small size this is a mechanical
impossibility, and hence there is no such precocious splitting
to form eight “‘ Vierergruppen.”
Hence it seems to follow that those who would see in this
precocious splitting a process especially brought about in
order to ensure a more varied combination of ancestral units,
are obliged to recognise that it may take place in one species
of the same group and not in another. In Phallusia at no
one stage of maturation\there /are \thirty-two chromosomes
“to choose from,’ so to speak, but the division takes place
exactly as in normal cell-division, except that the chromo-
somes divide transversely and not longitudinally (? in second
polar body). On the contrary, the two cases of Phallusia and
Styelopsis seem to point towards the phenomena of matura-
tion being nothing more than normal cell-division. Whereas
in the former case the large number of chromosomes prevents
precocious division, in the latter the small number allows it,
and it is possible that some good to the organism may be
gained thereby. The point to be found out is at what stage
does the reduction from sixteen to eight chromosomes in the
development of the sexual cells of Phallusia take place, which
is the only real ‘ reducing division” of the kind.
The fact that the process of maturation of the egg of Phal-
lusia resembles what has been described for the egg of certain
vertebrates, may be an additional point of evidence for a
phylogenetic connection between them and the Ascidians.
Narites; March, 1895.
ON FECUNDATION, MATURATION, AND FERTILISATION. 327
ADDENDUM.
SHortiy after the above notes were sent in to await publi-
cation an interesting and suggestive paper by Boveri! appeared,
which it is necessary for me here to briefly notice.
If I understand Boveri aright, his results and mine agree
up to the stage I have figured in fig. 3, but after that they
differ considerably. In the stage I have drawn in fig. 6 Boveri
finds the centrosome, not as one, or two, very minute inten-
sively staining granules, but as a hollow vesicle swollen to such
a size as to be separated from the radial striations by a very
narrow “‘heller Hof.” He finds no trace of any deeply-staining
central body.. In going carefully over my preparations for a
second time, I cannot discover the slightest reason for Boveri’s
interpretation. The one, or two, central bodies are so clearly
defined that I am surprised Boveri has not found them. Of
course it is possible that, as he suggests, this body, or bodies,
may be merely the “ Centralkorner” of the centrosome ; but if
this is so I am at a loss to know where the real centrosome is.
I must, however, state that in one or two preparations I have
distinctly seen a ring round the centrosomes, e.g. fig. 6, which
according to Boveri should be the centrosome in an earlier
stage of swelling up than he has figured. I consider, how-
ever, that this appearance is unimportant and may be an arte-
fact, as in many cases where the centrosomes were very plain,—
e.g. in figs. 3, 4, and 11,—there was no trace of it. It is, I
think, a matter of some regret that Boveri has as yet only
published a single figure illustrating his work. I hope, how-
ever, I may not be construed as in any way trying to depreciate
the accurate and valuable work of such a distinguished observer
as Professor Boveri, who has had such a far wider experience
in cellular morphology than myself, but in this particular case
I cannot help thinking that his interpretation is erroneous.
1 “Ueber das Verhalten der Centrosomen bei der Befruchtung des Seeigel-
ries nebst allegemeinen Bemerkungen tiber Centrosomen und Verwandtes.”
‘Verhand. der Phys.-med. Gesell. za Wiirzburg,’ N. F. Band, xxix.
328 M. D. HILL.
Finally, I may mention that the process I have described,
viz. the homogeneous middle-piece of the spermatozoon be-
coming first the granular, then the reticular, and finally again
the homogeneous central mass of the astrosphere (figs. 3, 4, 5,
and 6 for Echinus, and figs. 14, 15, 17, 18, and 19 for Phal-
lusia) is almost exactly the same as Vejdoffsky described for
Rhynchelmis. From these results he believes that protoplasm
is at first quite homogeneous and structureless; that then very
small granules appear which group themselves together to form
a reticulum, which may become once more homogeneous. I
do not pretend, however, to use this word “ homogeneous”
other than as before explained, in a purely relative sense.
With higher powers of the microscope I see no reason to sup-
pose that the middle-piece would not itself present a “ Waben-
structur,” and that the network is anything more than a coarse
protoplasmic reticulum. It is right, moreover, to mention
that I was not biased by Vejdoffsky’s work when noting the
above process, for my attention was not drawn to this parti-
cular point until reading his paper again after my own had
been finished and sent in for publication.
A point worthy of notice is that, at least in one particular
instance (fig. 14), the homogeneity of the central mass has
been arrived at before the division of the spermastrosphere.
M. D. Hitt.
June, 1895.
List oF WORKS REFERRED TO.
1. Boveri, Ta.— Befruchtung,” ‘Erg. der. Anat. u. Entw.,’ Merkel and
Bonnet, Band i, 1891.
2. Bovert, Tu.—‘Zellen Studien,’ i—iii, 1887, 1888, 1890, Jena.
3. Firox, R.—** Ueber die Reifung und Befruchtung des Axolotleies,” ‘ Zeit.
wiss. Zool.,’ Bd. lxv, 1893.
4. Fou, H.—“ Le Quadrille des Centres,” ‘Arch. Sci. Phys. et Nat.,’ xxv,
1891.
ON FECUNDATION, MATURATION, AND FERTILISATION. 329
5. Junin, C.—“ Structure et développement des glandes sexuelles ; ovogénése,
spermatogénése et fécondation chez Styelopsis grossularia,”
‘Bull. Se. de la France et de la Belgique,’ t. xxxv, 1893.
6. Ruckert, J.— Die Chromatin reduktion bei der Reifung der Sexual-
zellen,” ‘Ergeb. der Anat. u. Entw.,’? Merkel and Bonnet, Band iii,
1893.
7. STRASBURGER, E.—‘ Zellbildung und Zelltheilung.’
8. Vesporrsky, F.—‘‘ Entwickelungsgeschichtliche Untersuchungen,” Heft
1, ‘Reifung, Befruchtung, und Furchung des Rhynchelmis-Lies,’
Prag., 1888.
9. WuereteR, W. M.—‘ The Behaviour of the Centrosomes in the Fertilised
Egg of Myzostoma glabrum,” ‘Journal of Morphol.,’ vol. x, 1.
10. Witsoy, E. B., and Marruews, A. P.—‘‘ Maturation, Fertilisation, and
Polarity in the Echinoderm Egg,” ‘Journal of Morphology,’ vol. x,
No. 1.
DESCRIPTION OF PLATE 17,
Illustrating Mr. M. D. Hill’s “‘ Notes on the Fecundation of
the Egg of Spherechinus granularis, and on the
Maturation and Fertilisation of the Egg of Phallusia
mammillata.”
cent. Centrosome. chr. Chromosomes. f. prox. Female pronucleus.
m. pron. Male pronucleus. 1st p. 6. First polar body. 2d p. b. Second
polar body. Sp. ast. Sperm astrosphere. Sp. 2. Sperm head. Sp. cent. Sperm
centrosome. sé sey. sp. First segmentation. 1s¢ seg. x. First segmentation
nucleus. m.p. Middle piece. 1s¢ p. s. First polar spindle. 2xd p. s. Second
polar spindle.
Figs. 1—6.—Cross sections of eggs of Spherechinus granularis,
Fig. 1. Section of unfertilised egg.
Fig. 2. Section of egg ten minutes after fertilisation.
Fig. 3. Section of egg fifteen minutes after fertilisation. Sperm astro-
sphere has grown in size, and the centrosome is apparent.
Fig. 4. Section of egg twenty minutes after fertilisation. Sperm astro-
sphere is in contact with female pronucleus.
es
300 M. D. HILL.
Fig. 5. Section of egg twenty-five minutes after fecundation. Male pro-
nucleus has “fused”? with female, and astrosphere has divided. No
trace of sperm centrosome.
Fig. 6. Section of egg forty-five minutes after fertilisation. Centrosome
has appeared again and divided into two in each astrosphere. Central
mass has become entirely homogeneous.
Fics. 7—20.—Phallusia mammillata.
Fig. 7. Cross-section through ovary of Phallusia mammillata, with
ovarian ova.
Fig. 8. Cross-section of an unfertilised ovum directly after leaving ovi-
duct. Nucleus is enormously reduced in size, and chromosomes are
eight in number.
Fig. 9. Shows changes in nucleus in forming first polar spindle.
Fig. 10. Shows extension of first polar body.
Fig. 11. The first polar body has divided into two, and the second polar
body has also been formed. (Slightly less magnification than Fig. 10.)
Fig. 12. Shows the changes through which remaining chromosomes from
second polar spindle pass during formation of female pronucleus.
Fig. 13. Cross-section of egg a few minutes after fertilisation. Sperm
head has rotated through 90°.
Fig. 13a. Sperm head enlarged.
Fig. 14. Cross-section of egg ten minutes after fertilisation. Sperm
head and astrosphere have increased in size, and centrosome is evident
in the granular central mass.
Fig. 15. Cross-section of egg fifteen minutes after fertilisation, Sperm
head has split into two.
Fig. 16, Cross-section of egg twenty minutes after fertilisation. Sperm
head has broken up into eight chromosomes, and central mass has
(unusually early) become homogeneous.
Fig. 17, Sperm head has passed into the resting stage of the male pro-
nucleus. The astrosphere is in act of division, and central mass
reticular.
Fig, 18. Sperm astrosphere has divided into two, and two pronuclei are
in contact.
Fig. 19, First segmentation spindle with sixteen chromosomes. Central
masses of atrospheres have become nearly homogeneous, and centro-
somes divided into two.
Fig. 20. Cross-section of an unfertilised ovum, taken from oviduct of a
specimen living in aquarium.
Fic. 21. Shows transformation of middle piece of spermatozoon into central
mass of astrosphere. Slightly diagrammatic.
FURTHER REMARKS ON THE CELL-THEORY. Bol
Further Remarks on the Cell-theory, with a
Reply to Mr. Bourne,
By
Adam Sedgwick, F.R.S8.
In a paper published last autumn (this Journal, vol. 37),
I called attention to the apparent inadequacy of the cell-
theory. Recently a criticism upon my article has appeared
from the pen of Mr. G.C. Bourne, to which I may be allowed
to devote a few words. But before replying to Mr. Bourne, I
should like to state my position with regard to the theory a
little more fully than I have hitherto done. In my previous
communication I used the word “inadequacy”’ because it seemed
to me to express, as nearly as possible, my own views with
regard to the theory. A theory to be of any value must ex-
plain the whole body of facts with which it deals. If it falls
short of this, it must be held to be insufficient or inadequate ;
and when at the same time it is so masterful as to compel men
to look at nature through its eyes, and to twist stubborn and
uncomformable facts into accord with its dogmas, then it
becomes an instrument of mischief, and deserves condemna-
tion, if only of the mild kind implied by the term inadequate,
The assertion that organisms present a constitution which
may be described as cellular is not a theory at at all; it is—
having first agreed as to the meaning and use of the word cell
—a statement of fact, and no more a theory than is the asser-
tion that sunlight is composed of all the colours of the spec-
trum. The theory comes in when we try to account for the
cellular constitution of organisms; and it isthis theoretical
part of the cell-theory, and the point of view it makes many
of us assume, that I condemn. It is not the word “ cell” which
332 ADAM SEDGWIOR.
I am at issue with, for structures most conveniently called
cells undoubtedly exist, as the ovum, spermatozoon, lymph-
cells, &c.; and I fully agree that the phenomenon called cell-
formation is very general in organic life. But at the same
time I hold with Sachs and many others that it is not of
primary significance, but ‘merely one of the numerous ex-
pressions of the formative forces which reside in all matter.”
No one who has studied animal tissues could for one moment
deny that nuclei have in many cases a relation to the
surrounding protoplasm, a relation which is expressed in
the arrangement and structure of that protoplasm. They
have not always this relation, but it is usually present, and
the question is, how are we to interpret it? That we
cannot interpret it finally until we know the relative values of
nucleus and extra-nuclear protoplasm, and the functional re-
lation between the two, is clear; but we may form and hold
provisional theories. The hypothesis or idea which holds the
field at the present day is the cell-theory in its modern form.
This theory, recognising the cellular structure (while not ad-
miring the phrase, I must use it for want of a better one)
asserts that organisms of Metazoa are aggregations or colonies
of individuals called cells, and derived from a single primitive
individual—the ovum—by successive cell-divisions ; that the
meaning of this mode of origin is given by the evolution
theory, which allows us to suppose that the ancestor of all
Metazoa was a unicellular Protozoon, and that the develop-
ment of the higher animals is a recapitulation of the develop-
ment of the race. Thus the holoblastic cleavage of the ovum
represents the process by which the ancestral Protozoon be-
came multicellular, and the differentiation of the cells into
groups the beginning of cellular differentiation. According
to this view the order is: unicellular stage—multicellular
stage—differentiation of cells into tissue elements ; cellular
structure preceded cell-differentiation, and to get tissues you
must first have cells. And ten years ago it was commonly
held that these cells were primitively separate from one another,
and that the connections found between them in the fully
FURTHER REMARKS ON THE CELL-THEORY. B00
formed tissues were secondary. You had your neuro-epithelial
cell, and your musculo-epithelial cell, each derived from a
distinct cell produced by division of the ovum ; and the question
was, how do they find each other and become connected?!
Further, in studying the development of a tissue you had to
find a group of cells, each of which became modified into one
tissue element. Thus the primitive streak was a proliferating
mass of cells which eventually gave rise to a number of meso-
dermal tissues; the nerve-crest similarly was a mass of cells
which gave rise to nervous tissues; a nerve-fibre was one of
these cells elongated, and before you would get your nerve-cell
and fibre you must have your nerve-crest cell produced by
division from the cells of the nerve-cord, and subsequently
sending out a process which elongated and travelled to the
periphery as a nerve-fibre.
My work on Peripatus first led me to doubt the validity of
this view of the origin of the Metazoon body. In the first
place I found that in some forms there is no complete division
of the ovum, and on examining the facts I discovered that such
forms were more numerous than had been supposed. It
therefore appeared that in some Metazoa the ovum divided
into completely separate cells, while in others it did not so
divide. The question then arose, which of these methods is
primitive ? and the answer naturally was, the complete division,
because this fitted in with our ideas as to the supposed evolu-
tion of the Metazoa from a colonial Protozoon. But on
reflection this difficulty arose: the individuals of colonial
Protozoa are in protoplasmic connection, while the cells of the
completely segmenting ova are separate; so that the parallel
between the ontogeny and the phylogeny breaks down in an
important particular. To get over this difficulty it was
necessary to suppose that the isolation of the segments of
incompletely segmenting ova was apparent and not real, that
they were really connected by protoplasmic strands which had
1 For exposition of this view vide Flemming, ‘ Zell-Substanz, Kern u.
Zell-Theilung,’ Leipzig, 1882, p. 74, and Balfour’s Address to the Depart-
ment of Anatomy and Physiology at the British Association in 1880,
304 ADAM SEDGWICK.
escaped observation. But, on the other hand, there was the
possibility that the completely segmenting ova were secondary
acquisitions of ontogeny, and that the development in such
forms as Peripatus, Alcyonaria, &c., was more primitive, and
that the passage from a Protozoon to a Metazoon had taken
place by way of a form more resembling a multinucleated
cilated Infusorian than Volvox. In other words, that the
differentiation of the Metazoa had been effected in a continuous
multinucleated plasmatic mass, and that the cellular structure
had arisen by the special arrangement of the nuclei in reference
to the structural changes. This was the stage to which my
researches on Peripatus led me. Since then I have paid
attention to Vertebrata, and I have found that a number of
embryonic processes have been wrongly described, amongst
them such important matters as the development of nerves
and the origin of the mesoderm ; and I thought that I traced the
errors referred to to the dominating influence of the cell-theory
in its modern form, for the facts seemed so obvious in them-
selves that it would have been impossible to make any mistake
about them had they been examined without the prejudice
imparted by a preconceived theory. A theory which led to such
obvious errors must, I thought, be wrong, and I denounced it.
But my denunciation in no way implies that I fail to recognise
the so-called cellular structure of organisms or their origin
from the one-celled ovum. On the contrary, I was led toa re-
consideration of the question, what is the meaning of the pre-
dominance of the structure called cellular, which is characterised
by a definite relation of the nuclei to the functional tissues,
and of the fact that the organism so often passes through a
unicellular stage. With regard to the former I must say
that I have arrived at no conclusions which enable me to
formulate to myself any satisfactory hypothesis, and, as
I stated at the outset, I do not think it is possible
to do this until we acquire some more understanding of
the relative function of nuclei and protoplasm, But with
regard to the latter there are some facts which might
well be considered. In the first place, the unicellular origin
FURTHER REMARKS ON THE CELL-THEORY. 335
is only found in sexual reproduction, not in asexual. The
characteristic of the unicellular form is its simplicity of struc-
ture, and the essential feature of sexual reproduction is the
conjugation of the reproductive cells. Now in the Protozoa,
in which the amount of formed tissue is generally slight and
the structure of the body simple, conjugation can and does
often take place between the ordinary form of the species.
But in the Metazoa, in which conjugation is as necessary a
phenomenon in the specific cycle as in Protozoa, conjugation
is impossible between adult or ordinary individuals of a species
from mechanical causes. How is this difficulty got over in
nature? My answer is, by the formation of special individuals
of extremely simple structure—a structure so simple that conju-
gation between them is possible. To put the matter in another
way, I should regard the ordinary dicecious Metazoon as a tetra- ,
morphic species, consisting of male, female, ovum, and _ sper-
matozoon, the two latter being individuals which are specially
produced to enable conjugation to take place.
Mr. Bourne, in his criticism, begins by complaining that he
cannot ascertain from my article my own views on the subject
of the cell-theory. Why should he expect or wish to discover
them? My remarks were simply directed to show the short-
comings of the theory with regard to certain anatomical facts.
As explained above, my own view is that the ceil-theory is
inadequate to explain the facts, and that it is not possible at
present to explain them by any theory. He proceeds to state
that Iam abusive because I say that certain observers “ are
constrained by this theory with which their minds are saturated,
not only to see things which do not exist, but also to figure
them” (I am referring to embryonic mesoderm of verte-
brates). He calls this abuse, not argument. I venture to
differ with him—it is neither abuse nor argument ; it is merely
a statement of fact (unless, indeed, it be considered abusive
to say that a man accepts and believes in the cell-theory). If
you disbelieve it, consult the memoirs of the last twenty years
in which this tissue is referred to, and in most of them you
voL. 38, PART 2.—NEW SER. Y
336 ADAM SEDGWICK.
will find the mesenchyme described or figured as consisting of
branched, isolated cells.
Mr. Bourne then refers to certain remarkable researches
which emphasize the distinction and complete isolation of the
cells formed in the segmentation of the egg ; with what object
is not apparent, for he proceeds on the next page to condemn
those who hold that the organism is constituted of independent
and isolated units. He even maintains that no reputable bio-
logist holds such a view. However that may be, I do not
think that his quotation from Haeckel in support of his con-
tention is a happy one, for it is perfectly clear from the quota-
tion that Haeckel, who indeed goes so far as to call the units
individuals, holds the view which Mr. Bourne condemns.
Haeckel even calls them individuals of the first order, and
says that in the adults they frequently unite to form colonies ;
and he particularly implies that the loss of independence
caused by their colonial union is secondary. Mr. Bourne
has completely failed to grasp Haeckel’s meaning, else how can
he write as he does on the same page with the quotation from
Haeckel—* So that, as a matter of history, while plants used
to be considered to be colonies of independent life units,
animals were not.”
The most remarkable part of Mr. Bourne’s criticism
is that in which, after strongly animadverting on my
statement that it is difficult if not impossible to euunciate
the cell-theory in a manner satisfactory to every one,—indeed
he quotes from Schwann and Hertwig to show how precisely
it can be stated,—he proceeds to devote a dozen or more
pages of his paper to a consideration of the various views
which are held and which may be held as to what a cell
really is! If this amount of discussion is required to arrive
at the meaning of the word cell, is it likely that there will
be simple agreement as to the theory which is supposed to
explain and account for the so-called cellular constitution of
organisms ?
Again he says, referring to my description of the embryonic
mesoderm as a protoplasmic reticulum with nuclei at their
FURTHER REMARKS ON THE CELL-THEORY. 337
nodes: ‘ Does he accept the logical consequences of this, and
say of the epithelial cells of the salamander or of unstriped
muscle fibres, that they are protoplasmic reticula with nuclei at
their nodes ? ”’
Now, with all due respect to Mr. Bourne’s logical faculties,
may I ask him where logic comes in here? If I describe
London as a network of streets, with public-houses at many of
the street corners, am I obliged by logic to give the same
description of the Gog-Magog Hills ?
However, on the next page Mr. Bourne makes up for all the
hard strictures he has passed upon me; for he says that, after
all, reflection may induce us to abandon the cell-republic or
colonial theory ; thus he admits a very important part of my
contention, for the assertion that organisms present a consti-
tution which may be described as cellular is not a theory at all,
it is a statement of fact (having agreed to the use of the word
cellular). The theory comes in when we try and account for
the cellular constitution of organisms; and it is this theoretical
part of the cell-theory which I condemn, and which Mr. Bourne
after a great effort agrees with me in condemning. At the
same time it is possible that we might still disagree as to the
meaning of the word cellular.
May I call attention to Mr. Bourne’s remarkable faith in
the rapidity of evolutionary changes? He says (page 169)
that Schwann’s assertion that “the elementary parts of all
tissues are formed of cells, &c.,” is even more true to-day
than when it was written. Also I should like to know how he
reconciles the implication at the top of page 170, that
“specialisation is not possible in continuous tracts of proto-
plasm,” with the statement a few lines further on, that ‘in the
Protozoa there is differentiation within the limits of a single
corpuscle.”’
The criticism on page 172 as to my use of the word empty
is not quite fair. On reference to the context it will be seen
that the word empty clearly means “empty of structural
elements.”
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THE DEVELOPMENT OF ASTERINA GIBBOSA. 309
The Development of Asterina gibbosa.!
By
E. W. MacBride,
Fellow of St. John’s College ; Demonstrator in Animal Morphology in the
University of Cambridge.
With Plates 18S—29.
THE investigations which form the subject of the present
memoir were commenced with the object of seeking in Asterids
the results which the author (14) had already obtained from the
study of Ophiurids, viz. the development of the so-called heart
and its accompanying sinuses.
A study of the literature soon led to the conclusion that our
knowledge of the development of most organs in the Asterid body
was very defective, and that a thorough revision of the whole
embryonic and larval history would be most desirable. This
work has occupied my attention for the last two years, and I
am now in a position to give a fairly complete account of the
whole organogeny; an account which will, I hope, place our
knowledge of Asterid development on the same level as that
to which our acquaintance with Crinoid ontogeny has been
raised by the researches of Bury (1) and Seeliger (18) ; I have
to express my warm thanks to Mr. Sedgwick not only for the
suggestion of Asterina gibbosa as a proper type to investi-
gate, but also for much assistance and advice in revising the
proofs of this paper.
That there was an immense lacuna in our knowledge to be
1 A preliminary account of the observations recorded in this paper was the
subject of the successful essay in the competition for the Walsingham Medal
of the University of Cambridge in 1893.
VOL. 38, PART 3.—NEW SER. Z
3840 E. W. MACBRIDE.
filled up will become evident when I state in the first place,
that my researches have made it clear that the Crinoids are
only very distantly related to the other classes of Echinoderms,
and secondly, that our previous knowledge of the metamorphosis
of Asterids and their allies was confined principally to the
changes which take place in their external form.
It will be most convenient, I think, to give first a general
account of the development, and then to point out how far the
results of other workers have been confirmed, as by this means
needless repetition will be avoided.
Methods adopted.
My material consisted of a large number of larve of all
stages including those which had just completed the metamor-
phosis, and of a considerable number of young adults varying
from an age of about three weeks to several months from the
metamorphosis. Of these the former, with the exception of two
small collections made by myself in Plymouth, 1893, and Jersey,
1894, were collected for me and preserved according to my
directions by the authorities of the Naples Zoological Station ;
the latter were obtained for me and preserved by myself during
my stay in the Naples Station in 1892. I have to express my
deep sense of my indebtedness to Prof. Dohrn for his kindness
in meeting my wishes, and to Cav. Salv. Lo Bianco for the
extreme care and attention with which he carried out my
directions.
All the stages were preserved in osmic acid, followed by
14—24 hours in Miiller’s fluid, as this method had yielded me
the best results in the case of Ophiurids. It makes the speci-
mens exceedingly brittle, but at the same time gives the most
excellent preservation of the minute histology; preserved in
this manner the various tissues are differentiated as to their
staining capacities, so that the sections look almost like coloured
diagrams.
On account of their brittleness, and in order to avoid
shrinkage in the tissues, the larvee were embedded in celloidin,
and the celloidin block subsequently embedded in paraffin.
THE DEVELOPMENT OF ASTERINA GIBBOSA. 341
They were then cut into series of sections in most cases 4}
thick—in the case of the adults 7 w; these sections were
mounted on hot water on the slide to flatten them, and stained
in either Grenacher’s hematoxylin or Mayer’s carmalum. Two
points of interest in connection with this process may be
mentioned : first, I found that when the slide was transferred
from turpentine to absolute alcohol some of the sections were
sure to be lost, but that this could be avoided by placing the
slide for a minute or so, after taking it out of turpentine, into
oil of cloves, and thence into 90 per cent. alcohol; second, that
the readiness with which sections, especially when overcharged
with osmic acid, will take up either hematoxylin or carmalum
is greatly increased by immersing them for twenty-four hours
in borax-carmine, though they do not acquire a particle of stain
from it.
In the youngest stages the osmic acid produces too great
impenetrability for either celloidin or paraffin, and accordingly
my best results were obtained from some specimens preserved
for me by Sig. Lo Bianco in a mixture of three parts concen-
trated aqueous solution of corrosive sublimate, and one part
glacial acetic acid. This method also gives most excellent
preservation, though without that fine differentiation of the
tissues yielded by osmic acid and Miiller’s fluid; as during
the stages in question however the larve consist almost exclu-
sively of epithelial cells, this is not a matter of any importance.
This second method was recommended to me by Dr. Hisig.
The orientation of the specimens was one of the chief
difficulties to be overcome. I found that the best results were
given by horizontal sections perpendicular to the median
sagittal plane of the larva, and sections parallel to the disc and
perpendicular to the median axis of symmetry in the just
metamorphosed star-fish. The planes, to which in these two
cases the sections are cut parallel, viz. a median horizontal
plane in the larva and the plane of the disc in the adult, make
an angle of about 70° with each other ; and hence it is difficult
to correlate sections cut parallel to the one with those cut
parallel to the other. I shall call these planes the “larval”
342 E. W. MACBRIDE.
and “ adult’? planes respectively. A rudiment of the przoral
lobe of the larva is retained, as we shall see, until the close
of the metamorphosis, and by means of it I found it possible
to determine the direction of the “ larval” plane up till the
adult form has been almost attained. Hence, by cutting
sections parallel to the larval plane, one can follow the internal
changes of the metamorphosis step by step; then when the
metamorphosis is complete it is possible to correlate with less
difficulty sections cut parallel to the two planes, and the
further history may be followed via, so to speak, the adult
plane. This was the course which I adopted; and I also
penetrated back a considerable distance from the adult con-
dition into the stages of the metamorphosis by sections parallel
to the adult plane, and so confirmed results obtained by the
other method. For the youngest stages of all, which are
spherical, orientation is, of course, impossible, and one has to
trust to chance to getting sections in the proper direction ;
but it is fairly easy to recognise from their appearance when
this is so.
General Account of the Development.
The ontogenetic history of the Asterina gibbosa may be
conveniently divided into three parts: first, the development of
the bilaterally symmetrical larva from the egg; second, the
metamorphosis of this larva into the young star-fish ; and lastly,
the gradual development of what we may term the young adult
into the sexually mature form. I have made no observations
on the segmentation of the egg, nor on the gastrulation; my
work, properly speaking, commences with the completed
gastrula, and my material was not suitable for observing the
development of the calcareous plates. On all these points I
intend, however, for the sake of completeness, to say a few
words, and my authority will be Ludwig, who, in his classic
research (12), has on these subjects left nothing to be desired in
point of view of completeness. I may add also that the figures
illustrating the changes in external form are copied from
Ludwig’s memoir. The three figures illustrating the relations
THE DEVELOPMENT OF ASTERINA GIBBOSA. 343
of the Asterid and Crinoid to their common ancestor were
designed for me by my friend and colleague Mr. J. J. Lister,
of St. John’s: in their present form they were drawn for me by
a lady friend.
The Development of the Larva.
The eggs are laid by the parent on the under surface of
stones, to which they adhere by means of their vitelline
membrane. I have never discovered a male, though Ludwig
says that the male twists his arms round the female whilst she
is depositing her ova, and then pours out his spermatozoa
upon them; it is quite certain that in the English Channel,
at any rate, isolated females will lay eggs which develop with
perfect regularity up to the conclusion of the metamorphosis.
Cuénot (4) says that young females of a certain size develop
spermatozoa in their ovaries—a statement I have not been able
to verify. It may, indeed, be said that Ludwig’s statement that
a kind of sexual congress takes place, Cuénot’s observations,
and the experience of the authorities of the Jersey Biological
Station are irreconcilable, and that the whole subject demands
renewed investigation.
The eggs are larger than those of most other Echinoderms ;
they are about ‘5 mm. in diameter. This is a result of the
yolk which they contain, and which gives them their bright
orange colour. This yolk is so uniformly distributed, however,
that it does not alter the type of segmentation, which is total
and regular. The blastomeres, in consequence of their larger
size, are more closely packed than is usual amongst Echino-
derms; they are wedged into the interspaces between their
neighbours, and so the strict “ radial’’! type of segmentation
characteristic of the group is no longer maintained.
The result of segmentation is a hollow blastosphere or
blastula, which on the second day of development becomes
converted into a gastrula by embolic invagination, The embryo
1 For a discussion of the different types of regular segmentation see “ The
Cell-lineage of Nereis,” by Prof. E. B, Wilson, ‘Journal of Morphology,’
vol. vi.
344, E. W. MACBRIDE.
is not quite spherical, its long axis exceeding very slightly its
transverse axis, so that we can see that the blastopore is
situated in the centre of what afterwards becomes the ventral
surface. The gastrula has acquired a uniform covering of cilia,
and the blastopore is a round opening with well-defined lips.
This well-marked stage of development, which is easy to
recognise, I have called Stage A (P1.18, fig.1). The blastopore
narrows in a peculiar manner, one of its lips becoming reflected
over it (Pl. 18, fig. 2), and it is finally reduced to a minute pore
(P1. 18, fig. 3). This opening, which is identical with the larval
anus, gradually travels back to near the posterior end of the
embryo; this is effected by differences in the rate of growth of
surrounding parts. During this time the embryo has been
lengthening its long axis, and on the fourth day it ruptures the
vitelline membrane and escapes. It then has the form shown
in P1. 18, figs. 4—6, and as this stage is also a well-marked one,
I have called it Stage B.
The foregoing is Ludwig’s account ; my material was not
suitable for such observations, which ought to be made on the
living embryos, and I had not the opportunity of observing
these early stages alive. As far, however, as I could make out,
Ludwig is perfectly correct in his statements. I was able to
recognise Stage A, for instance, with ease.
Let us turn now to the internal changes which have gone on
during this time. Pl. 19, figs. 20 and 21, are two sections of
an embryo of Stage A, and they form the starting-point of the
changes we shall have to consider ; I may here say at once that
all sections which illustrate the development of the larva and
its metamorphosis are to be understood to have been cut
parallel tothe larval plane except the contrary is dis-
tinctly affirmed. Fig. 22 is a sagittal section of a slightly
older embryo ; here mesenchyme cells have appeared. The
large size of the archenteron is a remarkable feature, the
blastocoele or segmentation cavity, usually spacious in Echino-
derms, being reduced to a mere slit. Fig. 23 shows us that the
archenteron becomes differentiated into an anterior thinner-
walled vesicle, the coelom, and a posterior thicker-walled gut ;
THE DEVELOPMENT OF ASTERINA GIBBOSA. 345
and in fig. 24 we see that the ceelom has grown back in the
form of two tongues, Jpe., rpc., lying one at each side of the
gut. Fig. 25 shows us a more ventral section passing through
the blastopore of the same individual, and we see that in it
these coelomic lobes are absent ; they are therefore still con-
fined to the dorsal side of the embryo.
It has been mentioned above that the larva, immediately on
escaping from’ the egg-membrane, has the form of Stage B, and
it will be observed that its anterior end has the appearance of
being obliquely truncated, and that the anterior surface so
constituted is surrounded by a thickened rim, which is covered
with specially long cilia, and to which I give the name of larval
organ. The changes of form involved in acquiring this shape
are considerable, and are undergone whilst the larva is still
enclosed in the egg-membrane, though superficially the ovoid
shape is maintained, the larval organ and the neighbouring
ectoderm being to a large extent developed as invagina-
tions into the interior of the larva, exactly as the
Tenia head is developed on the wall of the cyst.
The histology of the embryo is illustrated in Plate 26, figs.
124 and 125. The first is a portion of section of a larva of
Stage A, the same specimen as that from which figs. 20 and 21
are taken. Both ectoderm and endoderm are seen to consist of
long narrow cylindrical ceils, and there is no mesenchyme.
Recent researches have gone to show that this is exceptional.
Field (5) has proved for Asterias, and it has been long known
in the case of Echinids, that mesenchyme is formed by the wall
of the blastula before any invagination has taken place. Fig.
125 is taken from a slightly older gastrula. It shows the forma-
tion of the mesenchymatous cells by the division of the endoderm
cells. I found no indication that mesenchyme continued to be
formed when Stage B is reached. The anterior wall of the
celom is the spot where its formation lasts longest, as in
Antedon (18). The coelomic epithelium consists of small cubical
cells (see Pl. 23, fig. 95).
We must now return to Stage B, up to which we have traced
the development. A stomodzeum is now developed just behind
3846 E. W. MACBRIDE.
the posterior wall and ventral edge of the larvalorgan. This is
well shown in the sagittal section, fig. 81. The larva increases
in size, and the prezoral portion and larval organ alter their
shape, the latter changing from a circular to an elongated
elliptical form, whilst the przoral lobe extends in a vertical
direction (Pl. 18, figs. 7 to 9). The whole larva has now the
form which Ludwig calls slipper-shaped, but which would be
more correctly termed boot-shaped, the dorsal lobe of the preoral
lobe representing the toe and the ventral one the heel of the
boot. In the centre of the larval organ appears an elevation
(fiz.). This structure, which Ludwig did not interpret, we shall
find to have a most important function during the metamor-
phosis; it is, in fact, the disc by means of which the animal
fixes itself. Possibly this disc also functions during free life
for temporary attachment, though in a different manner ; thus
when the larval organ is applied to the substratum, the retrac-
tion of this disc would cause a cupping action which would be
relieved by its again being protruded. It has been pointed out
by Ludwig, and I have myself confirmed it again and again,
that the larva is able to attach itself most strongly to the sub-
stratum. The mode of life of the larva Ludwig calls ‘‘creeping.”
This is not strictly correct; as far as I have seen, the larva
swims by means of the cilia of the larval organ. The latter is
directed downwards, and for this reason Ludwig calls what I
have termed the anterior surface of the animal the ventral, and
the posterior end becomes for him the dorsal end. I cannot
agree with this orientation ; the proper longitudinal axis of any
bilaterally symmetrical animal is the oro-anal one, and it is by
this that I discriminate between the dorsal and ventral, the
anterior and posterior surfaces. That the posterior end is held
upwards is no more reason for calling it dorsal than the fact
that the Cephalopod directs the apex of its visceral hump back-
wards is reason for calling that posterior. I should mention
that Ludwig calls the whole przoral portion of the body, the
preoral lobe in fact, the larval organ. I wish to avoid this,
since the preoral lobe has functions which Ludwig did not
suspect, and hence I confine the term “ larval organ” to the
THE DEVELOPMENT OF ASTERINA GIBBOSA. 34.7
thickened ridge with long cilia, which is the locomotor organ
of the larva, and is the first thing to disappear in the meta-
morphosis.
Stage C is the point which we have now reached, and it is
characterised by the appearance of this disc for fixation.
Ludwig compares the larval organ to the non-ciliated processes
of the Asterid larva, the Brachiolaria. This larva appears to
be merely a further stage in the development of the well-
known Bipinnaria, from which it differs in the development of
three stalked papille from the apex of the preoral lobe, which
are presumably used forattachment. These papillearise between
the anterior dorsal and the anterior ventral arms of the Bipin-
naria: one of them is median and more dorsally situated than
the other two, and to this arrangement Ludwig compares the
occasional bifurcation of the ventral lobe of the larval organ of
Asterina. Now, however, that we know the function of the
adhesive disc, it is, in all probability, this which is to be com-
pared to the papillee of the Brachiolaria; and the larval organ
with its long cilia (compare Pl. 27, figs. 133—135) in all proba-
bility represents some portion of the ciliated bands of the
Bipinnaria. Garstang (6) has, in fact, recently described a
Bipinnaria in which the dorsal arm of the preoral lobe exe-
cutes muscular movements in the same way as Ludwig asserts
for the Asterina larva. I repeat, however, that the latter can
swim by ciliary action alone, without any muscular move-
ment.
The internal changes which have occurred between Stages B
and C are numerous and important. We have already referred
to the appearance of the stomodzum or larval cesophagus.
About the same time the primary madreporic pore is formed ;
it arises by a pocket of the ccelom slightly to the left! of the
mid-dorsal line, meeting a thickening of the ectoderm (fig.
26, mp.) and a perforation taking place. The pocket of the
coelom is called the “ pore-canal” (pe., fig. 26), and is lined
by cylindrical ciliated cells. By this time the two posterior
1 This position is not shown in fig. 26; the figure represents a section which
was rather oblique.
348 E. W. MACBRIDE.
lobes of the ccelom have extended so as nearly to meet one
another in the mid-ventral line ; the mesentery formed by their
apposition is seen in fig. 80, posterior to the gut. The opening
of the gut into the ccelom has become closed ventrally (figs. 29
and 80); dorsally, however, it remains open for some consider-
rable time yet. On the left side the coelom becomes segmented
into an anterior portion, a., into which the pore-canal opens,
and a left posterior portion, Jpc., which we may call the left
posterior coelom (fig. 27); this second cavity includes a large
part but not all of the left coelomic lobe mentioned above ;
part of this latter is, as is seen in the figure, included in the
anterior celom. The septum between the two cavities is first
formed dorsally, and then extends in a ventral direction ;
fig. 28 shows it in process of formation.
At the same time one can notice the first indication of that
predominance of the organs of the left side which is the key
to the whole ontogeny of the star-fish. We see in fig. 30 that
the septum between the right and left coelomic sacs is pushed
over to the right, owing to the tendency of the left posterior
coclom to extend over to the right on the ventral side. At no
time, so far as I have seen, however, does this septum break
down. Some curious trabecule are in this stage stretched
across the left celom. They are easily distinguished from the
septum between the two sacs, as they consist of solid strings
of cells, whereas the septum has two layers of epithelium with
a slit of blastoceele between in this stage. These trabeculz
are very transitory; in figs. 28 and 29 (Stage B) we see them
being formed, and in fig. 33 is the last trace of them (Stage C).
As development proceeds the gut becomes more completely
separated from the coclom, the larval anus closes, and the short
rectum (fig. 81) disappears. Shortly before this, however,
the stomodeum opens into the gut, the main portion of
which constitutes the larval stomach (/. stom.), the rectum
being very short; but it is only for an extremely short time
that the larva possesses both mouth and anus.
Stage C is reached about the end of the fifth day, or the
commencement of the sixth day. The division of the left
THE DEVELOPMENT OF ASTERINA GIBBOSA. 349
posterior coelom from the ccelom of the przoral lobe, which
we may now call the anterior celom (a., figs. 32—35), is com-
plete. On the right side the separation of the posterior part of
the right coelomic lobe, the right posterior ccelom, from the
anterior coelom has just commenced dorsally (fig. 32). On the
left side the rudiment of the water-vascular system, or, as it is
convenient to term it, the left hydrocele, has appeared (as
will be related immediately a similar rudiment appears on the
right side, but ‘ hydrocele”’ alone means left hydroceele). It
originates as an outgrowth from the hinder end of the anterior
celom ; and whilst it is as yet but faintly marked off from
this cavity, indications of its five primary lobes are seen.
These are arranged ina curve open anteriorly, and throughout
all the figures they are denoted by the Arabic numerals; the
most dorsal being No. 1, the most posterior No. 8, and the
the most ventral No. 5 (see figs. 32—34). Their mutual re-
lations are well shown in the sagittal section (Pl. 20, fig. 47),
though this represents a somewhat later stage.
We have seen that the division of the right posterior ccelom
from the anterior ccelom has begun in exactly the same manner
as happened in the case of the left posterior ccelom at an earlier
stage. This division has not proceeded very far towards the
ventral surface, when the anterior coelom buds off a vesicle
from its right posterior extremity. This vesicle is homologous
to the water-vascular rudiment on the left side, for which
reason it will be termed the right hydroceele ; so we see that the
colom on the right side of the larva undergoes exactly the
same changes as that on the left, only that they are retarded
in their appearance. The first trace of the right hydroceele is
shown in PI, 238, fig. 95; we see that it consists of a small
vesicle of cubical cells arising as a thickening of the coelomic
wall. Its lumen is, in this stage, a minute slit; other pre-
parations show this slit in open communication with the an-
terior colom. It is important to observe that it originates
from the dorsal portion of the hinder end of the anterior
coelom, which extends further back ventrally to it, as would be
seen if a more ventral section than fig. 95 were shown.
350 E. W. MACBRIDE.
Later stages of this organ are seen in figs. 85 and 86. In
fig. 35 it is a conspicuous solid bud; in fig. 36 it has acquired
a lumen, and is connected with the anterior ceelom by a string
of cells, which soon atrophies, and it is then left as an isolated
vesicle in the midst of the mesenchyme. Bury (2), indeed, has
seen it in this stage, and called it ‘‘ a mesenchymatous vesicle ;””
and Field (5) has described what I believe to be an homologous
structure in the larva of Asterias. The right hydrocele persists
in the adult as a closed sac just under the madreporite, and
has been seen here by Cuénot (8), and Leipoldt (9) has described
a similar sac in Echinids. It may seem rather a rash assump-
tion to regard this organ as the fellow of the water-vascular
system, but a complete proof that this is really its nature will
be given when abnormal larve are described.
Stage D, the summit of the development of the larva, is
reached on the seventh day, according to Ludwig (PI. 18, figs.
10 and 11). The preoral lobe and the larval organ have
greatly increased in size, the former having acquired a large
ventral as well as a dorsal lobe. The internal changes are
more striking than the external. The separation of gut from
celom was practically complete in Stage C, the last trace of
connection being shown in fig. 86. The right posterior ceelom
is entirely separated from the anterior ccelom, but, strange to
say, the septum between the left posterior celom and the ante-
rior coelom has become broken down in two places. This occurs
by the two layers of epithelium of which it is composed fusing,
and then thinning out to a film. Of these two secondary com-
munications between the two sacs, one is situated dorsal to the
left hydroceele (Pl. 20, fig. 42), and the other ventral to it
(Pl. 19, fig. 41). Figs. 42 and 43 belong to the same series ;
we see that the dorsal opening is formed before the separation
of the right posterior coelom is complete ; the ventral opening
is formed at the same time. Not having had the opportunity
when I wrote my preliminary account (15) of observing younger
larvee than these, I imagined that the segmentation of the
ceelom of the left side was incomplete ab initio, a mistake
which was the more excusable as both the breaches in the
THE DEVELOPMENT OF ASTERINA GIBBOSA. 351
septum dividing the two portions of the ccelom from each other
become again closed during the metamorphosis.
The left hydroccele has become much more sharply separated
from the anterior coelom than in the last stage, though in the
region of the third lobe the hydroceele still opens widely into
the anterior ccelom (P1.19, figs. 388—41; Pl. 20, figs. 44—46).
We saw that the pore-canal in Stage B originated a little to
the left of the middle line; now, however, owing to the in-
creasing predominance of the left side, it is shifted to the right
of the median plane (pe., fig. 44). The stone canal (séc., figs.
45 and 46) arises as a groove along the anterior face of the
transverse septum forming the hinder wall of the anterior
celom. The central portion of this groove soon becomes closed
to form a canal, opening at one end into the hydroceele between
lobes 1 and 2 (fig. 46), and at the other into the anterior ceelom
(fig. 45); and this opening is in this stage entirely inde-
pendent of the opening of the pore-canal.
I have referred more than once to the predominance of the
organs of the left side. This is strikingly shown in the stage
we are considering by the narrowness of the right posterior
ceelom as compared with the left. Already in Stage B we have
seen that the left posterior ccelom has begun to sweep round
to the right on the ventral side of the right posterior ccelom ;
this occurs more and more, and in the stage we are considering
in the most ventral sections (fig. 41) the right posterior coelom
is entirely absent. The left not only passes under it, but toa
certain extent interposes between its anterior portion and the
gut (figs. 39 and 40), and here opens freely into the anterior
celom! (fig. 40) by the secondary ventral communication de-
scribed above. This portion of the left ceelom we may call its
right ventral horn ; it plays a most important part in the meta-
morphosis, and it is marked /‘p’c’. in all the figures.
Ludwig failed entirely to recognise the left posterior ceelom
1 T may anticipate a little by informing the reader that the anterior ccclom
gives rise to the axial sinus of the adult ; a space which opens to the exterior
by the pore-canal and into the left hydroccle (water-vascular ring) by the
stone-canal.
Soe E. W. MACBRIDE.
as a sac separate from the anterior ccelom; he states that the
mesentery between the right and left coelomic lobes is absorbed
ventrally. We have seen that only the posterior parts of the
right and left coelomic lobes are employed in the formation of
the right and left posterior cceloms respectively ; the anterior
parts of these lobes are continuous with the anterior ccelom,
and the longitudinal mesentery between them breaks down, as
Ludwig observed. Hence we see that the hinder part of the
anterior ccelom in Asterina is at first a double structure; in
the Bipinnaria larva the anterior ccelom is at first double
throughout its whole extent.
At the dorsal anterior angle of the left ccelom (fig. 37) an
invagination of its wall takes place, giving rise to a thick-
walled vesicle (07. c.), which communicates by a narrow slit
with the celom. This structure has been strangely misunder-
stood. Ludwig saw it, but not its origin, and supposed it to
arise as a ‘* schizoceele,” and regarded it as the rudiment of
the oral blood-ring. In my preliminary account I recognised
its true nature, but supposed that its upper end was the rudi-
ment of the so-called heart,! with which, as a matter of fact,
it has nothing to do. It is the rudiment of the oral celom, a
space closely surrounding the adult cesophagus, the relations of
which we shall study later.
Histology of the Larva.
The structure of the body-wall of the larva is shown in
Pl. 27, fig. 1388, and Pl. 28, fig. 144. In the first we see
that the peritoneum of the left posterior coelom consists of
1 Tt will be observed that Bury, in his last paper (‘Q.J.M.S.,’ September,
1895), makes the same mistake. This work appeared after the present paper
had been sent in for publication, and is therefore not referred to further here.
The best answer to Bury’s criticisms on my observations as recorded in the
preliminary account (15) is the publication of full details in the present paper.
Bury’s observations contain much interesting matter, but also in my opinion
many mistakes, which are due to the fact that the stages which he obtained in
the development of most of the larve he studied, did not form a series without
gaps; the orientation which he adopted seems to me also not that which yields
the best results.
THE DEVELOPMENT OF ASTERINA GIBBOSA. 353
small cubical cells; the ectoderm is made up of exceedingly
long and narrow cells bearing flagella, and the wall of the
hydroceele of similar cells, but I could not make out any
flagella there. Fig. 144 is taken from the posterior end of the
animal on the right side; the form of the ectoderm cells is
well seen, and one observes occasional goblet cells (god.)
amongst them. The section goes through a peculiar patch of
peritoneum, where the cells are actively engaged in budding
off the ameebocytes which float in the celom. So far as I can
make out, however, no cells are budded off at this stage into
the blastoceele (i.e. the space between the ectoderm and the
ceelomic wall), and the mesenchyme cells are as yet entirely
undifferentiated. The characters of gut cells are shown in
Pl. 26, fig. 126. Although this is taken from a larva in which
the metamorphosis has commenced, yet the characters of these
cells do not vary till the very close of the metamorphosis.
They have the same general form as the ectoderm cells, but
the bases of the latter are often contracted, and leave chinks
between them, whereas the endoderm cells are closely apposed
to one another. Fig. 126 also shows another point of interest:
here and there a small round amcebocyte may be seen applied
to the basal end of the gut cells, and one discovers amongst
the latter also one or two rounded cells, thus suggesting that
these amcebocytes may be able to pass between the gut cells
like the lymph cells in the Vertebrate intestine.
Plate 27, figs. 183—135, are three sections through the larval
organ which have already been alluded to. It is to be noted
that in this stage the adhesive disc has short cilia, just as See-
liger (18) has described for the adhesive disc of Antedon. Where
I have put ‘‘ nerv. Jarv.” a thin strand of pale fibrous matter is
observable with the highest powers. This is the only trace I
can discover of a larval nervous system, and I am not perfectly
satisfied about it, since it does not take the yellowish-brown
tone with osmic acid so characteristic of the adult nervous
system. Should my interpretation of it be correct, the larval
nervous system would consist of a layer of ‘ Punktsubstanz ”
underlying the larval organ.
354 E. W. MACBRIDE.
P|. 27, fig. 187, shows the character of the wall of the pre-
oral lobe. The peritoneal cells have developed fine muscular
tails (musc.larv.), and it is perfectly apparent to anyone looking
at sections of a number of larve that it is the peritoneum which
is the active agent in contraction. The ectoderm is often
wrinkled (fig. 38), but the peritoneum never, though its cells
vary in shape from cylindrical to flattened according to the
state of contraction ; thus in some cases the peritoneal cells on
the left side will be cylindrical, those on the right side flattened.
The coelomic wall is in this case short and straight on the one
side, and on the other bulged in to the lumen of the anterior
celom by a great accumulation of the fluid of the blastoceele,
or rather (as we must conclude from observations which have
been made on other Echinoderms) the blastoccelic semi-fluid
jelly. In fig. 137 we see some fine fibrils traversing the blasto-
coele; these, so faras I can make out, are not protoplasmic, but
of skeletal nature—of the same nature, that is, as the adult
fibrous tissue.
The Metamorphosis.
On the eighth day the larva fixes itself by the adhesive disc
by means of a thin secretion of mucilage (see Pl. 27, fig. 136,
which represents a much later stage), and remains fixed
during the whole of the metamorphosis. I had the
opportunity of observing this in Plymouth in 1893 and in
Jersey in 1894, and it was most instructive to observe the
difference between the larve which had thus definitely become
sessile and those which, being still able to move, had attached
themselves by the cupping action of the muscles of the pre-
oral lobe, the larval organ being applied to the substratum.
In the first case, that of truly sessile larve, if one attempted
to remove them with a pipette, one failed to move them unless
very strong suction was applied or they were displaced by a
needle; but once displaced they were perfectly helpless, those
even which had to all appearance almost completed the meta-
morphosis being unable to use their tube-feet (which as yet
were rudimentary) ; they could do nothing but feebly rotate by
THE DEVELOPMENT OF ASTERINA GIBBOSA. 355
the action of their general covering of cilia, and they had no
power of re-attachment. In the case, however, of larve which
were attached by what we may call voluntary muscular action,
if one brought the pipette cautiously near so as not to alarm
them, it was very easy to remove them from a stone, just as it
is easy to kick a limpet off a stone if it is taken unawares;
but if they were irritated they were excessively difficult to re-
move, and when one finally succeeded in getting them up into
the pipette, unless one promptly re-expelled them, they at-
tached themselves to the glass, and it was almost impossible to
detach them from it.
The metamorphosis of Echinoderms is probably the most
remarkable ontogenetic change known in the animal king-
dom; but our knowledge of its details has been up to the
present most insufficient. We possess a completely satis-
factory account of only one form, viz. Antedon, for which the
credit is due to the researches of Bury (1), which have been
amply confirmed by Seeliger (18). As I mentioned in the
introduction, I hope the account I am about to give of the
metamorphosis of Asterina will compare in completeness with
those I have just mentioned; and as it is of the utmost im-
portance for the comprehension of the meaning of the anato-
mical structure of the Asterid that its relation to the larva
should be thoroughly grasped, I shall anticipate a little what
I have to say in order to make the essence of the process per-
fectly clear. The metamorphosis of the Asterid, then, consists
in the following processes, which go on simultaneously :
(1) The constriction of the body into disc or body sensu
stricto, and stalk, the latter being formed from the preoral
lobe.
(2) The sharp flexure of the disc on the stalk [the former is
bent obliquely downwards and to the left. This is not well
shown in any of the figures copied from Ludwig ; it is better
seen in the diagram, Pl. 29, fig. 158 (Dec. 1895) ].
(3) The preponderating growth of the organs of the left side,
the left posterior celom and the left hydroccele both sending
out dorsal and ventral horns, which meet so as to form complete
VoL. 38, PART 3.—NEW SER. AA
356 E. W. MACBRIDE.
circles, whilst the right hydrocele and the right posterior
ceelom remain small.
(4) The gradual atrophy of the stalk.
(5) The outgrowth of the adult cesophagus and the formation
of the new mouth on the left side.
In the Crinoid the list would stand thus:
(1) The constriction of the animal into calyx and stalk.
(2) The displacement of the mouth and neighbouring organs,
i.e. the hydroceele, to the posterior end of the body by unequal
growth.
(3) The mutual displacement of the right and left posterior
celoms, the left becoming posterior and the right anterior,
both having a ring-shaped growth.
(4) The spiral growth of the intestine and formation of anus
close to primary madreporic pore.
It will be seen that the Asterid metamorphosis is very
different from that of the Crinoid, being much simpler: one
great difference which strikes one at once being that in the
former case the ends of the hydroceele grow so as to embrace
the stalk, which thus appears to spring from the oral surface ;
whereas in the latter case the hydrocele is carried far away
from the stalk to the posterior end of the body. Much diligent
search has been made in the centre of the aboral surface of
Asterids for traces of a stalk, but to anyone who has grasped
the foregoing explanation it will be at once obvious how futile
such search must prove. Pl. 29, figs. 158 and 159, though
intended to indicate ancestral forms, illustrate the two meta-
morphoses outlined above very well.
The sections about to be described illustrating the meta-
morphosis are nearly all cut parallel to the larval plane, and
as was the case with the sections of the larva, where two or
three sections from the same series are figured the most dorsal
is in every case placed first, and so one can clearly see their
relation to corresponding sections of the larva. As one always
thinks, however, of the organs of an Asterid as related to the
plane of the disc or adult plane, it will be well to repeat the
relation which these two planes bear to one another. The
THE DEVELOPMENT OF ASTERINA GIBBOSA. 357
adult plane makes an angle of about 70° or more with the
larval plane; but without any very serious error, it may be
regarded, for purposes of description, as at right angles to it:
thus the direction right to left, according to the larval plane,
becomes aboral to oral according to the adult plane, and dorsal
to ventral according to the larval plane is nearly parallel to
the adult plane. Here I may remark that the words “ dorsal”
and “ventral” will only be used with reference to the larval
plane; in speaking of the adult plane the words “ oral” and
“ aboral”’ will be used.
Pl. 18, figs. 12 and 13, show the appearance of a larva which
has only been fixed for a short time. On the left side we see
that the hydroceele lobes have become visible externally, since
they have raised the ectoderm into protrusions which, as we
shall find, are the rudiments of sensory terminal tentacles of
the radial water-vascular canals. Outside the curve of these
rudiments is another set of protrusions, also arranged in an
open curve. These are the rudiments of the arms: they are
all, as we shall see, outgrowths of the left posterior ccelom, and
their primary function is to form supports for the lobes of the
hydrocele, to which they later become apposed. The con-
striction of the przoral lobe or stalk from the body proper is
hardly as yet marked, but the rounded appearance of the dorsal
and ventral outgrowths of the preoral lobe is to be noticed.
This is due to the disappearance of the larval organ, the opposite
‘sides of which become approximated to each other and wrinkled,
and then broken up, portions of the organ becoming invaginated
into the interior and destroyed by histolysis. The appearance
of the remnants of it at this stage gave Ludwig the impression
that one had to do with the outgrowth of a series of protrusions
homologous to the adhesive disc. Thisis, of course, a mistake;
the adhesive disc remains single and unaltered to the end of
the metamorphosis. This well-marked phase of development
we may call Stage E. PI. 20, figs. 48 to 50, are taken from a
larva of this age; fig. 48 is of course the most dorsal section
(see explanation of plates). In fig. 50 we notice the great
srowth of the left hydroceele, lobe 3 reaching nearly to the
3808 E. W. MACBRIDE.
posterior end of the body, and we can also make out an arm
rudiment, which at this stage is a mere protrusion of ectoderm
filled with mesenchyme cells; it forms the extreme posterior
end of the section. The rudiment of the adult cesophagus
a. ce is also seen, and we notice the relation of the oral ccelom to
it, and we may remark that the larval esophagus is by this time
disrupted from the gut. Fig. 49 shows that dorsally the hydro-
coele is completely shut off from the anterior coelom, and shows
that the oral celom dorsally opens into the left poste-
rior celom. Fig. 48 shows that the opening of the oral ccelom
is in close relation to a process of the left posterior ccelom extend-
ing over to the right, dorsal to the gut. Thisis the right dorsal
horn (see p. 351 for the ventral horn) of the left posterior coelom,
and it is marked /’p”c” in all the figures. In later stages it
extends ventrally for a short way, insinuating itself between
the gut and the septum dividing the anterior coelom from the
left posterior one (Pl. 21, fig. 61). The opening of the oral
celom is later shifted so as to be connected only with the right
dorsal horn, and hence it came to pass that Ludwig regarded
oral coelom and right dorsal horn of the left coelom as one
structure, and described the oral ccelom as the oral blood-ring
and the dorsal horn as the “ heart.” In common with all other
growing spaces in the larve, this right dorsal horn has at its
growing tip an epithelial thickening, and it was this which in
my preliminary account I mistook for the rudiment of the
“heart.”
Figs. 51—53, taken from a slightly older larva, show the
appearance of the rudiments of the perihemal spaces. It may
be useful to refresh our memory of the arrangement of these
spaces in the adult; this the annexed woodcut is intended to
do. They are usually described as consisting of a canal situated
just aboral to each radial nerve, and divided by a longitudinal
septum (Pl. 29, fig. 155). These radial canals open into a
circular canal surrounding the mouth, inside which is another
inner ring-canal. The longitudinal septa of the radial canals
are inserted in the septum separating these two ring-canals.
Into the inner of the circular canals a vertical canal opens
THE DEVELOPMENT OF ASTERINA GIBBOSA. 309
which is the axial sinus, embedded in the wall of which is
the stone-canal (Pl. 25, figs. 110—118). This axial sinus
ie tle
ph. 1.2, &e. Rudiments of the outer perihemal ring. a‘, Axial sinus and
its outgrowth the inner perihemal ring. a. Aboral sinus. gev.r.
Genital rachis.
was supposed to open at its upper end into an aboral periheemal
ring or pentagon, from which in each interradius two canals
branched off to go to the genital organs. As is well known,
these spaces were called “perihemal” by Ludwig (10), because
he imagined that he had discovered the true blood-system in
the form of curious tracts of tissue embedded in the longitudinal
septa of the radial canals, and in the septum separating the
two circular canals. He further supposed that that curious
360 E. W. MACBRIDE.
so-called heart, which projects along with the stone-canal into
the axial sinus, was connected with this system, and that a
string of tissue lying in the aboral ring and connected with the
“heart”? was also part of the vascular system. We shall,
however, see later that these two latter structures (“heart ”
and aboral string) are of totally different nature from the oral
ring, being composed of primitive germ-cells, and have, as a
matter of fact, no connection with it. ‘The radial tracts are
absent in Asterina, but the oral circular tract is well repre-
sented, and we shall study its development later.
The woodcut shows us that the foregoing description is not
quite correct. In the first place, we see that one can hardly
speak of an outer perihemal ring, because this space is broken
up into five compartments by the prolongations of the longi-
tudinal septa of the radial canals; secondly, apart from the
mistake we just pointed out in reference to the nature of the
‘heart’ and aboral ring, we see that the axial sinus (a’) does
not open into the perihemal aboral ring ; and, further, that to
the upper end of the axial sinus is closely apposed a small
closed sac, the right hydroceele.
Returning to figs. 51—53, we see that each of the five
compartments of the outer oral perihemal ring arises
separately as a wedge-shaped outgrowth of the
ccelom. Ihave numbered these rudiments according to the
numbers of the lobes of the hydroceele between which they
occur—ph. 1.2, ph. 2.8, ph. 3.4, ph. 4.5, and ph. 5.1; the last,
however, arises later, and is not seen in these figures, and the
first is an outgrowth of the anterior coelom (Pl. 20, fig. 51, Pl.
21, fig. 54) : all the rest arise from the left posterior celom. The
shape and relations of these rudiments are well shown in the
enlarged drawing given of one of them (Pl. 27, fig. 139) ; we
see that the base of the wedge is directed outwards, and that its
basal angles tend to insinuate themselves between the ectoderm
and the hydrocele. As a matter of fact, each angle grows out
till it meets the adjacent one of the next rudiment. The two
then become apposed to each other, and their walls, which
meet, form the longitudinal septum of the radial canal, and
THE DEVELOPMENT OF ASTERINA GIBBOSA. 361
both spaces grow out together underneath the growing lobe of
the hydrocele, and thus the radial perihemal canal itself is
formed ; we shall find later that the inner perihemal ring arises
as an outgrowth from the oral end of the axial sinus or anterior
coelom, and hence it is marked a’ in the woodcut.
Fig. 53 shows us that the fourth and fifth lobes of the
hydroccele have extended over to the right; this being the
result of the tendency of the two ends of the hydroceele, which
have become entirely shut off from the anterior ccelom, to
approach one another. We also see from the obliquity of the
right posterior ccelom (compare figs. 44—46 with figs. 52 and 53)
that the lateral flexure of the body on the stalk has commenced.
The flexure in a downward direction cannot be well shown by
sections.
Pl. 29, figs. 54—57, are sections of a larva rather older than
Stage E. We see that the differentiation of the stalk from the
body has been initiated by the dorsal constriction of the neck
of the preeoral lobe. In consequence of this the anterior coelom
becomes divided into a stalk portion @, and a body portion a’,
the latter forming the axial sinus. We see, further, that the
ventral horn of the left posterior ccelom U'p’c’ has pursued its
growth, extending obliquely to the right under the gut, and then
upwards in a dorsal and anterior direction, and on its course
the last of the five arm rudiments appears, viz. V. Fig. 57
shows the outgrowth of septa destined shortly to close the
ventral communication between this right horn of the left
posterior coelom and the anterior coeelom. The primary lobes
of the hydroccele have each by this time given rise to two
lateral lobes, the rudiments of the first tube-feet, the primary
ones themselves being destined to form the terminal tentacles
of the water-vascular system.
Figs. 58 and 59 represent a larva about midway between
Stages E and F. We see the final division of the hydroccele
from the anterior coelom, the last connection being in the
neighbourhood of lobe 3, and also the separation of the axial
sinus from the stalk celom. We see also the remains of the
larval cesophagus (/e.), which already in Stage E has broken off
302 E. W. MACBRIDE.
from connection with the gut; the relative position of the adult
cesophagus (a.@.) is also well shown. Fig. 60 is from a larva
of about the same age; it shows the formation of the fifth
periheemal rudiment (ph. 5.1) as an outgrowth of the ventral
horn of the posterior celom: this lies beyond the fifth
hydroceele lobe, and will therefore come to lie between this
and No. 1 lobe when the two ends of the hydroceele meet. We
also see the process of destruction of the stalk going on, the
ectoderm of its anterior surface being invaginated in patches,
and, as we shall see, each patch as it is invaginated becomes
destroyed by histolysis. Fig. 61 is from a larva which has
nearly attained Stage F; it shows how the dorsal horn (/’p’c’’)
of the left posterior coelom wedges itself in between the gut and
the hinder wall of the anterior celom (a’). In this wall we see
running from left to right (i.e. from oral to aboral sides of the
disc) from the second lobe of the hydroceele, the stone-canal.
The ciliated cylindrical epithelium of this has now become
continuous with that of the pore-canal, but only on one side ;
the conjoined tubes still open to the anterior coelom, and this
opening persists in the adult, a fact which Ludwig did not
observe (to see this, a more dorsal section than fig. 61 would
have to be shown). The reader will remember that the pore-
canal is formed by a dorsally directed outgrowth of the anterior
celom fusing with the ectoderm, and a perforation occurring at
the point of contact, and that the stone-canal is at first a ciliated
groove running along the posterior wall of the anterior ceelom.
This groove we found became converted into a canal opening
into the hydroccele on one side, and the anterior celom on the
other just below the inner opening of the pore-canal (woodcut 2).
We have now arrived at Stage F, the external appearance of
which is shown in Pl. 18, figs. 14—16. We notice that the
preoral lobe or stalk has become very much reduced, and that
the two ends of both curves, that of the hydroceele lobes
(numbered in Arabic figures) and that of the arm rudiments
(numbered in Roman numerals), have become very much ap-
proximated to each other.
At the same time we see that oral and aboral parts of the
THE DEVELOPMENT OF ASTERINA GIBBOSA. 363
future star-fish are decidedly oblique to one another, being
closely apposed posteriorly, but anteriorly separated by the
thick base of the stalk. We see also that a lateral shift of the
arm rudiments has commenced, No. V having passed beyond
the hydroccele lobe No. 5, and so also in the case of the others.
A second pair of rudiments of tube-feet has grown out from
each lobe of the hydroceele, so that they are now 5-partite.
Figs. 62—69, Pl. 21, are taken from a most instructive series
of sections of a larva of this age, and are intended to give a
clear conception of its internal anatomy. We are struck at
once by the great reduction of the stalk, although ventrally
(fig. 66) the stalk ccelom still communicates with the axial
sinus. In fig. 65 we see the last trace of the secondary ventral
communication between the left posterior ceelom (/p’c’) and
the axial sinus a’ (anterior coelom) just closing. The secondary
dorsal opening persists much longer, but fig. 63 shows us that
it also is beginning to be closed. Comparing figs. 64 and 65,
we see that the adult cesophagus has acquired two lateral out-
growths, one directed anteriorly, the other posteriorly ; there is
also a third horn directed dorsally, which of course cannot be
seen in the sections. Fig. 67 shows how the oral ccelom (07.c.)
now half encircles the adult esophagus. As to the arm rudi-
ments, the most interesting thing is to notice the wide separa-
tion of No. V from the hydrocele lobe No. 1. When the
intervening tissue shrinks, a change which involves a reduction
in size of the axial sinus (compare a.’, Pl. 22, figs. 75 and 76) ,
the metamorphosis will be complete. The incipient shift of the
other rudiments is seen, especially in the case of Nos. II and
III, the latter falling between lobes 3 and 4.
By a continuation of the processes referred to above, viz.
the constriction of the base of the stalk, the increasing
flexure of the body on it, and the continued growth of the
hydrocele and left posterior coelom, we soon reach Stage G,
which is represented in Pl. 18, figs. 17 and 18. We notice the
great reduction of the stalk (which is now usually directed
downwards almost at right angles to the disc, though the
extent of the angle between the two varies) and the completion
364 E. W. MACBRIDE.
of the circle of arm rudiments, though No. I is not quite
adjusted to hydroceele lobe No. 2, and the hydroceele ring is as
yet incomplete. Here is a fitting place to give in a word or
two the gist of Ludwig’s observations on the calcareous plates.
On the oral side (fig. 17) we notice ten small calcareous stars,
two at the base of each primary hydroceele lobe, situated on
the inner side of the first pair of tube-feet rudiments. These
are the beginnings of the first ambulacral ossicles (amd.).
On the aboral side we notice eleven plates, one central (C.), five
situated in the arm rudiments and destined to form the
terminals (7’.) (the plates which protect the terminal tentacles
of the water vascular system), and five interradially situated,
the basals (B.), one of which becomes the madreporite. The
name ‘ basal” is given on account of an imagined homology
with the basals of Crinoids ; the groundlessness of this assump-
tion I shall point out later. All these plates make their first
appearance simultaneously, rather earlier than Stage F. Fig.
19 shows the aboral surface of a young star-fish about sixteen
days old. We see that the anus has been formed close to the
central; that a plate has been interposed between each terminal
and the central, the former maintaining its position in the
tip of the growing arm, and that finally a pair of plates has
appeared in each interradius, peripherally situated with regard
to the basals, the latter retaining their position in the centre of
the disc. These paired interradial plates are homologised by
Ludwig with the interambulacrals of Echinids.
Plate 22, figs. 70 and 71, are two sections of a larva of Stage
G. As in all the figures the stalk is placed as nearly as
possible in the same position, one can see at a glance the very
great lateral flexure which the disc has undergone with reference
to the stalk. We see the relation of the rudimentary larval
cesophagus to the permanent one ; we further see that the oral
celom is commencing ventrally to open into the left posterior
one (this is of course a secondary communication, and I may
say at once that the oral cwlom does not give rise to a separate
space in the adult, but merely forms the part of the caelom
abutting on the inner side of the buccal membrane), and finally
THE DEVELOPMENT OF ASTERINA GIBBOSA. 365
we observe the incipient bifurcation of the posterior end of the
pyloric sac (which is formed from the larval stomach) to form
the pyloric czca.
Fig. 79 is a section parallel to the adult plane of a slightly
younger larva; it shows beautifully the mutual relations of the
water-vascular ring (wv), the axial sinus, and the oral ccelom.
If one compares this figure with Pl. LV, fig. 53, in Ludwig’s
paper, one sees at once that his supposed rudiment of the oral
blood-ring is only the oral celom. Figs. 75 and 76 show the
completion of the metamorphosis by the apposition of arm
rudiment No. V covering the tip of the ventral horn of the left
ceelom (I'p’c’) to hydroceele lobe No. 1. As compared with
the larva represented in PI. 21, figs. 62—69, we notice the
much smaller size of the axial sinus (a’). Fig. 75 shows also the
bifurcation of the anterior end of the pyloric sac into two ceca.
Comparing it with fig. 76, which is a more ventral section
from a larva of the same age, we see also that the spaces
between the pyloric ceca (py) and the aboral body-
wall are continuations of the right posterior celom.
Fig. 76 shows also the first trace of ovoid gland (‘ heart”)
(ov.g.) arising as a ridge of epithelium including blastoccelic
jelly and fibres and amcebocytes, projecting into the axial sinus.
By comparing this figure with Pl. 21., fig. 61, the shift of arm
rudiment No. V can be clearly made out. Figs. 80 and 81 are
sections parallel to the disc of a larva rather older than Stage G.
Fig. 80 shows how the oral ccelom almost surrounds the ceso-
phagus, and also that the axial sinus is commencing to form
the inner perihemal ring by growth from its lower end (compare
woodcut). In fig. 81 we see at the point marked * the closing of
the water-vascular ring by outgrowths from the hydrocele lobes
Nos. 1 and 5 respectively. We also notice what we have already
seen in fig. 76, that the septum between the oral ceelom and the
left posterior coelom is breaking down ; and in fig. 82, which is
from a young star-fish in which the metamorphosis is just
complete, we see that from the remnants of this septum the
retractor muscles of the cesophagus or “ stomach ” are formed.
The remaining figures on the plate show the finishing touches
366 E. W. MACBRIDE.
of the metamorphosis. In fig. 72 the adult mouth is formed,
and the sessile mode of life has been given up, the stalk
being reduced to a small solid rudiment. We see also the first
trace of the eye as a small knob at the base of hydroccele
lobe No. 3. Fig. 78 shows the permanent anus; if we com-
pare its position with that which the larval anus occupied, we
find that they are by no means the same: the larval anus, if it
had persisted, would be situated at the point x, though both
occupy a position on the mesentery dividing the left from the
right posterior celoms. Fig. 77 from the same larva shows
that the left posterior celom now forms a complete ring by
the breaking down of the partition between its right ventral
and right dorsal horns (U‘p’c’. and l’p’c”.).
In fig. 73 a dorsal section, and in fig. 74 a ventral section,
we see the incipient bifurcation of the right posterior ccelom in
order to form the outgrowths connected with the two dorsal and
the ventral pyloric cxca respectively. We see, therefore, that
of the five pyloric ceca, two are formed from the dorsal end
of the pyloric sac or larval stomach, and two from its ventral
end, and that their suspensory mesenteries are outgrowths from
the mesentery separating right and left posterior celoms. The
fifth czecum is directed dorsally and posteriorly. In Pl. 22, fig.
82, and Pl. 23, figs. 83, 84, we have three sections parallel to
the adult plane of a specimen which had just completed the
metamorphosis. Once the mouth is open, the trifid form of the
adult cesophagus changes, we get the five slightly bifid lobes of
the adult “ stomach.” In fig. 83 we see the first trace also of
the bifurcation of the pyloric ceca; I remind the reader that
in each arm of the adult there are two ceca; the characteristic
appearance of the axial sinus, stone-canal, and right hydrocele
in a section parallel to the disc are also shown, the right hydro-
cele having a crescentic form. Fig. 84 shows us the relation
of the rectum and the rudiment of the rectal cecum to the
pyloric ceca; we see that the mesentery which binds the
bases of the pyloric ceeca together is only the original mesentery
between the right and left posterior (oral and aboral celoms) ;
and, further, that the mesenteric band connecting the inter-
THE DEVELOPMENT OF ASTERINA GIBBOSA. 367
radius of the stone-canal with the stomach is a part of this same
original mesentery, with which, however, is continuous a piece
of the wall between dorsal and ventral horns of the left ccoelom,
these two horns being still separated by this wall near their
right sides (aboral surfaces).
Histological Changes during the Metamorphosis.
Up to Stage G the histology has little changed from that of
the larva before metamorphosis. The most striking alterations
are those connected with the destruction of the preoral lobe.
Pl. 27, fig. 186, gives a specimen of them. This figure, which
is taken from the larva represented in figs. 62—69, shows that
the ectoderm becomes invaginated into pockets, and then these
pockets completely closed, so that no breach in the continuity
of the skin is made. The invaginated portion is then destroyed
by ameebocytes as shown in the figure. The peritoneum lining
the stalk ceelom contracts violently, the cells becoming cylin-
drical instead of flattened, and the larval muscles very appa-
rent. So far as I can make out, these cells are destroyed by
amcebocytes of the ccelom.
In the larva the whole hydroceele rudiment is lined by cylin-
drical cells (P1. 27, fig. 188); but as metamorphosis proceeds,
and the hydroceele increases in size, the cells are stretched so
as to become flattened (Pl. 27, fig. 189); they retain their
original character only in the rudiments of the tube-feet (PI.
28, fig. 149) and terminal tentacles. The first trace of the
adult nervous system appears in Stage F in the ectoderm
covering the water-vascular ring,—that is, the portion of the
hydroceele between the primary lobes. The ectodermal cells
become long and filamentous, with their nuclei set at different
levels, and amongst their bases (Pl. 28, fig. 140) appears a
tangle of fine fibrils of excessive tenuity, so that the highest
magnification is required to make them out; this is the first
trace of the adult nervous system.
Ludwig talks of cells stretched parallel to the surface under
the ectoderm, which he supposed to become the bipolar gan-
glion cells of the nerve-cord; but the cells in question, if I
368 E. W. MACBRIDE.
rightly identify what he means, are only the epithelial lining
of the perihzemal spaces which at a later period become closely
apposed to the ectoderm. The first trace of muscles in the
body-wall appears much earlier. Pl. 28, fig. 145, shows the
formation of a well-marked muscular band from the wall of
the right posterior ccelom of a larva of Stage E. We see that
it consists of indubitable myo-epithelial cells. I have traced
this band into the oldest specimen I have examined for histo-
logy ; and so far as I can see it appears to become a dilator
of the anus. It is very strange that it should appear long
before any other muscles of the body-wall ; it forms quite a
conspicuous feature in sections of all well-preserved metamor-
phosing larve. The same figure shows the first trace of histo-
logical differentiation in the mesenchyme; we see the first
formation of that fibrous intra-cellular substance which gives
_ firmness and tenacity to the adult body-wall.
The cells of the gut remain unchanged till the very end of
the metamorphosis, but in Stage G we can trace some differen-
tiation. Pl. 26, figs. 127, 128, show part of the lining of the
adult oesophagus and of the pyloric sac of such a larva. The
cells of the former are very long and narrow, and their outer
portions take a clear yellow tone with osmic acid; those of the
latter are ordinary cylindrical epithelium cells.
Abnormal Larve.
I mentioned above that the demonstrative proof that the sac
I have termed the right hydroceele is of that nature is obtained
from the study of abnormal larve. I suppose that about one
in thirty of the larvee I examined were abnormal, though in very
different degrees. The commonest abnormality results from
the unusually great development of the organs of the right
side, and the consequent checking of the metamorphosis.1 The
larva of which the two sections are given in figs. 85 and 86
had about attained Stage D. The left hydroccele is perfectly
normal, but the right, though not much larger than usual, is
1 The reader will remember that in the analysis of the metamorphosis which
I have given on p. 355, one of the main factors recognised is “the preponde-
rating growth of the organs of the left side.”
THE DEVELOPMENT OF ASTERINA GIBBOSA. 369
divided into distinct rounded lobes lined by cylindrical epi-
thelium (rhy.), in all respects similar to those of the left, and
the rudiment opens by a narrow but distinct slit into the
anterior celom. This larva also exhibits another very common
abnormality, which I do not in the least understand ; this con-
sists of the breaking up of the gut epithelium into a mass of
cells having the appearance of mesenchyme, which choke up
the lumen, but leave the walls almost denuded of epithelium,
consisting chiefly of the basement membrane. This curious
change can take place at any stage from the commencement of
the differentiation of the ceelom, up to young adults a month
old: in one such specimen it affected the pyloric ceca. As to
what its meaning is, I confess I am entirely in the dark.
Figs. 87 and 88 represent a most remarkable larva. The
development of the left posterior coelom would indicate that it
had reached Stage E, but the left hydroccele consists only of
four lobes, and is poorly developed. There are two rudiments
of a hydrocele on the right side; the more ventral has three
distinct lobes lined by cylindrical epithelium (7’hy’., fig. 88),
and opens by a distinct opening into the anterior coelom; the
more dorsal is perfectly normal (rhy., fig. 87); but, as if
to emphasise the fact that, in spite of the presence of the
other rudiment, it does in fact represent a hydroccele, we find
in connection with it a second small stone-canal and pore-
canal (p’c’. st’. c.). The relation of these to the right hydro-
coele may seem unusual; instead of the canal (conjoined stone
and pore-canal) leading from the hydroceele to the anterior
ccelom and thence to the exterior, it appears to lead from the
anterior ceelom to the hydrocele and thence to the exterior.
This apparent difference may be reconciled with the arrange-
ment on the left side by observing the angle which stone-
canal and pore-canal make with one another. Woodcut 8, p.
370, shows that this is an acute instead of an obtuse angle, and
hence that stone-canal and pore-canal have coalesced laterally ;
Woodcut 2 shows for the sake of comparison the normal stone-
canal and pore-canal and their relationship to the left hydro-
cele and the axial sinus or anterior celom.
370 E. W. MACBRIDE.
Fig. 89 is a section of a larva of Stage D; both hydrocceles
are well developed—the right, in fact, better than the left; the
Bre. HIG:
right hydroccele appears on the left side of the figure, since by
an oversight the section was drawn from the wrong aspect. It
took me some time in this larva to determine which side was
which, but the right hydroceele is rather more dorsally situated,
and opens by only a narrow slit into the anterior celom. It is
also curved somewhat differently, the most posterior lobe being
No. 4, not No. 3, as on the left side. Fig. 90 shows a most
remarkable variation. We see a pore opening directly from the
hydroceele to the exterior. If, as I shall attempt to show later,
the anterior coelom may be compared to the proboscis cavity of
Balanoglossus, and the two hydroceeles to the collar cavities of
that animal, we see that what we may terma collar-pore may
arise as a variation. Figs. 91—94 are sections taken from a
larva of Stage G. Its only abnormality is that in connection
with the right hydroccele, which is of normal character, a
second pore-canal and stone-canal are developed. Fig. 92
should show the opening of the second stone-canal into the
hydroceele, but the lithographer has unfortunately not brought
out the slit-like opening ; fig. 93 the opening of conjoined pore-
canal and stone-canal (compare woodcut 3) into the axial sinus.
Fig. 91 shows that the two pore-canals unite, to open by a
common median pore. The above are not by any means all
the variations observed, but they are sufficiently typical to in-
dicate their nature.
“THE DEVELOPMENT OF ASTERINA GIBBOSA. 371
The History of the Young Star-fish.
The just metamorphosed Asterina gibbosa has a disc of
about ‘6 millimetre in diameter; if we take R to denote the
length from the tip of the arm to the centre of the disc, then
R equals ‘36 millimetre. A larva such as that figured in
figs. 51—53 may be ‘8 millimetre from the tip of the adhesive
disc to the posterior end, and measured obliquely from the
dorsal end of the preoral lobe may exceed a millimetre in
length. There is, therefore, a considerable diminution in size
during the metamorphosis, the reason of which is evident when
we consider that no nutriment is taken during this time. A
full-grown specimen may have a diameter one hundred times
greater than that of the just metamorphosed star-fish,—that is,
it may exceed the latter one million times in bulk. The young
star-fish, however, rapidly increases in size, and by the time
R equals 3:7 millimetres the ovaries are visible. This is the
oldest stage I have examined; my account of the histology is,
however, taken from smaller specimens, in whichRequals ‘8mm.
The changes we shall have to consider are (1) the formation
of the primitive germ cells, the ovoid gland, genital rachis, and
ovaries ; (2) the dermal branchie; and (3) general histological
differentiation.
We have already in Fig. 76 seen the first trace of the ovoid
gland. It there appears as a ridge projecting into the axial
sinus ; inside this ridge there is as yet to be found only ameebo-
cytes, jelly and fibres, as is the case with the other blastoceelic
spaces in the larva. Later, a thickening of peritoneum takes
place on the wall of the left posterior ccelom opposite the aboral
end of this ridge—and from this thickened patch a cord of cells
grows into the ridge, gradually forcing its way in an oral
direction ; this is the characteristic core of the ovoid gland.
From this same thickening of peritoneum a cord of cells
grows out in a direction parallel to the disc ; this is the origin
of the genital rachis. By the outgrowth of a flap of peritoneum
it is enclosed in a space which is called the aboral sinus. The
genital rachis and the space enclosing it both give off branches
VOL. 38, PART 3.—NEW SER. BB
372 E. W. MACBRIDE.
one at each side of each arm. Local thickenings of these
branches of the rachis constitute the genital organs. The
surrounding spaces, the genital sinus (ad gon, figs. 122 and 123),
is shut off from the aboral sinus by the outgrowth of a septum.
Fig. 99 is the marginal portion of a section vertical to the disc
of a larva of StageG. We see the rudiment of the ovoid gland
(ovg.) as a fold projecting into the axial sinus. Further up we
notice a thickened patch of peritoneum, which is invaginated
into the septum separating the axial sinus from the left posterior
celom (pr. germ. inv.). This is the rudiment from which, on
the one hand, the genital rachis and, on the other, the core of
the ovoid gland are derived. Figs. 100—103, similar sections
to fig. 99, from a just metamorphosed star-fish, illustrate this.
We see that from this rudiment a cord of primitive germ
cells has grown out and filled the fold which is the
rudiment of the ovoid gland. The last two sections
cut a more oral portion of the fold, since they are slightly
oblique ; we see (figs. 102 and 103) that this core has not as yet
penetrated to the oral end of the fold, and, further, that the
fold is attached to the oral side of the inner perihemal ring,
or, in other words, that it traverses the lower end of the axial
sinus, and is attached to its lower side. The original invagina-
tion to form the germ cells is situated at the very tip of the right
dorsal horn of the left coelom, where it meets the right ventral
horn, but at this level the two horns do not open into each
other (see p. 367). Figs. 104—106, again representing sections
vertical to the margin of the disc, are taken from a young
star-fish, in which R equals ‘4 millimetre. Fig. 104 shows the
cord of cells which arises from the peritoneal invagination and
penetrates the dorsal organ, and the relation of this cord to
the right hydroccele and the axial sinus. We see that now this
core of cells reaches to the oral end of the ovoid gland, and
penetrates also a prolongation of the same, which is prolonged
as a fold, hanging from the aboral wall of the inner perihemal
canal (figs. 105 and 106).
Pl. 25, fig. 110, which represents a similar section to figs.
99—106, shows practically the adult condition of the ovoid
THE DEVELOPMENT OF ASTERINA GIBBOSA. ie
gland and neighbouring organs. We see that the madreporic
pore has commenced to be divided into two by the ingrowth of
a fold. It is not the case in Asterina, as far as I can make
out, that the numerous pore-canals found in the fully grown
adult are derived from fresh perforations, as Cuénot has stated
(3). Rather the statement which he quotes from Perrier seems
to give the actual method of their formation.1 We see that the
openings of the stone-canal proper and the pore-canal into the
axial sinus are still maintained. The ovoid gland with its core
is seen to reach right down to the oral end of the axial sinus,
and to be attached to its oral wall. Embedded in the septum
dividing the inner perihemal ring-canal (lower end of the axial
sinus—see woodcut 1) from the perihemal spaces proper is
the so-called oral blood-ring (sang. cire.). This is a ring-shaped
tract of peculiarly modified connective tissue; the section shows
that it is of a different nature from the ovoid gland, and has no
connection with it. In Asterias this ring gives off radial pro-
longations traversing the longitudinal septa of the radial
perihemal canals, but these do not exist in Asterina. The
development of this structure as far as its histology is con-
cerned is shown in PI. 24, figs. 107—109, which represent
small portions of sections parallel to the disc. The first two
sections are taken from the same specimen as figs. 82—84; in
this specimen as we have already learned (see above, p. 366)
the metamorphosis has just concluded. We see that the
mesenchymatous tissue between the outer and the inner peri-
heemal rings has undergone differentiation. Most of it has be-
come converted into fibrous tissue, but at one level no fibres
have been formed, the whole of the mesenchyme cells becoming
ameebocytes (sang. circ.); this part is the rudiment of the
blood-ring. In fig. 109, taken from a specimen in which R
equals ‘45 millimetre, we see that the ring is completely formed ;
1 Durham, in a paper on “ Wandering Cells in Echinoderms” (‘ Quart.
Journ. Mier. Sci.,’ vol. xxxiii), has described the communication of the axial
sinus and stone-canal in a young Cribrella. He also insists that we have no
blood-vessels, but rather “ haemal strands ” in Echinoderms, but makes the
common error of supposing the ovoid gland to belong to this category.
374 E. W. MACBRIDE.
the intercellular jelly or plasma has acquired staining properties.
To Leipoldt (9) is due the credit, in a careful paper on the
anatomy of *‘ the so-called excretory organ of the sea-urchin,”
of emphasising the fact that the ovoid gland and the oral blood-
ring are of totally different nature; he describes branches from the
blood-ring ramifying on the external surface of the ovoid gland.
The question arises, what is the true nature of this blood-
ring? Cuénot (8) answers that it is a lymphatic gland, or
centre for the formation of amcebocytes; and there is a great
deal to be said for this view. We must, however, remember
that structures of similar nature are found accompanying the
alimentary canal in Echinids and Holothurids. Ludwig (18)
has given a splendid description of their arrangement in the
last group. He brings out with great clearness that they are
tracts of connective tissue in which the fibres are sparse. The
close relation of these “ vessels ” to the alimentary canal suggests
forcibly that we may have here the first attempt at forming
blood-vessels. There is certainly no propulsive organ or proper
circulation, but the staining properties of the plasma show that
it has been chemically altered, and the idea is suggested of
some secretion from the gut-cells propelling itself along these
tracts by the vis a tergo force of secretion. In the Asterid
no close connection with the gut is observable,—the oral ccelom,
in fact, intervenes between the cesophagus and the ring, as we
have seen (p. 365); but the altered character of the plasma
suggests that perhaps here some substance is formed necessary
for the well-being of the organism, which then diffuses out into
the neighbouring coelomic spaces. The blood-spaces of the
higher animals are known in many cases to be remnants of the
blastoccele or segmentation cavity of the embryo; this has been
shown in the case of Balanoglossus with great clearness by
Spengel (21). Strictly speaking, therefore, the blood and
lymph spaces of other forms are represented in
Echinodermata by all the spaces in the body-wall
unoccupied by fibrous tissue and dermal ossicles, and
traversed by amcebocytes; but the blood-ring, gut vessels,
&c., may be a first attempt at specialisation.
THE DEVELOPMENT OF ASTERINA GIBBOSA. 370
Figs. 113—117 are intended to illustrate the formation of
the genital rachis; and they all represent portions of sections
cut parallel to the disc; those portions, in fact, which are
transverse sections of one of the five interradial folds of the
body-wall which in the star-fish project into the body-cavity.
As we see in Pl. 23, fig. 83, the axial sinus, right hydrocele,
and the stone-canal, are embedded in one of these folds. It
follows that the coelomic wall of this particular fold represents
the larval septum between the anterior ccelom and the pos-
terior cceloms; and its interradial position in the star-fish
becomes explained when we remember that the stalk with its
contained anterior ccelom lies opposite an interradius of the
water-vascular ring; which interradius is constituted by the
outgrowth of processes of the two lobes situated at the ends of
the hydroccele, which is as yet an imperfect ring. These out-
growths meet, so to speak, above the neck of the stalk. Figs.
118 and 114 are from the same specimen as fig. 109. We
see the appearance of the rudiment of the germ cells in a
section parallel to the adult plane, and notice the remains
of the cavity of invagination (fig. 114, pr. germ. inv.).
Fig. 113 shows us that one horn of the right hydroccle has
become embedded in the ovoid gland, and this is one reason
why it is extremely difficult to trace the continuity of the
primitive germ cells by sections taken parallel to the adult
plane, since the cord of cells is in some spots so narrow,
and is therefore difficult to distinguish from the epithelium
lining the right hydrocele. Longitudinal sections, such as
fig. 104, show it much better. In figs. 115 and 116 (taken
from a specimen in which R equals *7 millimetre) we see the
formation of the genital rachis; this takes place by a lateral
outgrowth from. the primitive patch of invaginated peri-
toneum, from which we have seen the core of the ovoid
gland originating as an orally directed outgrowth; the aboral
sinus which surrounds it (a@d.) is formed at the same time,
it is a portion of the ccelom shut off by the outgrowth
of a fold of peritoneum. Fig. 117, taken from a much
older specimen, shows the genital rachis in its complete form
376 E. W. MACBRIDE.
in continuity with the original rudiment of the primitive
germ cells.
It is, then, not quite correct to speak of the genital rachis as
being an outgrowth from the ovoid gland, as Cuénot has done
(3). This statement, nevertheless, marked a step in advance in
our knowledge, for it gave a hint as to the meaning of the ovoid
gland. Cuénot found specimens of Astropecten with the ovoid
gland, but without the genital rachis, and noting the identity of
the character of the cells in the two structures, stated that the
rachis was an outgrowth from the gland, though he found no
intermediate stages. These were first found by me (14) in
the Ophiurid Amphiura squamata, and at the same time I
demonstrated the epithelial origin of both gland and rachis.
It is the genital rachis which of course was formerly known as
the aboral blood-vessel; in most Asterids and Ophiurids it
later undergoes partial degeneration, giving rise to cells con-
taining violet pigment. Ludwig, however (11), and Haman
(7) have pointed out that the central core remains unaltered ;
the latter was the first to point out that in all Echinoderms,
except Holothurids, a genital rachis exists, of which the
genital organs are local outgrowths. In Amphiura squa-
mata, however, and in Asterina gibbosa, according to
Cuénot (8), the whole genital rachis remains unaltered through
life; this is only one of the many points in which Asterina
shows itself to be one of the most primitive of Asterids. In
the plans given in text-books of the blood system, two vessels
are shown proceeding from the aboral ring in the interradius
of the madreporite to the pyloric sac. These are two mesen-
teric bridles, remnants of the piece of septum left at this level
between the two horns (right dorsal and right ventral) of the
left celom. At this spot the right (aboral) celom breaks
through into the left (oral) ccelom, perforating the piece of tissue
referred to, and leaving only the mesenteries. The peritoneum
covering them seems to be peculiarly modified, and is possibly a
place where the amebocytes of the ccelomic fluid are formed.
The genital rachis gives off, as it passes each interradius,
two branches enclosed in corresponding branches of the aboral
THE DEVELOPMENT OF ASTERINA GIBBOSA. 377
sinus (gen. 7., woodcut 1); one of these branches runs in an oral
direction down each side of the interradial septum. This
septum is an ingrowth of the body-wall, which has by this time
become marked, though its first beginnings date back to the
end of the metamorphosis (Pl. 23, fig. 84).
A section of one of these branches in an older specimen is
given in Pl. 26, fig. 119). These genital branches are formed
as the rachis reaches each interradial septum before it has
formed a circle; in one specimen I have observed a rachis
reaching only to the next interradius, and there giving off one
genital branch. Figs. 120 and 121 (taken from the same
specimen as fig. 119) show the first rudiments of the genital
organs. The branch of the rachis ends in a swelling accom-
panied by a dilatation of the aboral sinus, and we see the begin-
ning of aseptum tending to shut off the main aboral sinus from
this dilatation. This septum was first described by Cuénot (8),
and in it the genital duct is formed. This is shown in fig. 123,
taken from the oldest specimen I examined, in which R equals
3:7 millimetres. We see that the genital duct is formed by
a core of primitive germ cells burrowing its way through the
body-wall. Fig. 122, from the same specimen, shows the con-
tinuity of the rachis and the ovary. We notice also the forma-
tion of follicles from the indifferent germ cells.
We are now in a position to compare the arrangement of
the ovoid gland and genital rachis and their accompanying
spaces in Amphiura squamata with that found in Asterina
gibbosa. In the former I described the genital rachis issuing
from the oral end of the gland and accompanied by three
spaces, which I named sinus a, sinus 6, and sinus ¢ (PI. 25,
fig. 112), This figure is a diagram of a section parallel to the
long axis of the stone-canal. Fig. 111 is a diagram of a
similar section of Asterina, but it is not quite accurate, since it
shows both the ovoid gland and the stone-canal, and these two
structures do not lie in the same radial plane in Asterina.
In order to avoid obscuring the opening of the stone-canal into
the axial sinus, it is necessary to indicate part of the ovoid
gland by dotted lines.
378 E. W. MACBRIDE.
Comparing figs. 111 and 112 we see that the axial sinus of
Asterina is represented in Amphiura by sinus ¢, the so-called
“ampulla.” The aboral sinus (ad, fig. 111, sinus a, fig. 112)
is also obviously homologous in both.
[Since my paper (14) was published, and since the present
work was sent in for publication, I have made a careful re-
examination of my sections of Amphiura squamata, and
have arrived at amore complete comprehension of the structure
and development of the ovoid gland and the neighbouring
spaces in that animal. The space marked sinus 0’ (fig. 112)
is not, as I formerly supposed, a part of sinus 4, but is quite
distinct. Sinus 0’ probably represents the right hydroceele ;
it is already present in the youngest specimens I examined.
Sinus 5* is a portion of the ccelom shut off by the outgrowth of
a flap of peritoneum; from the inner wall of this sinus the
cells which at the same time give rise to the ovoid gland and
to the genital rachis take their origin; it is obviously homolo-
gous to the cavity of the invagination of the primitive germ
cells (pr. germ inv., figs. 110 and 111), only in Asterina this
space disappears.—December, 1895. |
We observe that the arrangement in Amphiura might be
obtained from that in Asterina by rotating the stone-canal and
accompanying structures outwards and downwards through an
angle of 180°. That this is what has occurred in phylogeny
is indicated, not only by the fact that in the young Amphiura
the madreporite is near the edge of the disc and the stone-
canal almost horizontal, whereas in the adult the madreporite
is situated far in towards the mouth on the oral surface, but
also by the curious undulating course of the genital rachis,
which is aboral in the interradii and oral in the radii. This
points to the conclusion that the aboral parts of the interradii
* In my paper on this subject (14) sinus Jis referred to as the axial sinus—
it was formerly supposed to be continuous with sinus ¢, though Ludwig knew
this was not so. At that time the meaning of the axial sinus in Asterids
which Bury first suggested (2) was not generally known, and his interpreta-
tions were not accepted, and hence two different spaces were called axial
sinus, one in Asterids and the other in Ophiurids.
THE DEVELOPMENT OF ASTERINA GIBBOSA. 379
have greatly developed, and have grown in between the radii on
to the oral surface, forcing the original oral plates to the extreme
centre of the disc; and so the stone-canal has been swung round
and the genital rachis pulled out of shape. Nowin Asterina
gibbosa there is a trace of this process; the rachis does
not, as Hamann (7) has described in Asterias, lie in one plane,
but pursues an undulating course, being much more aboral in
the radii than the interradii. I am inclined to look upon this
as the primitive condition from which the Asterid and Ophiurid
arrangements have been derived. I may as well mention here
some other facts which indicate the primitive nature of Asterina.
Chief among them is, that in the family of which it is a member
we meet with the most rudimentary form of those characteristic
Asterid organs the pedicellariz. We have in Asterina the
aboral surface covered with small spines, arranged in twos and
threes, and acting on irritation like pedicellariz. It is true
that some Asterids have no pedicellariz, but here the evidence
from allied genera (cf. Luidia and Astropecten) suggests that
they have been lost; Asterina, however, shows us pedicellariz
in statu nascendi. The simple biserial tube-feet also con-
stitute a primitive character.
Fig. 118 represents ovoid gland and stone-canal in the
latest stage examined by me. The gland is attached by an
exceedingly narrow pedicle to the wall of the axial sinus.
Its surface is thrown into deep folds, and the peritoneal
lining of the axial sinus, which forms its outer covering, is
modified, consisting of cylindrical cells with projecting rounded
ends. The interior of the gland is filled with a mass of primitive
germ cells supported by fibres, doubtless of mesenchymatous
origin. I was unable to find any trace of a tube lined by
primitive germ cells, such as was discovered by Hamann in
the young Asterias.
What, we may finally ask, is the function of this strange
organ? Cuénot, as usual, maintains that it 1s a lymphatic
organ. This I am disposed to doubt very strongly; the
cells which it contains are of quite a different nature from
the amcebocytes of the oral blood-ring, and the evidence that
380 E. W. MACBRIDE.
Cuénot brings to show that they escape by diapedesis into the
axial sinus is quite insufficient. The cells of outer epithelial
lining are not flattened but cylindrical, and I strongly suspect
that he has mistaken their freely projecting ends for escaping
amoebocytes; and I may remark that this curious outer
epithelium shows its distinctive character from the time the first
rudiment of the ovoid gland appears. Whatever its function
may be now, there is no doubt that the ovoid gland was pri-
mitively a part of the genital organ, and probably is a remnant
of the arrangement of the reproductive cells before the radial
symmetry was acquired. It is interesting to notice that it
originates from the left posterior coelomic wall, whereas an
analogous organ in Crinoids arises in the right or aboral ceelom,
so that they are not strictly homologous.
If Hamann is, as there is strong reason to suppose, right
in stating that the primitive germ cells wander along the
rachis into the genital organ, it seems very probable that,
at any rate in the young adult, the ovoid gland is a centre
of formation of the primitive germ cells; and its relation to
the axial sinus may have to do with its aération, for it must be
remembered that the pore-canal opens into the axial sinus, and
the current in this is, as we shall see, inwards. In the fully
grown adult it no doubt undergoes, to some extent, the degene-
rative change noted above in the genital rachis of other genera.
What the meaning of this change is, is very obscure. Obser-
vations on the histology of the gland at different seasons might
elucidate its meaning.
Turning now to the stone-canal, we see, in fig. 118 (a section
transverse to the axial sinus and stone-canal), the beginning
of that curious T-shaped ingrowth which is so marked a feature
of the stone-canals of Asterids, but which is much less developed
in Asterina than in other genera. It is covered by short cilia,
the rest of the epithelium bearing long flagella.
Cuénot asserted that the stone-canal was a functionless rudi-
ment, the current being neither outwards nor inwards, Ludwig!
' Ludwig, “ Uber die Function der Madreporenplatte und des Steinkanals
der Echinodermen,” ‘ Zool. Anz.,’ 1890, p. 377,
THE DEVELOPMENT OF ASTERINA GIBBOSA. 381
subsequently showed that in the stone-canal of Holothu-
rids and KEchinids the direction of the current is inwards.
He examined the stone-canal cut out of the living animal; I
have confirmed his result by a somewhat more satisfactory
method. I kept Amphiura squamata living for several days
in sea water, carrying in one case carmine, and in another
lamp-black in suspension; and on cutting sections I found
these particles in the pore-canal, and in some cases apparently
ingested by the cells lining it. In view of Ludwig’s researches
Cuénot comes in a later paper (4) to what I believe to be the
correct solution of the question of function. He there suggests
that the flagella lining the stone-canal are always tending to
produce an inward current, and that thus the turgidity of the
whole water-vascular system is kept up. [This is practically
the old view; except that he does not assert a continuous
inward current.— December, 1895. |
It is obvious from the structure of the valves of the tube-
feet that, in consequence of the ambulatory movements, there
must be a slow loss of fluid. The ampulla and the tube-foot
are shut off from the canal leading into the radial water-vascu-
lar canal by a pair of valves opening only inwards. Conse-
quently during the contraction of either ampulla or tube-foot
the two act together as a closed system, since no fluid can escape
into the radial canal. The existence of the valves however shows
clearly that fluid occasionally enters the tube-foot, and this can
only be rendered possible by a slow loss of turgidity owing to
the osmosis of the contained fluid when under pressure. This
is confirmed by considering the case of Ophiurids, where, the
tube-feet having lost their ambulatory function, the madre-
porite has only one or at most two pores, and the calibre of the
stone-canal is exceedingly narrow.
The dermal branchiz arise when the star-fish has reached a
diameter of about 1°5 millimetres (R equal *85 millimetre).
We see that the branchia is only a very thin piece of the body-
wall produced into a finger-like process (Pl. 23, fig. 98).
Around the base of the branchia is a peribranchial space lined
by flattened epithelium: this space, as Cuénot has rightly
382 E. W. MACBRIDE.
observed, is the only one of the great ‘‘schizoccelic”’ spaces
which Hamann (8) has described in the body-wall which has
any real existence, the others being merely artefacts produced
by the process of decalcification. I have found one specimen
showing the first trace of a dermal branchia (figs. 96 and 97).
We see a slight thickening of the peritoneum, and above it the
peribranchial space. Fig. 96 shows that the latter is a diverti-
culum of the ceelom. As I have only one section illustrating
this I do not speak with absolute certainty on the point; but,
with this possible though very improbable exception, there is
no schizocele whatsoever in Asterina gibbosa: all
spaces lined by epithelium are of celomic origin.
Histological Differentiation.
The cells of the gut-wall have undergone some change since
the close of the metamorphosis. Specimens of the epithelium
from different regions are given in Pl. 26, figs. 129—132. These
are all taken from a young adult in which R equals ‘85 milli-
metre. The cells of the lateral walls of the stomach (i.e. the
adult oesophagus) have become exceedingly long and narrow ;
their outer ends are refracting and take a light yellow tone
with osmic acid (fig. 129). The cells of the aboral wall, on
the contrary, have developed numerous gland cells filled with
globules; interspersed amongst them are some very narrow
filamentous cells. Fig. 180 shows the spot marked x where the
stomach opens into the pyloric sac and the abrupt change
in the character of the epithelium. The pyloric sac is lined
by uniform columnar cells; the nucleus is generally near the
base of the cell, and is never placed further up than the
middle, and the protoplasm is uniformly granular (fig. 131).
The cells lining the rectal ceecum (fig. 132) are similar in form
but smaller, and the protoplasm is clearer, with the outer part
more refringent. It is at least a plausible suggestion that the
gland cells of the stomach secrete the poison which paralyses
the prey, and that the cells of the pyloric sac give rise to a
digestive ferment.
The differentiation of tissues which has gone on in the
THE DEVELOPMENT OF ASTERINA GIBRBOSA. 383
body-wall is illustrated in Pl. 28, figs. 146 and 147. These
sections are taken from young adults in which R equals ‘4 mm.
and ‘86 mm. respectively, and they pass through the same
region as fig. 145, which is from a larva in Stage H, and which
we have already described. In fig. 146 we see that the mus-
cular fibres of the muscle we may call the dilator ani are
still connected with the peritoneal cells; but in fig. 147 they
have become quite distinct, and the cells of the peritoneum
have become quite flattened. The ectoderm has entirely
changed its character, the numerous larval goblet cells have
almost disappeared, and thecells in general have become shorter;
many of them are inversely wedge-shaped, and are apparently
about to become converted into gland cells, probably of the
same histological character as those of the aboral wall of the
stomach. Here and there is a narrow cell ending in a fine
hair, one of the scattered sense-cells of the aboral surface ;
these are shown in fig. 148, a piece of ectoderm from another
individual of the same age. All observers agree in maintain-
ing that the ectoderm of the adult retains its ciliated covering;
but though I have been able to make out easily the cilia, or rather
flagella of the metamorphosing larva, I have not been able to
do so with any certainty in the aboral wall of these young
adults. Probably the cilia are very delicate and fragile. The
tissues of the mesenchyme have undergone marked differentia-
tion. So far as my researches have extended it seems that
three fates are open to mesenchyme cells, all of which are
illustrated in fig. 147. They may remain practically unchanged
as amoebocytes or wandering cells (amed.), or they may become
embedded in bundles of fibres so as to form connective-tissue
cells (the fibres being intercellular, not outgrowths of cells) ;
or, finally, they may fuse to form a syncytium having the form
of a meshwork (calc,). This is the skeletogenous tissue; lime
is deposited in the interstices of the meshwork. There is a
fourth fate, which is not reached by any as far as I have gone,
but which obviously must be the lot of some, and that is to
form the muscles moving the spines or rudimentary pedicellariz,
The superficial position of these muscles renders it exceedingly
384 E. W. MACBRIDE.
unlikely that their muscles are of peritoneal origin, and their
position in other Asterids where, as in Asterias, for example,
they occur on the skin covering the spines, growing even from
their tips, makes such a supposition almost impossible. There-
fore we must postulate some muscles of mesenchymatous origin
for Asterina, although all those which I have examined are of
epithelial origin.
The development of the nervous system has advanced greatly,
and has reached, as soon as the metamorphosis is complete,
its final form ; this is shown in fig. 141, taken from the same
specimen as fig. 146. The ectoderm cells have increased
immensely in number, and become excessively filamentous, so
that the nuclei are many layers deep; the fibrillar layer has
increased very much in thickness. It is traversed by vertical
fibres which sometimes branch and sometimes have small
nuclei on them; these are in continuity with the ectoderm
cells, but are probably of non-nervous character. Sections
parallel to the disc show that numerous little bipolar cells are
embedded in the mass of fibrils (Pl. 24, fig. 109, dip. gang.).
Since these cells are not present in the just metamorphosed
form, they must be ectoderm cells which have passed in, and
occasionally one sees a cell just at the boundary of the fibres
apparently in the act of passing in. The perihzemal spaces
become closely apposed to the nerve-cord, no mesenchyme
being left between (ph. fig. 141) ; the vertical fibres do not, how-
ever, arise in connection with the epithelium of these cavities,
since they are present before this close apposition takes place.
Cuénot states that all the ectoderm cells of the nerve-cord end
in the vertical supporting fibres described above. This is a
bold statement which it is quite impossible to prove by sections,
and which is most improbable. As a matter of fact these
vertical fibres are not present in nearly large enough number
to account for all the ectoderm cells ; and Hamann’s statement
(8) is probably correct, that many of these end in fine processes
which lose themselves in the mass of fibrils.
The sense-orgaus of Asterina are all developed in connection
with the appendages of the water-vascular system. The eye
THE DEVELOPMENT OF ASTERINA GIBBOSA. 385
arises at the base of the terminal tentacle of the radial canal ;
two stages in its development are given in PI. 28, figs. 142 and
143. In the first we see a simple ectodermic involution; in
the second we see a pit surrounded by columnar cells, pro-
bably retinal, and filled up by closely fitting polygonal cells,
which correspond to the layer of vitelligenous cells in an
Arthropod eye. The existence of these cells has been vigor-
ously denied by Cuénot (8), who maintains that we have only
polygonal cuticular plates. My sections, however, remove all
doubt on the subject; they show with perfect clearness that we
have to do with cells, and their nuclei can be made out. This
pit is the first only of the numerous pits which cover the “eye ”’
of the adult, which is really essentially a small rounded
swelling at the very tip of the radial nerve. The method
of preservation employed seems to have dissolved the pigment.
The remaining sense-orgaus are the tips of the
tube-feet and the terminal tentacle. A longitudinal
section of a tube-foot is given in Pl. 28, fig. 150. This is
taken from a specimen in which R equals ‘4 millimetre, but
it holds true for specimens of a radius of a millimetre or more,—
that is, for probably the first two months after the metamor-
phosis. Comparing it with fig. 149, a similar section taken
from a larva in Stage F, we see that the ectoderm at the tip
has become thickened, and underneath it we can make out on
each side a mass of nerve-fibrils. A powerful nerve leaves the
radial nerve-cord to supply each sense disc; it would be more
correct to speak of these branches as actual prolongations of
the nerve-cord with its cells and fibrils; they are, indeed, the
only conspicuous branches which it gives off. Some of the
ectoderm cells of the sense dise have a peculiar regular cylin-
drical form, which recalls that of the retinal cells.
The facts above related justify the view that the whole radial
canal with its tube-feet is to be looked on as one large branched
tentacle, the main function of which was probably originally
prehensile and therefore also sensory; and since a plexus of
nerve-fibrils is in the adult found under the ectoderm all over
the body, the central nervous system may be said to be a local
386 E. W. MACBRIDE.
concentration of this in the neighbourhood of a greatly deve-
loped sensory tentacle. The support of this tentacle by the
arm is a secondary matter, as we have already learned—a fact
which comes out still more clearly in Crinoid development.
There the primary hydrocele lobes develop into long free
tentacles covered with sensory hairs. At a very late period
(later than any which Seeliger observed) these primary tentacles,
according to Perrier (17) become applied to five outgrowths
of the body-wall; these latter immediately bifurcate to form
the ten arms, and so the free tips of the tentacles are situated
each in the angle between a pair of arms. Seeliger (18) adduces
this last fact to show that the primary tentacles are not the
same as the primary hydroceele lobes of Asterids, forgetting
that the point where a pair of arms diverge corresponds to the
tip of the Asterid arm, since in Antedon there are ten arms
which have arisen by dichotomy from five.
The epithelium of the water-vascular system in fig. 150
shows an interesting feature; the cells have developed muscular
tails which are arranged longitudinally, and the important point
is that these myo-epithelial cells persist as such for a
considerable period of free life.
Pl. 29, figs. 151—154, show us that the aboral wall of the
perihemal space also gives rise to muscles. These connect
one ambulacral ossicle with its fellow of the opposite side, and
serve, by approximating these to one another, to close the ambu-
lacral groove. Figs, 151 and 152 show us that here again we
have, in the first instance, to do with myo-epithelial cells.
Muscles connecting one ossicle with its successor and prede-
cessor are also present, but very much more feebly developed.
In Ophiurids, however, as is well known, they are most power-
ful, and this point gives the key to nearly all the peculiarities
of this group as compared with Asterids. Presuming, as we
fairly may, that these muscles are developed from the peri-
hzemal wall as in Asterids, we are brought face to face with a
most interesting effect which this produces on the nervous
system. Tig. 156 gives a section of the radial nerve-cord of an
Ophiurid. We notice two great masses of cells and fibres on
THE DEVELOPMENT OF ASTERINA GIBBOSA. 387
the aboral side of the nerve-cord, and Hamann (8) has shown
that the nerves for the ambulacral muscles arise entirely from
these.
Now it has been for a long time suspected, and Cuénot has
finally proved it (4), that there is a similar but feebler develop-
ment of what we may call “coelomic nervous tissue” takes
place in the Asterid. None of my specimens were old enough
to show this, though one can see (fig. 141) that the perihemal
epithelium has come into intimate connection with the nervous
matter. Pl. 29, fig. 155, represents a transverse section of
the nerve-cord of a young Asterias; we see in it that this
epithelium has become thickened on each side of the median
septum; one requires, however, a section of the nerve of a
fully grown adult to see the coelomic nervous fibrils. So we
may say that from their aboral wall the perihzmal spaces give
rise to muscles, and from their oral wall to the corresponding
nervous tissue. I ought to mention in this place that
Cuénot describes a canal leading from the perihemal space
into the celom at the level of each ambulacral ossicle; also
five pores leading from the outer perihemal ring to the celom.
If these communications exist, they are certainly secondary,
as there is no trace of them in my specimens; but as Cuénot’s
results were founded on injection I am exceedingly sceptical
as to the existence of such openings.
I have said above that the increasing importance of the
ambulacral muscles is the explanation of the evolution of
Ophiurids from Asterids. The Ophiurids have substituted
the quick powerful movements of these muscles for the slow
motions of the tube-feet. In correlation the nervous system
has become better developed, the radial cords becoming gan-
gliated, and the whole is removed from the exterior by invagi-
nation, and thus the subneural space is really a neural canal.
The ambulacral ossicles have become firmly united, each to its
fellow, to form a series of vertebre, and thus the cavity of the
arm is reduced, and this, with the simpler food, accounts for
the disappearance of the pyloric czca.
We have already pointed out that the lessened activity of
VOL. 38, PART 3.—NEW SER. (ote
388 E. W. MACBRIDE.
the tube-feet, consequent upon the loss of the locomotor
function, explains the reduced stone-canal and madreporite,
though probably their increased sensitiveness has helped in
developing the nervous system.
Literature consulted.
An account of the earliest publications on Echinoderm de-
velopment is not given here, since a résumé of them will be
found in the papers I quote; and I hold it to be a waste of
time to reiterate with each new paper the whole history of the
growth of our knowledge ab initio. I mention here only
those authors on whose results I have, so to speak, built, or from
whom I have found it necessary to differ. Ludwig’s work on
the anatomy of Asterids (10) laid the foundation of our know-
ledge of the heemal and perihemal systems ; though, as we have
seen, many of his ideas about these structures were incorrect.
Subsequently in treating of Ophiurids (11) he discovered the
genital rachis. Hamann (7) extended this result, and pointed
out the ameeboid nature of the primitive germ cells. Then we
had Ludwig’s great work on the development of Asterina
gibbosa (12), the first account of the metamorphosis of any
Echinoderm which had any pretence of completeness, and to
which I have constant occasion to refer. His account of the
changes in external form and of the developmeut of the
calcareous plates can hardly be improved upon. Owing,
however, to the imperfect methods in vogue at that time he
failed to penetrate with equal success into the course of the
internal changes. He saw nothing of the segmentation of the
celom or of the ring-like growth of the left coelomic vesicle ;
he saw nothing also of the origin of genital organs, ovoid
gland, or oral celom. He did not observe the right hydrocele
or find the origin of the perihzmal spaces. He missed the
fixed stage, and he does not seem to have had any clear con-
ception of the relation to each other of the larval and adult
planes of symmetry. We owe to him, however, the clear
distinction of pore-canal and stone-caual, and the recognition
of the fact that the pore-canal is completely independent of the
THE DEVELOPMENT, OF ASTERINA GIBBOSA. 389
hydrocele. Bury (1) may be said to have introduced modern
conceptions of Echinoderm development by his work on the
development of Antedon; there he distinguished between an-
terior celom and hydroccele, and showed that the stalk was the
preoral lobe. Then he made a series of observations on Echino-
derm larve (2), and showed that generally speaking the celom
on each side becomes segmented into two vesicles, an anterior
and a posterior. He, however, regarded the hydrocele as an
essentially unpaired structure, an outgrowth from the anterior
ceelom, and was greatly distressed to find that it originated
from the posterior vesicle in Ophiurids, and that in Asterina
the stone-canal, which in other forms represented the original
neck of communication between anterior celom and hydrocele,
was apparently an independent perforation. The Jast difficulty
has been answered by Ludwig;! as to the former, the proof I
have brought that the hydrocele is paired shows that there are
really three primary divisions of the coelom on each side, viz.
the anterior celom, single in Asterina, but primitively paired
in Asterias; the right or left hydrocele, and the posterior
ccelom (right or left as the case may be); the apparent forma-
tion therefore of the hydroccele from the anterior or posterior
vesicle is a mere question as to whether the septum between
the posterior coelom and the hydrocele or the septum between
the hydrocele and the anterior ccelom is formed first.
In speaking of the Bipinnaria, Bury says that in a future
paper he intends to prove that the anterior coelom becomes the
axial sinus, but up till now he has published nothing further
on the subject.” He made a few observations on Asterina
1 Bury had not seen the stage of development when the stone-canal is an
open groove.
2 Since the preliminary account (15) of the present paper was published, a
paper on the “‘ Organogeny of Stellerids,” by M. Achille Russo, has appeared
in the ‘ Atti della Accadema reale di Napoli’ for 1894. In this work (to
which I only obtained access some considerable time after the present paper
was finished) M. Russo gives a description of the ontogeny and anatomy of
the ovoid gland and axial sinus in Asterina gibbosa and an Ophiurid. He
combats my statements about the origin of these structures in Amphiura
squamata. ‘The origin of the axial sinus in Asterina has been correctly de-
scribed; it is about the only thing that is correctly described in the paper,
390 E. W. MACBRIDE.
larve of Stage D, and saw the completely closed ccelomic
vesicle on the right, and the imperfect transverse septum on the
left side, and was at a loss how to interpret these appearances ;
the right hydrocele he calls a mesenchymatous vesicle.
It is curious to see how unable many zoologists have been to
grasp Bury’s idea of the anterior celom; thus Seeliger, who
has confirmed his work on Antedon and amplified it till it
may be said that we have an exhaustive knowledge of the
subject, objects to consider the structure Bury named anterior
ccelom as such, on the supposition that Bury meant by that a
fellow of the hydrocele, which it obviously is not. Seeliger
calls it the “parietal canal,’ but the structural facts he so
accurately relates are convincingly in favour of Bury’s inter-
pretation. The weak point in Bury’s observations on Plutei
and other larve was that in no case were any more than a few
stages taken at random examined; but I hope the account I
have given in this paper will provide a more solid basis for the
idea of segmentation of the celom in Echinoderms. Field (5)
has published a short paper on the development of the Bipin-
naria; he carries it up only to a stage corresponding to midway
between Stages B and C of Asterina. The chief points of
interest in the paper are that many of the larve had two
madreporic pores, and he suggests that this is a normal stage
in the ontogeny; also that the two ciliated rings characteristic
of the Bipinnaria are derived from one, and that there is a
preoral sense-organ comparable to that in Antedon.
This paper does not contain the discovery that the water-
vascular rudiment is paired; for, as a matter of fact, in the
oldest larva examined no trace of the left hydroccle was
present. The “ schizoccelic space,” near the madreporic pore,
may represent the rudiment of the right hydroceele; needless
to say, it was not recognised as such.
Theel (22) has recently succeeded in following the meta-
morphosis in Echinocyamus pusillus so far as the external
features are concerned. He finds that already in the blastula
M. Russo’s technique was obviously not equal to dealing successfully with
such difficult subjects as Kchinoderm larvee.
THE DEVELOPMENT OF ASTERINA GIBBOSA. 391
a preoral sense-organ is present; this subsequently becomes
incorporated with the ciliated ring, and if this organ is homo-
logous with that of the Bipinnaria, we may conclude that the
ciliated band of the Pluteus corresponds only to the posterior
of the two bands of the Bipinnaria, since in the Bipinnaria
the sense-organ is situated between preoral and post-oral
ciliated bands, and this spot corresponds to a constriction in
the original longitudinal ciliated ring, not to a position on its
anterior edge.
Our knowledge of Echinoderm histology is largely due to
Hamann (8) and Cuénot (3 and 4). The latter, as we have
seen above, was the first to suggest that the ovoid gland gave
rise to the genital rachis. The first account of the development
of ovoid gland and rachis is given in my paper on Amphiura
squamata (14), and I have there collected the fragmentary
notices on this subject, which had till then appeared.
[I regret that when I sent in this paper for publication I did
not mention the well-known paper of Metschnikoff (“ Studien
tiber die Entwickelung der Echinodermen und Nemertinen,”
‘Mémoires de l’ Académie Impériale de St. Pétersbourg,’ tome
xiv, No. 8), in which he describes a right hydrocele in
Amphiuriasquamata. He there says that the right coelomic
vesicle becomes divided into anterior and posterior portions
just like the left ; the anterior portion sometimes atrophies but
sometimes develops into a regular five-lobed hydrocele. It
has been the fashion to ignore this work, since it was not
accomplished by modern methods; but after my experience
with Asterina I feel morally certain that Metschnikoff was
right, though of course he did not distinguish between
hydroceeles and anterior celom. Bury (2) seems to have missed
the importance of this observation.—Dec., 1895.]
General Considerations.
On reviewing the developmental history recorded in this
paper, two main questions present themselves: first, what
light does it throw on the affinities of the Asterids with other
Echinoderms? and second, does it suggest any direction in
392 E. W. MACBRIDE.
which we may look to find the origin of the group Echino-
dermata as a whole?
In answer to the first question, we must observe that the
stalks of Asterina and Antedon are morphologically equivalent,!
both being formed from the preoral lobe, and, so far as one
might judge from the different shape of the latter in the two
cases, the adhesive discs by which they fix themselves are
situated in precisely the same position. Now no one doubts
that Antedon had a fixed ancestor; it is, in fact, one of the
very few Crinoids which do not remain fixed throughout their
whole life. If Asterids ever had an ancestor in common with
Crinoids which could be called an Echinoderm at all, it must
have been one represented by the fixed larva of Antedon before
it has fully acquired radial symmetry, since, as we have already
pointed out, the metamorphoses of Antedon and Asterina
pursue different courses. In the first case the mouth is shifted
backwards and upwards, and a precisely similar thing happens
to the larve of Entoproct Polyzoa, Ascidians, and Cirri-
pedes when they fix themselves. In the second case, how-
ever, the disc is flexed obliquely downwards on the stalk, so that
the left coelomic sac and the hydroceele both come to encircle
the base of the stalk ; and as consequence the aboral poles in
the two cases are not homologous, for in the first case this pole
is the cicatrice left by the rupture of the stalk, whereas in the
second case the point where the stalk passes into the disc is
quite remote from the aboral pole. The apparent correspondence
of the calcareous plates of the calyx in Antedon and the so-
called calyx in Asterina is simply due, in my opinion, to the
' Since the present paper was sent in for publication, my attention has been
called to some observations of Perrier’s which I regret having overlooked. In
his account of the Echinoderms collected by the “‘ Mission Scientifique du Cap
Horn,” he describes the larve of Asterias spirabilis, which adhere to the
buccal membrane of the mother. They are attached by a pedicle which
Perrier compares to the stalk of the Antedon larva and to the preoral lobe of
the Asterina larva. He points out that both in the case of Asterias spira-
bilis and of Asterina gibbosa the pedicle arises fromthe oral surface, whereas
in Antedon it is aboral in its origin, but he offers no explanation of this dif-
ference in position.
THE DEVELOPMENT OF ASTERINA GIBBOSA. 393
fact that their arrangement is in both cases dominated by the
prevailing pentamerous symmetry of the adult.
The reason why the change in the position of the mouth
takes place in Antedon is that this animal, like the others in
which a similar change occurs, feeds on swimming or floating
prey, and, so to speak, turns the mouth upwards to receive it.
Asterids and their allies, on the other hand, find their food on
the substratum, and therefore we must suppose that in the fixed
ancestor of Asterids the body was flexed downwards so as to
bring the substratum within reach of the tentacles. The
difficulty suggests itself that a fixed form finding its food on
the substratum might very soon devour all within its reach ;
and the suggestion may be made that perhaps the ancestor of
Asterids never was fixed, but that the divergence from Crinoids
took place when the common ancestor was a creeping form,
since we may reasonably conclude that creeping habits formed
the transition stage between a free-swimming and a fixed mode
of life. In this case, however, the difficulty meets us of
accounting for that radial symmetry which is so deeply impressed
on the organisation of Asterids and other forms. It would
be rash to say that fixed life is the direct cause of radial
symmetry when we consider the case of Brachiopods, Cirripedes,
&c., but this symmetry is only, so far as our knowledge goes,
developed in connection with a fixed life.
The proximate cause of the radial symmetry of Asterids is
the immense preponderance of the organs of the left side, and
it is difficult to see how this could have gone on to the extent
it has done in an animal which moved about with a definite
part directed forwards. The motion of the Asterid when
metamorphosed is vague,—that is, any part is directed forwards;
and it seems to me that a fixed stage must intervene between
this and the mode of motion in which the head went first.
1 Some might object that Ctenophores and Meduse are radially sym-
metrical, but the first are not truly so; and as to the second, I hold very
strongly the view that the Medusa is only a specialised bud, which has secon-
darily acquired locomotive powers in order to disperse the ova. Its radial
symmetry has been inherited from fixed ancestors.
394 E. W. MACBRIDE.
Therefore I feel that we are shut up to the supposition that
Asterids had a fixed ancestor, and we must suppose that this
form lived under conditions where enough food drifted along
the bottom to meet its demands. PI. 29, fig. 157, represents
the characters which I consider the common ancestor of all
Echinoderms possessed when it became fixed. Figs. 158 and
159 show how these characters became modified in the cases of
the Asterid and Crinoid respectively.
It is probable that a fixed stage occurs in the life history
of all Asterids. The larve of Echinaster and Asterias
Miilleri, which are carried in brood-pouches, certainly possess
one, and the three papille on the Brachiolaria larve are
generally interpreted as an apparatus for fixation.
The fixed stage has, however, been lost so far as we know in
all other Echinoderms; and it is instructive to note in this
connection that Asterids alone retain the great preoral lobe.
This has completely atrophied in the Plutei both of Ophiurids
and Echinids ; and in the latter case, as I have indicated above,
(page 391) there is some evidence to show that a preoral ciliated
band has likewise disappeared. The Auricularia still retains
a trace of the preoral lobe, and it has been regarded as an ex-
ceedingly primitive form because it retains the undivided lon-
gitudinal ciliated band, and because the larval mouth becomes
the adult one. The internal anatomy of this larva shows that,
except in these two points, it is the most modified of all; the
anterior coelom so conspicuous in the Bipinnaria is represented,
as Bury has shown (2), by a bud of cells which forms the
secondary madreporite on the stone-canal, and the whole mode
of segmentation of the ccelom is most erratic.
I have dwelt on this subject at some length because some
have regarded the Holothurids as the primitive group of the
Echinoderms, and Sémon (19) has even attempted to show that
the primary hydroceele lobes in them became the oral tentacles,
whilst the so-called radial canals were really interradial out-
growths. Ludwig (13) has, however, shown the incorrectness of
this; in the Synaptidz alone do the oral tentacles spring
direct from the ring-canal, and it was the development of
THE DEVELOPMENT OF ASTERINA GIBBOSA. 395
Synapta on which Sémon based his theory. In all Holothu-
rids the buccal tentacles spring like the buccal tube-feet of
Echinids from the proximal portion of the radial canals. It is,
however, difficult for me to see how anyone can doubt that the
Asterids are the least modified group of the Echinoderms, I
have already dealt with their relations to Ophiurids, and have also
pointed out that the Asterid central nervous system is really a
concentration of the diffuse nervous plexus in connection with
what must be regarded as a great sensory tentacle,—that, in fact,
the whole radial water-vascular canal is to be regarded as a
pinnately branched tentacle for which the arm is a secondary
support. Sémon himself has suggested this (20), and it comes
out even more clearly in Crinoid development than in the case
of Asterids. Now the long radial canals in Echinids, ending
in degenerate sense tentacles, clearly at one time had arms to
support them; but these supports have been drawn back into
the body. The Holothurids have been probably derived from
the primitive Echinids; their calcareous nodules are most
likely plates and spines atrophied in order to allow of free
muscular movement. The terminal sense tentacles of the
radial canals have entirely disappeared, and the forward shift
of the madreporite and genital opening is no more difficult
to understand than the varying position of the anus in Echi-
nids. In the Asterids alone is locomotion entirely dependent
on the tube-feet, and in them only we have the nervous system
exposed.
On the second question, viz. that of the affinities of the
Echinodermata as a whole, much light is thrown by the
development of Asterina gibbosa. It is of course well
known that the Tornaria larva of Balanoglossus shows a strong
resemblance to the Bipinnaria in the course of its ciliated
bands, and in possessing a preoral celom opening by a pore on
the left. The adult Balanoglossus has five celomic cavities,
and Bateson has shown that these arise as separate pouches of
the gut, The question arises whether it is legitimate to
homologise with these the five coelomic cavities of the Asterina
larva which arise by division of pouches already formed, but
396 E. W. MACBRIDE.
which still persist in the adult as sharply separated cavities,
only the most posterior pair, viz. the right and left posterior
celoms (oral and aboral) of the adult having partially fused
with each other. The development of Antedon seems to answer
this question in the affirmative. In its case the hydroceele is
budded off quite independently of the posterior ccelomic sacs.
Adopting, then, the view that the ccelomie sacs of the Ente-
ropneusta and Asterids correspond, we find that the hydrocele
represents the collar cavity. Now in Cephalodiscus the collar
cavities are produced into long pinnately branched tentacles,
comparable to the radial water-vascular canals, and further a
branch from the central nervous system accompanies each
tentacle, just as the radial nerves accompany the radial canals
in Echinoderms. Now, if we suppose that the two hydroceles
of Asterina were equally developed and approximated in the
mid-dorsal line, the fusion of the anterior portion of the two
nerve “‘rings,”’ which of course would in this case be only open
curves (since a ring-form is attained through the preponderating
srowth of one side) would give rise to a mid-dorsal nervous
system like that of Cephalodiscus. Nor is that all; Professor
Spengel (21) has shown in his monograph of the Enteropneusta
that the currents in the proboscis-pore and collar-pore are
inwards, and that by this means the animal inflates the proboscis
and collar so as to render them efficient locomotor organs.
We have seen that the function of the stone-canal is a similar
one.
We conclude, then, that the free-swimming ancestor of
Echinoderms, for which we may adopt the name Dipleurula,
and the Tornaria ancestor of Balanoglossus, were closely allied,
This involves the assumption that they were allied to the Pro-
tochordata, for, as I have elsewhere stated (16), Professor
Spengel’s attempt to refute the Chordate affinities of Balano-
glossus has been, in my opinion, futile. Although it may seem
somewhat fanciful, I cannot help seeing hints of Vertebrate
peculiarities in the anatomy of Echinoderms. Where else
among all animals of higher grade than the Coelenterates do
we find calcareous ossicles in the dermis? Where else
THE DEVELOPMENT OF ASTERINA GIBBOSA. 397
is the removal of the nervous system from the surface effected
by invagination leading to the formation of a neural canal ?
When we come to try and picture the characters which the
Dipleurula possessed, we see at once that it must have been
far more primitive than any existing form. In point of fact
an Asterid is about the most undifferentiated animal above the
level of Coelenterates which exists. No proper blood-vessels,
no specialised excretory organ, a central nervous system which
is really a local concentration of a diffuse skin plexus, perfectly
simple generative ducts,a most feebly developed muscular sys-
tem, the fibres being for a considerable time simply myo-epi-
thelial cells,— where is such a state of things to be found outside
the Coelenterata? When we further add that in the Crinoid the
ambulacral nervous system nearly atrophies in the adult, and
is replaced by a new system developed in a totally different
position, we see that we are at about as low a level as one could
well imagine, since the central nervous system in all higher
forms is a most persistent structure.
Assuredly Platyhelminths, which have been usually regarded
as the basal group in the Ceelomata, or better, Triptoblastica,
are far more highly specialised. To say nothing of their
cephalic ganglia, we have their highly developed muscular
wall and their complicated excretory and genital organs to
prove this.
We shall not, then, go far astray in assigning the Dipleurula
and the Tornaria to a group, the Protoccelomata, which were
not far removed from the Celenterates ; the colom was
divided into three parts on each side, but of these the most an-
terior were usually fused to form an unpaired vesicle. The
Dipleurula differed from the Tornaria chiefly in possession of an
aperture, the stone-canal, in the wall separating the proboscis
ccelom from the collar ccelom. This may have been the primi-
tive arrangement, or it may have been a secondary arrangement
acquired in consequence of the Dipleurula having lost the collar-
pores, one of which may, however, as we have seen, be developed
as a variation in the Asterid larva. At the apex of the preoral
lobe was a more or less developed sense-organ with associated
398 E. W. MACBRIDE.
nervous tissue. The collar cavities were probably prolonged
into tentacles with which nervous tissue was associated.
If this supposition is correct, the group Protoceelomata was
a pelagic cosmopolitan one, and it isin accordance with what we
know of wide ranging groups that some of its members should
adopt changed habits and modified structure. The Echino-
dermata, then, represent the earliest offshoot which took to a
sessile life and acquired radial symmetry. A little later the
Hemichordata branched off, a burrowing life being adopted
and consequent degeneracy resulting. The main stem, how-
ever, remained pelagic and gave rise to the Chordata. The
Ascidians were the next offshoot, and then came Amphioxus,
We see, therefore, that the track of the great Chordata
phylum through past ages is traced by examining those
of its members who at very different periods of its history,
and at different stages in its evolution, have forsaken their
high vocation, and taken to a sessile or burrowing life, with
the inevitable consequence—degeneracy.
The following diagram may represent these relationships a
little more clearly :
Protocclomata
Dipleurula
Hemichordata (Tornaria).
Fixed ancestor Balanoglossus, Cephalodiscus,
of Echinoderms.
Crinoids. Protochordata.
Asterids. Ascidians.
Protoechinids, Ophiurids. | Amphioxus.
Echinids. Holothurids. Vertebrata.
THE DEVELOPMENT OF ASTERINA GIBBOSA. 399
I hope in a future paper to be able to show that the
Trochophore larva is also related, though much more distantly,
to
the Dipleurula.
Zoological Laboratory,
March 8th, 1895. Cambridge.
List oF WoRKS REFERRED TO IN THIS MeEmork.
. Bury, H.—‘The Early Stages in the Development of Antedon
rosacea,” ‘ Phil. Trans. Roy. Soc.,’ 1888.
. Bury, H.—‘‘ Studies in the Embryology of Echinoderms,” ‘ Quart.
Journ. Micr. Sci.,’? 1889.
x
. Cuinot, L.—“ Contributions & lEtude anatomique des Asterides,”
‘Arch, pour Zool. Exp.,’ 2me series, tome v bis.
. Cusvor, L.— Etudes morphologiques sur les Echinoderms,” ‘ Arch. de
Biol.,’ tome xi, fascicles 1 and 2.
. Fretp.—* The Larva of Asterias,” ‘ Quart. Journ. Mier. Sci.,’ 1892.
. Garstanc, W.—“On some Bipinnarie from the English Channel,”
* Quart. Journ. Mier. Sci.,’? 1894.
. Hamann, Orro v.—‘ Die Wandernden Urkeimzellen und ihre Reifungs-
statte bei den Echinodermen,” ‘ Zeitsch. fiir wiss. Zool.,’ 1880.
. Hamany, Orro v.—‘ Beitrage zur Histologie der Hchinodermen,’ 4
Hefte, Jena, 1889.
. Lerrotpt, H.— Das angebliche Excretiensorgan der Seeigel,” ‘ Zeitsch.
fir wiss. Zool.,’ 1893:
. Lupwic, H.—*‘ Beitrage zur Anatomie der Asteriden,”’ ‘Zeitsch. fiir
wiss. Zool.’
. Lupwic, H.—‘“‘ Neue Beitrage zur Anatomie der Ophiuriden,” ‘ Zeitsch.
fiir wiss. Zool.,’ Bd. xxxiv, 1880.
. Lupwie, H.—“ Entwickelungsgeschichte der Asterina gibbosa,
Forbes,” ‘ Zeitsch. fiir wiss. Zool.,’ Bd. exxxvii.
. Lupwic, H.—‘ Bronn’s Thierreich,’ new edition, article “ Echino-
dermata.”
. MacBripz, E. W.—“ The Development of the Genital Organs, &., in
Amphiura squamata,” ‘ Quart. Journ. Micr. Sci.,’ 1892.
. MacBripe, EK. W.—‘ The Organogeny of Asterina gibbosa,” ‘Proce.
Roy. Soc. London,’ vol. liv, 1898.
400 E. W. MACBRIDE.
16. MacBripe, E. W.—“ Review of Professor Spengel’s Monograph on
Balanoglossus,” ‘Quart. Journ. Mier. Sci.,’ 1894.
17. Perrrer.—‘ Mémoire sur l’organisation et le développement de la Coma-
tula de la Mediterranée,’ Paris, 1886.
18. SrELIGER.—“ Studien zur Entwickelungsgeschichte der Crinoiden,”
‘Zool. Jahrbiicher, Abt. fir Anat. und Ontog. der Thiere,’ Bd. vi.
19. Simon.— Die Entwickelung der Synapta digitata und ihre Bedeu-
tung fiir die Phylogenie der Hchinodermen,” ‘Jenaische Zeitschrift,’
1889.
20. Simon.—“ Die Homologien Innerhalb des Hchinodermenstames,”
‘Morph. Jahrbuch,’ 1889.
21. SrenceL, J. W.—‘ Die Enteropneusten des Golfes von Neapel und der
angrenzenden Meeresgebieten’ (Fauna and Flora of the Gulf of
Naples), 1894.
22. Totrt.— The Development of Echinocyamus pusillus,” ‘ Trans-
actions of Roy. Soc. of Upsala,’ 1892.
EXPLANATION OF PLATES 18—29,
Illustrating Mr. E. W. MacBride’s paper on ‘‘The Develop-
ment of Asterina gibbosa.”
(The outlines of all the sections figured were drawn with the camera lucida.)
List of Abbreviations used.
a, Anterior body-cavity and the part of it persisting in the stalk. a’. Axial
sinus. a. Aboral sinus. ad. gon. Dilatation of branch of aboral sinus round
the gonad. amb. Ambulacral ossicle. amb. Ameebocytes. a.@. Adult
cesophagus or “stomach.” 2. Basal plate. dip. gang. Bipolar ganglion-
cells. ranch. Dermal branchia. C. Centro-dorsal plate. cade. Calcigenous
tissue. ect. Ectoderm. zd. Endoderm. jidr. Fibrous tissue. jix. Dise
on preoral lobe for attachment of larva. gez.7. Genital rachis. gob. Goblet
cells. ist. Inyolution of ectoderm to facilitate histolysis. dhy, Left water-
vascular rudiment or hydroceele ; its lobes are numbered with Arabic numerals.
Roman numerals I, U1, UI, IV, V, denote the arm rudiments. Jo, larval organ.
le. larval cesophagus. dpc. Left posterior celom. Jp'c’. The right ventral
horn of the same. 2'p"c'. The right dorsal horn of the same. @. s¢om.
Larval stomach. mes, Mesenchyme. mp. Madreporic pore. musc. Muscular
THE DEVELOPMENT OF ASTERINA GIBBOSA. 401
tissue. musc.amb. Ambulacral muscles. muse. /arv. Larval muscles. muse.
retr. Retractor musclesof adult cesophagus or stomach. xerv. Nervous tissue.
nerv. circ. The nerve-ring, zerv. darv. Larval nervous tissue. or. c. Oral
ccelom. pdr. Peribranchial space. p.c. Pore-canal. p’'c’. Additional pore-
canal in abnormal larva. yer. Peritoneum. pf. Perihemal space. The
perihemal rudiments are numbered 1.2, 2.3, 3.4, 4.5, and 5.1, according as
they originate between the hydroccele lobes 1 and 2, 2 and 3, 3 and 4, 4 and 5,
and 5 and Lrespectively. pr. germ. Primitive germ cells. pr. germ. inv. Invo-
lution of the peritoneum whence these cells arise. py. Pyloric sac and its
ceca. vet. Retinal cells. shy. Right hydrocele. Its lobes when they exist
are numbered like those of the left hydroccele, &c. r’Ay'. Extra right hydroccele
present in abnormal larva. spe. Right posterior celom. sazg. cire. Oral
blood-ring. séec. Stone-canal. sé/c’. Extra stone-canal present in abnormal
larva. 7. Terminal plate. ¢7. Trabecula. vi¢. Cells forming crystalline
body (Glaskorper). wv. Radial water-vascular canal. wor. Water-vascular
ring-canal. .
PLATE 18.
All the figures are reproduced, though ina somewhat simplified form, from
Ludwig’s memoir on the development of Asterina gibbosa. The various
figures have, however, been enlarged or reduced as the case demanded so as to
bring them to one uniform scale of magnification, viz. 85 diameters.
Fic. 1.—A gastrula with wide blastopore. Stage A. This stage is reached
on the second day.
Fie, 2.—A slightly older gastrula. The blastopore is commencing to be
narrowed, and one of its lips is reflected over it.
Fig. 3.—A still older gastrula.
Fic. 4,—Lateral view of larva three days old which has just escaped from
the egg membrane. The “larval organ” (/. 0.) or preoral ridge of ectoderm
with long cilia has appeared. Stage B.
Fie. 5.—Ventral view of the same larva.
Fie. 6.—Dorsal view of the same larva.
Fic. 7.—Lateral view of larva of six days. The disc for adhesion (/iz.) has
appeared in the centre of the larval organ. Stage C.
Fig. 8.—Antero-lateral view of the same larva of six days.
Fie. 9.—Anterior view of the same larva of six days.
Fie. 10.—Left view of fully developed larva of seven days. Stage D.
Fic. 11.—The same drawn in the position it assumes in life.
Fic. 12.—Left view of larva in which metamorphosis has commenced, and
which has fixed itself. The Arabic figures denote the primary lobes of the
water-vascular system or hydroccele, the Roman figures the rudiments of the
arms. The larval organ has disappeared. Stage H.
402 E. W. MACBRIDE,
Fic. 13.—Right view of the same larva.
Fic. 14.—Ventral view of larva of about nine days. The arm rudiments
form a nearly complete circle. The lobes of the water-vascular system have
developed two pairs of accessory lobes each. Stage F.
Fie. 15.—Right view of the same larva of about nine days.
Fie. 16.—Left view of the same larva of about nine days.
Fie. 17.—Oral view of just metamorphosed star-fish about ten days old.
amb. Ambulacral ossicles. Stage G.
Fie. 18.—Aboral view of another specimen of the same age. C. Central
plate. JB. Basal. 7. Terminal. The curve of the arm rudiments has become
a circle, No. V coming to be apposed to the lobe No. 1 of the water-vascular
system. mp. Madreporic pore. Stage G.
Fig. 19.—Aboral view of a young star-fish sixteen days old. Notice the
anus, the additional calcareous plates, and the spines.
PLATE 19.
All the sections represented in this plate are magnified 80 diameters, and,
except where otherwise stated, they have been cut parallel to the ‘larval
plane,” i.e. they are horizontal longitudinal sections. Where several sections
from the same series are figured, the most dorsal is in every case put first.
The darkest shade represents the epithelium of the gut; the intermediate
shade represents ectodermic and ccelomic epithelium, including the lining of
the derivatives of the ccelom ; the lightest shade represents the cavity of the
blastoccele with all its contained structures, jelly, fibres, cells, &c., and also
the muscular tails of the epithelial cells lining the water-vascular system. In
Fig. 27, however, a portion of the gut opening into the celom, and in Figs.
30, 31, 34, 39, 40, and 41 the larval cesophagus, have been printed (through
oversight) in the intermediate tint.
Fics. 20 and 21.—Two sections of a gastrula a little older than Stage A.
No mesenchyme has as yet appeared.
Fic. 22.—Sagittal section of a gastrula about the same stage as Fig. 3.
mes. Mesenchyme cells.
Fic. 23.—Section of an embryo older than that shown in Fig. 3. It shows
the differentiation of the archenteron into gut and cclom.
Fics. 24 and 25.—Two sections of an embryo somewhat younger than Stage
B, and still enclosed in the vitelline membrane. The ccelom has grown back
at each side of the gut, forming two posterior lobes, /pe., rpc. Fig. 25
shows, however, that these lobes do not as yet extend ventral to the gut.
Fic. 26.—Section of larva rather older than Stage B, to show the forma-
tion of the madreporie pore. pe. Pore-canal ending blindly in contact with
the ectoderm. /. stom. Larval stomach. mp. Thickening of ectoderm where
the primary madreporic pore will be formed.
THE DEVELOPMENT OF ASTERINA GIBBOSA. 403
Fics. 27—29.—Three sections of a larva slightly older than the preceding.
Fig. 27 shows that on the left side the ccelom is divided into an anterior
ceelom a, and a left posterior coclom Jpe. Fig. 28 shows that this division
only extends about halfway to the ventral side. Fig. 29 shows that the
separation of the ccelom from the archenteron commences venirally, since here
the ceelom is shut off from the gut. ¢r. First trabecula.
Fie. 30.—Section of a larva rather older than that shown in Figs, 27—29.
le. Larval cesophagus. ¢r. Trabecule cords of cells spanning the left pos-
terior celom.
Fic. 31.—Sagittal section of larva about Stage B, to show the formation of
the larval esophagus. It is clearly seen that this is a stomodzum which has
not as yet joined the gut.
Figs. 82—34.—Three sections of a larva younger than Stage C. The
segmentation of the clom on the left side is complete; on the right side it
has begun dorsally (Fig. 32). The left water-vascular rudiment or hydroccele
(/hy.) has appeared as an outgrowth of the anterior ccelom, its lobes numbered
asin Pl. 18. Fig. 32 shows Nos. 1 and 2; Fig. 33, No. 3; and Fig. 34, Nos.
4 and 5.
Fie. 35.—Section of a larva of Stage C. The first trace of the right hydro-
cele (riy.) has appeared.
Fig. 36.—Section of a slightly older larva than preceding. The development
of the right hydroccele is more advanced.
Fies. 37—41.—Five sections of a larva of Stage D, or slightly younger.
In Fig. 37 we see a section of the pore-canal (pe.) and the origin of the rudi-
ment of the oral celom (or.c.). In Fig. 38 the fully developed form of the
right hydroceele (r/y.) is shown. In Figs. 39 and 40 we see the left posterior
ceelom extending obliquely beneath the right posterior ccelom (7pe.); this is
the right ventral horn (/'p'c’.) of the left posterior celom. In Fig. 41 we
see it opening into the anterior ceelom.
PLATE 20.
‘The same remarks apply to this as to Plate 19, but- in addition it is to be
remarked that the epithelium of the pore-canal and of the stone-canal is
distinguished by a cross-striation.
Figs. 42 and 43.—Two sections of a larva rather younger than Stage D.
ste. Rudiment of the stone-canal. Fig. 42 shows the septum between the
anterior ccelom and the left posterior ccelom broken down dorsally ; and Fig.
43 shows that the septum between the anterior ccelom and the right posterior
ccelom is still incomplete ventrally.
Figs. 44—46.—Three sections of a larva of Stage D. Fig. 44 shows the
opening of the pore-canal into anterior celom; Fig. 45, opening of the stone-
canal into the same; aud Fig. 46, the opening of the stone-canal into the
VOL. 38, PART 3.—NEW SER. DD
4.04 E. W. MACBRIDE.
hydroceele. It shows also that the hydroccele has a wide opening into the
anterior ccelom independent of the stone-canal.
Fic. 47.—Sagittal section of a larva of Stage D, to show the relations of
the lobes of the left hydroccele to each other.
Fies. 48—50.—Three sections of a larva of Stage E. The larva has suffered
an injury, a piece of ectoderm in the preoral lobe indicated by the dotted line
being missing. Fig. 48 shows relation of rudiment of oral ccelom (or. c.) to
the right dorsal horn of left posterior cceelom (/"p''e’.). Fig. 50 shows the
great growth of the left hydroccele (compare Fig. 40). a. @. Adult cesophagus ;
rudiment of the ‘‘stomach” of the adult. In Fig. 49 the o of ov.c. has failed
to print.
Fies. 51—53.—Three sections of a larva slightly older than the preceding,
to show rudiments of the perihemal spaces (p/.). These are numbered ac-
cording to the lobes of the hydroccele between which they occur: ph. 1.2,
ph. 2.3, ph. 3.4, and ph. 4.5. ph. 1.2 arises from the anterior celom, the
rest from the left posterior celom. The lobes of the hydroccele are com-
mencing to be trifid.
PLATE 21.
The same remarks apply to this plate as to the two foregoing.
Fics. 54—-57.—Four sections of a larva about midway between Stages E
and F. Fig. 54 shows the incipient dorsal constriction of the anterior ccelom
into a stalk portion (@.) and a body portion or axial sinus (a’.); also the origin
of the perihemal rudiment (pA. 1.2) from the anterior celom. Fig. 55 shows
the growing tip of the right ventral horn of the left posterior ccelom, and over
it the arm rudiment No. V; it shows also the stone-canal opening into the
hydroccele and the perihemal rudiment insinuating itself between the hydroccele
and the ectoderm. Fig. 56 shows the axial sinus and the stalk ccelom con-
tinuous with each other, and also the anterior ccelom opening into the right
ventral horn of the left posterior cwlom. Fig. 57 shows that this right ventral
horn is commencing to be again divided from the anterior ccelom ventrally
by the outgrowth of a septum.
Figs. 58 and 59.—Two sections of a larva slightly older than the preceding,
to show the separation of the axial sinus ventrally on the one hand from the
stalk ceelom, and on the other hand from the hydrocele. Py. Rudiment of
the pyloric sac of adult. de. Last trace of the larval cesophagus.
Fic. 60.—Section of larva about the same age as preceding, to show the
fifth perihemal rudiment (p/. 51) which intervenes between lobes 5 and 1,
as yet widely separated.
Fie. 61.—Section of larva about Stage F, to show the mutual relations of
the stone-canal, the axial sinus (a’.), the right dorsal horn of the left poste-
rior ceelom (/"p"'c’.), and the right hydroceele (7Ay.).
THE DEVELOPMENT OF ASTERINA GIBBOSA. 405
Fics. 62—69.—Hight sections of a larva slightly older than Stage F, to
show the relation of the arm rudiments to the lobes of the hydroccele. Fig. 68
shows the incipient healing of the breach in the septum between the anterior
ccelom (axial sinus) and the left posterior ceelom. Figs. 64 and 65 show that
arm rudiment No. V is still widely separated from hydroccele lobe 1 by the
base of the stalk, and also that the right ventral horn (/'p'c!.) of the left
posterior ccelom is not completely separated from the axial sinus (a’.). Fig.
65 also shows the complete separation of the hydroccele from the axial sinus.
Figs. 66 and 67 show relation of the oral ecelom (07. c.) to the adult cesopha-
gus (a. @.). Fig. 69 shows the adhesive disc of the stalk (jiz.) attached toa
piece of Alga (x), and the rest of the ectoderm of the preoral lobe being
invaginated (Azs¢.) to undergo destruction. It also shows that each primary
lobe of the hydroccele has developed two pairs of secondary lobes.
PLATE 22.
The same remarks apply to Figs. 70—78 as to the contents of the three
foregoing plates. Figs. 79—82 are sections cut parallel to the dise of the
star-fish or ‘‘adult plane,” the magnification being the same, viz. 80 dia-
meters.
Fies. 70 and 71.—Two sections of a larva of Stage G. Fig. 70 shows the
relationship which the adult and the larval cesophagus occupy with regard to
one another, the latter being a mere rudiment unconnected with the gut ; it
also shows the outgrowths from the adult cesophagus. Fig. 71 shows the
oral ccelom opening into left posterior ccelom ventrally by breaking down of
partition between them; also the first trace of the pyloric ceca as outgrowths
from the pyloric sac.
Fic. 72.—Section of larva rather older than Stage G. The adult mouth is
formed, and the oral ccelom opens widely into the left posterior celom. The
stalk has become a small solid rudiment. The dotted line shows the boundary
between the pyloric sac and the adult ‘‘ esophagus” or “‘ stomach.”
Fic. 73.—Section of a larva of the same age as the preceding; it shows the
two dorsal pyloric cxca already formed, also the so-called heart or “ovoid
gland” (ovg.), as a fold projecting into the axial sinus (a’.).
Fic. 74.—Another section from the same series as Fig. 72. Shows the two
ventral pyloric ceca; it is seen also that their suspensory mesenteries are
derived from the mesentery separating the right posterior ccelom from the left
(compare Fig. 75). Note also that the tube-feet have acquired their suckers,
The animal has broken loose from its attachment, which accounts for the
rudimentary condition of the stalk.
Fie. 75.—A section of a larva of Stage G. Shows the dorsal pyloric ceca
and their suspensory mesenteries.
Fig. 76.—A section of another larva of Stage G. Compare with Pl. IV,
fig. 61, and note that the arm rudiment No. V (not marked in the figure) has
4.06 E. W. MACBRIDE.
now become applied to the lobe No. 1 of the hydroccele. The stone-canal is
seen opening into lobe No. 2, and the perihemal rudiment 1.2 has grown out
into a canal insinuating itself between the ectoderm and the hydroccele.
Fics. 77 and 78.—T wo sections of a rather older larva. Fig. 77 shows that
the right ventral and right dorsal horns (/'p'c’. and /p"'c'.) of the left
posterior ccelom have coalesced, and that the left posterior ccelom has thus
acquired a ring-like form. Fig. 78 shows the formation of the anus of the
adult.
Fie. 79.—Section parallel to the adult plane of a larva of Stage F. Shows
the relationships of the axial sinus, oral ccelom, and water-vascular ring (wvr.),
the last being still incomplete; also four perihemal rudiments alternating
with the five hydroccele lobes.
Fies. 80 and 81.—T wo sections in same plane as Fig. 79 of a larva of Stage
G. Fig. 80 shows the axial sinus (a’.) in process of growth to form the inner
periheemal canal. Fig. 81 shows the completion of the water-vascular ring at
the spot marked by the asterisk between the hydroccele lobes Nos. 1 and 5; it
also shows the trifid form of the adult cesophagus before the mouth is formed,
and the oral ccelom opening into the left posterior ccelom.
Fic. 82.—Similar section of older larva in which mouth is formed. The
five interradial lobes of the ‘‘stomach” are present, the trifid shape having
disappeared ; and the retractor muscles of these lobes are formed from remnants
of septum between oral and left posterior celom. The distance (R) from tip
of arm to centre of disc ‘36 millimetre.
PLATE 23.
Fies. 88 and 84.—Two more sections from the same series as Fig, 82.
Fig. 83 shows the pyloric sac with its five ceca just beginning to be bifid,
and the mutual relations of the right hydroccele and axial sinus; also the stone-
canal opening into the latter. Fig. 84 shows the point of origin of the rectum
and the rudiment of rectal cecum and the relation of right posterior ccelom to
the pyloric ceca. In Fig. 83 (pr. germ. inv.) is the involution of peritoneum
from which the primitive germ cells are formed.
Fies. 85—94 represent sections of abnormal larve. These sections are cut
parallel to the larval plane, except Fig. 90, which is rather oblique to that
plane. Magnification the same as before.
Fics. 85 and 86.—Two sections of a larva of Stage D, or slightly
younger. rhy. Right hydroccele developed into two distinct lobes
lined with cubical epithelium.
Figs. 87 and 88. Two sections of a larva between Stages D and BE.
p'c'., st'c. Pore-canal and stone-canal of right side in connection
with normal right hydroceele, 7.4y. Their openings into this are
in another section. 7'/y'. A second, more ventrally situated hydro-
THE DEVELOPMENT OF ASTERINA GIBBOSA. 407
cele rudiment on the right side, with a distinct opening into ccelom:
The left hydroccele is feebly developed for the stage which the larva
has reached, and has only four lobes.
Fig. 89. Section of a larva of Stage D, in which the right hydroccele has
five lobes, and is larger than the left. This section is drawn from the
ventral aspect, and hence appears reversed.
Fig. 90. Section of a larva of Stage G, showing a “collar pore’ opening
from the left hydroccele between lobes 2 and 3, directly to the exterior.
Figs. 91—94. Four sections of an almost normal larva of Stage F, or
somewhat older. p.c. Normal pore-canal, opening into axial sinus,
the septum between the latter and the left posterior ccelom being
still incomplete dorsally. p’.c’. Pore-canal, s¢’. c’., and stone-canal in
connection with the right hydrocele. Fig. 93 shows the opening of
the second pore-canal into the axial sinus. Fig. 92 ought to show
the opening of the second stone-canal into the right hydroceele, but
the slit-like opening has not come out in the figure. Fig. 91 shows the
two pore-cauals uniting to open bya common pore. (Compare Wood-
cut 3.)
Fig. 95.—Section parallel to the larval plane from larva of Stage C, showing
the first trace of right hydroccele. (Compare Plate 19, fig. 35.) Note its
relationship to the anterior ceelom, which extends obliquely beyond it pos-
teriorly, passing under it and to the right of it. Magnification 1000 diameters ;
Leitz’s immersion 5.
Fies. 96 and 97.—Two sections of body-wall of young star-fish, cut per-
pendicular to disc, in which R equals ‘8 millimetre. Fig. 97 shows first trace
of “ papula” or dermal branchia (4ranch.). Fig. 96, origin of its peribranchial
space, p.dr. Magnification 400 diameters.
Fic. 98.—Section of body-wall of young star-fish, in which R equals
‘88 millimetre, showing dermal branchia and its peribranchial space. Mag-
nification about 400 diameters.
9
PLATE 24.
Fries. 99—106 illustrate the development of the so-called heart or “ ovoid
gland.” The sections represented are perpendicular, or nearly so, to the
disc of the star-fish, and the magnification is 850 diameters.
Fig. 99. Section of larva of Stage G. ov.g. Fold projecting into the
axial sinus, the rudiment of the ovoid gland. pr. germ. inv. Invagina-
tion of peritoneum, whence the primitive germ cells are formed. Cue.
Calcigenous tissue in the body wall.
Figs. 100—108. Four sections of a specimen older than preceding. Fig,
101 shows the growth of the primitive germ cells into the rudiment
of the ovoid gland. Figs. 102 and 103 show that they do not yet
extend through its whole extent. Fig. 103 shows that the ovoid gland
408 E. W. MACBRIDE.
rudiment is at one point attached to the oral wall of the axial sinus.
(Compare Plate 25, fig. 110.)
Figs. 104—106. Three sections of a young star-fish, in which R equals
‘4 millimetre. Fig. 104 shows the primitive germ cells arising from
the involution of the peritoneum. Figs. 105 and 106 show that they now
extend throughout the whole extent of the ovoid gland; these figures
also show the relation of the oral end of the axial sinus to the peri-
heemal spaces.
Fics. 107 and 108.—Two sections from same series as Figs. 82—84,
magnified 350 diameters. They show the development of the oral “blood ”
ring, sazg. cire., aS a modification of the mesenchymatous tissue of the
blastoccele. id. Fibrous tissue.
Fie. 109.—Similar section of a young star-fish, in which R equals °45
millimetre. Same magnification. The blood-ring is fully formed. Notice
also the minute cells amongst the nerve-fibres (dip. gang.).
PLATE 25.
Fic. 110.—Longitudinal section of the stone-canal of young star-fish, in
which R equals ‘8 millimetre. sang. cire. Oral “blood” ring. wvr. Water-
vascular ring-canal. musc. amb. Muscles of ambulacral ossicles. Notice the
incipient division of madreporic pore into two, and entire independence of
ovoid gland and blood-ring. Magnified 350 diameters.
Fic. 111.—Diagram showing the relative positions of the ovoid gland,
stone-canal, and various sinuses in proximity. gez. 7. Genital rachis. ad,
Aboral sinus (or sizus a.). pr. germ. inv. Primary peritoneal involution to
form germ cells. The cavity of this is probably the same as sizus 6 in next
figure. The axial sizws a' is sinus c. The dotted lines show the continuity of
two parts of the ovoid gland in a different plane to that of the stone-canal.
Fic. 112.—Similar diagram of Amphiura squamata. Accompanying
spaces, sinus a, sinus b, and sinus c, as in the author’s paper (14).
Fies. 113—118 illustrate the development of the ovoid gland and genital
rachis. They are all taken from sections cut parallel to the disc; they are,
in fact, transverse sections of the interradial septum in which the axial sinus
is embedded.
Figs. 113 and 114. Two sections from a star-fish, in which R equals °45
millimetre. Fig. 113 shows the manner in which the right hydro-
ccele is enclosed in the upper part of ovoid gland; Fig. 114, the
primitive peritoneal involution, the pore-canal, and the crescentic form
of right hydroccele. Magnification 350 diameters.
Figs. 115 and 116. Two sections from star-fish, in which R equals
‘7 millimetre. ad. Aboral sinus containing the rudiment of genital
rachis. Fig. 115 shows that the sinus is a portion of the celom
shut off by the outgrowth of a flap from the body-wall.
TILE DEVELOPMENT OF ASTERINA GIBBOSA. 409
Fig. 117. Section from star-fish, in which R equals 2°2 millimetres,
showing the continuity of rachis and ovoid gland, and that the rachis
now extends in both directions. Magnified 150 diameters.
Fig. 118. Section from star-fish, in which R equals 3 millimetres, show-
ing fully developed ovoid gland and changed form of stone-canal.
Magnified 350 diameters.
PLATE 26.
Fics. 119—121.—Portions of three sections from the same series as Fig.
118. Fig. 119 shows the genital rachis enclosed in the branch from the
aboral sinus, ad. Fig. 120 shows the passage of the genital rachis into the
rudimentary genital organ, and the outgrowth of septum which cuts off the
perihemal space surrounding this rudiment from the “ genital vessel.” ad. A
branch of the aboral ring. Fig. 121 shows the development of the cavity of
the genital organ. Magnification 350 diameters.
Fies. 122 and 123.—Two sections of young ovaries, from a specimen in
which R equals 3°7 millimetres. Fig. 122 shows the continuity of the ovary
and rachis, Fig. 123 the outgrowth of germ cells to form the genital duct.
Fic. 124.—Portion of body-wall of gastrula figured in Plate 19, fig. 21.
Notice the absence of mesenchyme. ezd. Endoderm. Magnification 600
diameters.
Fic. 125.—Similar view of the body-wall of slightly older embryo, to show
the formation of mesenchyme. Same magnification.
Fic. 126.—Portion of the gut epithelium of larva figured in Plate 20,
figs. 51—53. Same magnification.
Fic. 127.—Epithelium of the adult cesophagus of the larva shown in
Plate 22, fig. 76, Stage G. Magnified 480 diameters.
Fic. 128.—Epithelium of the pyloric sac (larval stomach), from the same
section as foregoing.
Fie. 129.—Epithelium of the lateral wall of the stomach of a star-fish, in
which R equals -8 millimetre. Magnified 480 diameters.
Fie. 1380.—Epithelium of aboral wall of the stomach from the same section
as foregoing. At X it passes into the epithelium of the pyloric sac.
Fies. 131 and 132.—Epithelium of the pyloric sac and of the rectal cecum
respectively. From the same section as fig. 130.
PLATE 27.
Fries. 133—135.—Three sections of the ectoderm of the anterior surface
of preoral lobe of larva of Stage D. Fix. Disc for fixation. 7. o, Larva
organ. serv. darv. Larval nervous tissue. Fig. 133 is through the dorsal part
of preoral lobe; Fig. 135 through its ventral tip. Magnified 480 diameters,
410 E. W. MACBRIDE.
Fie. 136.—Section of the adhesive disc of larva shown in Plate 21, figs.
62—69. wz. A small piece of alga, to which it adheres by a secretion of
mucus. ist. Involutions of neighbouring portions of ectoderm to undergo
destruction by amcebocytes, ameb.; by this means the preoral lobe is reduced
in size. Magnified 480 diameters.
Fic. 187.—Section of the lateral wall of preoral lobe of larva of Stage D.
musc. larv. Larval muscles derived from the peritoneal cells. Magnified 1000
diameters.
Fic. 188.—Section through the ectoderm and hydroccele wall of a larva of
Stage D, to show the characters of the various larval epithelia. Magnified
1000 diameters.
Fie. 139.—Similar section from a larva between Stages EH and F. A peri-
hemal rudiment is shown. Magnified 1000 diameters.
PLATE 28.
Figs. 148, 149, and 150 are magnified 600 diameters, the rest 1000 dia-
meters (Leitz’s immersion 35).
Fic. 140.—Similar section from a larva of Stage F (that shown in Figs.
62—69). mxerv. The incipient nervous tissue developing as a fine plexus
amidst the bases of the ectoderm cells.
Fic. 141.—Similar section from a young star-fish, in which R equals
‘4 millimetre. Nerv. circ. Nervous ring. calc. Calcigenous tissue. jibr.
Fibrous tissue. re¢r. musc. Retractor muscles of stomach.
Fie. 142.—Developing eye of same star-fish. A simple ectodermic pit is
seen.
Fic. 143.—Eye of star-fish from which Figs. 129—182 are taken. ret.
Visual cells. vit. Cells functioning as ‘ Glaskorper.”
Fics. 144—148 illustrate the differentiation of tissues in the body-wall.
Fig. 144. From the right side of a larvaof Stage D. At * a cell is
seen in the act of dividing, to form one of the amebocytes of the
ccelom.
Fig. 145. From larva of Stage E (that shown in Figs 51—53). god.
Goblet cells. mzasc. Developing muscles; as yet they are simply tails
of the celomic epithelium. jidr. First rudiment of fibrous tissue.
Fig. 146. From the young star-fish from which Fig. 141 is taken
calc, Small portion of calcigenous tissue.
Fig. 147. From the young star-fish from which Fig, 148 is taken, and
also Figs. 129—182.
Fig. 148. Ectoderm of another specimen of same age, to show the sense-
cells.
Fic. 149.—Tube-foot of the larva shown in Figs. 62—69.
Fic. 150.—Tube-foot of the star-fish from which Figs. 141 and 146 are
THE DEVELOPMENT OF ASTERINA GIBBOSA. All
taken. xerv. Nervous tissue under the sensory epithelium at the tip. muse.
Muscular tails of hydroccele epithelial cells.
PLATE 29.
Figs. 151—154 show the development of the transverse muscles, which
extend from one ambulacral ossicle to its fellow of the opposite side.
Figs. 151 and 152.—Two sections perpendicular to the disc from a star-
fish, in which R equals ‘4 millimetre. sang. etre. Oral “blood” ring.
muse. amb. Ambulacral muscles ; the reference line (in Fig. 151) is too
long. ph. Perihemal space; the reference line (in Fig. 151) is too
short. Magnified 350 diameters.
Fig. 153. Similar section from star-fish in which R equals ‘63 milli-
metre.
Fig. 154. Similar section from star-fish of the same size as the preceding,
but more advanced in the development.
Fic. 155.—Transverse section of the radial nerve-cord of a young Asterias,
to show the feeble development of coelomic nervous system.
Fic. 156.—Similar section of nerve-cord of an Ophiurid, to show the great
ganglia of the coelomic nervous system.
Fie. 157.—Diagram of the hypothetical ancestor of Asterids and Crinoids.
The hydroccele is a paired structure.
Fig. 158.—Diagram of a stage in the evolution of Asterids from this an-
cestor. Notice the growth of both left hydroccele and left posterior ecelom
to form rings. The hydroccele encircles the base of the stalk. This drawing
does not properly represent the oblique position which the disc acquires in
reference to the stalk. The mouth ought to be half turned towards the
observer.
Fic. 159.—Diagram of stage in evolution of Crinoids. Notice that the
hydroceele is carried entirely away from the stalk.
These last two diagrams are only hypothetical, in so far as they represent
as co-existing structures which succeed one another in ontogeny ; otherwise
they represent the actual fixed stage in both Asterid and Crinoid ontogeny.
VOL, 38, PART 3.—NEW SER. EE
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THE DEVELOPMENT OF ASTERINA GIBBOSA. 411
taken. xerv. Nervous tissue under the sensory epithelium at the tip. muse.
Muscular tails of hydroccele epithelial cells.
PLATE 29.
Figs. 151—154 show the development of the transverse muscles, which
extend from one ambulacral ossicle to its fellow of the opposite side.
Figs, 151 and 152.—Two sections perpendicular to the disc from a star-
fish, in which R equals ‘4 millimetre. sang. circ. Oral “blood” ring.
muse. amb. Ambulacral muscles ; the reference line (in Fig. 151) is too
long. ph. Perihemal space; the reference line (in Fig. 151) is too
short. Magnified 350 diameters.
Fig. 153. Similar section from star-fish in which R equals °63 milli-
metre.
Fig. 154. Similar section from star-fish of the same size as the preceding,
but more advanced in the development.
Fic. 155.—Transverse section of the radial nerve-cord of a young Asterias,
to show the feeble development of ccelomic nervous system.
Fig. 156.—Similar section of nerve-cord of an Ophiurid, to show the great
ganglia of the cceelomic nervous system.
Fig. 157.—Diagram of the hypothetical ancestor of Asterids and Crinoids.
The hydroccele is a paired structure.
Fig. 158.—Diagram of a stage in the evolution of Asterids from this an-
cestor. Notice the growth of both left hydroccele and left posterior ceelom
to form rings. The hydroccele encircles the base of the stalk. This drawing
does not properly represent the oblique position which the dise acquires in
reference to the stalk. The mouth ought to be half turned towards the
observer.
Fic. 159.—Diagram of stage in evolution of Crinoids. Notice that the
hydroceele is carried entirely away from the stalk.
These last two diagrams are only hypothetical, in so far as they represent
as co-existing structures which succeed one another in ontogeny ; otherwise
they represent the actual fixed stage in both Asterid and Crinoid ontogeny.
VOL, 38, PART 3.—NEW SER. EE
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THE EARLY DEVELOPMENT OF AMIA. 413
The Early Development of Amia,
By
Bashford Dean, Ph.D.,
Columbia College, New York.
With Plates 30—32.
Ganorps, or more accurately Crossopterygians and Chondro-
stean Actinopterygians, must be looked upon as, in many ways,
a transitional and intermediate group. For, on the one hand,
the evidence is becoming conclusive that the Teleosts are to
be regarded as its highly differentiated descendants; and,
on the other hand, its most primitive members have certainly
the closest ties of kinship with both the early sharks and lung-
fishes.!
Amia calva is doubtless, at the present day, the sole
survivor of the race of Mesozoic Ganoids of which Caturus
or Megalurus may be taken asa type. It claims, therefore,
an especial interest as most nearly the ancestral form of some,
if not all, of the recent Teleosts; for its structures are pecu-
liarly Teleostean, and its closely kindred forms occurring from
the Oolite to the Cretaceous provide the actual stepping-stones
to the Clupeoids.
But in embryology the Ganoid and the Teleost still stand
widely separate, and there has even been a tendency to look
1 The writer refers to the structural nearnesses of the early Crossoptery-
gians (e.g. Gyroptychiids), Phaneropleurid Dipnoans, and Pleuracanth
sharks. He also notes that decidedly shark-like features are now found to
be present in the early development of the sturgeon, and especially of the
gar-pike.
VoL. 38, PART 4,—NEW SER. FE
414, BASHFORD DEAN.
upon these kindred forms as representing distinct phyla, early
divergent from a primitive chordate ancestor. And it is there-
fore evident that, before Teleosts can be conclusively shown to
be of Ganoidean descent, it becomes necessary to demonstrate
that well-marked transitional characters exist not only in their
structures, but in their ontogeny.
It has accordingly been my object in the study of Amia to
determine its developmental relationships. For in the ontogeny
of this most Teleostean Ganoid there seemed evidently the key
to the solution of the entire problem—on the embryological
side—of Teleostean descent; and, conversely, the degree of its
developmental unlikenesses to the types of sturgeon and gar-
pike could not fail to prove suggestive.
Embryonic material of Amia was to be obtained at Pewaukee,
Wisconsin, a locality which has long been known as an ex-
ceptionally favorable spawning ground. It was here that
Allis, Ayers, Strong, Ecclesheimer, and Filleborn had suc-
ceeded in securing developmental material, and this locality
appeared, therefore, far more reliable than Black Lake, St.
Lawrence County, New York, where, from my preceding studies
on Lepidosteus, I was but moderately sure of success. In
order to undertake the collecting trip, I was enabled through
the kindness of President Low, of Columbia College, to leave
my duties as early as May 14th of the present year. Proceed-
ing directly to Wisconsin, I was fortunate enough to secure
eggs and larve by May 17th, and on May 19th had the
opportunity of observing the spawning fish and to secure the
earliest cleavage stages. Cold and rainy weather then proved
favorable to my studies, for it retarded the development of
the eggs, and gave me an opportunity to observe the living
material, and to prepare the figures of those stages especially
which in surface view (as my studies of Acipenser and Lepido-
steus! had taught) could not well be examined in the fixed
material.
By the time of my visit, however, the spawning season had
1 1895, Dean, ‘On the Early Development of Gar-pike and Sturgeon,”
‘Journal of Morphology,’ vol. xi, No, 1.
THE EARLY DEVELOPMENT OF AMIA. 415
practically ended. Indeed, so late was the time of my arrival
at the spawning ground that it was altogether due to the kind-
ness and skilful efforts of Mr. Henry Meyer, of Oconomowoc,
that my trip proved successful. I found it impossible, accord-
ingly, to secure at the same time both male and female fish in
spawn for purposes of artificial fertilisation, and I was unable
to employ the method of caring for the eggs which had proved
so helpful in the studies of Acipenser and gar-pike. Fortunately
the eggs of Amia were found to be especially hardy ; they
might be removed from the nests and retained in floating hatch-
ing cases, often even kept in the laboratory in pans and trays
without serious losses. From their adhesive membrane, how-
ever, | have no doubt that the same method of procedure would
have succeeded as in the case of the other Ganoids. In fixing
the eggs, alcoholic (50 per cent.) picro-sulphuric mixture was
generally used.
As to the general habits of Amia, but little need be said in
the present paper; the notes of Dr. Fiilleborn’ regarding
them have been fully confirmed. The fish is especially abundant
in the Wisconsin lakes, and as it is worthless as food, and
persists in taking any and all baits, it isnot looked upon kindly
by the local fisherman—especially as it not infrequently breaks
both his rod and line. Luckily for him, perhaps, it feeds mainly
during the evening and night,—but even then he meets it con-
tinually when using the jack-light. The strength and apparent
clumsiness of the fish are to be emphasised ; when disturbed in
the shallows it will break through the water noisily in its
strong efforts to escape.
The themes of the present paper have been arranged as
follows :
1 1895, Fiutezorn, “ Berichte tiber eine zur Untersuchung der Entwicke-
lung von Amia, Lepidosteus, und Necturus unternommende Reise nach
Nord-America,” ‘Sitzungsberichte der Akademie der Wisseuschaften zu
Berlin,’ Bd. xl, ss. 1057—1070.
416 BASHFORD DEAN.
PAGE
I, The breeding habits of Amia . ‘ . 416
II. The early development 3 ; : . 421
A. Egg and egg membranes : : : . 421
B. Rate of development. : : . 423
C. Segmentation (first to eighth dheseeny. : . 424
D. Blastula . : 429
EK. Gastrula: its relations 46 WiSpidostens hubeeane said
Teleost . ; : ; . 429
F. Mode of formation of the amber : 436
G. Origin of the germ layers and early embryonic Gees 437
III, Conclusions . : : ‘ : : . 440
I. Tar Breepinc Hapsits oF AmIa.
The account of the spawning habits of Amia, as recently
given by Fiilleborn (op. cit.), has been entirely confirmed by
the present writer. A number of additional notes! are pre-
sented in the following pages.
In the beginning of the spring Amia makes its way from
the deeper water, where it has remained sluggish during the
winter season, to the shallows in the vicinity of its spawning
ground. This is usually in the swampy end of a lake, where
the water is well filled with reeds, stumps, Chara, Potamogeton
rootlets, here and there broken by little clear channels or
inlets several feet in depth. In this neighbourhood the fishes
are early seen, often in numbers, sunning themselves near the
surface. They are at this period active, and may not be closely
approached. Like the gar-pike, they are then usually in com-
panies, the fish well separated.
The spawning season is an early and somewhat extended
one, and is apparently induced by the first warm days of spring.
The time of spawning in 1894, as recorded by Fiulleborn, ex-
tended from the beginning of May to the first days of June.
In the spring of 1895, however, although the previous winter
had been unusually severe, a few warm days in April appear to
have been the cause of earlier spawning. As with the gar-pike
1 These include observations made by the present writer at Pewaukee,
Black Lake, and in the South Carolina rivers.
THE EARLY DEVELOPMENT OF AMIA. 417
the spawning then appeared, and has been almost simul-
taneous,—a general “run,” after which the season was con-
cluded by an intermittent spawning, with a nest here and
there. At the height of a “run” as many as a half-dozen
nests, as fishermen stated, were found to occur within the space
of a few square yards. The fish were observed depositing their
eggs as early as April 25th, and before the Ist of May the
spawning appeared to have been generally completed. By the
middle of this month larvae were abundant, and from their
uniform size in the different localities, and in different lakes,
the spawning time could have been varied but little throughout
the entire region.!
Immediately before spawning it is said that the fish divide
themselves into parties, each comprising a female and several
males, and that they then circle about nearer and nearer the
shallows. A spawning place is selected—a well-sheltered spot
with a water depth of about a foot—and a nest is there pre-
pared. And it seems evident that nests are prepared some-
times well in advance of spawning, for several were noted by
the writer which were occupied by the fish for a number of days
before the eggs were deposited. The mode of building a nest
is in some ways doubtful: fishermen state that the spawning
party prepares it by constant circlings before the time of
spawning, and this view seems entirely corroborated by a care-
ful examination of the newly made nest; the soft weeds and
rootlets appear bent and brushed aside in a way that gives it
somewhat the appearance of a crudely finished bird’s-nest.
The mode of depositing the eggs appears to be entirely
similar to that described by the present writer in the case of
the gar-pike. The spawning fish leave the nest from time to
time, returning in company. The eggs and milt are emitted
simultaneously. The fishes apparently rub closely together,
since scales are found scattered in the nest bottoms, as noted
by Filleborn, and now confirmed by the present writer. The
eges become instantly adhesive, sticking to any portion of the
1 The present writer finds the spawning region very general: nests were
found in La Belle Lake (cf. Filleborn, op. cit. p. 1059).
418
BASHFORD DEAN.
weedy nest which at the moment they happen to touch (e.g.
Potamogeton or bulrush rootlets, Fig. 1). The writer has seen
a nest in which—judging from the wide difference in the
cleavage stages—the oviposition must have taken place during
Fic. 1.—Eggs of Amia.
a period of about twelve hours.
Shown as collected, attached to root of bulrush.
x about 2.
Another nest, on a somewhat
warmer day, appeared to have been filled with eggs within
about half an hour, since all cell stages were notably uniform.
The number of eggs the writer roughly estimates as in the
neighbourhood of a million.
Shortly after oviposition a single male takes his position on
the nest—whether by driving the others away or not the
writer has been unable to determine.
Here he remains until
the eggs are hatched, sometimes in the nest, circling slowly
about, sometimes in the adjoining “ runway,” as in Fig, 2, his
THE EARLY DEVELOPMENT OF AMIA. 419
head and pectorals projecting over the nest. It is evident
that his constant breathing is an important source of the eggs’
aération: his movements, moreover, aid, no doubt, in sweeping
the nest free of sediment, from which, on account of their
position, the eggs might otherwise suffer.!
Shortly after the eggs are hatched the entire swarm of larvze
/
hs \y Ni
: fay aed lely
Oconomowoc, Dis
Fic. 2.—Nest of Amia.
leaves the nest. A fine nest of eggs, from which the writer had
expected to get the young just before the time of their hatch-
ing, was found to be entirely deserted at a time when the
young could not have been older than twenty-four hours. The
closest search in and about the nest revealed no trace of their
whereabouts, although from their larval habits it was thought
that they should surely be found attached to the neighbouring
weeds or deep in the mass of root-fibres and detritus of the nest
bottom. They had evidently left the nest in a body, and were
1 The few remaining eggs of nests which had been “ robbed,” and to which
the male did not return, were found to become destroyed by fungus.
4.2.0 BASHFORD DEAN.
nowhere in the immediate neighbourhood. It was plausibly
suggested by Mr. G. W. Kosmak, who then accompanied the
writer, that they had been taken away by the male fish, attached
to him by their sucking dishes,
my " rl Fe
ih AAS f
(lly
Fic. 3.—Young of Amia attended by male.
It is certain that when the male reappears it is with a swarm
of nestlings; but they are now well grown (3—1} inches).
With these he remains for a time in the fereunmunhond of the
spawning ground ; then he appears to gather them together with
constant circlings and slowly takes his way to the neighbouring
shallows (Fig. 3). The fish is courageous in the protection of
his charges, remaining with them, facing the danger, until the
boat approaches within a couple of yards; in one instance the
writer has seen the fish actually pushed aside with the handle
of the spear.
THE EARLY DEVELOPMENT OF AMIA, 421
II. Toe Earty DeveLorMEnT or AMIA.
A. The Egg and Egg Membranes.—Theegg, shortly after
its deposition (15’—80”), presents the general appearance of
P1.30, fig.1. It has assumed an oblong form, averaging 2°2 x 2°8
mm. The germinal area, even in freshly deposited eggs, was well
defined as a whitish cap reaching down to about one third of
the ege’s longer or vertical axis. Its yolk pole region is pale
greyish in colour (resembling that of the freshly laid sturgeon
egg), and retains this colour throughout earlier development,
appearing dingier and browner, however, as the outer mem-
branes become soiled.
A single micropyle probably occurs, as in Lepidosteus and
the Teleosts. Of this, however, the writer is by no means sure,
as his only observation was made hastily during a collecting
trip, and he neglected to immediately harden his material.
The egg membranes are essentially similar to those of Lepi-
dosteus and Acipenser ; they appear, however, relatively thin-
ner and more intimately associated with the egg. They are
not to be removed by needles until the embryo has become
well established ; even then the process isa difficult one. There
is present a well-marked zona radiata and villous layer; these,
in the younger stages, are approximately of equal thickness.
The radiata is more compact in structure than in the other
Ganoids; the villous layer, on the other hand, is of a far
looser texture, its elements crumpled and intertwined, the
heads of the villi oblong and swollen. At the point of the egg’s
attachment, e. g. the stem of waterweed, furthermore, as may be
seen in the adjoining figure (Fig. 4), the villi become enormously
elongated, their heads firmly fixed to the attaching object. A.
granulosa occurs irregularly ; it sometimes appears as a cell
tract of considerable size, at other times it is almost wanting ;
it has certainly no such important relation asin Acipenser. In
Pl. 30, fig. 1, the egg is shown attached to the waterweed ; its
membranes show a broad jelly-like base consisting of the elon-
gated villi, and the mottling roughnesses of the egg surface re-
present patches of the granulosa.
422 BASHFORD DEAN.
The germinal area, as above noted, is readily to be determined
by the unaided eye, even at a very short time after the egg is
Z RADIATA
VILLOUS LAYER.
Fig. 4.—Egg membranes of Amia. x about 180.
deposited ; its margins fade gradually into the yolk somewhat
above the equatorial region of theegg. Its marked appearance
is doubtless the reason of the oblong form which the egg early
assumes. This shape, moreover, would seem to bear with it a
specialised developmental character in that it does not, in the
event of the egg’s displacement, permit the germinal region to
rotate backward into its vertical position, as it so readily does
in the case of the sturgeon and gar-pike. The writer has
found, however, in the early stages of segmentation that the
cleavage planes occur in the normal way when the position of
the egg was reversed. In these instances there was a slight
attempt at the rotation of the germ disc, but in no case was
this complete as far as the present observations went. The
power of rotation in the eggs of Amia, it might here be noted,
becomes more perfect at the subsequent stages of development ;
toward the close of gastrulation the egg’s outline becomes more
THE EARLY DEVELOPMENT OF AMIA. 423
nearly spherical, and the writer has observed that the blasto-
pore of inverted eggs could slowly rotate downward.
Deutoplasm and germ region are more early to be distin-
guished in Amia than in other Ganoids. Studied in vertical
sections (P1.31, fig. 21) the line of union of yolk and germ may
be traced in the plane passing through the lower rim of the
germ area; this plane of demarcation, however, on closer
examination, proves to be broader than it at first appears; the
yolk spherules may be traced, becoming smaller and smaller,
well into the germ area. The nucleus, small in size, takes its
position in the lower portion of the germ.
“«‘ Pigment ” is practically absent in the late as well as in the
earlier stages; in this regard the eggs of Amia and the gar-
pike closely correspond.
B. The Rate of Development.—The following summary
of the rate of development relates to a particular set of embryos.
It is evident, however, that the time-proportion between the
different stages is relatively uniform for a longer or shorter
period of development. The position with regard tothe upper
and lower egg poles assumed by the growing embryos was
observed by the writer to correspond with those he has already
recorded in the Lepidosteus. Disturbance in the actual posi-
tion of the egg, notably during the stages earlier than gastru-
lation, is attended with variations of position; the lack of the
power to rotate backward into its normal position has already
been recorded, p. 422.
Hours after oviposition. Hours after oviposition.
Hours. Hours.
First cleavage . : . 1 | Early gastrula . : AE,
Second cleavage . 2 | Blastopore closes . 80
Third cleavage . : . 3 | Embryo’s length 90°. 220/68
Fourth cleavage . 4} a a SOR eid)
Fifth cleavage . ae 7 pao . 124
Sixth cleavage . ; . 6% ms Pao On. los
Seventh cleavage ; . %% | Egg hatches . ; . 192
The rate of hatching, as recorded by Filleborn, varied between
eight to fourteen days. The present writer finds, however, that
424, BASHFORD DEAN.
the eggs hatched far more rapidly than this during the latter
part of the present season,—in one case (May 24) within four
days, in all cases not longer than eight. A sudden increase in
the water temperature, as in the Lepidosteus, and, for that
matter, doubtless as in all other fishes, hastens the rate of
development.
C. Segmentation.—The segmentation stages of Amia
are readily studied in the living egg, the transparency of its
membranes permitting the cleavages to be followed. They are,
however, far more obscure than those of the other Ganoids, and
impress the observer with their marked Teleostean characters.
They are readily reduced to the plan of those of Lepidosteus ;
and the accompanying figures (PI. 30, figs. 1—9) may be in-
structively compared with those in the ‘ Journal of Morphology,’
vol. xi, pl. i, figs. 1—9. And, on the other hand, they closely
suggest the plan of segmentation of the Teleost, e.g. that of
Serranus.’ In the following description, accordingly, it will
be the writer’s purpose to emphasise these more important
comparisons,
In general it may be said that the egg of Amia is mero-
blastic, that its (earlier) cleavages are confined entirely to the
germinal area, that the compact blastomeres are Teleost-like,
and that the segmentation cavity is practically wanting.
First Cleavage (Pl. 30, fig. 2) —In the living egg the first
cleavage passes at once vertically through the germ area,
causing the resulting blastomeres to be closely opposed. In
the preserved material, however, the rim of contact (as in Pl. 30,
fig. 3) appears somewhat rounded. At this stage the lower rim
of the germ disc presents a more definite line of contact with
the yolk, and the cleavage plane rounds off the corners of the
blastomeres. In the transverse vertical section shown in
(Pl. 31, fig. 21) are illustrated the depth and the character of
the cleavage plane ; it has passed slightly deeper than the
niveau of the nuclei, but leaves below it a well-marked layer
of the germinal protoplasm; the germ disc in this region has
1H. V. Wilson, “The Embryology of Serranus atrarius,?’ ‘ Bull.
U.S. Fish Comm.,’ 1891, pp. 209—277,
THE EARLY DEVELOPMENT OF AMIA. 425
become slightly deeper. The first cleavage was observed in
several instances to divide the germ disc into blastomeres of
unequal size ; this abnormality, however, as in Lepidosteus,'
Teleosts,? as well as in other Chordates, was found to in no way
influence the subsequent developmental stages.
The writer, it might be here noted, has taken especial care to
verify his observations on the meroblastic character of the
cleavages of Amia. During the first few cleavages several
hundred living eggs were examined with a view of determining
holoblastic variations. These, however, did not occur, nor
were there found even by the most favorable means of
illumination, traces of what might be construed as surface
furrows traversing the yolk region of the egg. In no case did
a marginal cleavage pass below the rim of the germinal disc.®
Second Cleavage (Pl. 30, fig. 3) passes in a vertical plane
at right angles to the first cleavage. This it closely resembles
in depth and marginal limits; and in this stage the nuclei
retain the same niveau with similar relations to the yolk.
Immediately below animal pole the blastomeres slightly
separate, giving rise to the beginnings of the segmentation
cavity. In an examination of a number of eggs at this stage
but very few (2 per cent.) variations were observed, the second
plane in these cases intersecting the first at an angle of about
70°. Polflucht was in no instance noteworthy.
Third Cleavage (Pl. 30, fig. 4).—This cleavage plane is
RTT
Fic, 5.—Variations in the stage of third cleavage.
again vertical, and, as in Lepidosteus and Teleosts, at right
angles to the preceding plane (i. e. parallel to the first cleavage).
In this stage variations were found to be common (20 per cent.),
1 Dean, op. cit., p. 16.
2 Ryder (cod), H. V. Wilson (Serranus), Whitman, and others.
3 Cf, the somewhat different view of Fiilleborn, op. cit., p. 1061.
426 BASHFORD DEAN.
and noteworthy Polflucht occurred. Several variations are
shown in the adjoining figure (Fig. 5), of which the most
frequent, as in Lepidosteus, is the symmetrical meridional
form shown at the left. The segmentation cavity takes its
definite origin at this stage ; in the region of the animal pole
the blastomeres are separated from the underlying yolk—the
germ disc by a narrow fissure, which has been found to arise
in the cleavage planes of the animal pole. ‘Thus in a section
of the germ disc passing through the points *—* of Pl. 30,
fig. 4, it will be seen (PI. 31, fig. 23) that the blastomere, which
is cut nearly transversely (a), is separated below from the yolk
region of the germ disc (yg.) ,and at the sides partly, from the
adjacent blastomeres by the fissure-like segmentation cavity
(sc.). And it will be further noted that the yolk region of the
germ disc (yg.) is still in common, marginally, with the
blastomeres. With these conditions should be contrasted the
more shark-like features of the corresponding stage of
Lepidosteus (Dean, op. cit., p. 17, and pl. il, fig. 26).
Fourth Cleavage (PI. 30, fig. 5).—The plane of the fourth
cleavage is again vertical, resembling the third cleavage in
essential regards. Its general direction is parallel to the
second cleavage. This stage usually results in the division of
the germ area into blastomeres of approximately equal size ;
often, however, as in the figure (PI. 30, fig. 5), the cleavage
passes nearer or further from the animal pole than in the
preceding stage: in this event the central blastomeres appear
at the surface rectangular instead of square. Many cleavage
variations at this stage were recorded, in which noteworthy
Polflucht occurred, and in which meridional planes often
replaced the transverse cleavages ; in no instance, however,
were horizontal cleavages observed. The segmentation cavity
in this stage does not markedly differ from that noted in the
earlier stage. In Pl. 31, fig. 24, a section is shown passing
through the central blastomeres in the direction of the (first or)
second cleavage plane ; its direction is slightly oblique, and it is
for this reason that five blastomeres appear. In this section
the extent of the segmentation cavity may be followed, and by
THE EARLY DEVELOPMENT OF AMIA. 427
a study of serial sections it may be determined that the central
blastomeres are now separate from the underlying germ-yolk,
but that the marginal blastomeres are unseparated from it; the
nuclei still remain in the low region of the blastomeres. The
dilated spaces separating the sides of the blastomeres might
perhaps be regarded as artefacts.
Fifth Cleavage (Pl. 30, fig. 6).—The stage of thirty-two
cells is the first in which horizontal cleavages have been noted.
These occur, however, only by variations in the divisions of
the central blastomeres, and are by no means common. The
typical conditions of this stage are shown in the above figure.
From the study of living eggs the fifth cleavage was observed
to take place in the following manner:—The five central
blastomeres of the sixteen cells divide vertically in somewhat
meridional planes, forming together a compact mass in the
region of the animal pole, separated from the marginal
blastomeres by a sharply cut trench ; a few minutes later the
marginal blastomeres undergo vertical division in meridional
planes. That this is the normal plan of cleavage has been
verified by serial sections of the late sixteen-cell stages where
the nuclear figures have been clearly followed. Thus it
appears that there occurs a noteworthy difference from the
normal mode of the fifth cleavage in Lepidosteus (Dean, op.
cit.,p. 17). Variations, however, are numerous ; the horizontal
cleavage of the central blastomeres has been already noted,
and the tendency of the cleavage planes of the marginal
blastomeres to pass obliquely seems to suggest an approach to
the conditions of the gar-pike. This cleavage of Amia, there-
fore, is not as nearly of the Teleostean plan as in the latter
form. An interesting condition in this stage is the mode of
origin of the newly formed marginal blastomeres ; these appear
to be budded out directly from the germ-yolk (Pl. 31, fig. 25,
gy.), their nuclei at first lying below the plane of the segmen-
tation cavity ; the central blastomeres as before are separated
from the germ-yolk by the segmentation cavity.
Sixth Cleavage (Pl. 30, fig. 7).—The lineage of the cells
of this stage could not be definitely followed; numberless
428 BASHFORD DBSAN.
variations were found, and every attempt to reduce them toa
common type was unavailing. Numerous horizontal cleavages
occur in all blastomeres; increments to the cell disc are not
lacking from the floor of the segmentation cavity. In the
section in Pl. 31, fig. 26, a cell is seen to be budding out of
the germ-yolk region at m, and at m’ a dividing nucleus in
the same region is clearly comparable to a merocyte. A study
of living material demonstrates one of the steps in the transi-
tion from the fifth cleavage, i.e. the usual mode of division of
the marginal blastomeres. These may be seen to bud off their
polar ends, which, in turn, join the central cell mass, and leave
as their outer boundary a furrow similar to that outlining the
central blastomeres in the preceding stage. It is evident from
the section of Pl. 31, fig. 26, that the area of the segmenta-
tion cavity, sc., has greatly enlarged, although as a cavity it
is no longer marked as in Pl. 81, fig. 25; the blastomeres are
now directly apposed to the germ-yolk area.
Seventh Cleavage (Pl. 30, fig. 8).—Horizontal and verti-
cal cleavage planes pass irregularly through the cells of the
germinal disc. Nuclei occur (PI. 31, fig. 27) in the germ-yolk
area(m, m,m.) and bud off blastomeres to the overlying cell disc,
and are seen to be undergoing direct division. The segmentation
cavity (sc.) has now a flooring of a single layer of irregular
blastomeres derived mainly from the germinal yolk.
Eighth Cleavage (PI. 30, fig. 9).—By this stage the blas-
tomeres have so subdivided that in surface view they can be
but obscurely defined; marginally, however, they extend no
further than in the earlier cleavages. Sections of this stage
show that the cell-cap has increased in thickness (PI. 31,
fig. 28) ; anumber of irregular blastomeres, yolk-laden, are seen
in process of being budded off from the floor of the segmentation
cavity, and numerous yolk nuclei are apparent. The segmenta-
tion cavity (sc.) extends irregularly among the blastomeres.
Subsequent stages of segmentation correspond closely with
that last described, the blastomeres continuing to subdivide,
aud at the same time to encroach slowly on the yolk region of
the egg.
THE EARLY DEVELOPMENT OF AMIA. A29
D. Blastula.—A typical stage of the blastula is figured in
vertical section in Pl. 31, fig. 29, and a somewhat later stage
in surface view in Pl. 80, fig. 10, and in section in Pl. 31,
fig. 30. The latter, contrasted with the sections of figs. 28
and 29, indicates clearly the downgrowth and the greater
depth of the blastoderm ; its cells have greatly increased in
number, and build a dome-shaped cell cap of nearly twice
the thickness of that of fig. 28; its cells are small, spherical,
and of uniform size; those of the surface layer, however,
have compacted into a firm cell stratum, e’, and those in the
lowermost part of the blastoderm are slightly larger, yolk-
laden. In this region the space between the loosely associated
cells (sc.) is evidently to be compared with that (sc.) of the
former figures. The segmentation cavity will be noted to extend
irregularly between the blastomeres as far as the outermost
cell stratum. Its floor is flattened, and bears a tier or more
of irregular, upwardly projecting yolk-cells, and below them
a merocyte-bearing zone of yolk.
The conditions of the blastula of Amia present interesting
resemblances to those of Lepidosteus, and especially to those
of the Teleosts.
The resemblances to the Teleostean blastula (e.g. cf. H.
V. Wilson, op. cit, fig. 27) include—the lenticular shape of
the blastoderm, the general uniformity of its elements, the
differentiation of the outermost cell stratum, the apparent re-
lations of the yolk nuclei to mode of growth of the blastoderm.
In the latter regard it cannot be doubted that the closest func-
tional kinships to the periblast are present; cell increments
are being constantly made in the plane of the base of the
blastoderm through the agency of a layer of nucleated elements
derived from the yolk region.
E. Gastrula.—Typical conditions of the gastrula have
been figured in surface views in Pl. 30, figs. 12, 13; an early
stage in fig. 11, and two of the closing stages in figs. 14, 15.
Like the blastula, it proves of considerable interest in com-
parison with the conditions of the older Ganoids on the one
hand, and of the Teleosts on the other.
VOL. 38, PART 4,—NEW SER. GG
430 BASHFORD DEAN,
The transition from the stage last described to that of the
early gastrulation may first, however, be followed. In Pl. 30,
fig. 10, is figured in surface view a late blastula; the light
coloured dome-shaped blastoderm appears sharply marked off
from the yolk ; its marginal rim, as a somewhat irregular line,
seems as if in bold relief against a somewhat dark-coloured
zone of the yolk; in surface view, in fact, it appears to
overhang the yolk, and thus to represent the beginnings of
gastrulation. That this observation, however, is incorrect is
demonstrated both by sections and by a closer examination of
the surface view of the object; the darker colour of the yolk
zone is probably due to its merocyte-bearing character. It is
in the stage figured in PI. 30, fig. 11, that gastrulation actually
begins. The downgrowth of the blastoderm is accompanied
by the slight overlapping of its rim at one side—at the left in
the figure ; the remainder of the clearly defined margin has
not as yet separated from the yolk. By the stage of Pl. 30,
fig. 12, the downgrowth of the blastoderm has separated its
entire rim from the yolk; and that portion which initiated
the process of separation is now separated most widely as the
dorsal lip of the blastopore. This region at a very similar
stage has been shown in the following figure, Pl. 30, fig. 13,
as exhibiting a variation! of considerable interest ; at the rim’s
dorsalmost point a slight indentation is present, which may be
supposed to correspond to the true blastopore of the ancestral
Ganoid and Elasmobranch, the remaining portion of the rim
representing the circumcrescence margin (O. Hertwig).
Later surface views of the gastrula are seen in Pl. 30, figs.
14,15. In the former the continued growth of the blasto-
derm has greatly reduced the size of the blastopore; this now
appears as a circular opening, its margin slightly nicked on
both ventral and dorsal sides, and shows dorsally the whitened
tract which marks the appearance of the embryo. In the
1 Variations in the outline of the closing blastopore are not uncommon
(cf. Lepidosteus, Dean, op. cit., p. 22). Oval blastopores were noted: in
some one or both of the marginal indentations had disappeared; the dorsal
one, however, is usually persistent.
THE EARLY DEVELOPMENT OF AMIA. ASL
latter is figured the stage of the closing blastopore ; the embryo
is here faintly outlined as a white flattened cell mass; at its
hindmost region the blastopore, as a funnel-shaped pit, is en-
closed by its thickened and constricting margin.
It is, however, from the study of gastrulz in sagittal sections
that the most interesting comparisons may be made with the
conditions in gar-pike, sturgeon, and Teleosts. Some of these
have been figured in Pl. 32, figs. 31 (=PIl. 30, fig. 11); 32
(=Pl. 30, fig. 12); 33 (=PI. 30, fig. 14) ; 34 (slightly earlier
than Pl. 30, fig. 15). But before comparisons may be estab-
lished, the advancing changes should briefly be reviewed.
The early gastrula, Pl. 32, fig. 31, it is to be noted, occupies
approximately the area of the egg’s surface as the blastula
of Pl. 31, fig. 830; it has, however, the following advancing
characters :—the loose cells of the blastoderm, now of minute
size, have flattened into a compact cell mass, ectoblast (0)
still presenting a well-marked surface layer (0’) ; the segmenta-
tion cavity (sc.) has accordingly become greatly depressed, and
is now fissure-like ; below it are several tiers of loosely com-
pacted cells, which, by regular transition, appear to take their
origin in large, vaguely defined yolk cells (yc.) arising from
merocytes (m’); the rim of the blastoderm at di. is the region
where the blastoderm is early separated from the yolk; there
is here, however, no cavity apparent separating the dorsal lip
of the blastopore (d/.) from the yolk (y.); both are closely
apposed, and at the point * their elements can no longer be
distinguished; the entoblast (7.), arising from the undiffer-
entiated tissue of the dorsal lip, is here composed of compact
elements, but in the central region of the blastoderm becomes
equivalent to the loose cellular layer already noted as con-
tinuous with yolk-cells and merocytes; at the opposite point
of the blastoderm’s margin, the blastopore’s ventral lip, no sepa-
ration of the germ layers from the yolk has as yet occurred.
In a following stage (Pl. 32, fig. 32) the blastoderm is shown
enclosing about 285° of the egg’s vertical circumference. The
ectoblast has greatly thinned out, is thickest at its perimeter
aud at its dorsal lip; the segmentation cavity is fissure-like ;
432 BASHFORD DEAN.
the ventral lip of the blastopore is now separate from the yolk,
but is closely apposed to it, as in the case of the dorsal lip,
making the ceelenteron (c.) fissure-like; the inner germ layer
is in contact with the yolk-cells near the point*; in the
central region of the blastoderm it no longer exhibits the
entoblast cells; their well-marked layer has apparently be-
come merged with the yolk-cells.'
In a still later gastrula (Pl. 32, fig. 33) the growth changes
include :—the greater thickness of the lips of the blastopore,
the retraction of the yolk-mass, the appearance of the cclen-
teron as a distinct cavity (c.) and its extension forward under
the dorsal lip (as far as the point *), the origin of the ectoblastic
head mass, and of the middle germ layer. In the last regard
this stage merits special attention: the mesoblast is found to
arise peristomal (m.); on the dorsal side it arises from the un-
differentiated tissue (of the tail mass), thence extends forward
as a separate cell layer, and finally appears to be blended with
the loosely associated cells of the entoblast ; ventrally the me-
soderm (m.), although distinctly to be recognised, is not to be
separated from the cellular elements of the entoderm.
And finally, in the stage of the closing blastopore (Pl. 32,
fig. 34), the embryo having surrounded about 180° of the egg’s
circumference, the following characters appear:—The greatly
increased size of the ectoblastic head mass (/.), and of the ceelen-
teron (c.) together with the complete differentiation beneath
the embryo ofthe middle germ layer (m.). The latter appears
to have been differentiated in situ from the loosely associated
cell mass shown in the preceding figure; it is separate
anteriorly as far as the region immediately below the head
terminal. The celenteron, now a deep cavity beneath the
dorsal lip, extends forwards below the entire head; its hinder
dilation (k.), immediately below the dorsal lip, is to be in-
terpreted as representing Kupffer’s vesicle ;! beneath the ven-
1 No critical attempt has ever been made to follow the actual mode of the
growth of the embryo, as, for example, has been done by Morgan in the case of
Ctenolabrus (1895, ‘ Journal of Morphology,’ vol. x, No. 2).
2 Dean, op. cit., p. 42.
Fic. 6.
NTGaSs
Fre. 10.
ite, 2
Sagittal sections of early and late gastrule of Ganoids and Teleosts. 6, 7.
Lepidosteus. (In Fig. 7 the blastopore has just closed.) 8,9. Acipenser.
10, 11. Amia. 12, 13. Teleost. c. Coelenteron. px. Dorsal lip of blasto-
pore. H. Head (= cephalic thickening of neuron). 1. Inner germ layer.
Kv. Kupffer’s vesicle. m. Middle germ layer. m’. Merocytes (= periblast
in Fig. 12). 0. outer germ layer. sc. Segmentation cavity, y. Yolk
region, yp. Yolk plug,
BASHFORD DEAN,
434,
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THE EARLY DEVELOPMENT OF AMIA. 435
tral lip the celenteron is no longer prominent. The closure
of the blastopore is effected very much as in Lepidosteus
or Acipenser, the constricting of its margin is the apparent
cause of a greatly enlarged Randwulst; this ingrowth is at-
tended by the protrusion of a slender yolk-plug (yp.), which
on the blastopore’s closure appears to be largely withdrawn.
A comparison of the gastrulation of Amia with similar
stages of kindred forms may now be made. And this will be
seen to become of especial interest since the intermediate
characters of its gastrulation enable a far clearer understand-
ing of this complicated growth stage of fishes than has yet
been given. On the one hand the gastrula of Amia present
decidedly Ganoidean features, while on the other its structures
are clearly to be compared with those of the Teleost. An
accompanying series of figures (figs. 6—13) enables compari-
sons to be more readily drawn ; they present sections (nearly
sagittal) of the earliest and of the latest stages of the gastrula
of Lepidosteus, Acipenser, Amia, and Teleost; in the earliest
stages the dorsal lip is coming to be formed; in the latest,
the blastopore is closing.
From the foregoing comparison it seems to the writer evident
that well-marked transition exists in the structures of cor-
responding stages. These he believes may, as in his figures,
most conveniently be followed, starting with the archaic plan
of gastrulation of Lepidosteus, passing to that (somewhat
divergent) of the ancient sturgeon, thence to the more modern
type of Amia, and finally to that of the highly specialised and
recent Teleost. And it seems clear to him, furthermore, that
the puzzling features of the gastrula of the specialised bony
fishes (Teleocephah) may be interpreted as altogether due to
a process of advancing or accelerated (precocious) development.
Thus the advancing growth changes appear to be indicated in
the following processes.
1. The invagination tends to begin at an earlier stage when
the blastodisc covers a smaller area of the egg’s surface, and the
elements of the early gastrula tend to become relatively larger,
and the roof of the segmentation cavity relatively thicker.
436 BASHFORD DEAN.
2. The segmentation cavity tends to become flattened and
to recede centrad.
3. The embryo tends to become larger, and its structures
more precociously differentiated, concentrating its substance
more and more early in the sagittal plane of the dorsal lip
(and leaving, therefore, the remaining region—e. g. ventral
lip undifferentiated), and growing notably in the head region.
4, The embryo, pari passu with its more precocious de-
velopment, has come to acquire more perfect relations with
the yolk. The growth of the embryo in the generalised con-
dition is derived from deep merocyte layers of the yolk ; in the
more specialised conditions, however, the more peripheral
layers of deutoplasm become of service. Cellular increment
is derived in Amia from a double tier of cellular zone of the
floor of the segmentation cavity, which in the Teleost becomes
clearly homologous with the periblast. In such an event the
celenteron becomes clearly confluent with the segmentation
cavity.
From the foregoing discussion the writer’s views as to the
homologies of the structures of the Teleostean gastrula are
clearly apparent; and they will be found to correspond with
those which he had formerly expressed in his paper on the
gar-pike and sturgeon (p. 52). His interpretation requires,
accordingly, the Ziegler-Wilson conception of the gastrula to
become modified as follows:'—The ccelenteron extends under
the rim of the blastoderm, from the free end of the “ primitive
hypoblast ” to that of the “ ventral mesoderm ” of H. V. Wilson,
the periblast yolk losing its ancestral cellular connection with
the embryo, on account of acquiring its indirect but more
highly important (and specialised) method of furnishing its
cellular increments.
F. The Mode of Formation of the Embryo.—The
embryo has already made its appearance by the time of the
closure of the blastopore. It is there recognised in the flat-
tened and opaque cell mass extending in front of the blastopore,
and enclosing about 180° of the egg’s circumference (Pl. 30,
1H. V. Wilson, op. cit., p. 264,
THE EARLY DEVELOPMENT OF AMIA, 437
fig. 15, and Pl. 32, fig. 34). Its substance is insunken in the
egg.
The mode of establishment of the early embryo’s outward
form may be followed in PI. 30, figs. 16—20. In the first of these
figures (fig. 16) the embryo surrounds about 185° of the egg’s
circumference, and the blastopore has disappeared; a whitened
line on the egg’s surface represents the neural axis, its en-
larged terminal the brain tract ; a slight darkening in the axial
line is due to a shallow trench-like insinking of the neural plate.
The embryo is not as yet noticeable above the surface curva-
ture of the egg. In PI. 30, figs. 17 and 18, the head and tail
regions of a slightly older stage, the following changes have
taken place:—The embryo, a rod-like surface thickening,
surrounds about 195° of the egg’s circumference; its head,
prominent and enlarged, rises slightly above the surface curva-
ture of the egg; its trunk tapers hindward, ridge-like in form,
closely apposed to and slightly insunken in the egg mass; two
mesoblastic somites have appeared. In the stage of Pl. 30,
figs. 19 and 20, six somites are present, and the embryo has
enclosed about 220° of the egg’s circumference; the neural
axis, now more sharply marked, rises slightly above the egg;
the tail in its growth is now separating from the egg surface ;
the brain regions are defined, optic vesicles are appearing ; and
the head region, growing slightly forward, has in front the
beginnings of the stomodzum.
In the features of early outward growth Amia again presents
marked transitional characters between Ganoids and Teleosts.
Like the latter, its form growth is early concentrated in the
sagittal plane, and the embryo deeply sunken in the egg mass.
It later resembles the Ganoids in its uplifting from the yolk
and in the details of organogeny. <A few further notes re-
garding the origin and growth of its structures are given in
the following section.
G. The Origin and Early Differentiation of the
Germ Layers.—The outer germ layer has been already
noted (p. 431) in the early gastrula as a compact cell mass of
comparatively uniform thickness. Its subsequent growth has
438 BASHFORD DEAN.
also been reviewed in the table and figuresof pp. 483-434. By
the time of the blastopore’s closure it has given rise to the
sagittal thickening out of which the central nervous system is
shortly to be formed. This, in its early characters, suggests
closely the neuron of the Teleost (Pl. 82, figs. 34 and [trans-
verse section] 86). Its deep keel-like thickening resembles
closely as well the earliest conditions of Lepidosteus, while
diverging from the type of Acipenser. To the conditions in
this form, however, an apparent similarity exists in the stage
already noted in Pl. 30, fig. 16, where a slight axial groove is
for a short time present; this condition, of interest, accord-
ingly, from its sturgeon-like feature, is further illustrated in
the transverse section of Pl. 82, fig. 87; the axial groove is
never deeper than here figured, and shortly passes away,
flattening as the neuron increases in size and depth. The
lumen which the neuron later acquires takes its definite origin
in the dissociation of cells in the vertical plane of its axis
(Pl. 32, fig.39). This condition is clearly to be compared with
that of the Teleost, as described by Hoffman, v. Kupffer, H.
V. Wilson, and others. It might, moreover, be regarded as
confirming conclusively the position of v. Kupffer in regard to
the morphological importance of the neural furrow of the
Teleost, i.e. that it is homologous to the primitive neural
furrow of Amniotes; and, on the other hand, it certainly
removes the ground for believing that the neural axis of the
Teleost was primitively solid, as Minot has maintained.'! For
there can be little doubt that Elasmobranchian characters are
present in the origin of the neuron of Acipenser, and it follows,
therefore, that the neural furrow of Amia, its ally, must
represent, if only in a transient way, the early Amniote con-
dition. In the Teleost, accordingly, it is reasonable to expect
that abbreviated growth stages have greatly reduced the
prominence of the ancestral medullary folds, while perfecting
the newly acquired mode of securing a neural canal.
A further and striking similarity to Teleostean conditions is
found in Amia in the early development of the optic vesicles.
1 « American Naturalist,’ November, 1889.
THE EARLY DEVELOPMENT OF AMIA. 439
Absenceof neurenteric canal is,again, Teleostean. In this regard
its features are notably transitional between those of Lepi-
dosteus and the Teleost. At the closure of the blastopore,
Pl. 82, fig. 35, there is no trace of the neural groove in the
hinder region, the outer germ layer here fusing in a solid plug,
merging below into the undifferentiated tissue, w; at a later
stage, Pl. 32, fig. 88 (cf. H. V. Wilson, op. cit., pl. xeviii,
fig. 84), the ventral lip of the blastopore has become largely
merged with the growing tissue, w, of the tail region, and the
conditions are markedly Teleostean.
The inner germ layer has already been noted (p. 481) in
its intimate relationship to the yolk mass. In PI, 82, fig. 31,
the entoderm is clearly defined, extending forward from the rim
of the dorsal lip to about the position of the point®*, having in
its texture all the characters of the similar stage in gar-pike and
sturgeon. Beyond this point, however, it merges into the
loose layer noted on p. 431, whose periblast-like characters have
already been indicated (p, 434). The growth in extent of the
entoderm may befollowed in Pl. 32, figs. 31—86, 38, 39; under
the ventral lip of the blastopore it appears for a short distance
(fig. 35) as a separate layer, but in a later stage (fig. 39) it
becomes merged with the yolk entoblast; under the blasto-
pore’s dorsal lip the darm-entoblast has separated from the yolk
region (apparently by differentiation of the cells in situ) as far
as the point * in fig. 33, and as far as the most anterior head
region in fig. 84; at its periphery the cells become indistin-
guishable from those of the neighbouring yolk. The gut arises
in a manner closely resembling that of Acipenser ; its cavity,
narrow and deep (fig. 34) tailward, flattens out broadly in the
head region, as is shown in the marginal limits of the parietal
zone of PI. 30, figs. 17—20 ; its mode of formation, it should be
noted, although of clearly marked Ganoidean character,
diverges, nevertheless, toward the plan of the Teleost; it has
not, however, acquired the yolk-apposed characters of the
latter. The notochord arises as in the sturgeon or gar-pike
(Pl. 32, fig. 38): it separates directly (i.e. delaminates) from
the entoderm; in the region of the hind brain, as seen in
44.0 BASHFORD DIAN.
Pl. 32, fig. 36, it is undifferentiated from the loosely associated
cells of the lower layer.
The mesoblast is notably peristomal (Pl. 32, figs. 38—
36, 88, 39); it is hardly to be distingushed in the blastopore’s
ventral lip (cf. figs. 35, 39): its appearance is first noted in the
gastrula of fig. 33. In its early growth it extends forward as
a wide and flattened cell mass, thinning distally, and becoming
confluent with the inner germ layer. As in the Teleosts, gas-
tral mesoderm is absent, and the division of the middle layer
into its somatic and splanchnic layers is not apparent until
comparatively a late stage ‘of development. A contrast with
the conditions of the mesoblast in Acipenser and gar-pike may
be made by reference to the table in Dean, op. cit., p. 47.
TII. Concivustons.
The early development of Amia, as outlined in the foregoing
paper, must certainly be regarded as furnishing abundant evi-
dence of intermediate characters. To the Ganoids on the one
hand, and the Teleosts on the other, these ontogenetic near-
nesses become, accordingly, of the greatest interest, since they
confirm the results of the structural study of recent and fossil
forms upon the Amioid descent of Teleosts.
A comparison of the developmental characters of Amia with
those of the gar-pike and sturgeon need be but briefly reviewed.
Its type of development is in many ways curiously Lepidos-
teoid, as in meroblastic segmentation, relations of blastoderm
to yolk, flattened segmentation cavity, late gastrulation, early
neuron, and absence of neurenteric canal. It certainly re-
sembles that of the sturgeon in some of its advancing cha-
racters, as in the mode of closure of the blastopore, decreased
prominence of its ventral lip, and in the embryo’s sagitally
accented growth. From either of these older Ganoids the
developmental type of Amia is nevertheless sufficiently different
to warrant any definite conclusions as to its descent; one
might, the present writer believes, safely infer that, like that of
the sturgeon, it is in general Lepidosteoid.
But the early development of Amia is clearly to be recognised
THE EARLY DEVELOPMENT OF AMIA. 4.4]
as of an advancing type. It bridges over in many and important
characters the gap which has always been pointed out as
separating the Ganoids and the Teleosts. Its abbreviated
development indicates, in short, the very stages which are most
abbreviated in the cenogenesis of the latter group. Its mero-
blastic mode of cleavage is decidedly Teleostean, especially in
the relations of its segmentation cavity and yolk nuclei,
although in these regards it also closely resembles Lepidosteus.
Its transitional characters are most clearly marked during
gastrulation, and during the early growth of the embryo.
These Teleostean features might, in conclusion, he briefly
summarised.
Small area of blastoderm at the beginning of invagination
(?). Flattened segmentation cavity. Early relations of inner
germ layer of dorsal lip with periblast-like conditions of the
yolk cells; coelenteron is then practically confluent with the
segmentation cavity. General thinness of the down-growing
blastoderm, whose Randwulst corresponds to the germ-ring ;
close apposition of blastoderm to yolk mass. Early appearance
of the embryo ; and in general early differentiation of the germ
layers of the blastopore’s dorsal lip, attended by a corresponding
lack of differentiation of the ventral lip. The mode of the
closure of the blastopore; the presence of Kupffer’s vesicle
and the absence of neurenteric canal. The early growth of the
neuron as an insunken tract thickest in the sagittal plane.
The early prominence of the brain mass. The evanescent
medullary groove ; the solid character of the early neuron, and
its secondary mode of acquiring a neural canal. The peristomal
mode of origin of the mesoblast ; its late differentiation; the
absence of gastral mesoblast. The early mode of establish-
ment of the embryo’s outward form.
44.2 BASHFORD DEAN.
EXPLANATION OF PLATES 30—32,
Illustrating Dr. Bashford Dean’s paper on “The Early
Development of Amia.”
PLATE 30.
Figs. 1, 2, 4, 15, 19, and 20 drawn by Bashford Dean from the living
eggs, the remaining figures from eggs hardened in alcoholic (50 per cent.)
picro-sulphuric mixture, by Dr. Arnold Graf. x about 50.
Fic. 1.—Living egg shortly before the appearance of the first cleavage
furrow. About 4 hour after fertilisation.
Fig. 2.—First cleavage ; it sharply separates the halves of the germ-disc,
but extends no further marginally than its limits. 1 hour.
Fic. 3.—Second cleavage, seen from above. 2 hours.
Fic. 4.—Third cleavage; similar in marginal extension to the first and
second. 3 hours.
Fic. 5.—Fourth cleavage, 44 hours.
Fic. 6.—Fifth cleavage, 53 hours.
Fic. 7.—Sixth cleavage, 63 hours.
Fic. 8.—Seventh cleavage, 74 hours.
Fie. 9.—LHighth cleavage (early blastula), 83 hours.
Fic. 10.—Early gastrula, 46 hours.
Fie. 11.—Early gastrula, 48 hours.
Fic. 12.—Gastrula, about 50 hours.
Fic. 13.—Gastrula showing indented rim of blastopore at the dorsal lip.
54 hours.
Fic. 14.—Late gastrula, showing mode of closure of the blastopore ; the
embryo’s appearance is faintly indicated in the light-coloured area imme-
diately above the centre of the figure. 71 hours.
Fic. 15.—Closure of the blastopore, and the appearance of the embryo.
78 hours.
Fic. 16.—Early embryo showing neural folds ; its length surrounds about
185° of the egg’s circumference. About 93 hours.
Fic. 17.—Early embryo, showing head region ; its length surrounds about
195° of the egg’s circumference. Two somites present. 95 hours,
THE EARLY DEVELOPMENT OF AMIA. 4.43
Fic. 18.—Karly embryo; tail region of embryo of Fig. 17.
Fic. 19.—Early embryo showing head region; its length surrounds about
220° of the ege’s circumference. Six somites present. 100 hours.
Fie. 20.—Early embryo; tail region of embryo of fig. 19.
PLATE 31.
Vertical sections of cleavage stages and of blastula. x about 55.
Fre. 21.—Second cleavage; sectioned parallel to a cleavage plane through
the resting nuclei.
Fie. 22.—Third cleavage; sectioned near and parallel to the plane of the
second cleavage.
Fie. 23.—Third cleavage; sectioned in a plane passing through the points
*—* of Pl. 1, fig. 4.
Fig, 24.—Fourth cleavage; sectioned in a plane (slightly oblique) near and
parallel to the first (or second) cleavage.
Fic. 25.—Fifth cleavage.
Fie. 26.—Sixth cleavage.
Fic. 27.—Seventh cleavage.
Fic. 28.—Kighth cleavage.
Fie. 29.—Harly blastula. Eleven hours after first cleavage.
Fig. 30.—Blastula. Thirty-three hours after first cleavage.
a. Central blastomere. e. Entoblast. e’. Epidermic stratum of ectoblast.
gyb. Germ yolk blastomere. m. Merocyte. m’. Blastomere arising from
merocyte-bearing germ-yolk. x. Nucleus. sc. Segmentation cavity. y. Yolk.
yg. Germ-yolk.
PLATE 32.
Vertical sections of gastrulation stages and of early embryos.
Fie. 31.—Early gastrula (47 hours), nearly sagittal section. x 35.
Fie. 32.—Gastrula (50 hours), nearly sagittal section.
Fic. 33.—Gastrula (55 hours), nearly sagittal section.
Fic. 34.—Late gastrula (or early embryo) (75 hours), nearly sagittal
section.
Fig. 35.—Early embryo (78 hours); section slightly oblique to embryo’s
axis, passing through the region of the closed blastopore. x about 70.
Fic. 36.—Early embryo (78 hours) ; transverse section passing through
the region of the hind brain. Xx about 70.
44,4, BASHFORD DEAN.
Fic. 37.—Early embryo (77 hours); transverse section of neuron of the
hinder trunk. x about 160.
Fie. 88.—Early embryo (95 hours), transverse section ; somewhat in front
of the undifferentiated tissue of the tail mass.
Fie. 39.—Early embryo (95 hours), sagittal section of the region of the
closed blastopore. x about 80.
Fic. 40.—Early embryo (95 hours); transverse section of neuron in a
region somewhat behind the hind brain.
ec. Celenteron. ch. Notochord. dl. Dorsal lip of blastopore. 2. Head
region (of neuron). 7. Inner germ layer. 4. Kupffer’s vesicle. m. Middle
germ layer. m’. Merocyte. o. Outer germ layer. 0’. Epidermic stratum of
the outer germ layer. sc. Segmentation cavity. . Undifferentiated tissue
of the lip of blastopore. o/. Ventral lip of blastopore. yp. Yolk-plug.
* denotes the innermost limit of ccelenteron.
ON KYNOTUS CINGULATUS. 445
On Kynotus cingulatus, a New Species of
Earthworm from Imerina in Madagascar.
By
W. Blaxland Benham, D.Sc.Lond., Hon. MW.A.Oxon.,
Aldrichian Demonstrator in Comparative Anatomy in the University of
Oxford.
With Plates 33 and 34.
Tue subject of the present paper was handed to me by
Professor Bell, of the British Museum, in 1898, for identifi-
cation. The delay of more than a year in completing the
description of this worm, which presents points of novelty
deserving earlier publication, is due to press of other work. I
have once more to acknowledge my indebtedness to my friend
Professor Jeffrey Bell, and to tender my thanks to the autho-
rities of the British Museum for their generosity in permitting
me to “ work my will” on the specimens handed to me.
The genus Kynotus was founded by Michaelsen! in
1891, and in addition to the original species, three have since
been described, two by himself? and one by Rosa.® All
these, like the present new species, were collected in Mada-
gascar.
One of the most striking characters of the genus is the great
number and small size of the segments composing the body,
quite apart from the total size of the worm. The species of
this genus are of considerable length, though none are of any
1 «Arch. f. Naturgesch.,’ 1891 (K. madagascariensis).
2 «Jahrb. Hamburg. Wiss. Anst.,’ ix, 1891 (K. longus); ‘Arch. f,
Naturgesch.,’ 1892 (K. kelleri).
3 K. michaelsenii, ‘ Boll. Mus. Zool. Torino,’ vii, 1892.
VOL. 38, PART 4,—NEW SER. HH
4.4.6 W. BLAXLAND BENHAM.
great thickness,—being, in fact, relatively thin. A second
feature, and one that leads to some difficulty in assigning the
organs to their proper position, is the amount of secondary
annulation presented by the segments in the anterior part
of the body, and in the assumption by these annuli of the
appearance of true segments; so great, indeed, is the resem-
blance, and so deep are the interannular grooves, that an ex-
ternal examination alone is absolutely insufficient to enable
one to tell what are ‘‘ annuli” and what are “‘ segments.” This
secondary ringing of the primary segments deceived Michaelsen
in his descriptions of the two earlier species, K. madagas-
cariensis and K. longus; and he attributed to certain in-
ternal organs a position so different from that occupied by
these organs in all known earthworms, that I was led to
suggest that he had mistaken ‘“‘annuli” for ‘ segments” or
“somites.”! Almost at the same time Rosa showed, from an
examination of a new species, K. michaelsenii, that Michael-
sen had indeed fallen into this error; and Michaelsen himself,
in describing a fourth species, acknowledged that this had been
the case. But, as we shall see below, we are even now in some
doubt as to the extent and limits of this ‘‘ annulation of the
segments ”—at any rate, in four out of the five species—so far
as the first few segments of the body are concerned.
In the bottle sent to me were three portions, each about nine
inches (225 mm.) in length; each is the anterior part of a
worm of much greater length, probably at least eighteen or
twenty inches. Each piece consists of some three hundred or
more segments.
One of these three pieces is of especial interest, as it is
genitally mature, and is provided with a clitellum—a structure
observed, hitherto, only in K. michaelsenii, and there of
much less extent than in the present species, the specific
name of which refers to the great extent of this organ. This
specimen, and one of the other two, also possessed a large
everted copulatory organ of relatively enormous dimensions—a
1 Beuham, “ Description of Three New Species of Karthworms,” ‘ Proc.
Zool. Soc.,’ 1892, p. 149.
ON KYNOTUS CINGULATUS. 44,7
fact, too, which is of interest, as adding to our knowledge of
the varied modes of copulation amongst earthworms.
External Anatomy.—The details refer to the specimen
provided with a clitellum, and preserved in the British Museum,
which I have not opened or otherwise injured.
This worm contains three hundred and fifty-six rings,! most
of which are true segments, though some of the anterior rings
are only “annuli.”’ Its length is 225 mm., its diameter about
12 mm. in front of, but scarcely half this behind the clitellum.
The posterior end was truncated, and the worm had evidently
been cut through, probably at about half its length. The
pre-clitellar region measures about 57 mm. (one and a half
inches); the clitellum itself is 50 mm. (two inches) long.
After the first dozen rings the body narrows slightly, then
at the twenty-third ring gradually widens again, whilst at the
thirty-first ring the diameter increases suddenly ; this diameter
is retained throughout the clitellum, behind which, at the
fifty-seventh ring, the body suddenly diminishes in diameter.
The body is cylindrical and strongly contracted, so as to
feel quite hard in the anterior region.
The general colour of the worms, in spirit, is dull buff;
the dorsal surface behind the clitellum is black, and similar
black pigment occurs on alternate rings in front of this organ.
I have been unable to detect a prostomium ; there is no trace
of it externally, and on slitting open the buccal cavity of one
of the specimens I saw no trace of it in a retracted condition.
This is the more curious, since K. longus is provided with a
large prostomium, and Rosa mentions it in his species.
The first two rings and part of the third are marked by
longitudinal grooves, as in other species (Pl. 33, fig. 1). The
first ring is obscurely divided into two by a furrow, which in
one specimen was so distinct as to cause me on first counting
to reckon this ring as two rings.
The fourth and following rings are marked by a distinct
ridge round nearly their middle, dividing each ring into two.
1 Tuse the term “ring” to indicate the apparent segment: that is, what
anyone would at first sight regard as a “‘ segment.”
4.48 W. BLAXLAND BENHAM.
This secondary ringing becomes more marked as we pass back-
wards, and the thirteenth ring is marked by two slight grooves,
dividing it into three well-marked secondary rings. This same
phenomenon is presented by the following rings up to about the
twenty-second, after which it becomes less and less distinct, till
it is scarcely recognisable on the thirtieth ring. These rings, up
to about the twenty-fifth, are all of nearly the same size. The
rings of the clitellum are not thus marked, but the post-clitellar
rings are biannulate dorsally. Any zoologist, even one familiar
with earthworms, examining these rings would regard them,
I do not doubt, as “segments ;” but such is not the case in
the anterior part of the body. The twenty-third ring and every
subsequent ring is a “segment” (a somite), as is shown by the
internal anatomy—septa, nephridia, blood-vessels, as well as by
the chetz,—but anteriorly to this most of the rings are
“annuli,” or secondary ringings of the body, two of which go
to form a “segment” (fig. 1). This, again, is shown by
internal anatomical arrangements, and the only doubtful rings
are the most anterior three or four. From the position of the
nephridiopores, I believe that the first (sometimes annulated)
ring is a segment; the second and third rings make up the
second segment. The segment 111 is also biannulate, but the
fourth segment is not annulated; each of the following seg-
ments, v to x11 inclusive, are biannulate, behind which every
segment consists of only one ring or annulus (see fig. 6).
Rosa construes the anterior rings rather differently. He
believes the first two rings constitute the first segment, and that
each of the next two rings is a segment, and that the fourth
segment is biannulate. Beyond this point we are in accord.
The point of difference, then, is that we have six rings at
the anterior end to fit into four segments. Now, as we shall
see, the most anterior nephridium opens at the anterior margin
of the second ring, the second nephridium between rings 3 and
4, the third between rings 5 and 6. At this point also is the
first septum.
Proceeding now with the description of the external anatomy
of K.cingulatus. As in three of the other species, there
ON KYNOTUS CINGULATUS. 44.9
are no chetz in the anterior segments of the body ; the first
cheetze to be recognisable are those on the twenty-sixth ring (seg-
ment xvr), though in sections I have recognised them in the pre-
ceding ring. On segments x1II, xiv, xv, the ventral couples
are replaced by long penial cheetz, which do not project in the
preserved specimen, but the apertures of the penial sacs are
seen (fig. 2). The chete are all lateral; the individuals of a
couple are very close together. The two couples of one side are
separated by a space of 3 mm.on the clitellum, and in front of
this space is only 2 mm.; behind it the ventral space, separat-
ing the right and left inner or lower couples, is 11 mm.!'; the
dorsal space, separating the right and left outer or upper
couples, is rather less.
These cheetz are relatively small, measuring only 0°84 mm.
(fig. 10); they have the usual shape, and possess a swelling or
node nearer the freeend. (In K. longus and K. kelleri, to
which my species presents several points of similarity, the
swelling is absent and the chetz are smaller.) These chete
are ornamented, as in some of the other species, by very incon-
spicuous transverse groups of short lines (fig. 11). Is this
ornamentation here and in some other cases, where it is so
simple and ill-defined, a mere wearing away of the surface of the
cheta, showing the broken ends of groups of fibres of which
the cheta is composed? In some worms, as in Rhinodrilus
and Trichocheta, the markings on the chetz are much
more distinct than in the present instance. Here the “ orna-
mentation” has to be carefully looked for; and as the tip of
the cheta is greatly worn down, it is just possible that the
“ornamentation”? may be the result of wear and tear. On
the other hand, a similar ornamentation occurs in the penial
cheetze, which presumably would not be worn down. On the
clitellum, too, each couple lies in a pit in the epidermis, the
cheetze themselves not reaching the surface.
1 These measurements were taken when the body-wall (of one specimen)
had been flattened out.
2 The lithographer has emphasised these markings: they should have been
very much fainter and at the same time more regular,
450 W. BLAXLAND BENHAM.
The clitellum occupies twenty-six segments; namely,
xxi to xLv1 inclusive (i. e. rings 31—56) (fig. 1). There
is not the slightest doubt that these rings are here true
segments and not annuli; for the cheetee and nephridiopores
are perfectly visible—indeed, better marked than on ordinary
segments,—and the intersegmental grooves are deep. This
clitellum is complete; its anterior and posterior boundaries
are very well marked, and there are no lateral ventral ridges or
markings analogous to tubercula pubertatis. The glandular
thickening is continued right across the ventral surface, as in
Diacheta and Pontoscolex.
The extraordinary length of the clitellum is approached
only by Allolobophora gigas (Dugés), where it occupies
segments xxx to LI inclusive, 1. e. covers twenty-two segments.
Amongst the Geoscolicide (Rosa) the clitellum is usually
pretty extensive, covering some eight to ten segments, and in
Diacheta thirteen segments. The great length of this organ
in the present worm is the more noteworthy since in the only
other member of the genus in which it has been observed, viz.
in K. michaelsenii, it only covers seven segments, x1x to XXv
(rings 27—33). Rosa states that it is quite evident, but finds
that the inter-segmental grooves are reduced to simple lines,
and are not as deep as in the non-clitellar segments. Again,
in this species the longitudinal margins are evanescent, and do
not pass beyond the ventral chetz, i.e. it is ‘‘incomplete.”
The clitellum in Rosa’s species appears, then, to differ from
that in mine—a difference that is not difficult to explain, as it
is possible that the worm was not quite mature.
We must wait till we can obtain a more abundant supply of
these worms before we can settle this point; for although four
species have hitherto been described, each species is repre-
sented by only a single specimen, and of my three specimens
only one has the clitellum fully developed. In a second I can
detect traces of it in the smoother surface of a number of
segments corresponding to the area occupied by the organ in
the specimen just described, whilst in the third I can detect no
trace of it. It is possible that, as in Moniligaster, which
ON KYNOTUS CINGULATUS. 451
was for so long believed to be without a clitellum, till Bourne
described it in M. sapphirinaoides, it is a very temporary
structure in Kynotus.
Generative and other Pores.—As in the other species,
there are four pairs of openings leading into more or less
extensive internal sacs (figs. 1,2). Of these the largest lies
in segment xv (ring 25) in line with the ventral chete ; this is
an ‘‘eye-like’’ opening, with the margins marked by radial
grooves, and presenting, within, a rounded papilla, sunk below
the general surface, and not visible in all the three specimens.
Immediately in front of this pore (which Rosa and Michaelsen
describe as the ‘‘ male pore’’) lies a second on segment xIv
(ring 24) of smaller size, and having rather a slit-like shape.
In front again, on segment x11 (ring 33), another of the
same appearance, but lying rather more laterally than the two
posterior pores. The most anterior pair of pores lies on
ring 21, the anterior annulus of segment x11; these have the
same appearance as the others, but lie very much nearer the
ventral mid-line than they do. In arrangement these pores
agree with what has been described by previous authors.
Of these four pairs of pores the three anterior pairs are the
openings of “ prostates,” or “‘sphermiducal glands,” as Beddard
has recently proposed to term these structures, and of the sacs
containing penial chete. The fourth and largest pair gives
exit to large copulatory organs of very characteristic appear-
ance and of relatively enormous size, through which the sperm-
duct opens. These organs I shall speak of as “ claspers,” for
they are evidently not capable of insertion into any sac during
copulation: they are not penes in the ordinary sense of the word.
In two of my specimens the organ on the left side was pro-
truded—in one case fully, in the other only partially. The
fully protruded clasper is represented from below in fig. 2,
and from above in fig. 3. It is somewhat circular in outline,
flat, or even slightly concave dorsally or outwardly, convex
ventrally,i.e. medially. On the latter face is a “ semicircular
ridge ” (fig. 2, 7 and 3) near the free margin, from which it is
separated by a groove. From nearly the middle of its course
452 W. BLAXLAND BENHAM.
this ridge sends down a branch (2) crossing the convex surface
and ending abruptly, like the main ridge, close to the body-
wall. This ridge throughout its extent is traversed by a very
narrow furrow.
The histological structure of this peculiar organ, so far as it
can be determined on my specimens, is fairly simple; it is a
solid mass of muscle, covered by an epithelium of a single layer
of cells (fig. 17), as will be more fully described below.
Presumably this pair of organs serves to hold two animals
together during copulation, the organs of each clasping the sides
of the other, somewhat in the same manner, no doubt, as the
peculiar “ penial appendages,” or, as I would call them, claspers
of Siphonogaster (Alma); the chief differences between the
two organs being the presence of chet and the absence of
any power of withdrawal of the organ into the body in the
case of the latter.
We know little of the mode of copulation even in our native
earthworms, but we can distinguish at least four kinds of
apparatus for holding the two worms together :—
1. The penis-like terminal duct of the spermiducal gland of
Pericheta, Acanthodrilus, and other worms, which appears
tobe capable of pleurecbolic eversion, and is presumably received
by the copulatory sac, a portion of the spermatheca; tosuch an
apparatus the term “ penis ” appears applicable.
2. “ Suckers,’ such as I have described in Microcheta
papillata; and under this head we must include probably
the terminal “atrium” of the sperm-duct of Criodrilus, and
perhaps of Geoscolex.
3. The “ claspers” of Kynotus and of Siphonogaster,
and perhaps of the Eudrilide.
4, The tubercula pubertatis of the Lumbricide, Sparga-
nophilus, Rhinodrilus, &c., which secrete a fluid and help
to “ stick ”’ the two worms together.!
In the case of the first three, specialised chet, copulatory
1 The external muscular organ of Stuhlmannia variabilis is very ex-
ceptional, and it is not quite clear in which group we should place it;
possibly in the first,
ON KYNOTUS CINGULATUS. 453
or penial cheetze may be associated directly or indirectly with
the various organs, and aid materially in holding the two
worms together during copulation.
To return to Kynotus, the second of my three specimens
indicates the manner in which this clasper is protruded, for
here the organ is protruded to just half of its extent (fig. 4, 5).
The first portion to make its appearance on protrusion is
the hinder border (fig. 4, 7), and in this case the dorsal or
outer surface is strongly curved so as to be much more con-
cave than when fully protruded (see fig. 5).
I was unable to observe the oviducal or the spermathecal
pores on external examination.
The nephridiopores become visible only on the twenty-
sixth and following rings; they exist anteriorly to this, but
owing to the great amount of contraction of the body-wall
they are invisible. They lie nearly midway between the couples
of cheetze on each side, and are especially distinct on the cli-
tellum (fig. 1, mp.).
There are no dorsal pores.
The Internal Anatomy.—The position of the eight thick
septa (a—h) which exist in the present species is a most im-
portant point to determine, and is not quite so easy as it would
appear (fig. 6). The most anterior septum is quite thin, and
lies behind the pharynx ; it is inserted between the fifth and
sixth rings.
The first thick septum, a, is inserted between rings 6/7.
The thick septum 2 3 a sf ESI:
> » 4 a so LOPS
” ” d 22 »” ” 12/13.
, tere the hinder
3 % margin of 14th ring.
“ Ce i . pet acaeolG ble 5,
55 ed, vs in the 18th ,,
= » A, somewhat thinner than the preceding
seven, near the hinder margin of the 20th ring.
The next, i. e. tenth septum, is inserted near the hinder
margin of the 22nd ring.
The eleventh, between 23/24 rings,
454 W. BLAXLAND BENHAM.
The following septa are inserted behind every successive ring
in the normal manner. My diagram shows the position of
these septa in relation to rings and to segments.
In Rosa’s species the strong septa are seven in number, and
are fixed at the segments v/vi to x1/x11, corresponding to
those in the present species labelled 6 to h.
In K. madagascariensis, too, the septa 6—h (Michael-
sen’s septa ii to viii) agree with those in mine, but the most
anterior one, which should correspond with a, is placed between
rings 7/8,
In K. longus (if we make the allowance suggested by
Michaelsen himself, that he counted a portion of the everted
buccal region as the first ring, and therefore subtract one from
all his numbers) there is a perfect agreement with mine.
We may, then, use these strong septa as characteristic of the
genus. Both in number and position, seven of them agree
in four of the known species.t
The alimentary canal is provided with a gizzard lying in
segment iv, thence the cesophagus passes back as a narrow
tube without diverticula, merging into the sacculated intes-
tine behind the genitalia in about the twenty-fifth segment ;
there is no typhlosole. That part of the gut which passes
through the segments xi—xvi is particularly narrow,—not
much wider, indeed, than the dorsal blood-vessel.
With regard to the vascular system, I only noted the
following points :—The dorsal vessel is very distinctly moni-
liform in this anterior region; there is a supra-cesophageal
vessel passing through segments v to x, which appears, how-
ever, to unite with the dorsal vessel at each septum, and from
these points of union the hearts are given off. Of these there
are six pairs lying in the segments just mentioned, the first
one being smaller than the rest.
The nephridia commence far forwards; there are three
1K. kelleri appears to be exceptional, for Michaelsen states that the
eight septa begin at segments vi/vi, and end at x1u/xtv, and places the
first prostate in segment xiv. It is just possible that some mistake, so easily
made, has oceurred in reckoning the “rings” in the anterior part of the body,
ON KYNOTUS CINGULATUS. 455
pairs lying in front of the first thick septum (a). Of these,
the first forms a large “ peptonephridium” lying below the
pharynx; its duct was traced to the body-wall, which it
penetrates in front of the second ring, i.e. segment 11 (see
fig. 6, np.). The second nephridium is also of considerable size ;
its duct opens between rings 3/4, i.e. at the anterior margin of
segment m1. The third nephridium lies behind the thin
septum in segment rv, and its duct was traced to its opening
between rings 5/6.
From these facts I conclude, as I have stated above, that
the second segment is two-ringed; the third is two-ringed ;
whilst the fourth consists of one ring only (the sixth).
The following nephridia are all of fairly large size, and
quite of the Geoscolicid pattern (fig. 23); the more posterior
ones behind the segment xv are provided with a “ cecum”
(fig. 24), those more anterior are not. The tubule of the latter
is spirally coiled, forming corkscrew-like bunches of peculiar
character.
The generative system is but insufficiently known in the
genus. Rosa, who has contributed most to our information
on the subject, found the testes in segments x and x1, in which
were masses of spermatozoa free in the coelom, and he traced
the male duct to the organ in segment xv; I can confirm him
in both these statements.
The copulatory organs, whose external features have
been described above, lie in segments XII, XIII, xIv, and xv
(fig. 7). In each of the first three segments are paired
spermiducal glands or prostates (pr.) in connection with sacs
containing penial chete (p.ch.) The gland is a convoluted
white tube of considerable size, and in well-developed worms
extends backwards into the neighbouring segments. Each
gland is enveloped in a thin sheath of muscle (see fig. 15,
sh.); its muscular duct receives, just before joining the body-
wall, the neck of the chetigerous sac or ‘chztophore.”
This is of considerable size, and has a thick muscular wall;
it contains three (or four) chete, one of which is much
longer than the others which appear to be reserve cheetee, and
456 W. BLAXLAND BENHAM.
have same shape as the first. The fully developed penial
cheta (fig. 8) is 49 mm. in length, measuring as accurately
as possible along the curve; the inner end is sharply bent
and somewhat enlarged: the free end (fig. 9) is quite similar
to that described by Rosa and Michaelsen. Its tip projects
freely into the duct of the gland, the rest of it lying in a sac
of its own with thick bundles of longitudinal muscles forming
its walls (figs. 12—14). Each of the reserve chet lies
in its own sac (figs. 12, 18, ch®., ch®.), but their pointed ends
are enveloped in this sac, and do not project into the lumen
of the duct. This seems to indicate that the penial chetz are
lost from time to time.
These features can be well seen in following serial sections.
A section somewhere about the middle of the chztophore
shows three chztz, each surrounded by a coat of circular
muscles, with a certain amount of longitudinal muscle. Passing
towards the body-wall, first one and then the second reserve
cheeta disappears as the section passes beyond their points,
while the functional cheta still persists. Now the wall of
its follicle presents a more distinct epithelium, and approaches
the duct of the prostate ; it becomes wrapped in the same coat
of circular muscles as the duct (fig. 12); and further onwards
(figs. 18, 14) the lumen of the chetophore communicates with
the duct of the prostate.
The penial cheetzee when mounted in glycerine appear to be
hollow, and the transverse lines figured by Michaelsen seem to
me to be confined to the inner surface of the apparent wall
(fig. 9, 7.), and are not of the nature of “ornament.” The
pointed end of the cheta is, however, ornamented in the
same kind of way as the ordinary cheta. In transverse sec-
tions it is seen that the axis of each penial cheta is of a
different character from the cortical zone: the latter is yellow,
and has the usual appearance of a cheta (fig. 14, cor.) ; it is
evidently brittle, for it exhibits cracks across it, and is fre-
quently torn away in sectionising. But the “ medulla” (med.)
stains pink in borax carmine, is homogeneous, and evidently
softer than the cortex.
ON KYNOTUS CINGULATUS. AD57
The penial cheta of Kynotus, in fact, resembles the
cheetze of several Polycheta in this respect, and I am not
aware that this feature has been noted previously in Oligo-
cheeta.
With regard to the prostate, or spermiducal gland, it has
the structure so frequently described for many earthworms ; its
lumen is surrounded by a layer of narrow columnar cells: this
is surrounded by a very thin coat of circular muscle, which
is traversed by the narrow necks of the great club-shaped
gland-cells (fig. 16). In a transverse section of the entire
gland (fig. 15), the limits of the various loops of the coil
are not by any means well defined; one sees two or three
ducts apparently embedded in a mass of glandular tissue, and
the whole surrounded by a sheath of muscle, in which run
blood-vessels. Closer examination generally enables one to
trace the outline of the groups of gland cells belonging to
each section of the duct.
The clasper (figs. 17—20).—I have already described the
appearance of this peculiar organ when protruded.
In the worm which was dissected, in which the “ clasper”
was entirely retracted, there was seen lying in segment xv
(the 25th ring) an irregularly oval body, convex upwards,
with a very irregular surface; its long axis is parallel with
that of the worm’s body (S., fig. 7). This is the structure
which Michaelsen terms “bursa propulsoria.””? What appear-
ance it would present from within when completely everted I
am unable to say: whether the whole structure is capable of
being protruded or not is at present unknown.
This “bursa propulsoria,” or retracted ‘“ clasper,” is larger
than the segment to which it belongs, and pushes apart the
septa in front and behind. Its external opening is of course
invisible from within, as it lies below the organ.
From its outer side there arises a muscle (fig. 7, m.) which
passes forwards, and becomes continuous with the longitudinal
muscles of segment xtv. This is called by Rosa the “ re-
tractor muscle.”’ At the hinder end of this organ there arises
from its ventral surface, a gland (fig. 7, pr*.), exactly like those
458 W. BLAXLAND BENHAM,
of the preceding segments except that it is much larger, and
extends through some five or six segments. Rosa terms this
the “ pseudo-prostate.”’
The minute structure of the clasper has been described by
Rosa, and my observations agree very closely with his, though
I cannot distinguish so definitely as he does the divison of the
enclosed chamber into two by an imperfect horizontal septum.
Nevertheless two regions of the cavity are readily dis-
tinguished in transverse sections (figs. 17, 18); one portion
of the lumen (C) is lined by close-set columnar cells, the
other (A) by gland cells intermixed with ordinary cells, some
of which are empty, and others filled with secretion (fig.
19, gl.).
The rest of the organ is muscular, with more or less abundant
blood-vessels distributed through it. When the cavities are
traced out it is found that the portion C is continuous with
that surface of the protruded organ which is directed inwards
(ventral), whilst the epithelium lining A covers the outer
surface of the organ. The epithelium is everywhere one cell
deep; there is no basement membrane, and the blood-vessels
(fig. 19) pass up between the cells. In the section through
a part of the protruded chamber the part labelled B (fig. 17)
is, of course, the prominent organ seen in figs. 4 and 5, which
was the specimen sectionised, and corresponds to Rosa’s
*‘gyande scudo ovale,” which projects downwards from the
roof of the upper chamber.
The duct of the gland enters that region of the lumen
marked C; its secretion, therefore, is discharged on the inner,
ventrally directed face of the organ which is presumably
used to grasp the other worm during copulation. Deeper
in the organ the two cavities become continuous as in
fig. 18.
The two sperm-ducts of one side, after passing backwards
along the body-wall on the medial side of the prostates, reach
the “bursa,” and pass along its medial border. They then
bend round it posteriorly, and enter the muscle surrounding
the neck of the gland (pr*.); here they turn forwards and
ON KYNOTUS CINGULATUS. 459
enter the duct, before the latter communicates with the
‘* bursa-propulsoria.”
Of the female organs I have only observed the sperma-
thece (fig. 7, spth.). These lie, as in other species, in Seg-
ments XIII, XIV, xv (rings 23, 24, 25), opening along the
anterior margin, though I was unable to detect the pores on
external examination. The spermathece are somewhat pear-
shaped sacs, variable in size and in number. In one specimen
dissected there are, on the right side, three in a row in each of
the above-mentioned segments; on the left side the numbers
were one, two, and two in these segments.
A second specimen gave the following numbers :—on the
right side none in the segment x111, three in xiv, and two in xv;
on the left side two sacs in each of the three segments. The
number is evidently variable in this species. We have no
information, of course, as to the extent of variability in the
other species.
There are one or two peculiarities in the structure of
the body-wall worthy of mention. Below the epidermis is
a layer of connective tissue, especially thick on the ventral
surface, where it has the appearance of a homogeneous matrix,
with spindle-shaped nuclei embedded in it (fig. 22, b¢.), which
recalls the ‘‘ basement tissue” of Nemertines. Deeper down
the longitudinal muscles are separated into blocks by incursive
fibrous connective tissue, which forms a fairly thick layer in-
ternal to the longitudinal muscles (fig. 21, ct.) A similar but
much thicker connective tissue exists also in Brachydrilus.
Each “ block” of muscle is made up of several bundles,
each of which is probably derived from a single cell, as
Vejdovsky has shown to be the case in Lumbricus. Further,
the connective tissue between the blocks appears to pass
through the circular layer of muscles and to terminate in
the basement tissue (fig. 21).
The blood-vessels in this region are very well developed, and
enter the epidermis, between the cells of which they ramify
(fig. 22), as in Moniligaster, Pericheta, Criodrilus,
and other earthworms.
460 W. BLAXLAND BENHAM.
Affinities.—The present worm, which I have regarded as
a new species, presents certain resemblances to K. longus and
to K. kelleri; but from the former it differs in the number
and arrangement of the spermathece, which are there in two
groups of eight on each side, belonging to the rings 25 and
26 (probably to segments x1v and xv). It has, further, an
elongated prostomium, the bursa propulsoria is “ disc-like,”
and flattened in the longitudinal direction, and the prostates
are pear-shaped (“ birnformige ”).
From K. kelleri the prostate of the bursa (Rosa’s “ pseudo-
prostate ”’) is ‘‘ zipfelformige ; ” those of the penial cheetophores
are “birnformige.’? As mentioned above, the ordinary
cheetz of these two species are without a “ node.”
From K. madagascariensis the present worm differs
in that this species has numerous small spermathece in
three rows on each side, as well as in other matters of detail,
such as the form of the prostates, &c.; whilst K. michaelsenii
is evidently a different species, for it only has two pairs of
penial chetophores and prostates.
Postscript.—Since this paper was written, two new species
of Kynotus have been described by Michaelsen (‘‘ Zur Kennt-
niss der Oligochaeten,’’ in ‘Abhandl. aus dem Gebiete d. Natur-
wisseuschaften,’ Bd. xiii), in which he makes some further
observations on the peculiar annulation of the body wall.
The species are K. oswaldi and K. distichotheca, both
of which differ in various points from the above-described
form.
K. oswaldi presents a clitellum of considerable length,
occupying eighteen segments, viz. x1x to xxxvil. In both, the
male pore is on the 26th ring; but from observations on the
relation of nephridia and septa he believes this to be the six-
teenth segment, whilst Rosa finds it on the fifteenth (ring 23)
in K. michaelsenii, which he has re-examined. His enume-
ration of the segments is different from that given by me for
K. cingulatus. The first strong septum lies between the rings
7 and 8, and in front of it are three pairs of nephridia, the first
pore being in front of the 3rd ring; he therefore considers
ON KYNOTUS CINGULATUS. 461
each of the first three rings to be a segment, so that there are
five segments (instead of four, as in my species) in front of the
first strong septum. At first sight one might be inclined to
suggest that Michaelsen had overlooked the first nephridio-
pore, but as he stripped the cuticle from the worm there can
be no mistake in the matter; the first nephridium of K. cin-
gulatus is therefore absent in these two species. It is still
possible thus to regard two of these rings as forming one seg-
ment, as I have described for the present species. He finds the
nephridiopores to lie in front of the rings 3, 4, 6, 8, 10, 12, &c.;
whereas I find them in front of 2, 4, 6, and so on. It is just
possible that I have made an error in observation, for I did not
strip the cuticle for my specimen, which was too hardened for
this manipulation, but I traced the duct to its pore.
Nevertheless he shows that K. michaelsenii differs in the
amount of annulation from K. longus and others in that
the segment 111 is biannulate, whilst in other forms the
biannulation commences on the next segment, and that it
ceases at segment x; whereas in K. longus it ceases at seg-
ment x111, and in K. oswaldi, as in my species, at segment xII.
This annulation, like the number of thick septa, appears to
be a specific character, as well as the segment on which the
cheetee commence.
EXPLANATION OF PLATES 33 and 34,
Illustrating Dr. Blaxland Benham’s paper on “Kynotus
cingulatus, a new species of Earthworm from Imerina,
in Madagascar.”
(Throughout the figures ‘‘annuli” are indicated by Arabic, and
‘**segments” by Roman numerals).
Fic. 1.—Ventral surface of Kynotus cingulatus (x 2). The annuli are
marked with Arabic numbers mostly on the right side. The Roman numerals
VoL. 38, PART 4,—NEW SER. II
462 W. BLAXLAND BENHAM.
on the left side indicate the true segments. The worm is represented as if
the hinder part were slightly twisted round its long axis, so as to bring the
side into view. B. The protruded clasper; the pores of the four pairs of
male organs are shown (see also Fig. 2). m. Mouth. zp. Nephridiopore
i. ch. Inner couple of cheete. 0. ch. Outer couple of chet.
Fic. 2.—A portion of Fig. 1 enlarged (x 4), showing annuli and tertiary
ringing of the segments. Annuli and true segments are indicated as before.
B. The clasper. Z, 2,3. The curved ridge on its ventral face. 6. The
pore on the left of the figure through which this organ is protruded. p', p?, p’.
The three pores of the prostates or spermiducal glands and penial cheetophores.
i. ch. Tuner couple of chete.
Fie. 3.—The clasper of the same specimen seen from the outer surface,
rather dorsally; the annuli are marked. 0. ch. Outer chete.
Fic. 4.—View of the clasper of another specimen in a partially protruded
condition ; the animal is seen partly from the side. 7, 2, indicate those parts
of the ridge similarly numbered in Fig. 2. &. The pore through which the
clasper is protruding. zp. Nephridiopore. %.ch., 0.ch., as before. The
annuli are here numbered.
Fic. 5.—The same partially protruded clasper seen antero-laterally ; a view
nearly corresponding to Fig. 3.
Fic. 6.—Diagrammatic representation of the relations of annuli, segments,
and septa. The annuli are marked by Arabic numerals on the left; the seg-
ments by Roman numerals on the right; the septa by thick lines; and the
interannular furrows by fainter lines. The eight strong septa are indicated
by small letters, a—A. The three anterior nephridiopores are represented
as dots (zp.) on the right side. The characteristic copulatory organs are
represented in their true position on both sides.
Fic. 7.—The characteristic copulatory organs of the right side considerably
enlarged. The annuli are indicated on the right, the segments on the left
side. Pr, Pr?, Pr’, The three spermiducal glands, each a convoluted tube
enveloped in a muscular sheath. p’. ch’. Penial chetophore connected with
the first prostate. S. The “bursa propulsoria,” or sac containing the clasper.
pr’. Its gland. m. Retractor muscle. sp¢h. Spermathecs. er. Position of
the nerve-cord.
Fic. 8.—The fully developed penial cheta (xX 35). Camera drawing.
Fic. 9.—The free end of the penial cheta (x 180). Camera drawing.
c. The denser cortical portion. m. The medulla. 7. The ring-like markings
on the inner surface of the cortex. N.B.—The outline has suffered in reduc-
tion of the drawing.
Fic. 10.—An ordinary cheta (x 35).
Fic. 11.—The tip of an ordinary cheta greatly magnified. The markings
are represented too strongly.
ON KYNOTUS CINGULATUS. 463
Fic, 12.—Transverse section of the penial cheetophore near its junction with
the duct of the spermiducal gland. d.pr. Duct of gland. ch', Sac of the
functional penial cheta. cir. Coat of circular muscles enveloping these.
lg. Longitudinal muscles. sh. Sheath. ch?, ch3. Reserve chete.
Fic. 13.—Transverse section of the penial chetophore lower down, where
the lumen has opened into the duct of the gland. Letters as in Fig. 12.
Fie. 14.—The common duct enlarged. Zp. Epithelium. Cuz. Cuticle. Cir.
Circular muscles. ch!, The penial cheta. Cor. Its cortical, and med. its
medullary portion.
Fic. 15.—Transverse section of a prostate. The lumen (/./.) is cut
through four times, and appears embedded in a mass of gland-cells (pr.).
ep. Epithelium. sh. Muscular sheath surrounding the whole gland. d.v. Blood-
vessels.
Fic. 16.—A portion of the preceding, enlarged. ep. Epithelium. g/.
Gland-cells. m. Muscular coat round the epithelium.
Fie. 17.—Transverse section of the bursa propulsoria and partially protruded
clasper (Figs. 4 and 5). The section does not go through the most projecting
region, but rather obliquely through the side (x 35). #B. The protruding
portion of the clasper. 4. C. The two regions of the bursa; the former lined
by gland-cells, the latter by columnar epithelium. ep. Epidermis of body-
wall. cir, Circular coat of muscles. 7g. Longitudinal muscles, m.
Muscular substance of the clasper. 7. Retractor muscle.
Fie, 18.—Another section through the same further backwards, so as not
to involve the pore(X 35). The regions 4. C. are here continuous. Other
letters as before.
Fic. 19.—A portion of the epithelium lining the region 4. of the bursa,
gl. Gland-cells. 4.v. Blood-vessels. m. Muscle-fibres.
Fic. 20,—A portion of the epidermis of region C.
Fic. 21.—Portion of the body-wall in the ventral region. Zp. Epidermis.
b.t. Thick basement tissue. cir. Circular muscles. ct. Connective tissue
separating the longitudinal muscles (/g.) into bundles, 4.v. Blood-vessel.
Fic. 22.—Portion of the above sections more highly magnified, to show the
peculiar basement tissue (4.¢.) with nuclei (#.) embedded in it, and the
vascularity of the epidermis.
Fie. 23.—One of the anterior nephridia, showing its peculiar spirally-coiled
tubule (¢.) and long duct (d.).
Fic. 24.—One of the posterior nephridia with a cecum (c) to the duct,
and a different coil to the tubule.
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CILIATION OF ECTODERM OF AMPHIBIAN EMBRYO. 465
Notes on the Ciliation of the Ectoderm of the
Amphibian Embryo.
By
Richard Assheton, M.A.
With Plate 35.
Tue fact that the embryo and larva of Amphibia possess a
ciliated ectoderm has been frequently noticed, but since, as far
as I know, a description of the distribution of the cilia, and of
the currents of water over the surface of the body, which
result from the action of the cilia, has not been hitherto
published, my notes upon the ciliation of the tadpoles of
Rana temporaria and Triton cristatus may perhaps be
of interest.
Quite recently the fact of the existence of a ciliated embryo
among craniate Vertebrates seems to have been doubted or
overlooked. Osborne (10), in his preface to Willey’s ‘‘ Am-
phioxus and the Ancestry of the Vertebrates,” with respect to
the ‘‘ real invertebrate and vertebrate affinities” of Amphioxus,
writes thus :—“ For example, among the real invertebrate ties
of the Protochordates are the ciliated embryos of Balano-
glossus and Amphioxus, the Tornaria larva and ciliated ecto-
derm of Balanoglossus.”
Willey (17), in the text of the book, page 113, says, ‘The
fact that Amphioxus has a free-swimming, ciliated embryo is
important as providing a general connecting link between the
Vertebrates and the Invertebrates, since the possession of a
ciliated ectoderm is very common among invertebrate embryos,
but entirely unknown among the craniate Vertebrates.”
466 RICHARD ASSHETON.
Again, a little further on, the same author expresses his
views still more strongly. On page 175 he writes: “The
ciliation of the ectoderm in the larva of Amphioxus continuing,
as it does, long after the muscles have been fully differentiated,
and when the cilia are therefore no longer required for purposes
of locomotion, should be especially noted as evidence of a very
archaic organisation. We shall find in the last chapter that
the possession of a ciliated ectoderm is a prime characteristic
of Balanoglossus and many of the lower worms (e.g. Nemer-
tines). In none of the craniate Vertebrates is the ectoderm
at any time ciliated.”
Eycleshymer (4), discussing the question of the cause of the
continuous rotatory movement of the vertebrate ovum, writes,
on page 355: “ Clarke states that in Amblystoma puncta-
tum the surface of the body is covered with cilia at the time
the neural folds close, by means of which it keeps up its rota-
tory motion. I have endeavoured to detect cilia by teasing in
normal saline solution also by osmic acid fixation, but without
success.”
Clarke’s (3) actual words are—
“The entire surface of the body is now” (i.e. when both
the anal or caudal and cephalic ends are becoming more
definitely indicated as they grow away and stretch out from
the body of the embryo) ‘‘ covered with cilia, by aid of which
it keeps up a horizontal rotatory motion upon its axis.”
Balfour (1), on page 141, says of the newt that “ the skin is
ciliated, and the cilia cause a rotation in the egg.”
Again, of the frog the same author says, page 127: “ The
outer layer of epiblast-cells becomes ciliated after the close of
the segmentation, but the cilia gradually disappear on the
formation of the internal gills. The cilia cause a slow rotatory
movement of the embryo within the egg, and probably assist
in the respiration after it is hatched. They are especially
developed on the external gills.”’
Marshall and Bles (7) also notice the fact, page 42: “The
whole surface of the tadpole is, as in the earlier stages, ciliated;
CILIATION OF ECTODERM OF AMPHIBIAN EMBRYO. 467
the cilia working from head to tail, causing the animal when
perfectly quiet to move forwards slowly in the water.”
Preyer (12) and Carriere (2) also describe the presence of
cilia upon the external gills of Salamander and Siredon re-
' spectively.
Stohr (15), in a very brief account of the “sogen Haft-
organe” of the Anura, describes the action of the cilia in
connection with these organs, to which I must refer again.
The ciliated ectoderm of Rana temporaria.
The first signs of the presence of cilia in the frog embryo
occur shortly before the closure of the neural folds. At the
time when the folds are first visible, and even when they are
commencing to fold, there is still no trace of ciliation.
Fig. 1, Pl. 85, represents the earliest stage at which I have
detected any ciliary movement. I have not at this stage seen
the cilia, but have observed a streaming of carmine granules
along certain regions.
It will be seen that the edges of the neural plate are raised
up as prominent ridges, but as yet they have not met at any
point. The anterior or cranial portion is marked by its great
lateral expansions, which do not become infolded, and which
give rise to the ganglia of certain of the cranial nerves. It is
upon these lateral expansions and the actual edges of the
neural plate that the cilia are first developed. The arrows in
fig. 1 indicate the direction of flow of the current set up by
the action of the cilia upon the surrounding fluids.
Fig. 2 shows the extent and course of the current when seen
from the side a few hours later. The neural folds have not as
yet met.
As the neural folds bend upwards the current becomes more
marked and extends somewhat further back, and also rather
further ventral-wards.
Just before the neural folds close—when they have closed
anteriorly, but are sufficiently open along the back to allow a
view of the neural groove—a distinct motion is visible upon the
anterior ventral surface of the embryo,—that is, over the area
468 RICHARD ASSHETON.
in the centre of which the mouth occurs subsequently. The
direction of the flow here is, as in the case of the dorsal current,
from before backwards (vide fig. 3).
I have tried most carefully to find any indication of a ciliated
ectoderm upon that portion of the neural plate which is folded
up and becomes the neural tube. I can find no indication of a
ciliation.
The cavities of the brain and spinal cord of the adult are said
to be lined by a ciliated epithelium. Although doubted by
some authors, this has been satisfactorily demonstrated by
others (vide Wightman, ‘ Studies of the Johns Hopkins Uni-
versity,’ vol. iv). At what stage the cells that bound these
cavities became ciliated I cannot say, but of every specimen
which I have examined I can say that at the time of closure of
the neural folds there is no motion of suspended particles at
any spot along the neural groove, although a rapid current is
produced along the external portion of the edges of the neural
plate.
Klein (6) has described a ciliation of the neural groove
while still open in the chick, in embryos with about seventeen
protovertebre.
By the time that the neural folds have closed, except at
quite the posterior end, the whole of the dorsal surface is
ciliated, including the groove formed by the junction of the
neural folds. By far the most rapid movement of particles
takes place along the line indicated by the large arrows in
fig. 4 (N.B.C.), which is along the extreme outer limit of the
neural plate, or rather over that portion of the epiblast which
subsequently gives rise to the sensory ganglia of the 5, 7, 8,
9, 10 cranial and the spinal nerves.
The ciliation now spreads very rapidly, and by the time the
embryo with seven or eight mesoblastic somites measures about
3 mm. in length, which is about the time of the perforation of
the anus, the whole surface of the embryo is ciliated, but the
currents vary very much in intensity.
Fig. 5 is a diagram of the currents produced by the cilia as
seen from the side.
CILIATION OF ECTODERM OF AMPHIBIAN EMBRYO. 469
The action of the cilia is strongest at the anterior end, and
causes the water to be driven backwards as from a centre, the
centre being the most anterior end of the embryo; in fact, just
as would occur if the embryo was swimming rapidly forward.
The cilia on the anterior surface can at this stage be seen
distinctly with a Zeiss D objective. There are, however,
certain tracks along which special currents of water flow,
which we may take to mean that along these paths the cilia
are especially large, more numerous, or more active.
A very well-defined and strong current (fig. 5, N.B.C.) passes
over the bases of the developing branchial arches, which would
seem to correspond to that marked by the large arrows in fig. 4.
Along the ventral surface the motion is extremely slack—it is
rather a series of eddies. The currents at the hinder end are
interesting. The action of the cilia of the whole of the hinder
region is to tend to cause a current of water to flow towards
the blastopore and anus. The most rapid current is that along
the back.
At one time—when we may presume both blastopore and
anus are open—there is a strong exhalent current from the
positions of both of the openings. The water brought by the
action of the cilia may be seen to curl over the edges of the
blastopore and anus, and apparently is immediately shot out
again with considerable violence (fig. 6).
Whether this indicates any interchange of fluid between that
within the archenteron and the medium in which the embryo
lies I cannot say. The anal current becomes stronger while
the blastoporic becomes rapidly weaker, and soon ceases with
the final closure of the blastopore.
Fig. 7 illustrates the direction of the currents of water over
the posterior end a few hours later, at which time the tail has
begun to grow out.
Special Currents along the Ventral Surface.
Until about now the ventral and antero-ventral currents
have been quite simple; there is a general flow along the
470 RICHARD ASSHETON.
ventral surface from before backward, although it is very
much more rapid around the region of the future mouth.
About the time the tadpole measures 4 mm. two curious
areas, known variously as “ suckers,” “ Haftorgane,” “ crochet
de Rusconi,” &c., below the region of mouth (which is now
represented by a slight depression), have become prominent
features, and in connection with them a considerable modifica-
tion in the ciliation of that region occurs.
The term “ sucker” is hardly suitable, for it seems doubtful
whether attachment is ever in Auura effected by true suction.
With regard to the functions of these organs, which perhaps
might be more rightly called cement glands, Marshall and
Bles, in their paper upon the development of the blood-vessels
of the frog, made a suggestion which seems to me to be entirely
unsupported by evidence.
They wrote thus on page 215 :—“ Though the mouth is not
yet open, the tadpole shows a distinct increase in bulk as
compared with the 8 mm. stage. It has occurred to us as
possible that the suckers may be used for absorbing food, and
that in this way the increase may be explained; ... and
sections of the sucker show that the greatly elongated and
columnar cells of the sensory layer of the epiblast covering
them are often produced at their free ends into protoplasmic
processes, that would seem well fitted for absorbing the jelly.”
The “increase in bulk” noticed by them between the
64 mm. stage and 5 mm. stage may be as well accounted for by
the large increase of the meshes of the mesoblast which occurs
at this period. To effect this conversion of a compact mesoderm
into a network of finely drawn-out stellate cells, no doubt it is
necessary that water should be absorbed. It seems, however,
hardly likely that absorption by the skin should take place at
the spots where the epiblast appears four to eight times
thicker than anywhere else.
The true function of these organs has been correctly stated
by Stohr (15) in these words:
“Sie bestehen aus langgestreckten einzelligen Driisen (bei
Bufo cinereus) die sich durch starke Pigmentirung auszeichnen
CILIATION OF ECTODERM OF AMPHIBIAN EMBRYO. 471
und ihr Klebriges Sekret in einem Hohlraum ergiessen, aus
welchem dasselbe durch Flimmerharre nach Aussen befordert
wird.”
Fig. 8 is a semi-diagrammatic section across these organs of
a tadpole of Rana temporaria 8 mm. long, at which time
they reach their greatest development.
They are essentially of the nature of mucous glands, bor-
dered by strongly ciliated ridges.
They secrete a very sticky substance, by means of which the
tadpole can anchor itself to any convenient object. The “pro-
toplasmic processes” alluded to by Marshall and Bles are
probably masses of exuded mucous or cement, and are thus the
product of the cells rather than processes of the cells them-
selves.
Both the ciliated cells and the mucous cells are developed
from the outer or epidermic layer of epiblast. The nervous
layer of epiblast forms a single layer of cells as elsewhere.
Fig. 9 is a semi-diagrammatic figure of a section taken
rather further back than that represented in fig. 8. In this
the ridges are more prominent. The long cement-secreting
cells, C.C., really le with their bases much further forward
than their necks and openings.
Fig. 10 shows the ventral surface of a tadpole of 34 mm.
The cement-secreting cells are in the depressions M.
The sides of the ridges surrounding the glands are com-
pletely covered with long cilia.
The cilia here are the most conspicuous of all upon the body.
These cause very violent currents of water, as indicated in
the figure. The central current passes over the stomodzal
depression (S) along the groove leading from the mouth be-
tween the two glands.
In an older stage, 64 mm., fig. 11, the glands have so in-
creased in size as now to be practically joined across the mid-
ventral line. This necessitates an alteration of the course of
the central current, which is now thrown outwards, and is then
caught by the strong lateral currents and passes round the
4.72 RICHARD ASSHETON.
ridges. A small portion passes straight on, making a stream
which becomes very prominent a little later.
When the tadpole measures about 8 mm. the cement-glands
are at their greatest development. Fig. 13 is a ventral view
of this stage.
The two glands appear as though raised upon a horseshoe-
shaped platform. Each gland, which is elongated in an antero-
posterior direction, is bordered on both sides by a high ridge
richly ciliated (fig. 9). The shape of these ridges and direc-
tion of action of the cilia upon them is such as to produce the
following currents in the adjoining water, as indicated by the
arrows in fig. 13.
(i) Externally, strong currents, C.C., which skirt round the
ridge towards the mid-ventral line.
(ii) Centrally, a strong stream, S.C. (the deeper portion of
which passes first into the stomodzal cavity, in which particles
may be seen to revolve in eddies for some minutes), runs up
the groove from the stomodeeal depression, and turns over the
hinder border of the inner ridges of the glands, and drives the
water in a strong stream partly straight outwards and away
from the larva, and partly along the ventral surface (vide fig. 14) ;
the effect being to sweep clean the hinder part of the glands.
(iii) The cilia on the crests of the ridges and on the sides of
the ridges facing the glandular cells cause water to be drawn
into the glands, which sweeps any secretion from the gland
backwards until it comes into the very strong stream described
last. These currents are indicated by the small arrows in
fig. 13.
When the tadpole first leaves the egg membrane, and
wriggles or is driven outwards from the jelly mass by the
action of its cilia, it may perhaps be said to adhere to the jelly
by suction, for the jelly is sufficiently fluid to be drawn into
the hollow of the glands by the currents of water over the
ridges, and too viscid to pass easily through and out again.
But suction plays no part at all in the mode of adhesion to any
object of a more rigid nature, such as a plant leaf, piece of
root or stick. The tadpole is seen to be anchored by the sticky
CILIATION OF EOTODERM OF AMPHIBIAN EMBRYO, 473
mucous secretion, which, produced by the glands, is driven out
by the system of water-currents described above.
Special Currents upon the Dorsal and Lateral
Surfaces.
Although I cannot speak with certainty with regard to the
earliest stages, yet as far as I have been able to determine I
have never found a stage in which every cell of the epidermic
epiblast bears cilia.
In 6 mm. tadpoles ciliated cells are scattered thickly over
the whole surface, but amongst them are other cells which bear
no cilia. On certain regions the ciliated cells are more
numerous, and the cilia on certain spots are longer.
I described above a very well-defined current, marked N.B.C.
in fig. 5. There is still in older tadpoles a current which is
swifter than any other (excepting those connected with the
glands described above), and very well indicated, which seems
to be identical with that marked N.B.C. in fig. 5. I have simi-
larly named it N.B.C. in fig. 12, of which I am now writing.
This current dips into the nasal depression which is pro-
vided with long cilia, and flows rapidly over the developing
external gill-filaments.
This conspicuous current exists until the posterior nares are
formed and the gill-filaments are covered over by the oper-
culum, whereupon the current is no longer distinguishable as
a special stream, but is merged in the general much-reduced
flow of water which sweeps slowly from before backwards over
the whole body. This is an interesting current, for it will be
noticed that the greater part of the water which washes the ex-
ternal gill-filaments until the tadpole is about 9 mm. long has
previously passed over the developing olfactory epithelium.
Whether this epithelium at this stage plays any part in testing
the water before it reaches the gills must be left to conjecture.
Fig. 15 is drawn from a horizontal section of a 6 mm. tad-
pole. It represents the ectoderm along the path of the current
just described, and is between the olfactory pit and the base of
the external gills. The ciliated cells are here very numerous,
474, RICHARD ASSHETON.
being only very slightly less abundant than those which do not
bear cilia.
Ciliated cells occur more sparsely over all the rest of the
sides, back, and ventral surfaces, and cause a steady flow of
water, which is rather more rapid over the tail.
The gill-filaments are provided with about one ciliated to
every two non-ciliated cells (fig. 16).
The ciliation after about the 7 to 8 mm. stage begins to
become less effective. A tadpole of 6 or 7 mm. will progress,
if placed upon its side in water along the bottom of a flat glass
vessel, at the rate of one millimetre in from four to seven
seconds.
The Ciliation of the Later Stages of Larval Life.
In tadpoles of 12 mm. in length, ciliated cells are still to be
found on all parts of the body. The general flow is from
before backwards. The motion is, however, much less rapid,
and there are no longer any special currents.
For instance, the streams connected with the cement-glands
are now hardly distinguishable from the general flow. Areas
wherein at an earlier stage every cell was ciliated, now contain
many cells without cilia.
The cement-glands have become much reduced. They are
mere circular bosses. The high lateral ridges have entirely
disappeared. Nor is the flow of secretion nearly as copious,
and the tadpole makes but little use of it. .
The action of the cilia now seems to be no more rapid along
the dorsal surface than ventrally.
The mouth and posterior nares are open, and the tadpole
draws the water by muscular as well as by ciliary action into
the pharynx by all three apertures. The exhalent flow by the
opercular spout is extremely rapid and quite regular, and
exhibits no signs of a muscular expulsion.
At 18 mm. there is still a flow of water over the whole sur-
face of the tadpole, but there are now regions which do not
bear cilia. For instance, the fringe of tentacles which have
grown round the lips of the mouth is quite destitute of cilia.
CILIATION OF ECTODERM OF AMPHIBIAN EMBRYO. 475
There are no. cilia upon the extreme dorsal and ventral edges
of the tail. There are none on the eyes.
The motion of water over the body becomes slower, and in
a 20-mm. tadpole it can hardly be termed a flow. The ciliated
cells are now so few and far apart, and so feeble, that a series
of eddies takes the place of a regular streaming. This, how-
ever, is not the case with the tail. Scattered all over the sides
of the tail are cells which in surface view appear elongated
(fig. 18, C.), and bear long and very active cilia, which work in
the direction of the shortest axis of the cell.
The general result of the action of these cells is the produc-
tion of a rapid flow of water from about the level of the noto-
chord in a diagonal direction, both dorsally and ventrally. A
granule of carmine may be seen to be dashed from one cell to
another like a shuttlecock, and made to take a zigzag course
across the tail fins, as indicated in fig. 17.
As the tail expands these cells become more and more sepa-
rated, and so become less numerous to a given area where the
tail expands most, and remain more numerous towards the
base (fig. 17).
Fig. 18 is a camera drawing of the surface of the tail of a
tadpole 19 mm., showing the ciliated cells, which are at this
stage more deeply pigmented than the majority of the other
surface cells. On the development of the hind limbs and
diminution of the tail the cilia disappear from the tail, as from
the rest of the epidermis.
The Newt (Triton cristatus).
I have not observed any cilia or currents of water presum-
ably due to cilia before the complete closure of the neural folds
in Triton cristatus. Ido not, however, wish to assert that
there is never any ciliation prior to that time, as my observa-
tions have been few in number. Clarke (8), as quoted above,
also seems not to have noticed the cilia until after the closure
of the neural folds. As the head and tail become distinct the
whole of the anterior end of the embryo is richly ciliated, and
4.76 RICHARD ASSHETON.
certain distinct currents are produced thereby. Posteriorly,
however, the ciliation is very scattered and feeble, and pro-
duces no distinct flowing.
Figs. 19 and 20 represent the lateral and ventral views of
such an embryo.
Over the dorsal and lateral surfaces there is a steady flow
along the longitudinal axis of the embryo. Ventrally there is
an especially rapid stream from the pre-oral region into the
stomodeal pit, whence the water passes over the ventral wall
of the embryo. The water from the lateral parts of the pre-
oral area also flows towards the stomodzal pit, and then passes
outwards and upwards towards the locality of the branchial
arches. This current is more markedly present in a 6-mm,
newt, as shown in fig. 22.
In fig. 21 a lateral view is given, showing how the action of
the cilia at this stage is to bring as much water as possible
into one stream, which may be said to start about the position
of the olfactory epithelium and skirt below the “ balancers ”
(M.), and pass very rapidly over the spots where the external
gill-filaments are about to develop. Water from the dorsal
region is also swept down into this same stream.
Over the posterior end of the embryo the ciliation is very
slight. After the complete development of the external gills
the ciliation over the greater part of the body becomes less
active, and by the time the newt tadpole measures 16 mm. it
has entirely disappeared excepting upon the gill-filaments
themselves.
The special ciliated region which is present in the frog be-
hind the stomodeum in connection with the cement-glands is
absent in the newt. Ciliated cells occur here as elsewhere,
but no special streams are produced. The balancers, which
are placed much further from the mid-ventral line than are the
cement-glands of the frog, are similar to the cement-glands in
that at their extreme points there are cells which secrete a
similar sticky cement by which the young newt can attach
itself to weeds. They differ in that the cement-glands, which
are very much smaller, are borne upon processes of the body-
CILIATION OF ECTODERM OF AMPHIBIAN EMBRYO. 477
wall which contain blood-vessels and connective tissue. In the
frog they are formed of the two layers of epiblast only.
Clarke describes these organs, which he terms balancers, as
being used to support the larva when it hatches and falls into
the mud. This may be so, but they are certainly also used
for the suspension of the larva from weeds. Their walls are
not ciliated.
Comparison of the Ciliation of the Frog and Newt.
In both animals the anterior and dorsal regions are more
richly ciliated than the ventral and posterior.
In both animals the stomodzal pit and area immediately
surrounding the stomodeum are especially rich in ciliated
cells, which cause a strong flow of water into the stomodeum,
where particles suspended in the water may be seen whirling
in eddies for a time and then passing out over the posterior lip.
In both animals a very distinct stream of water is produced,
which passes first over the olfactory epithelium, and subse-
quently washes rapidly over the developing external gills.
In the newt this stream appears to be the main flood, into
which tributary currents flow from both the dorsal and ventral
regious (vide figs. 21 and 22).
In the frog this stream is in the main parallel with other
currents, but is distinguishable by its very much greater
rapidity (vide figs. 12 and 14).
In the frog currents are set up by the ciliary action at an
earlier stage, and are maintained to a later stage than in the
newt. In the newt the tail soon loses its ciliation, whereas in
the frog it remains active almost up to the time of the meta-
morphosis.
If a ciliated ectoderm really is ‘ evidence of a very archaic
organisation,” a consideration of the exact distribution of the
cilia, and the determination of any special areas or bands of
cilia at the several stages of development of the embryo, may
be of great morphological interest.
Although I am inclined to think that at no time is every
VOL. 388, PART 4—NEW SER. KK
478 RICHARD ASSHETON.
cell of the ectoderm of the frog’s tadpole ciliated, yet for a
certain period ciliated cells are so numerous as to render it
legitimate to speak of the whole surface as being ciliated, e.g.
tadpoles 3 mm. to 10 mm. This, however, is not the case
from the beginning.
The first sign of ciliation is along the edges of the neural
plate. This is followed by a ciliated patch on the antero-
ventral surface, in the centre of which there arises later the
stomodzeal depression. Shortly afterwards the whole surface
of the embryo becomes ciliated, but the above-mentioned areas
remain recognisable by reason of the greater intensity of the
ciliation upon them.
If for the moment we omit the consideration of the func-
tion of these specially ciliated tracts, it is possible to draw a
comparison between the condition just described and the dis-
tribution of the cilia upon the free-swimming larvee of certain
Kchinoderms.
The ciliated edges of the neural plate (which tract is subse-
quently to be recognised as the cause of the naso-branchial
current of water) might be compared with the longitudinal
ciliated band, while the antero-ventral patch of the stomodezal
region might be compared with the adoral ciliated band of the
Auricularia larva of Synapta digitata described by Semon
(18). The comparison between the edge of the neural plate
of Vertebrates and the longitudinal ciliated band of the Auri-
cularia and Tornaria has been made by Garstang (5), and the
fact that the edges of the neural folds are ciliated in at any
rate one Craniate certainly supports his suggestion.
Garstang concluded that the longitudinal band of cilia were
“ practically homologous with the medullary folds of the
Vertebrata.” I am not sure what he means exactly by the
“ medullary folds.” In Rana temporaria, up to the time of
the closure of the neural folds, it is only the edges—that is to
say, that part of the neural plate which does not actually form
part of the tubular nervous system—that are ciliated. The
actual neural plate is not ciliated until later.
If, therefore, the ciliated ridges of the frog embryo may be
CILIATION OF ECTODERM OF AMPHIBIAN EMBRYO. 479
regarded as homologous with the longitudinal ciliated band of
Auricularia, it is the ectodermal space between the bands of the
latter which may be regarded as the homologue of the neural
plate of Vertebrata, and the longitudinal ciliated bands would
perhaps be more exactly represented in the craniate embryo
by the system of branchial sense-organs. ‘The possibility of a
connection between the Chordate and Echinoderm phyla has
been very often suggested; and in view of the remarkable
agreement of the actual ontogenetic fate of the blastopore in
both these groups the idea of a possible relationship between
the phyla would be much more favorably considered were it
not for the very general belief in the concrescence theory of
vertebrate development. It is only by some form of concres-
cence in development that it is in any way possible to bring
embryological evidence in favour of the theory of vertebrate
descent from a gastrula with an elongated mouth, one end of
which gave rise to the present vertebrate mouth, the other the
vertebrate anus.
It should of course be remembered that cilia are of very
general occurrence in the animal kingdom, that they are found
in the simplest as well as the most highly differentiated types,
and that they appear at the later as well as the earliest stages
of development.
These facts lead one to suppose that cilia may be with great
ease acquired by an organism. The ciliation of the Amphibian
may be only an ontogenetic adaptation. If the ciliation of the
tadpole is a purely coenogenetic feature, what purpose does it
subserve ?
There can be very little doubt that, on the whole, it is re-
spiratory. As regards the three special systems described in
this paper, one, that connected with the cement-gland, has a
very obvious use, and may very probably be a recent modifica-
tion of a general ciliation of the ectoderm.
The second, that connected with nasal epithelium and gill-
filaments, has also a very obvious use in producing a very rapid
flow over the gill-filaments, and very possibly its connection
with the olfactory epithelium may be advantageous.
480 RICHARD ASSHETON.
This system might very well also be a comparatively recent
adaptation; but, on the other hand, its very early development
in the embryo, and its appearance in connection with the edges
of the neural epithelium before there is any sign of gill-fila-
ments, suggest a much more archaic origin.
The third system, that which produces a flowing into the
stomodzal pit, has certainly no obvious use to the embryo,
although, in a small free-swimming form with open mouth and
complete alimentary canal, such a current of water would have
had an important function.
Although I do not lose sight of the possibility of the whole
ciliation of the amphibian tadpole being ccenogenetic, yet the
occurrence of ciliated tracts, which may be compared in posi-
tion and relation to such important morphological features as
the blastopore and mouth, with certain ciliated tracts of Tor-
naria and Echinoderm larve is, at any rate, worthy of notice.
A ciliated ectoderm has never been described, as far as I know,
for any Elasmobranch. I have examined living specimens of
one member of this group, Scyllium canicula, at various
ages during the first four months of development, and have
never found any trace of ciliation. Although presumably a
more ancient type than the Amphibians, the great difference in
the condition of the egg and surrounding fluids may be suffi-
cient to account for the disappearance of a primitive ciliation ;
for of what use could a ciliation be immersed in the very
viscid, almost jelly-like albuminous fluid surrounding the ovum
in an Elasmobranch egg-capsule ?
It is interesting to note that in default of a ciliation to pro-
duce a constant flow of water over the gill-filaments and skin,
the Elasmobranch embryo maintains an incessant undulating
movement of its body from the time it is sufficiently folded off
from the yolk until the time of hatching.
Similar conditions at first present the same objection to the
occurrence of a ciliated ectoderm in the Amniota, in whose case
a special organ of respiration is subsequently developed.
Tn none of these cases is it surprising to find a ciliation absent.
In the case, however, of embryos developing from holoblastic
CILIATION OF ECTODERM OF AMPHIBIAN EMBRYO. 481
eggs under conditions similar to those of amphibian develop-
ment, one would certainly expect to find a ciliated ectoderm.
I do not know whether this is the case in Petromyzon, in
Ganoids, or Ceratodus.
In connection with Sedgwick’s theory of the respiratory
nature of the neural groove, it is interesting to find that the
neural groove is not ciliated as long as it remains open to the
exterior. As stated above, I have not determined at what
time it becomes ciliated. Certainly I have never succeeded in
seeing cilia in sections of the neural tube at any stage of tad-
pole life.
CAMBRIDGE ;
June, 1895.
List oF LITERATURE REFERRED TO.
1. Batrour, F. M.—‘A Treatise on Comparative Embryology,’ second
edition, 1885.
2. Carrier, J.—‘ Die postembryonale Entwickelung der Epidermis des
Siredon pisciformis,” ‘ Arch. f. mikros. Anat.,’ vol. xxiv.
3, Cuarke, 8S. F.—‘‘ Development of Amblystoma punctatum,”
‘Studies from the Biological Laboratory of the Johns Hopkins
University,’ vol. ii, 1880.
4. Hyciesuymer, A. C.—‘‘ The Early Development of Amblystoma, with
Observations on some other Vertebrates,” ‘Journal of Morphology,’
vol. x.
5. Garstanc, W.—“ Preliminary Note on a New Theory of the Phylogeny
of the Chordata,” ‘ Zoologischer Anzeiger,’ vol. xvii.
6. Kirn, E.—“ Histological Notes,” ‘Quart. Journ. Micr. Sci.,’ vol. xx.
7. Marsuatt, A. M., and Buus, HE. J.—‘‘ The Development of the Blood-
vessels in the Frog,” ‘Studies from the Biological Laboratories of
the Owens College,’ vol. ii.
8. Morean, T. H.—‘‘On the Amphibian Blastopore,” ‘Studies from the
Biological Laboratory of the Johns Hopkins University,’ vol. iv.
9. Morean, T. H.—‘‘The Growth and Metamorphosis of Tornaria,”
‘ Journal of Morphology,’ vol. v.
10. Oszorn, H. F.—Preface to ‘Amphioxus and the Ancestry of the Verte-
brates,’ by Willey.
482 RICHARD ASSHETON.
11. Pritznrr, W.—‘ Die Hpidermis der Amphibien,” ‘ Morphologisches
Jahrbuch,’ vol. vi.
12, Preyer, W.—“ Verlangerung der Embryonalzeit bei Wirbelthieren,”
‘Jen. Zeit. f. Naturwiss.,’ 1881.
13. Semon, R.—“ Die Entwickelung der Synapta digitata,” ‘Jen. Zeit.
f. Naturwiss.,’ vol. xxii.
14. Sepewick, A.—‘‘ The Original Function of the Canal of the Central
Nervous System of Vertebrata,” ‘Studies from the Morphological
Laboratory in the University of Cambridge,’ vol. ii.
15. Stour, Po.— Ueber die Haftorgane der Anurenlarven,” ‘Sitzungsber.
phys.-med. Ges. Wurzburg,’ 1881, No. 8, p. 118.
16. Wicutman, A. C.—‘“‘On the Ventricular Epithelium of the Frog’s
Brain,’ ‘Studies from the Biological Laboratories of the Johns
Hopkins University,’ vol. iv.
17. Wittny, A.—‘ Amphioxus and the Ancestry of the Vertebrates,’ 1894.
EXPLANATION OF PLATE 35,
Illustrating Mr. Richard Assheton’s paper, ‘‘ Notes on the
Ciliation of the Ectoderm of the Amphibian Embryo.”
List of Reference Letters.
A, Anterior end of embryo. AN. Anus. BP. Blastopore. C. Ciliated
cell. €.C. Stream of water in connection with the mucous or cement gland.
C.GL. Mucous or cement-gland cells. C.GZ'. The necks of the mucous or
cement cells. DG. Dorsal groove formed by the junction of the edges of
the neural plate. 2P.2#. Epidermic layer of epiblast. #P.N. Nervous layer
of epiblast. 1. Nasal depression. V.B.C. Naso-branchial stream. .G.
Neural groove. JZ. Mucous or cement gland (sucker). P. Posterior end
of embryo. P.S. Primitive streak. £&.G@. Rudiment of Gasserian and other
ganglia of the cranial nerves. AM. Ridges, richly ciliated, bounding the
cement-glands. S. Stomodeum. SC. Stomodzal stream.
Fic. 1,—Surface view of the embryo of Rana temporaria. The neural
plate is folding upwards. The arrows indicate the only area upon which
cilia are at this time developed, and the direction of the flow of water pro-
duced by their action.
CILIATION OF ECTODERM OF AMPHIBIAN EMBRYO. 483
Fie. 2,—A few hours later than Fig. 1. The cross indicates the furthest
point backwards upon which cilia may be present.
Fic. 3.—A slightly older stage. In this a second area of ciliation has
appeared, as indicated by the arrows, along the antero-ventral surface.
Fic. 4.—Embryo of frog, showing current along the dorsal groove formed
by the junction of the neural folds.
Fig. 5.—Embryo of frog, about 3 mm. long. The whole surface is now
ciliated, although ventrally the ciliation is very scanty. Three well-marked
streams are indicated by the arrows V.BC., SC., and CC., respectively.
Fic. 6.—Diagram showing the currents of water produced by the ciliation
about the region of the blastopore and anus.
Fie. 7.—Diagram showing the currents of water produced by the ciliation
at the posterior end of the embryo, after the closure of the blastopore and
growth of the tail.
Fic. 8.—A semi-diagrammatic figure of a transverse section across the so-
called sucker of the frog embryo of about 63—7 mm. ‘The epidermic layer of
epiblast, #P.#., is a layer of one cell in thickness, which cells at two points,
C.GL., become enormously lengthened, and secrete a very sticky kind of
mucus. The neighbouring cells bear cilia. Those upon the walls of the
ridges nearest the cement or mucus-secreting glands bear very short
cilia; those more remote, and especially those between the two bases, bear
very long cilia. Hach long mucous gland-cell is broad at its base, contains
a large nucleus surrounded by “granular” protoplasm at that part, and
narrows into a long neck as it reaches the surface, filled with the sticky
secretion, which stains slightly with most stains, but with plain hematoxylin
it stains deeply.
Fie. 9.—A semi-diagrammatic figure of the “sucker” at a rather later
stage, namely, a tadpole 8—9 mm. in length. In this the ridges bounding
the glands are much more prominent. In reality the gland-cells lie diagonally
from before backwards, so that their necks and openings are much more
posteriorly situated than their protoplasmic bases.
Fie. 10.—Ventral view of a 34-mm. tadpole, showing the currents of water
produced by the ciliation of the ectoderm.
Fic. 11.—Ventral view of a 6-mm. tadpole. The arrows indicate the
general flow of water and the special streams on the ventral surface.
Fig. 12.—Lateral view of the same embryo as preceding.
Fic. 13.—Ventral view of anterior end of an $-mm., tadpole. The arrows
indicate the main currents produced by the special ciliation connected with
the stomodeum, the mucous glands and olfactory epithelium, and branchial
filaments.
Fig. 14.—Side view of the same embryo as preceding.
484. RICHARD ASSHETON.
Fie. 15.—Section of the epidermis of a 6-mm. tadpole, taken horizontally
between the nasal depression and the developing gill-filaments.
Fie. 16.—A piece of a gill-filament of an external gill of an 8-mm. tadpole.
The ciliated cells are darker, and project slightly above the remainder.
Fic. 17.—Diagram of the tail of a 12-mm. tadpole, to show the general
arrangement of the long ciliated cells drawn in Fig. 18. The zigzag lines
terminating in arrows show the course taken by particles as they are dashed
from cell to cell across the tail fins.
Fic. 18.—This shows three of the ciliated cells (C.), which are oblong and
darker in colour than the surrounding polygonal non-ciliated cells.
Fic. 19.—Side view of the embryo of Triton cristatus, showing the
direction taken by currents of water due to the ciliation of the anterior end
of the embryo. The two stars indicate a spot where the motion is very rapid—
the stomodeum.
Fic. 20.—The same embryo of Triton cristatus. A ventral view.
Fic. 21.—An embryo 6 mm. in length of Triton cristatus, seen from
the side. The arrows indicate the flow of water produced by the ciliation.
Fic. 22.—A ventral view of the anterior end of the same embryo.
DIFFERENTIATIONS OF ECTODERM IN NECTURUS. 485
Ontogenetic Differentiations of the Ectoderm in
Necturus.
Study II—0On the Development of the Peripheral Nervous
System.
By
Julia B. Platt.
a
With Plates 36—38.
INTRODUCTION.
IT unpErTooK the study of the development of the lateral
line system in Necturus at the suggestion of Professor C. O.
Whitman, and am indebted to Professor K. Brooks for access
to the literature of my subject. As the questions of chief
interest to me are connected with the relation of this system
to vertebrate segmentation, and to the formation and distribu-
tion of the cranial nerves, I turned to the earlier stages of
embryonic development to discover there, if possible, the first
indications of ectodermic differentiation which might serve as
the source either of cranial ganglia or of sensory specialisations
in the skin. This led to the discovery that large numbers of
cells, arising in the ectoderm and migrating below the surface
of the body, take part neither in the formation of ganglia nor
nerves. They are, however, distinguished in Necturus from
the surrounding tissues by marked difference in the size of
the yolk granules they contain.
I grouped together the ganglionic and connective-tissue
cells which thus migrate inwards from the ectoderm under a
common term, “ mesectoderm,” and in a former paper (82)
486 JULIA B. PLATT.
traced the lines of their origin and the path of their migration
to the time when the nervous contingent separates from the
connective-tissue residue in which later cranial cartilages form.
Here my work naturally diverged in two directions, one Jead-
ing to the further development of the peripheral nervous
system, the other to the formation of the cranial cartilages,
and I consequently closed my first study at this point, pre-
ferring to consider separately the two topics it served to
introduce.
Since the lateral line system dates its origin to an earlier
period of embryonic development than that with which this
paper properly opens, I shall review briefly the observations
recorded in my former paper in regard to those ectodermic
thickenings, or ridges, which are its precursors, but in so doing
shall take little notice of the connective-tissue cells that con-
stitute part of the mesectoderm. They are the subject-matter
of a following study.
1. In Review.
As the neural folds develop in Necturus, the ectoderm be-
comes deeper in the line that marks the uplifting of the folds
from the surface of the egg, thus forming a rather wide band
of deep ectoderm, which begins to be differentiated at the
anterior end of the embryo and gradually extends backwards.
When the neural folds close the band on each side of the trunk
of the embryo is replaced by three narrow longitudinal ridges,
of which the median is the deepest. These three ridges extend
backward from the line of the third intersegment posterior to
the ear. Anterior to this line the ectoderm at the side of the
head continues deep, but becomes marked by two longitudinal
ridges, the dorsal of which passes through the auditory epithe-
lium, and is the source of the dorso-lateral proliferation of
mesectoderm (v. Kupffer’s lateral ganglia). This ridge continues
the line of the dorsal ridge on the trunk.
The lower of the two primitive ridges on the head is the
source of the epibranchial proliferation of mesectoderm. It is
the continuation of the median of the three trunk ridges,
DIFFERENTIATIONS OF ECTODERM IN NECTURUS. 487
namely, of that ridge which marks the separation of the proto-
vertebra from the nephrotome. The most ventral of the ridges
on the trunk marks the line at which the nephrotome separates
from the remaining parietal plate. This line is continued later
on the head by a ridge which, beginning at the oral invagina-
tion, extends backwards below the gill clefts as these succes-
sively form, and finally unites with the ventral ridge of the
trunk.
As the protovertebree are cut off from one another, the
median longitudinal ridge becomes differentiated by a series of
intersegmental elevations which correspond to intersegmental
expansions of the alimentary canal. ‘This appears to favour
Boveri’s (5) theory that gill clefts once extended throughout
the length of the body, for in the branchial region these inter-
segmental elevations on the median longitudinal ridge mark the
beginning and the dorsal limit of the gill clefts, while on the
trunk of the embryo they extend towards, although they do not
touch, the corresponding intersegmental expansions of the
alimentary canal.
The longitudinal ridges soon become connected with one
another by transverse intersegmental ridges. On the head of
the embryo these transverse ridges are formed from the separate
elements of the line of dorso-lateral proliferation of mesecto-
derm, which severally unite with a corresponding epibranchial
thickening of the ectoderm, which, in turn, blends with the
upper limit of that transverse ridge of ectoderm which meets
the endoderm prior to the formation of the gill clefts.
If the skin of an embryo at this stage be removed and
viewed by transmitted light, it is seen to be divided into a series
of small squares, each of which is bounded by the longitudinal
and transverse ridges I have just described, and each of which
corresponds to the outer surface of a protovertebra. This
method of determining the position of the ectodermic ridges
cannot be applied in the branchial region or in the anterior
part of the head, since the rapid proliferation of mesectoderm,
which is here taking place, renders it for the time impossible
to separate the ectoderm accurately from the tissues beneath.
488 JULIA B. PLATT.
Beginning at the anterior extremity of the dorsal longitudinal
ridge, we find that it gives rise to cells which join the trige-
minal portion of the neural crest, and take part in the formation
of the Gasserian ganglion and its anterior continuation—the
ramus ophthalmicus profundus. This nerve is formed from the
ectoderm in the same manner as are the cranial ganglia.
The migration of cells into the trigeminal mesectoderm
extends from the anterior limit of the dorsal ridge to the inter-
segment between the mandibular head cavity and the anterior
of the two somites which in Necturus as in Scyllium (van
Wijhe, 37) lie above the hyomandibular cleft. In this inter-
segment the proliferation of mesectoderm extends downwards
to the epibranchial ridge, which here passes over the hyoman-
dibular cleft, and curves towards the oral invagination in
conformity to the cranial flexure.
In the continuous band of deep ectoderm above the eye, 1
have been unable to distinguish any limited area, which in
giving rise to ganglion cells on the profundus would be the
homologue of what is described by Beard (8) as the “ sense-
organ” of the ciliary ganglion in the Elasmobranchs.
This area, which is distinct in Elasmobranchs, appears in
Necturus to have fused with the dorso-lateral thickening of the
ectoderm connected with the Gasserian ganglion, so that we
here have but one band of deep ectoderm, which proliferates cells
continuously to the Gasserian ganglion and to the profundus.
I do not find that the band of deep ectoderm above the eye
disappears at any time throughout its entire length, yet it
becomes greatly reduced in width by the migration of cells
into the trigeminal mesectoderm, and shortly previous to the
stage with which this paper begins the band is interrupted by
spaces, from which the cells have migrated so rapidly as to
leave the ectoderm above scarcely, if any, deeper than on the sur-
rounding surface of the head. This condition is but transitory,
and other cells soon occupy the place of those that have gone,
restoring the depth and continuity of the ridge.
Anterior to the hyomandibular cleft the lens arises, as
described by von Kupffer (23) in Petromyzon as a specialisation
DIFFERENTIATIONS OF ECTODERM IN NECTURUS. 489
of the epibranchial ridge. It would be possible to associate
that proliferation of cells to the ophthalmicus profundus,
which constitutes the ciliary ganglion with the lens as dorso-
lateral and epibranchial differentiations in the intersegment
between the mandibular and premandibular head-cavities.
These cavities are not distinct in Necturus.
My description of the changes that take place in the ectoderm
prior to the stage with which this study begins may be more
easily followed by referring to the figures given with the first
study (82).
Between the two somites above the hyomandibular cleft we
find at an early stage two circular areas, in which the ectoderm
becomes deep in both the dorso-lateral and epibranchial lines.
The two areas unite with one another later in an intersegmental
ridge, which meets the intersegmental proliferation of cells to
the trigeminal mesectoderm above the hyomandibular cleft.
Thus the hyomandibular cleft is the ventral continuation of
two intersegmental lines, one connected with the trigeminus,
the other with the facialis. The specialised areas connected
with the facialis increase greatly in depth and extent, and at
the time when the migration of cells to the facial mesectoderm
is most rapid the dorsal thickening becomes continuous, on
the one hand with the auditory epithelium, and on the other
with the trigeminal portion of the dorso-lateral ridge.
The specialised areas of ectoderm in the next intersegment
are the ear on the dorso-lateral line, and the epibranchial
thickening above the hyobranchial cleft. The two areas
become sharply separated from one another only as the ear
becomes constricted off as a closed vesicle. From the auditory
epithelium cells migrate into the auditory ganglion, and from
the epibranchial thickening into both facial and glosso-
pharyngeal mesectoderm.
The two following intersegmental ridges are above the first
two branchial clefts, and are connected with the migration of
cells into the vagus mesectoderm. In these intersegments it
is not easy to distinguish the dorso-lateral from the epibranchial
thickening of the ectoderm, so deep is the entire ectoderm in
490 JULIA B. PLATT.
this region, in preparation for the formation of the large vagus
ganglia.
I may mention here that while the hyobranchial and the
first two vagus clefts arise in the line of a corresponding inter-
segment, after the second vagus cleft has appeared the entire
branchial apparatus begins to change its position in rela-
tion to surrounding structures, and the second vagus cleft in
consequence pushes forwards, so that when the third cleft
appears it lies in the intersegment that was originally occupied
by the second cleft. Hence the primitive somatic and branchial
segmentation do not entirely correspond.
The migration of cells from the ridges in the ectoderm,
which I have described, is not by the wandering of cell after
cell into the tissue below, but by the peeling or splitting off
of the cells en masse, leaving the ectoderm above for the
time no deeper than on the surrounding surface of the head.
A new limiting membrane forms; and where we once found
an area of deep ectoderm we now find a mass of mesectoderm
cells, covered by ectoderm of no unusual depth.
The primitive longitudinal and the transverse ridges of the
trunk J described in my first study (82) as transitory differen-
tiations of the ectoderm, which disappear and leave no clue to
the cause of their existence unless interpreted in the light of
later events, when it is found that the three lateral lines of
sense-organs occupy the position once held by the primitive
longitudinal ridges.
The result of further study in regard to the primitive ridges
of the trunk is recorded in this paper; the supposition, how-
ever, that the ridges indicate sensory differentiations in the
ectoderm, which are subsequently to be developed in the lateral
line system, is not vitiated by their temporary disappearance,
for ectodermic thickenings and ridges on the head of which
many of the cells are undoubtedly sensory, inasmuch as they
develop into ganglia, disappear completely for a time in giving
rise to cells of the mesectoderm, then reappear as the Anlagen
of lateral line organs, while other similar lines on the head
give rise in part of their length to sense-organs, and in part
DIFFERENTIATIONS OF ECTODERM IN NECTURUS. 491
disappear, leaving neither sense-organ nor ganglion to testify
to their previous existence.
The plan of the lateral line system in Necturus, as thus
early laid down, is that of three Jongitudinal lines on each side
of the embryo, connected by intersegmental cross-lines, with
special differentiations at points of intersection. To show what
portions of these lines are retained in the final system, what
their modifications are, and what their relations to the sensory
nerves, is the purpose of this study.
2. The Embryo of PI. 38, fig. 1.
In Pl. 38, fig. 1, I have represented an embryo in which the
final lateral line system has begun to develop. The ridges or
thickenings in the skin are left white in the drawing, as if
raised above the surface of the embryo. Some of the ridges
are actually visible on the surface, but their appearance is
exaggerated in the drawing, which has further supplemented
the visible ridges by others that become evident only in
section.
The position of ear and eye is outlined, and the contour of
the cranial ganglia is indicated by a flat shade. The posterior
part of the Gasserian ganglion, which is in no way connected
with the lateral line system, has been omitted to avoid con-
fusion with the facial ganglion. The fine lines in which the
ganglia terminate indicate the root of the ganglion or the small
sensory nerves that have already separated from the skin.
In the primitive dorso-lateral line we find above the eye the
ridge from which the supra-orbital sense-organs are about to
be formed. Just below lies the Gasserian ganglion and the
ramus ophthalmicus profundus, both in part composed of cells
that have but now migrated from the primitive supra-orbital
ridge which occupied the position of the present ridge. As in
an earlier stage, this ridge still forms an unsegmented line ex-
tending backwards to the anterior of the two intersegments
that meet in the hyomandibular cleft. The primitive thick-
ening in the dorso-lateral line which belonged to the posterior
492 JULIA B. PLATT.
of these two intersegments has disappeared entirely in giving
rise to part of the facial mesectoderm.
The ear has developed from the dorso-lateral thickening in
the hyobranchial intersegment, and it will be noticed that
although the ear undoubtedly belongs in the lateral line
system, and is in fact the centre of that system, it is not
properly a “ branchial sense-organ,” as Beard suggests (2), for
this term cannot be accurately applied to sense-organs above
the epibranchial line.
The dorso-lateral thickening of the following intersegment
has for the time disappeared in giving rise to cells that now
form part of the vagus mesectoderm. In the second inter-
segment posterior to the ear a knob-like structure at the ante-
rior end of a longitudinal club-shaped ridge is the beginning
of the most dorsal of the lateral lines of the trunk. Posterior
to this intersegment the primitive dorso-lateral ridge has dis-
appeared, and anterior to the intersegment, as has just been
pointed out, two of the primitive intersegmental differentia-
tions in the dorso-lateral line are missing, namely, those
connected originally with the facial and glossopharyngeal
ganglia.
It is evident that in following the later distribution of sense-
organs one must look to the most dorsal of the lateral lines of
the trunk for representation or omission of sensory differentia-
tions in intersegmental lines posterior to the first intersegment
back of the ear.
At its anterior extremity the ridge of the epibranchial line,
about to give rise to the infra-orbital sense-organs, blends with
the wide and deep nasal epithelium which has now begun to
invaginate. Following the line upward, one finds that at the
first of the intersegments meeting in the hyomandibular cleft
the ridge fuses with that of the dorso-lateral or supra-orbital
line, while above the hyomandibular cleft a round area of
sensory epithelium has been cut off from the infra-orbital
ridge as the primitive epibranchial sense-organ of the hyo-
mandibular cleft.
The epibranchial thickening in the posterior of the two
DIFFERENTIATIONS OF EOTODERM IN NECTURUS. 4985
intersegments above this cleft has disappeared, like the dorso-
lateral, in giving rise to facial mesectoderm.
A distinct sensory area is found above the hyobranchial cleft,
but posterior to this intersegment the epibranchial ridge is
only interrupted where the region from which the sense-organs
above the gill clefts are to be formed is separated from the
club-shaped ridge that begins the main lateral line of the
trunk. It will be noticed in fig. 1 that the posterior extension
of the vagus ganglion lies between these two divisions of the
epibranchial line. At an earlier stage this extension of the
ganglion formed with the lateral line ridge and the ridge above
the gill arches one deep ectodermic thickening. The two
ridges here separated are consequently morphologically parts
of the same ridge out of which a section has been cut to form
the ganglion.
Pl. 36, fig. 4, represents a cross-section though the region
just described in a younger embryo than that of fig. 1. The
plane of the section is indicated in fig. 1 by a corresponding
number. In fig. 4 the large vagus ganglion is being cut from
the primitive epibranchial ridge (p. ep. 7.), leaving a few deep
cells above, which later increase in number and rearrange
themselves to form the beginning of the lateral line of the
trunk. Below the ganglion, cells are migrating from the
ectoderm into the ectodermic connective tissue, but, although
the whole ectoderm is here deep, that special elevation which
constitutes the beginning of the sense-organs above the bran-
chial arches has not yet appeared, the original ridge being
temporarily obliterated in this region by the formation of
mesectoderm.
The most ventral of the three primitive longitudinal ridges
(fig. 1) begins near the oral invagination, and extends backwards
touching the ventral margin of the successive gill clefts. The
ridge ends at the transverse ridge in the posterior margin of
the last gill cleft. It therefore extends as far as the second
intersegment posterior to the ear. I have previously called
attention to the fact that the branchial clefts do not correspond
to the intersegments, inasmuch as the second and third vagus
VoL. 38, PART 4.—NEW SER. LL
4.94 JULIA B. PLATT.
clefts arise successively in the second intersegment. The poste-
rior continuation of the ventral ridge on the trunk of the embryo
has disappeared with the other longitudinal ridges of the trunk.
Of the transverse ectodermic ridges at the gill clefts but two
remain, that at the posterior margin of the last cleft and the
hyomandibular ridge. The other ridges were lost as the clefts
formed, but although the ectodermic ridge at the hyomandi-
bular cleft touched the wall of a corresponding pocket from
the alimentary canal no cleft formed, and the endoderm after-
wards receded, leaving the ridge of ectoderm in the position it
originally occupied. This ridge of deep ectoderm, once the
rudiment of the hyomandibular cleft, is now the Anlage of
the hyomandibular line of sense-organs. I prefer with Ewart
(10) the designation ‘‘ hyomandibular ” for this line of sense-
organs, rather than “ operculo-mandibular,” which Allis em-
ployed to designate an homologous branch of the lateral line in
Amia (1).
In fig. 1 a short ridge is seen to extend forward from the
middle of the hyomandibular ridge. This is the beginning of
the mandibular line of sense-organs, which thus branches off
from the hyomandibular line and grows towards a point near
the mid-ventral axis of the body, posterior to the oral invagi-
nation, where it fuses with the ventral longitudinal ridge. It
is worthy of note that while two primitive intersegmental
ridges meet above the hyomandibular cleft, below, two sensory
lines form from that cleft.
Pl. 36, fig. 2,a@, is a section through the supra-orbital ridge
of an embryo at the stage of development represented in fig. 1,
where the plane of the section is indicated. The cells of the
supra-orbital ridge are seen to have the radial arrangement
peculiar to the sense-organs of the lateral line. The section,
however, does not cut a sense-organ. The cells of the sensory
ridge arrange themselves radially about the long axis of the
ridge before dividing into groups arranged about a point above
the centre of a sense-organ. In cross-section, therefore, the
sensory ridge presents the same appearance as the median plane
of a sense-organ.
DIFFERENTIATIONS OF ECTODERM IN NECTURUS. 495
Fig. 2,0, is a cross-section through the same region as fig.2,a,
but in a younger embryo, in which as yet but one vagus cleft
is found. The section shows the primitive ectodermic ridge
connected with the trigeminal ganglion and ramus ophthal-
micus profundus. Comparing the two sections a and 6, one
sees that in 4 the ectodermic ridge has been produced by
increase in the cell layers of which the ectoderm is composed,
with no tendency to radial arrangement; while in section a the
ridge is produced by the radial arrangement and depth of a
group of cells in the lower of the two layers here composing
the ectoderm.
Pl. 36, figs. 3 and 4, are both from an embryo with two
vagus clefts, and pass through planes given in fig. 1. But few
cell outlines are drawn, since they are rendered indistinct by
yolk granules, which I have omitted as unimportant in the
present study. The plane of fig. 3 lies anterior to the hyoman-
dibular cleft, and at the beginning of the facial ganglion, a few
cells of which touch the ectoderm dorsally in the section, while
below a few connective-tissue cells of ectodermic origin sepa-
rate the ectodermic ridge from the wall of the alimentary
canal. Only those mesectoderm cells which come in contact
with the ridge are reproduced. The section shows a primitive
ridge about to add its cells to the facial mesectoderm. In the
dorsal part of the ridge may be seen a slightly radial arrange-
ment of the cells. This appearance might indicate either the
formation of a secondary sensory ridge from the primitive
ectodermic thickening, or that a round mass of ganglion cells
is about to be cut from the skin. Which of the two processes
is actually taking place is to be discovered by comparing the
next stage of development with the present. Such a com-
parison shows that the section lies posterior to the branchial
sense-organ above the hyomandibular cleft, and illustrates the
initial stage in the excision of the epibranchial part of the
facial ganglion. Fig. 4, which cuts the primitive ridge that has
just given rise to part of the vagus ganglion, shows a similar
excision at the point of completion.
The three sections (figs. 2, 3, 4) give the relative depth of
496 JULIA B. PLATT.
the primitive ridges connected with the trigeminal, facial, and
vagus Anlagen. These ridges are formed not alone by increase
in the number of cell layers composing the ectoderm, but also
by increase in the depth of the cells themselves. The latter
fact would be more evident in the figures had a longer strip
of ectoderm at either side of the ridge been included in
the drawing. The change in the depth of the cells is a
gradual one.
The long axis of a nucleus usually corresponds to that of the
cell, and the long axis of an ectoderm cell lies usually either in
the direction in which the cell is migrating if it be migratory,
or in the direction of the transmission of energy if it be
nervous. As both the path of migration and the line of trans-
mission of energy in the posterior part of the vagus ganglion,
through which fig. 4 cuts, are parallel to the long axis of the
embryo, the cells composing the ganglion changed the direction
of their axes on leaving the ectoderm, and the section which
passes through the long axis of the cells in the deeper layers
of the ectodermic ridge cuts across that of the ganglion
cells.
In Pl. 38, fig. 1, a commissural connection between the
glossopharyngeal and vagus ganglia is seen a short distance
below their respective roots. This commissure is formed from
cells of the neural crest, which at first extended as a con-
tinuous sheet from the beginning of the glossopharyngeus to
the posterior limit of the vagus Anlage. The cells of the crest
in migrating down the side of the brain divided into two
groups, in each of which the long axis of the cells corresponds
to the direction of the migration, and is consequently vertical.
The two groups remain connected with one another, however,
by a short commissure, which, serving possibly to transmit im-
pressions from one ganglion to the other, is composed of cells
with axes parallel to the long axis of the embryo, or at right
angles to the axes of the cells composing the main vagus and
glossopharyngeal ganglia.
The fusion of the vagus ganglion with the ectoderm begins
immediately posterior to the commissure, and a mass of ecto-
DIFFERENTIATIONS OF ECTODERM IN NECTURUS. 497
derm cells is here cut off to form the ganglion of the lateral line.
As I have mentioned above, the long axis of the cells compos-
ing this ganglion is at right angles to that of the cells in the
neural part of the vagus ganglion, but in the same plane as that
of the cells in the commissure. The consequence is that when
the cells of the lateral line form nerve-fibres, the
greater part of the fibres, following the path of
least resistance, pass over the commissure and enter
the brain with the root of the glossopharyngeal
ganglion. Cells which lie on the inner side of the lateral
line ganglion and in immediate contact with the primitive
vagus ganglion send fibres to the brain by the vagus root,
but the ninth nerve is the chief root of the lateral line
ganglion.
Pl. 36, fig. 5, is from a horizontal section through the
dorsal and median lateral lines of the trunk at the time of their
beginning. The plane of the section is shown in fig. 1. One
who has seen the growing lateral line plough its way through
the skin, leaving a row of sense-organs in the wake, would
perhaps imagine the structure at the right of the section to be
a large sense-organ which the advancing line, cut longitudi-
nally at the left of the section, had formed in its path. Such,
however, is not the case. The section drawn is from the series
reconstructed in Pl. 38, fig. 1, and the position of the lateral
ridge in relation to the somites has been carefully determined.
There is no sense-organ at a later stage in the place now occu-
pied by the group of cells in question. The first sense-organ
of the dorsal lateral line does not lie anterior to the third
intersegment back of the ear. The group of cells now found at
the second intersegment consequently either push their way
farther, or leave the skin in giving rise to ganglion and nerve.
Moreover the fate of that part of the main line which has as
yet developed is not different, and the cells of this deep ridge
also either advance in the skin, or give rise to ganglion or
nerve, for the first sense-organ of the main lateral line does
not fall anterior to the fourth intersegment. The entire
ectodermic thickening represented in fig. 5 ultimately dis-
498 JULIA B. PLATT.
appears, leaving the skin where it now is apparently un-
modified.
There is in Necturus a qualitative as well as structural
difference between the primitive and secondary ectodermic
ridges. The primitive ridges, formed by the multiplication of
layers in the ectoderm without radial rearrangement of the
cells, may disappear, leaving no apparent modification in the
structure of the embryo; or they may be directly modified into
secondary ridges, as is the case with the primitive ridge of the
hyomandibular cleft ; or, lastly, they may be the source of large
additions to the mesectoderm, as in the dorso-lateral and
epibranchial lines on the head. The mesectoderm thus pro-
liferated may be either exclusively ganglionic, as when the
lateral line ganglion is cut from a primitive epibranchial ridge ;
or may be composed of elements partly ganglionic and partly
connective tissue, as in the primitive dorso-lateral proliferation
connected with the trigeminus; or, finally, the proliferation
may be exclusively of connective-tissue cells, as at the margin
of the gill clefts below the epibranchial line.
Where the proliferation of mesectoderm is composed of both
nervous and connective-tissue elements, it often appears impos-
sible to decide whether cells not closely grouped in the body of
a ganglion are to be classed with the nervous system, or are to
form the basis of a branchial cartilage, until the prolongations
of some of the scattered and apparently homogeneous mesec-
toderm cells show the fine fibrillar striation peculiarly nervous.
The secondary ridges, however, composed of cells radially
arranged, are the source of neryous structures alone,
and their radial arrangement may be co-ordinated with the
fibrillar differentiation of nerve-cells forming part of the
mesectoderm, as indicating an exclusively nervous character.
In fig. 1 it will be seen that the chief nerves of the lateral
line system are beginning to develop. From the vagus-glosso-
pharyngeal group, fibres in a dorsal bundle lose themselves in
the main lateral line of the trunk at the point where fibres of
the dorsal lateral line, following the growth of the knob of cells
at the head of the main line, will soon appear. Another bundle
DIFFERENTIATIONS OF ECTODERM IN NECTURUS. 499
of fibres belonging to this group enters the intersegmental
ridge on the posterior margin of the third vagus cleft. This is
the beginning of the nerve of the ventral lateral line of the
trunk.
There is a small nerve connecting the glossopharyngeal
ganglion with the branchial sense-organ below the ear, which
is not represented in the drawing.
Fibres of the ramus ophthalmicus superficialis facialis have
just left the skin at the posterior limit of the supra-orbital
ridge, while the ramus buccalis begins to be formed at the
dorsal extremity of the infra-orbital ridge. A small branch not
represented in the drawing connects the primitive sense-organ
above the hyomandibular cleft with the facial ganglion, while a
prolongation of the facial ganglion fuses with the dorsal margin
of the hyomandibular ridge, although I find as yet no nerve-
fibres here.
3. Comparative and Critical.
v. Wijhe (87) first called attention to the fact that the cells
of the neural crest in the Selachii fuse with the ectoderm in
two planes. Misses Johnson and Sheldon (21), in their “‘ Notes
on the Development of the Newt,” extend this observation to
the Amphibia, The first fusion occurs, they tell us, “ above
the level of the notochord ;” and in the cases of facial and
glossopharyngeal ganglia a second fusion takes place “ in the
dorsal wall of the corresponding gill cleft.”” Like v. Wijhe,
they find the dorsal fusion connected with the development of
the lateral Jine.
Froriep (138) believes the ventral fusion found in the Selachii
to be the homologue of a similar union of ectoderm and
ganglion which he discovered in the Mammalia, and first
associated with the gill clefts as ‘‘ Kiemenpaltenorgan.”
v. Kupffer (22, 23) shows that the neural Anlagen in
Petromyzon also fuse with the skin in both dorsal (lateral)
and ventral (epibranchial) lines, receiving at each place of
fusion large ganglionic additions from the ectoderm. These
fusions between ectoderm and ganglion, which v. Kupffer finds
500 JULIA B.) PLATT:
in Petromyzon, I believe to be homologous with those I have
described in Necturus.
The more dorsal of the two fusions, however, which v. Wijhe
and Misses Johnson and Sheldon mention, are not homologous
with those found on the dorso-lateral line in Necturus ; for, as
fig. 1 shows, the primitive sense-organs connected with the
glossopharyngeal and vagus ganglia lie in the epibranchial
line, while in those segments immediately posterior to the ear
the primitive dorso-lateral ridge is entirely reduced by the
formation of mesectoderm, i. e. the vagus and glosso-pharyngeal
ganglia with associated connective tissue, and can consequently
not be directly associated with the formation of the lateral
line. The same conclusions hold in regard to the facial
segments.
The section given in Pl. 36, fig. 5, chances to pass through
several cells (a, a’, b,c) in the act of dividing; and as these
cells surely take part in the formation of the nervous system,
it is worthy of note that their planes of division lie in each of
the three dimensions of space. We consequently have here
not merely presumptive but positive evidence against Mall’s
(27) statement that “the primitive growing point of all ver-
tebrate nerves is in the layer of cells on the outermost side of
the ectoderm, and the axis of division is parallel with the
ectoderm.” Neither is the primitive growing point of the
lateral line nerve in the layer of cells on the outermost side of
the ectoderm, nor is the axis of division always parallel to the
surface of the ectoderm. Yet Mall studied Necturus !
The series of sections (Pl. 36, figs. 2—5) serve also to de-
monstrate the inaccuracy of Beard’s (4) statement that the
ganglion splits off from the deeper layers of the ectoderm,
leaving an external sense-organ. This does not happen in
Necturus. The large dorso-lateral and epibranchial ganglia
are formed from cells which split off en masse, leaving the
ectoderm external to them for the time thin. A sensory ridge
may appear later in the exact place where the ganglion arose,
as happens in the supra-orbital line, or sense-organs may form
at either side of the ganglion Anlage, as in the vagus region,
DIFFERENTIATIONS OF ECTODERM IN NECTURUS. o01
or again, as on the path of the lateral line of the trunk, cells
may migrate individually from the sensory ridge into the
ganglion; but just that relation of ganglion and sense-organ
which Beard describes I have never found in Necturus.
In designating the several divisions of the lateral line system
I shall adopt the plan of Ewart (10) rather than that of Allis
(1), as I also think it wise to associate each division of the
system with the particular nerve it supplies. The infra-orbital
line is thus limited to that part of the system which gives rise
to the ramus buccalis. For the organs that develop from the
sensory epithelium above the hyomandibular cleft Ewart
suggests the appellation “otic,” and “ glossopharyngeal” or
“temporal ” for those developing from the primitive sensory
epithelium in connection with the glossopharyngeal nerve.
The line of organs formed from the longitudinal ridge
external to the vagus ganglion I shall call the epibranchial
line, as they give rise to an independent nerve in Necturus,
and do not form part of one of the sensory lines of the trunk.
I must take exception, however, to Ewart’s statement that,
from the contact of the buccal and superficial ophthalmic
ganglia at their proximal ends, “‘it might be inferred that
there has been a splitting of the original epidermic thickening
above the spiracular cleft, the splitting resulting not only in
the formation of two ganglia, but also of two sensory canals—
the supra-orbital above and the infra-orbital below the eyeball.”
The development of these lines in Necturus does not support
this view. The supra-orbital line of sense-organs traces its
origin to the anterior part of the primitive dorso-lateral ridge,
which developed in approximately its present length when the
cells of the neural crest were still connected with the mid-
dorsal wall of the brain, at a period consequently long preceding
that in which the proper lateral line system begins. The cells
of the ramus ophthalmicus profundus trigemini are connected
with the anterior part of the primitive ridge at the time
when the posterior part of the ridge begins to assume a radial
structure, and to give rise to fibres of the ramus ophthalmicus
superficialis facialis,
502 JULIA B. PLATT.
The original epibranchial ridge to which the infra-orbital
line dates its origin is hardly less primitive. In fact, the otic
part of the system which Ewart views as the primitive, undi-
vided part of the severed infra- and supra-orbital lines, is, in
Necturus, itself cut off from the infra-orbital ridge very shortly
before the stage represented in fig. 1.
The description given by Mitrophanow! (29) of the develop-
ment of the supra- and infra-orbital lines in Acanthias accords
better with Ewart’s prognostication, inasmuch as Mitrophanow
finds that these lines grow in directions nearly at right angles
to one another from a thickening of the ectoderm above and
anterior to the hyomandibular cleft; but Mitrophanow also
finds that the thickening connected with the otic nerve takes
its rise from the infra-orbital line. It consequently cannot
be viewed as representing in Acanthias the undivided rudi-
ment from which the supra- and infra-orbital lines have
parted.
Mitrophanow does not mention the early connection of the
primitive supra-orbital ridge with the trigeminal ganglion and
ramus ophthalmicus profundus, nor does he lay stress on the
extensive additions which the peripheral nervous system re-
ceives from the skin along the lines of ectodermic thickening.
In fact, instead of viewing the skin as the source of the sensory
nerves, the author speaks of the branchial sense-organs above
the first and second branchial clefts as following in their
development the small supra-branchial branches of the glosso-
pharyngeal and vagus nerves; and again, in stating his con-
clusions (loc. cit., p. 211) Mitrophanow says of the several
branchial sense-organs that “leur formation est simultanée
avec le développement des petites branches nerveuses supra-
branchiales.” Evidently the author does not recognise the
precedence of the sensory ridge.
Since Mitrophanow claims as the result of his study that the
segmentation of the lateral line system is entirely secondary, I
shall be interested to discover when I again have my Acanthias
material with me whether traces of the primitive segmentation
1 T regret that the Russian publications of this author are inaccessible to me.
DIFFERENTIATIONS OF ECTODERM IN NECTURUS. 503
so evident in Necturus cannot also there be found, for it is
difficult to believe that the great similarity which exists in the
position and direction of the main lines of sense-organs in
Necturus and Acanthias should not be the result of a similar
course of development.
Noticing that in Amia the lateral line nerve innervates a
continuous canal beginning with the sensory differentiation
above the first vagus cleft, Ewart (loc. cit.) infers that the
embryonic sense-organ found here gave rise to the pre-com-
missural, commissural, and trunk portions of the canal, “ with
or without involving the branchial sense-organs lying above
the second, third, and fourth vagus clefts,” which probably
assisted in forming the several vagus ganglia, but have taken
little or no part in forming the lateral line.
In Necturus the sensory ridge above the vagus clefts is not
formed, as are the lateral lines of the trunk, by the prolongation
of a ridge developing from a given point, but is formed by the
direct modification of a band of deep ectoderm that lies below
the ganglion cut from the centre of the primitive epibranchial
ridge, This sensory line is therefore to be homologised se-
rially with the sensory differentiations above the hyobranchial
and hyomandibular clefts, and its anterior extremity is not the
beginning of the lateral line of the trunk.
Where, then, are the dorsal nerves which in Amia and Lemar-
gus should innervate the separate branchial organs above each
vagus cleft? For, as v. Wijhe (87) tells us, every typical head
segment should contain on each side, besides its somite, a
dorsal and a ventral nerve-root. Yes, but it is also true that
every typical vertebrate segment should include a muscula-
ture supplied by a motor nerve and a sensitive outer covering
giving rise to a sensory nerve. The two nerves in a typical
segment should undoubtedly be connected with that part of the
central nervous system which their segment includes.
Consider how far the segments of the vertebrate head have
departed from their type.
Beginning with the premandibular segment, we f
ffelion,
covered by a sensory epithelium which sends its fibres saditory.
504 JULIA B. PLATT.
ramus ophthalmicus profundus to that segment of the brain
which supplies motor fibres to the mandibular somite.
Part of the sensory skin covering the mandibular somite sends
its fibres to the brain by the same root.
At a slightly later stage in the development of the embryo,
a band of sensory epithelium on both premandibular and
mandibular segments, which first sent its fibres to the brain in
the second (mandibular) segment, now sends them by the ramus
ophthalmicus superficialis facialis to that segment of the brain
which supplies the motor fibres of the third and fourth
somites.
Again, the intersegmental sensory ridge above the hyo-
branchial cleft gives rise to two sense-organs lying primarily
in the same transverse line. One of these organs, the ear,
sends its sensory fibres to the brain segment which supplies
the motor fibres of the third and fourth somites, while the
lower sense-organ in the transverse line sends fibres to the
brain in the segment supplying motor fibres to the fifth,
glossopharyngeal, somite.
The sensory epithelia on the vagus segments send a large
part of their fibres to that segment of the brain which gives
rise to the motor root of the glossopharyngeus; part of the
fibres, however, are distributed to the brain in the first vagus
segment.
Behold the heterogeneous mixture, which omitted facts
would still further complicate; and yet we are told that there
is a segmental value in the dorsal nerve-root !
Each primitive ganglion may be, indeed, a segmental struc-
ture, so also is the motor nerve, but the value of that ana-
lytical division of the several cranial nerves which ascribes a
separate segmental root to each sensory branch is not ap-
parent.
The “root”? of the ophthalmicus profundus is primarily
supplied by part of the skin covering two or more segments—
the mandibular, preemandibular, and other anterior segments,
™ ““h there be. The “root” of the ophthalmicus super-
' I regreycialis is supplied by fibres from the same sensory area
DIFFERENTIATIONS OF ECTODERM IN NECTURUS. 505
covering the same segments. The root of the buccalis derives
its fibres from a more ventral band of sensory tissue on these
very segments. The otic branch of the facialis contains fibres
arising originally from the median part of the anterior of the
two intersegmental ridges meeting in the hyomandibular cleft.
The sensory fibres in the hyomandibular root of the facialis
come from the ventral portions of the two intersegmental
ridges here united. The following primitive intersegmental
line of sensory epithelium supplies from its dorsal region
branches to the root of the auditory nerve, from its median
region fibres to the so-called ‘dorsal root” of the glosso-
pharyngeus ; and from the incomplete account of the innerva-
tion of sense-organs on the gular plate given by Allis (1) in
his description of the lateral line in Amia, we must conclude
that the ventral portion of the intersegmental ridge at the
margin of either the hyobranchial or first branchial cleft is
also represented in the ‘‘ root” of the glossopharyngeal nerve.
A band of tissue on the successive vagus segments sends its
fibres to the “root” of the lateral line nerve in the glosso-
pharyngeal segment, but also sends fibres to the ‘‘ roots” of
the vagus nerve. I will go no further than to add that, as far
as the lateral line organs are concerned, their fibres choose the
nearest and most direct path to the auditory centres in the
brain, which seem to be also the centres of the entire lateral
line system, yet both development and comparative anatomy
tend to show that it is a matter of little moment whether these
fibres enter the brain by one nerve-root or another.
4. The Development of the Spinal Nerves, and the
Relation of the Vagus Ganglion to its Myotome.
In Pl. 38, fig. 1, I have indicated the position of the anterior
spinal ganglia. There is in Necturus one ganglion for each
segment of the head and trunk, if one regards the auditory-
facial group as composed of two primitive ganglia. As I have
already mentioned, the preemandibular segment is not distinct
from the mandibular which contains the Gasserian ganglion.
To the following two segments would belong the auditory-
506 JULIA B. PLATT,
facial ganglia. The first protovertebra posterior to the ear lay
below the Anlage of the glossopharyngeal ganglion, but gives
rise to no myotome, being apparently crushed out of existence
by the growth of the ear. The second segment posterior to
the ear gives rise to the first myotome, and contains the vagus
ganglion. The third segment is that of the first spinal
ganglion, &c.
The two anterior spinal ganglia possess no dorsal root, but
consist each of a small group of cells at the base of the motor
nerve. It was of interest to know from what source these
ganglia came, and with the solution of this problem in view I
turn to earlier stages in the development of the peripheral
nervous system of the trunk.
The posterior division of the neural crest begins with the
facial Anlage, and from it successively the neural portions of
the facial, glossopharyngeal, and vagus mesectoderm are
separated. The facial and glossopharyngeal portions of the
crest lie above protovertebre that develop no myotomes, but
immediately disappear in giving rise to mesenchyme over
which the cells of the neural Anlage migrate beneath the skin
to the epibranchial ridge.
The vagus segment, however, develops its proper myotome,
and when the cells of the neural crest migrate downwards only
the anterior part passes over the dorsal wall of the proto-
vertebra. The posterior part of the Anlage lies, as in the
spinal region, between the brain and the protovertebra. When
the protovertebra begins to extend dorsally as its muscle-plate
forms, the growth of its anterior part is checked by the vagus
Anlage, which passes over the protovertebra from the brain to
the epibranchial ridge. Therefore only the posterior part of
the myotome can grow upwards. This it does, and then ex-
tending forwards replaces the missing dorsal part of the an-
terior half of the myotome. In consequence the anterior part
of the vagus ganglion appears at this stage of development to
cut half through the myotome.
Pl. 36, figs. 6 and 7, illustrate this relation. Fig. 6 is
anterior to fig. 7 by one third of the width of a myotome, and
DIFFERENTIATIONS OF ECTODERM IN NECTURUS. 507
shows the dorsal and ventral portions of the myotome parted
by the vagus ganglion, which originally lay entirely dorsal to
the protovertebra. Fig. 7 shows the posterior part of the
vagus Anlage, the neural cells of which have migrated from their
original position above the dorsal wall of the brain to their
present position between the brain and muscle plate, where
they finally become attached to the brain by a nerve-root. The
difference in relation to the muscle plate between the position
occupied by cells of the neural crest in the head, and that occu-
pied by cells of the neural crest in the trunk, has been regarded
as one of the essential distinctions between cranial and spinal
ganglia. It therefore seemed of interest to note that a gan-
glion on the border line between head and trunk develops in its
anterior part like other cranial ganglia, and in its posterior part
like the ganglia of the trunk. The position of the neural out-
growth, therefore, seems to me of little value in distinguishing
the two groups of ganglia.
There is another peculiarity connected with the development
of the vagus segment to which I would call attention. The
protovertebral divisions posterior to the ear are at first of about
the same size; but as the third vagus cleft forms in the inter-
segment that bounds the vagus segment posteriorly, the proto-
vertebra of this segment increases in width, and, when the
muscle plate develops, a vertical division of the protovertebra
occurs in a plane corresponding to the present position of the
second vagus cleft, which, as will be remembered, has pushed
forwards from its original intersegmental position, now occu-
pied by the third cleft, and consequently lies beneath the vagus
protovertebra. The two parts of the protovertebra thus severed
are each smaller than the following protovertebre, and it is
through the anterior of the two that the vagus root apparently
cuts its way in the manner I have above described.
To find a dorsal as well as ventral segmentation interpolated
in a region where one looks for reduction and consolidation
was unexpected.
From the vagus segment the neural Anlage continues back-
wards, becoming gradually reduced in size, until it consists in
508 JULIA B. PERATT.
cross-section of but two or three cells above the dorsal wall of
the spinal cord (figs. 8, 10, 11, 13). These cells appear to
arise in the angle where the spinal cord separated from the
skin, and a thickening in the ectoderm which forms the slight
dorso-lateral ridge of the trunk seems occasionally about to
add its deeper cell to the cells that have already migrated into
the neural Anlage. Should this really take place, the dorso-
lateral ridge of the trunk would be not potentially, but actually
the homologue of the primitive dorso-lateral line of ectodermic
proliferation on the head, of which it is the posterior continua-
tion. Fig. 11 passes through such a cell above the fifth per-
manent (i. e. myotome-forming) protovertebra.
The dorsal ridge is very slight and soon disappears, but the
median longitudinal ridge is well marked and continues com-
paratively long. It is most conspicuous in those sections which
pass through or near an intersegment, where the median longi-
tudinal ridge unites with an intersegmental ridge. Figs. 9 and
12 show the median longitudinal ridge between the fourth and
fifth permanent protovertebre at two stages of development.
Fig. 9 is from the younger embryo. It is seen that at an early
stage the median ridge may be three cells deep near an inter-
segment, while the surrounding ectoderm is composed of but
two layers. In the later stage, given in fig. 12, the ridge is
seen to extend to a point that reaches far in between the
muscle plate and the pronephros.
The appearance of the ridge as represented in figs. 9 and 12,
though typical, is by no means constant. Neither is the ridge
three cells deep at every intersegment in the younger embryo,
nor does it extend so far into the lower tissues at each inter-
segment of the older embryo.
The boundary of the ectoderm, elsewhere true as if formed
by a limiting membrane, often appears frayed on the ridge as
if cells had just pulled away. Had they done so, however, it
would be difficult to obtain positive evidence of the fact, for the
reason that at this very time cells begin to wander towards the
notochord from the dorsal extremity of the lateral plates, form-
ing a scattered mesenchyme in which cells wandering from the
DIFFERENTIATIONS OF ECTODERM IN NECTURUS. 909
ectodermic ridge would be lost. In the head, cells that mi-
grated from the neural crest or thickened ectoderm were easily
distinguished from surrounding tissues by difference in the
size of the yolk granules they contain; but in the trunk, at
this early stage, cells of the skin and spinal cord are not more
free from yolk than those of the protovertebra, nephrotome, or
lateral plate.
Fig. 11 gives the comparative distribution of yolk granules
through the tissues of the trunk. The first cell differentiation
occasioned by reduction of the yolk granules occurs in the
ventral part of the spinal cord, and gradually extends through
the cord dorsalwards. Some of the cells from the neural crest
which lie in the loose mesenchyme at the side of the spinal
cord also soon become clearer than the more ventral mesen-
chyme cells that have possibly come from the lateral plates,
but there are always intermediate cells in regard to whose
origin the yolk granules furnish no clue. Therefore, should
cells from the median ridge join those migrating at the same
time from the lateral plates, I know of no means by which the
ectodermic cells may be identified.
On reviewing my sections, however, I find that in the region
where the intersegmental ridge joins the median longitudinal
ridge, and where as yet no mesenchyme cells from below have
wandered, cells may be observed to migrate from the ectoderm.
In Pl. 36, fig. 8, I have represented such a cell. The section
is from the same series as fig. 9, but its plane is two segments
anterior to that of fig. 9. The migration of cells from the
neural crest above the second permanent protovertebra (fig. 8)
has not extended as far as the fourth protovertebra represented
in fig. 9. The intersegmental ridge is a dorsal extension from
the median longitudinal ridge, and being deepest where it
meets the median ridge, gradually fades away as it rises be-
tween the protovertebre. As development proceeds, however,
the longitudinal ridges of the trunk become less distinct, while
the intersegmental ridges gain in prominence, thus repeating
in the trunk the sequence of the ectodermic ridges occurring
in the head. The cell migration actually observed in favor-
VoL. 38, PART 4.—NEW SER. MM
510 JULIA B. PLATT.
able positions confirms the evidence given by the depth of the
median ridge (fig. 9), by its far-reaching prolongation into the
underlying tissues (fig. 12), and its frayed edge, making it
probable that the median and transverse ridges of the trunk
throughout their length are the source of cell proliferation
from the ectoderm, and of addition to the mesectoderm.
When the neural Anlage has extended over several segments
of the trunk, and lies above the spinal cord as a band of tissue
five or six cells wide if measured from one lateral margin to
the other, the even surface of the spinal cord is interrupted at
the point where the motor nerve is to be formed by the out-
growth of a fine protoplasmic prolongation, which soon after
its appearance becomes attached to one or more of the neigh-
bouring mesenchyme cells. These cells, moreover, send
prolongations to meet the spinal outgrowth, as seen
in fig. 10, which passes through the root of the third spinal
nerve.
Somewhat later, but before the cells of the neural crest in
their downward course touch the root of the motor nerve, a
condition represented in figs. 16 and 17 is found. There is as
yet no indication of the formation of a “‘ Randschleier,” and in
both sections cells are seen to migrate from the spinal cord.
Fig. 17 passes through the root of the third spinal nerve in the
younger embryo, and fig. 16 through that of the tenth in an
older embryo. Fig. 13 shows a further stage in the develop-
ment of the motor nerve in which its main path is already
established by the bipolar prolongations of a medullary cell.
The section passes through the root of the fifth nerve in the
same series from which figs. 14, 15, and 16 are taken.
Figs. 14 and 15 cut respectively the right and left roots of
the fourth spinal nerve, and show that the cells which mi-
grated from the spinal cord have now taken on the fibrillar
striation that belongs to nerves. The nucleus of the cell
which lies in the nerve path is entirely enclosed by the striated
protoplasm of the nerve, in the threads of which the yolk
granules seem entangled. Although, in this later stage, the
cells of the neural mesectoderm have come in contact with the
DIFFERENTIATIONS OF ECTODERM IN NECTURUS. 511
mesenchyme cells below, and form with them a loose con-
nective tissue, in which I am unable to distinguish the cells of
one source from those of another, yet comparison of sections
14 and 15 with sections 18, 16, and 17, in which the neural
Anlage has not as yet reached the level of the motor root,
shows most clearly that the first nuclei found in the motor
nerve have migrated from the spinal cord through the motor
nerve-root. Ganglion cells have frequently been observed on
motor nerves, and although His (20) affirms that no medullary
cell migrates permanently from the cord into a motor nerve,
Dohrn (6), claiming to have observed the passage of such cells
from the spinal cord to the motor nerves of the trunk, supports
this view by observations recorded in a paper on the origin and
development of the eye-muscle nerves in the Selachii (7). The
description there given of the development of the trochlearis
is at variance with observations simultaneously published by
Froriep (13) and myself (80). Dohrn’s account of the origin
of the oculo-motorius differs no less from the account I gave
(loc. cit.), which has since been confirmed by Mitrophanow
(29) and Sedgwick (34). Froriep, Mitrophanow, Sedgwick,
and the author find that the first ganglion cells of the troch-
learis or oculo-motorius in the Selachii are of peripheral, and
not central origin. It is therefore with the greatest pleasure
that I now confirm Dohrn’s observations in regard to the origin
of the ganglion cells on the motor nerves of the trunk, and
add this evidence in support of his view that an actual and
permanent migration of medullary cells takes place.
The cells of the neural crest do not immediately take part
in forming the spinal ganglia, bnt wander in a continuous
sheet down the sides of the spinal cord, and are there lost in a
loose connective tissue, of which at first only those cells that
come directly in contact with the motor nerve appear to develop
nervous properties. Shall we then say with Goronowitsch
(17, 18) that the cells of the neural crest have become “meso-
derm”? By no means. I do not for a moment imagine the
actual disparity of the cells dependent on my ability to dis-
tinguish them.
O12 JULIA B. PLATT.
In the anterior segments of the trunk the cells of the neural
Anlage form two layers at its ventral edge before coming in
contact with the mesenchyme below. As the Anlage, in this
early stage, lies between the sharply bounded spinal cord on
the one hand, and no less distinct myotome on the other, it is
easy to determine the number of cells composing it by counting
the nuclei on successive sections. The result shows that there
is no early accumulation of cells in definite regions pointing to
the formation of ganglia, and consequently no primitive seg-
mentation. Comparing segment with segment, one finds a
gradual diminution of the Anlage as one approaches the tail.
My purpose in counting the cells was chiefly to determine the
number of neural cells taking part in the formation of the
connective tissue in the posterior portion of the vagus segment
which contains no spinal ganglion, and in the following seg-
ment where the ganglion, which develops at a relatively late
stage, consists merely of a small group of cells at the root of the
motor nerve.
Counting the neural cells between the spinal cord and the
first myotome, before the neural Anlage meets the mesenchyme,
I find on the left side of the embryo 166 cells. The order on
successive sections is—10, 8, 8, 7,5, 5, 5, 7, 4, 6, 9, 6, 4, 4, 5,
6, 4, 5, 5, 9, 6, 4, 5,4, 6, 4, 5, 4, 6. In the following segment
there were 126 cells, scattered quite as irregularly. In the
third segment 123, and in the fourth 125. The slight increase
in the number of cells in the fourth segment is probably due
to preparation for the large brachial ganglion that lies in this
segment. ‘The following segments show rapid reduction in the
number of neural cells.
I then counted all of the connective-tissue cells above the
level of the motor nerve-root in the first trunk segments of
the series from which figs. 13, 14,15, 16 are taken, where
the neural crest of the anterior segments has united with the
mesenchyme, and forms a loose tissue of two layers at each
side of the spinal cord.
The result gives for the posterior part of the vagus segment
on the left side of the embryo 150 cells. Their order in section
DIFFERENTIATIONS OF ECTODERM IN NECTURUS. 513
is—=7,'9);,6; 106; 7,6, 4, 3,115 6; 55-574, 55:3, 6, 23 7, 7, 6; 8,
7, 4,6. The sections in the two series compared are of the
same thickness, although it takes twenty-nine sections to pass
through a segment in the younger embryo, which twenty-five
sections cover in the older embryo. For the second segment,
I found above the level of the motor nerve 138 cells, an
increase of only twelve cells. The third segment gave 137
cells, an increase of fourteen cells. In the fourth segment I
found 186 cells, showing rapid increase, probably due, as above
mentioned, to the formation of a large brachial ganglion,
although as yet no grouping of the cells into a ganglionic
mass is found.
These figures are merely of relative value, as the number
of neural crest cells varies in different embryos at about the
same stage of development, and varies also on opposite sides of
the same embryo.
Comparing the number of cells in the first segment of the
two series, we find that the neural cells of the younger embryo,
even had there been no increase by division, which is impro-
bable, more than suffice to account for all of the connective
tissue above the level of the motor nerve-root in the older
embryo. I have not extended the enumeration, as the figures
given demonstrate the continuity and extent of the tissue of
ectodermic origin at the side of the brain in segments in which
either no spinal ganglion or but a small one is formed. Part
of the neural cells, however, that lie in the posterior division of
the vagus segment form the posterior vagus root, the two
vagus roots thus corresponding to the divisions of the primi-
tive segment.
There is not only a rapid multiplication of neural cells to
form the spinal ganglia, but also an additional migration of
cells from the spinal cord through the dorsal nerve-root. The
neural crest cells of the trunk resemble those of the head in
having at first no special connection with one another, or with
the central nervous system, and only on reaching the level at
which the spinal ganglion is to develop do they send prolonga-
tions in two directions, one constituting the peripheral nerve,
514 JULIA B. PLATT.
the other the dorsal root. The cells that secondarily migrate
into the spinal ganglia are, however, like the primitive cells of
the motor nerve, from the first bipolar. Thus the cells of the
neural crest are but potentially nervous, while the cells migrat-
ing into the peripheral nervous system through a dorsal or
ventral root are from the first differentiated nerve cells.
The ganglia at the base of the first motor nerves in Necturus
are, therefore, composed in part of cells that have migrated
from the spinal cord through the motor nerve-root, and in
part of neural crest cells that have come in contact with the
motor nerve on their downward path. There is, however, little
increase in the neural cells of the first two segments, such as
helps to form the following spinal ganglia, and no secondary
additions from the cord through a dorsal nerve-root.
5. The Embryo of Pl. 38, fig. 18.
In fig. 18 I give a second stage in the development of the
lateral line system. The supra-orbital ridge has become wider,
and the cells of which it is composed begin to arrange them-
selves about two parallel lines in anticipation of the double
row of sense-organs about to form. ‘The infra-orbital ridge
has become distinctly separate from the nasal epithelium.
The hyomandibular ridge is little changed. The sensory
thickening above the hyobranchial cleft has elongated verti-
cally, and from the anterior extremity of the epibranchial
ridge above,the vagus clefts a bit of sensory epithelium, that has
parted from the rest, has also elongated dorsally. Thus two
sensory ridges now replace portions of two intersegmental
ridges that were lost in giving rise to ganglia, and to the ear.
The posterior part of the ventral ridge below the gill clefts
begins to disappear, but the vertical ridge at the margin of the
last cleft shows a slight ventral extension which is the rudiment
of the ventral trunk line of sense-organs that is about to grow
backwards from this point. Both the dorsal and median trunk
lines have lengthened, and it will be noticed that no ridge is
now found (fig. 18) where the rudiment of the median and
dorsal trunk ridges is seen in fig. 1.
DIFFERENTIATIONS OF ECTODERM IN NECTURUS. 515
The growth of the lateral line of the trunk is chiefly through
division and migration of cells that originally formed the
anterior extremity of the line, and were in contact with the
vagus ganglion. The cells that compose the growing lateral
line are much more free from yolk than are the cells of the
skin through which the line ploughs its course. The sensory
ridge is thus sharply distinguished from neighbouring tissue. I
believe, however, that the ridge is not exclusively composed of
cells foreign to the segments through which it passes, but that
a few cells at each side of the ridge join those that have
advanced from anterior segments.
Pl. 36, fig. 19, from an embryo at the stage of fig. 1, on
which the plane of the section is indicated, shows the relative
amount of yolk in the cells of the lateral line ridge and in
those of the adjacent skin. Fig. 20 is a cross-section through
the skin in a plane given on fig. 18, and shows the present ap-
pearance of the skin where in the earlier stage (fig. 1) a deep
lateral ridge was found. I would call attention to two facts:
first, that the lateral nerve (fig. 20, /. n.), consisting in cross-sec-
tion of one nucleus and a small bundle of fibres, is far too
small to account for the disappearance of a ridge once as deep
as that of fig. 19; secondly, the even distribution of yolk
granules in fig. 20 shows that the deeper cells of the ridge,
which were free from granules, have not remained in the skin
after ceasing to form a sensory ridge.
What, then, became of these cells? The answer is given in
fig. 5, which illustrates one of the most peculiar phenomena in
vertebrate development with which I am acquainted. Those
lateral line cells that find themselves in a position with which
for some reason they are dissatisfied, leave that position, and
making their way over the heads of their neighbours, between
the outer and inner layers of the skin, crowd themselves down
into a front place in the advancing line, with a self-seeking in-
dependence that is almost human. ~
In describing the development of the sea-bass, Wilson
(39, p. 239) also calls attention to the strangely independent
action of individual cells, which are evidently under no common
516 JULIA B. PLATT.
pressure. The circumstance which gives occasion for this
comment is connected with the folding off of the alimentary
canal, and Wilson ascribes the apparently independent action
of the cells to inherited tendency to follow ancestral lines of
migration. The action of cells, however, in the path of the
lateral line in Necturus seems the more peculiar since we cannot
ascribe it altogether to heredity, because of the irregularity
with which the sense-organs are formed. In asegment on one
side of the embryo a sense-organ often appears that is omitted
on the other side. Now one, now two segments are omitted.
Here two, there three sense-organs are allotted to a given seg-
ment. The inherited tendency is evidently one that allows
wide range to individual variation, and this fact renders the
independent action of individual cells most striking.
I am convinced that we shall never have even approximately
accurate knowledgé of the course of vertebrate development
until we are by some means enabled to follow the migration of
individual cells. We recognise the advancing mass, or the
elongating cord, but shut our eyes to the fact that cell after cell
moves on its independent mission, wandering alone—who
knows how far? or in what direction ?
Merkel (28), in commenting on Mall’s (27) statement that
the direction of the transmission of an impulse is already
determined by the position of the cell in the ectoderm, the
receptive pole being that originally on the surface of the body,
says (loc. cit., pp. 299, 300) that it would be interesting to
know if this view be really of general validity, for Merkel
finds it conceivable that the direction of a nerve-current
might change; at least “such a possibility must be first ex-
cluded before one can speak with certainty even in regard to
the retina.” The migration of cells in the lateral line, which
I have just described, seems to me to demonstrate that the
law Mall states is not generally applicable, for the impulse
which induces three of six cells lying in a continuous line, and
equally exposed to the surrounding water, to migrate while
the rest remain, must surely be received from within. A
transmitting pole has therefore become a receiving pole, and
DIFFERENTIATIONS OF ECTODERM IN NECTURUS. 517
the universality of the law of polarity, as Mall states it, is
destroyed.
6. The Embryo of Pl. 38, fig. 21.
Fig. 21 shows the nerves and lateral line system of an
embryo in which pigment has begun to appear in the skin, and
when the stem of the external gills has become distinctly
visibie. The length of the embryo is 15 mm., but the varia-
tion in length at the same stage of development is frequently
as much as two millimetres.
On the supra- and infra-orbital lines sense-organs can now
be distinguished in the skin, when removed and studied by
transmitted light, although sections still show a continuous
deep ridge, as in fig. 22, which passes through the infra-orbital
line. The supra-orbital line has extended ventrally, and now
passes around the nasal epithelium. The dorsal part of the
line has given rise to a double row of sense-organs, as has also
the median part of the infra-orbital line, where this line crosses
the space between the eye and nose. The number of sense-
organs is not constant, and gradually increases as the embryo
grows older. Moreover where the lines are double a continuous
area of deep sensory ectoderm connects the two rows of organs,
in which further sense-organs may develop later between the
rows now found.
On the hyomandibular lines distinct sensory spots have not
yet appeared. The dorsal part of the primitive ridge has
become double, and has also extended in an anterior direction,
as indicated by the two small branches that run forward from
the hyomandibular nerve. The mandibular ridge now meets
the anterior extremity of the ventral longitudinal ridge near
the median plane of the embryo, and posterior to the mouth.
At the corner of the mouth the mandibular line passes from a
direction nearly horizontal to one nearly vertical. The line, how-
ever, curves on to the ventral surface of the embryo, and thus
approaches again the horizontal plane, although in a direction
at right angles to that of the dorsal part of the line. The
posterior or hyoid part of the hyomandibular line is little
518 JULIA.E. PLATT.
changed, but has annexed to itself, as the innervation shows,
the anterior part of the ventral longitudinal line. That part
of the original ventral ridge, which bounded the branchial
clefts ventrally, has now disappeared, and the ridge can be
followed but little beyond the plane of the hyomandibular
ridge.
The glossopharyngeal ridge has divided into two sensory
areas. ‘The long axis of the upper, like that of the undivided
ridge, is vertical, while the long axis of the lower division is
now longitudinal. In the interval between the two areas the
deep sensory cells of the original ridge have entirely dis-
appeared from the ectoderm.
The posterior intersegmental ridge is little changed. The
nerve which supplies it appears at this stage of development
to enter the vagus ganglion by a root distinct from that of the
nerve supplying the epibranchial or horizontal part of the
ridge. The two nerves, however, enter the ganglion by a
common root in a slightly older embryo.
The ridge at the posterior margin of the last branchial cleft
no longer exists. The slight ventral extension of the ridge
seen in fig. 18 has grown backwards as the ventral trunk line
which is now about to pass beneath the arm, and has conse-
quently reached the region in which the anterior sense-organs
of this line are to form. No trace of the ventral ridge is
found in the space through which it has just passed from the
posterior margin of the gill cleft to its present position. The
anterior cells of this ridge have migrated as did those of the
dorsal and median trunk ridges.
Four distinct organs lie in the path of the dorsal line, and,
as fig. 21 shows, their position is not strictly segmental. There
is, however, a strong tendency in the dorsal trunk line to form
a sense-organ at each intersegment, and the anterior organ of
the line most frequently lies between the third and fourth
myotomes (no account being taken of the division of the vagus
myotome, the anterior part of which is rudimentary). In the
embryo represented in fig. 21 the anterior sense-organ of the
dorsal trunk line lies above the fourth myotome.
DIFFERENTIATIONS OF ECTODERM IN NECTURUS. 519
A long row of closely and irregularly scattered sense-organs
mark the path of the median trunk line—one, two, or three
organs falling apparently as chance determines to each seg-
ment.
In fig. 21 the facial and vagus nerves are coloured red, the
trigeminal and glosso-pharyngeal black. The eye muscle
nerves are not represented. I have been unable to find the
trochlearis in these early stages. The abducens may be easily
followed in a slightly older embryo, and possibly already exists.
The oculo-motorius passes from the floor of the mid-brain to
the ramus profundus, which it meets at a point near the most
dorsal of the branches which fig. 21 represents as leaving the
profundus. The ganglia are represented in a flat shade as in
the preceding reconstructions, and the position of ear, eye,
and nose is indicated. The embryo from which the lateral
line organs are reconstructed is the embryo drawn. The
nerves, however, in this and in the following reconstruction
have been traced from transverse, horizontal, and sagittal
sections through embryos at the same stage of development as
the embryo drawn, but killed and stained with formic acid and
gold chloride, which I have found satisfactory and reliable re-
agents for the topography of the nervous system, Lee (25
p. 143) to the contrary notwithstanding.
From the Gasserian ganglion three branches arise; the
ramus ophthalmicus profundus, the ramus mandibularis, and a
branch which almost immediately unites with the ramus
buccalis facialis. The anterior part of the ganglion forms the
posterior part of the ramus profundus, and lies close to the
median wall of the optic vesicle. Several small branches, of
which the two larger have been drawn, pass upward between
the eye and the brain, connecting the profundus with the
sensory ectoderm of the supra-orbital ridge, which, it will
be remembered, originally contributed to the origin of the
nerve. Between the eye and nose the profundus sends a
branch directly outwards, and then divides into its two chief
branches, which closely embrace the thick-walled olfactory
vesicle. The anterior of these branches anastomoses with the
,
520 JULIA B. PLATT.
olfactory nerve, which is not represented, and with the ramus
ophthalmicus superficialis facialis.
The homologue of that branch of the trigeminus which
fuses with the ramus buccalis is described by Dohrn (8) in the
Selachii as the ramus “ infra-maxillaris,” by v. Wijhe (38) in the
Ganoidei as the ramus “ maxillaris superior,’ while Strong
(36) mentions the nerve in the tadpole as an accessory branch
of the trigeminus.
In Necturus this nerve is one of the chief primitive
branches of the trigeminus, although fig. 22 shows its entire
present length from the Gasserian ganglion above to the
point where the nerve is lost in the ramus buccalis, which lies
immediately below the ectodermic ridge of the infra-orbital
line. The stage represented is but a transitory one in the
splitting off of the nerve, which takes place throughout the
length of the common Anlage. In a slightly older embryo one
finds two distinct nerves side by side, one belonging to the
facialis, the other to the trigeminus. The relation of these
nerves to one another seems to me of interest, because so
closely resembling that of the ramus ophthalmicus superficialis
to the profundus. In the one case the supra-orbital ridge gives
rise successively to two distinct nerves; in the other the infra-
orbital ridge gives rise to a nerve from which the inner part
splits off as a trigeminal branch, while the outer part remains
as a branch of the facialis. The difference in the manner of
formation is therefore chiefly one of time. A longer interval
separates the two nerves formed from the supra-orbital ridge
than that separating those formed from the infra-orbital ; but
the close similarity of origin suggests that this branch of the
trigeminus might well be called the ‘ buccalis profundus,” and
so I have ventured to name it in Necturus under the shelter of
dissident authorities.
Dohrn (8, p. 267) says of the “ N. infra-maxillaris ” in Pris-
tiurus, that many branches pass to it from the infra-orbital
canal, that these branches have a much more oblique and a
longer course than those of the buccalis, “ und,—was noch
auffallender ist—sie gehiéren einem Nervenstamm an, der von
DIFFERENTIATIONS OF ECTODERM IN NECTURUS. 521
Hause aus vor den Facialiscomponenten liegt, wahrend die
Zweige doch aus Schleimcanalen herstammen, die hinter dem
Buccalis-Schleimcanalsystem liegen. Diese auffallende Ver-
bindung hinterer Ectodermpartien mit vorderen Nerven machte
mich argwohnish ob iiberhaupt eine Regel in diesen Verbin-
dungen zu erkennen sei; desshalb verfolgte ich sehr sorgfaltig
die beginnende Zweigbildung all’ dieser Schleimcanalnerven.
Ich konnte dabei constatiren, dass aus derselben
Schleimcanalanlage Zweige an verschiedene Ner-
venstiamme abgegeben werden, und dass derselbe
Nerv Zweige aus verschiedenen Schleimcanilen emp-
fangt. Dies scheint darauf zu deuten, dass ausser den
Zweigen, welche von vorn herein bei dem Auseinanderweichen
der Nerven und der zugehorigen Ectodermpartien als Briicken
zwischen beiden bestehen bleiben und sich allmahlich in die
Lange ziehen, noch andere Zweige selbstandig vom Ectoderm
gegen das Innere zu wachsen und sich mit denjenigen Nerven
secundiar verbinden, welche sie aufihrem Wege finden.”’
V. Wijhe (loc. cit., p. 312) holds it one of the important
results of his study to have established the independence of the
two nerves, “‘ maxillaris superior” trigemini and buccalis facialis,
since these nerves lie so near to one another in many of the
Ganoidei as to have been mistaken by earlier investigators for
a single nerve. Hence neither Dohrn nor v. Wijhe reached
the conclusion towards which the development of the nerves in
Necturus points, namely, that each represents part of a single
nerve, originally a branch of the trigeminus, from which the
lateral line component separated, making use of the more
direct path offered by the contact of facial and trigeminal
mesectoderm to send the sensory impressions received from
the organs of the lateral line through the root of the facialis
to the cranial centres of that system.
The ramus mandibularis breaks up into several branches on
the mandibular muscle, which I can follow into the muscle,
but not far beyond.
The ramus ophthalmicus superficialis facialis still les
throughout its length immediately below the sensory ectoderm.
Span JULIA B. PLATT.
A branch supplying the short dorsal row of sense-organs may
be distinguished in the nerve-plexus which here underlies the
skin. The ramus buccalis has been mentioned. A _ small
branch, the ramus oticus, connects the solitary supra-branchial
sense-organ of the hyomandibular cleft directly with the facial
ganglion. .
The ventral branches of the facial, glossopharyngeal, and
first vagus ganglia closely resemble one another. Hach ganglion
rests upon the dorsal wali of the corresponding branchial cleft,
and sends a large branch downwards, from the proximal end
of which another branch runs backwards and outwards into
that branchial arch on the anterior wall of which the main
nerve lies. A third and smaller branch extends from the
ganglion inwards and forwards on the dorsal wall of the
branchial cavity. In the facial group this anterior branch,
the ramus palatinus, is already fairly developed, but the fibres
running forwards from the glossopharyngeal and vagus ganglia
can hardly as yet be called nerves. They resemble a bush of
tiny fibres, leaving the ganglion in a bundle, but almost
immediately scattered and lost on the adjacent pharyngeal
wall.
The vertical branch of the facialis, the ramus hyomandibu-
laris, begins like the following branchial nerves close to the
posterior wall of the branchial pocket, but almost immediately
crosses the dorsal corner of the pocket, and comes in contact
with the skin at the point where the endoderm of the hyoman-
dibular pocket last touches the ectoderm. Here for a short
distance the nerve is bounded within by the endoderm, without
by the ectoderm; but as the branchial pocket recedes from
the surface of the embryo the nerve clings to the ectoderm,
and divides into its two sensory branches, which closely underlie
the corresponding sensory ridges.
The vertical branchial nerves of the glossopharyngeus and
vagus begin at relatively the same point as the hyomandibularis,
but continue as they begin close to the endodermic wall of the
gill cleft, which they follow downwards. Then, turning
inwards with the gill cleft, the distal part of the nerve occupies
DIFFERENTIATIONS OF ECTODERM IN NEOTURUS. 525
a nearly horizontal position, passing through the mesectoderm,
with the cartilage Anlage above and the branchial wall below,
as seen in fig. 23, Pl. 36. This figure shows the distal part of
the glossopharyngeal nerve. Near the median plane of the
embryo the nerve ends in small branches. Fig. 21 does not
represent the distal part of these glossopharyngeal and vagus
nerves.
The lateral branchial nerves of the glossopharyngeus and
vagus, which run backwards and outwards, and which appear
to be serially homologous with the ramus hyoideus facialis,
are chiefly distributed to the branchial muscles which lie
against the anterior wall of the posterior cleft, although
branches also go to the skin or are lost on the walls of the
blood-vessels. As the hyoid muscle is very large, and fills the
posterior half of the hyoid arch, its nerve is correspondingly
large, and to reach the muscle measures but half the width of
the arch, while the following smaller homologous nerves reach
their respective muscles near the posterior wall of the arch.
The branchial nerves of the second vagus arch arise at this
stage from the root of the nerve of the first arch, and only
later do these nerves acquire independent connection with the
ganglion. The course of the nerves in the posterior arch is
similar to that of those in the first vagus arch. One nerve
passes downwards against the posterior wall of the branchial
cleft, the other passes into the posterior arch supplying its
lateral musculature and the external gill. The secondarily
acquired independence of the nerves of the posterior vagus
arch seem to me significant in connection with the manner in
which the third vagus cleft forms and the primitive vagus
myotome divides.
Two small branches, that enter the ganglion by a common
root, supply the two divisions of the glossopharyngeal sensory
ridge. The nerves connecting the following intersegmental
(vagus commissural) and epibranchial ridges with the vagus
ganglion appear to enter the ganglion at this stage by separate
roots. I may be mistaken in this, however, for in an embryo
but little older the nerves can be traced to a common stem,
524 JULIA B. PLATT.
The proximal parts of the nerves of the dorsal and median
trunk lines now lie some distance below the surface. The
median parts of the nerves closely underlie the skin, and the
distal parts run for some distance in the ectoderm. Near the
base of the nerve of the ventral trunk line several large branches
are given off, and soon lost in surrounding muscular tissue.
The first spinal nerve sends its chief branch immediately
downwards to the ventral wall of the muscle plate, along which
the nerve runs for a short distance backwards, thus avoiding the
branchial region; then taking again a vertical direction, it
meets the second spinal nerve, which passes downwards near
the anterior wall of the pronephros. The two nerves here
unite with one another and with the ventral lateral line nerve.
Beyond the point of union the spinal nerves may still be
followed forwards for a short distance. The next three spinal
nerves form the brachial plexus. From each of the spinal
nerves dorsal and lateral branches arise, which are not repre-
sented in the reconstruction, and the discussion of which I
shall postpone.
Goette (16) tells us that in the “ Unke” the first spinal
nerve arises from the second trunk segment, and passes over
the sterno-hyoideus muscle to the genio-hyoideus, and that the
second and third spinal nerves form the brachial plexus.
Hence the first spinal nerve in Necturus has apparently no
homologue in Bombinator.
Ecker and Wiedersheim (9) speak of the hypoglossus as
represented in the Amphibia in general by the first spinal
nerve, which in the frog arises by two roots, an anterior larger
root, and a posterior smaller root, supplied with a small
ganglion. The nerve follows a course similar to that of the
two pre-brachial nerves in Necturus, which therefore probably
together represent the hypoglossus of other forms.
I was interested to find that the first two spinal nerves in
the Ganoidei, according to Stannius (35) and v. Wijhe (loc.
cit.), have only anterior roots. V. Wijhe regards these nerves
as lower vagus roots, a supposition which is not supported by
the development of the nerves, if homologous, in Necturus.
DIFFERENTIATIONS OF ECTODERM IN NECTURUS. 525
7. The embryo of Pl. 38, figs. 31, 82, 38.
The embryo represented in figs. 31, 32, 33, is 19 mm. long.
The lines of the lateral line system are complete, and with this
stage the present study closes. The number of sense-organs
gradually increases as the embryo grows, but the main lines
are not different in the oldest embryos I have—at eight months,
or 40 mm.,—and from the superficial examination of the adult,
made near the collecting ground, I believe the lines to be
those of the full-grown Amphibian.
When the skin of larger embryos is examined, it is found
that the sense-organs open to the surface by a slit-like scissure
in the outer layer of the ectoderm. The long axis of the
opening lies in different planes on the several sensory lines.
Thus, on the main lateral line of the trunk the slit is horizontal,
on the dorsal line of the trunk vertical, and on the ventral
line the opening is often round, or may be elongated in any
direction. In the groups of organs at the end of the snout
the direction of the elongation also varies. In the supra-
orbital, infra-orbital, and mandibular lines it is usually parallel
to the direction of these sensory lines. In the ventral or
anterior part of the double hyomandibular line the long axes
of the openings in the outer row of sense-organs are at right
angles to those of the inner row. These differences in the
direction of the long axes of the openings to the sense-organs
seem to be in no way related to their development, and are
possibly co-ordinated with the direction in which currents of
water flow as the animal moves. At the stage with which this
study closes slit-like openings to the sense-organs are not
found.
The histology of the sense-organ resembles that of the lateral
line organs in fishes. At the present stage of development,
however, the difference between supporting and sensory cells
is not sharply marked, although the external cells of the organ
are in general somewhat flattened against the internal pear-
shaped cells.
vou. 38, PART 4,—NEW SERIES. NN
526 JULIA B. PLATT.
At each side of a mid-dorsal fold in the skin a row of mucous
glands is found, composed of a few cells invaginated from the
deeper layer of the ectoderm, and now lying below the surface,
tiny balls of cells surrounding a central cavity that opens to
the surface by a small pore. Similar glands are found on the
ventral surface of the body between the fore-limbs, and on the
tail. Although these glands are about the size of sense-organs,
nothing in their structure or in the manner of their develop-
ment suggests that genetic relation of sense-organ and mucous
gland on which Leydig (26) insists.
Pl. 38, fig. 33, represents the ventro-lateral surface of the
head, showing the position of the anterior sense-organs and
the distribution of pigment, which, as the figure demonstrates,
makes the position of the dorsal sense-organs less apparent.
I have omitted the pigment in fig. 32, which shows the dorsal
sense-organs of the head and the sense-organs of the three trunk
lines. The pigment cells are so grouped that a light band with
irregular outline extends on each side of the embryo throughout
the length of the body. Its position and relative width are
shown in the small area over which, in fig. 32, the pigmenta-
tion is reproduced. The anterior sense-organs are also out-
lined in fig. 31, which gives their innervation.
Comparing the sensory differentiations of the ectoderm at
this stage with those of the younger embryo represented in
fig. 21, one finds that a cluster of sense-organs has developed
on the antero-ventral surface of the snout at the anterior
extremity of the supra-orbital line. The two groups on oppo-
site sides of the head lie near to one another, but do not meet.
At the anterior extremity of the infra-orbital line a similar
cluster of sense-organs is found, resulting from the continued
multiplication of sense-organs between the eye and nose, which
had already begun in the younger embryo. The organs at
the posterior extremity of the supra-orbital line have extended
in a postero-dorsal direction beyond the point where this line
meets the infra-orbital, and the angle at which the two lines
diverge is now more acute.
The change in the relative position of the hyomandibular
DIFFERENTIATIONS OF ECTODERM IN NECTURUS. 527
and mandibular lines is considerable; and without the inter-
mediate stage given in fig. 21, one would hardly identify these
lines with the primitive ridges seen in figs. 1 and 18, from
which they have developed. The changes, however, are chiefly
connected with the forward growth of the mandible and the
conversion of a head originally deeper than wide into a broad
flat head. The dorsal part of the hyomandibular line has
given rise to a double row of sense-organs; and although the
line was originally nearly vertical, the dorsal part of the line
is now horizontal, the change of direction begun in fig, 21
having further increased. The anterior (once dorsal) extremity
of the line, moreover, now meets the infra-orbital line back of
the eye.
The mandibular line, which originally made an obtuse angle
with the dorsal part of the hyomandibular line, now leaves
that line at an acute angle, and at the corner of the mouth
closely approaches the base of the hyomandibular line. Here
one or two sense-organs lie between the mandibular and infra-
orbital lines, but anterior to this point no sense-organs, save
those of the supra- and infra-orbital lines, are found on the
upper lip. The mandibular line then bends on to the lower
lip, near the middle point of which it ends in close proximity
to the mandibular line of the opposite side. The two lines are
distinct, however.
Although the median lines of sense-organs on the lower jaw
do not primarily belong to the hyomandibular ridge, but to
the ventral longitudinal, as the organs are innervated by a
nerve that directly continues the main hyomandibularis, I
prefer to speak of them as the anterior part of the hyoman-
dibular line rather than as independent lines. PI. 38, fig. 33,
shows that each of these two median lines has now given
rise to a double row of sense-organs, and although they are
no longer united at their anterior extremity with the outer,
mandibular, lines, as an index of the former union, seen in
fig. 21, the innervation of the inner row of sense-organs at the
auterior extremity of the mandibular line comes from the
nerve supplying the hyomandibular line (fig. 31). From
528 JULIA B. PLATT.
the point where the primitive hyomandibular line meets the
ventral longitudinal part, one, or sometimes two sense-organs
extend towards the median surface of the throat.
The otic sense-organ has not changed.
From the more dorsal of the divisions of the glossopharyngeal
ridge, seen in fig. 21, two or three sense-organs have developed.
They do not usually lie in a vertical line, but lie often at an
angle with one another, and may even be in a horizontal line,
parallel to that occupied by the two sense-organs formed from
the lower division of the glossopharyngeal ridge.
Since the vertical line connected with the vagus is “ com-
missural” in many fishes, I retain this designation, and would
call attention to two sense-organs seen in fig. 32 posterior to
the dorsal extremity of the line. These organs appear here
exceptionally, for the vagus commissural line usually consists
of a vertical row of organs, as in fig. 31. I consider the two
organs just mentioned of interest, because they suggest a ten-
dency to continue the dorsal lateral line of the trunk on to
the head. The epibranchial line is now represented by four
to six sense-organs.
Not only is the number of sense-organs inconstant, but their
position on both head and trunk is often unsymmetrical, and
not infrequently an organ is found at some little distance from
the line in which it properly belongs.
The dorsal line of the trunk (fig. 32) does not extend beyond
the anterior part of the tail. At the point where this line
ends the median line leaves its position opposite the notochord,
and as it grows backwards assumes a position similar to that
occupied on the segments of the trunk by the dorsal line. The
median line continues to the end of the tail. The ventral
trunk line ends below the hind limb with a few sense-organs
that lie much nearer to one another than do the organs in the
median path of the line. The position of the organs on the
dorsal line is often intersegmental, although an intersegment is
occasionally omitted, or occasionally an organ lies above the
myotome.
To avoid confusion, in fig. 31, which shows the innervation
DIFFERENTIATIONS OF ECTODERM IN NECTURUS. 529
of the lateral line system, merely the proximal part of several
nerves not connected with that system has been outlined. In
the trigeminal group the peripheral distribution of the ophthal-
micus and buccalis profundus is shown, while the mandibularis
is cut short, and its maxillary and mandibular branches are re-
presented as arising much nearer the root of the main stem
than is actually the case. The ophthalmicus profundus is seen
to send several branches to the skin, which are lost in the
vicinity of the supra-orbital line, thus connecting the nerve
with the ectoderm of its origin. Asin the younger embryo, the
ophthalmicus profundus divides distally into three main
branches; one, of which only the beginning is represented in
fig. 31, extends directly outwards between the nose and eye.
The two remaining branches enclose the nasal epithelium, and
are finally distributed to the skin of the snout. The anterior
of these branches anastomoses with the ramus ophthalmicus
superficialis, the posterior branch with the ramus buccalis
facialis. The buccalis profundus is now distinct from the
buccalis facialis throughout its entire length, and its branches
are lost in the skin posterior to the infra-orbital line, as de-
scribed by Dohrn (loc. cit.) in the Selachii.
The two dorsai branches of the ophthalmicus superficialis
facialis, which are seen in fig. 31 to supply the posterior and
dorsal sense-organs of the supra-orbital lines, are of constant
occurrence. At the end of the snout the superficialis breaks
up into terminal branches, supplying the anterior cluster of
sense-organs.
In connection with the ramus buccalis facialis I would call
attention to a dorsal branch which I have marked by a star in
fig. 31. This branch supplies four sense-organs that probably
belong genetically to the hyomandibular line. The dorsal part
of the infra-orbital sensory ridge gives rise to a single line of
sense-organs, and the nerve supplying organs in the same
region as the four in question has been traced in another em-
bryo to the ramus hyomandibularis. Thus the irregularity in
the position of the sense-organs in Necturus is apparently cor-
related with irregularity in their innervation, which is especially
530 JULIA B, PLATT.
liable to occur where, as in the present case, one sensory line
meets another.
I have traced the nerve twigs to each one of the terminal
cluster of organs on the infra-orbital line, and find that four
of the organs, which I have marked in the reconstruction, are
supplied by nerve twigs composed in equal parts of fibres com-
ing from the buccalis facialis, and from the ophthalmicus pro-
fundus. These fibres unite in a common twig that goes
directly to the heart of the sense-organ.
Dohrn says (8, p. 274) that he has observed in the Selachii no
mingling of the fibres of the ophthalmicus superficialis with
those of the profundus, but considers such a union not impos-
sible; while Strong (386, p. 179) speaks more positively of
*‘the fact that the trigeminus proper does not participate in
the innervation of the lateral line system,” as “‘ brought out by
Allis (Amia), by Ewart (Lemargus and Raja), and by the
writer (tadpole).”’
In this connection, the section given in Pl. 36, fig. 26, is
significant. The figure shows one of the dorsal branches of
the profundus, seen in the reconstruction (fig. 31), as it passes
directly through the ramus ophthalmicus superficialis to the
supra-orbital line, a sense-organ of which the section cuts
tangentially. The following section shows the diameter of the
ophthalmicus superficialis to be as great in the plane traversed
by the profundus branch as it is in this section just posterior
to that plane. In other words, a few fibres of the ophthal-
micus superficialis lie outside of the branch from the ophthal-
micus profundus, and, as the section shows, fibres from the
superficialis join those from the profundus on their way to the
sensory ectoderm.
When one considers that the facialis invades a territory
originally trigeminal, one is not surprised to find at every hand
indications of the usurpation, showing that the separation of
the specialised sensory tract, that finds its co-ordinating centre
through the root of the facialis, is incomplete. Ido not, for
this reason, include the trigeminus among the lateral line
nerves, but should nevertheless hesitate to say that the “‘ tri-
DIFFERENTIATIONS OF EOTODERM IN NECTURUS. 531
geminus proper does not participate in the innervation of the
lateral line system.”
The innervation of the hyomandibular line shows that the
surface covered by the sense-organs of this line has extended
in a posterior as well as anterior direction, and that the length-
ening of the main stem of the hyomandibularis has not kept pace
with the growth of the sensory line. While in fig. 21 we find
that the hyomandibularis closely underlies its sensory ridge,
in fig. 81 the sense-organs that have developed from the ridge
are seen to be connected with the main nerve by long slender
stems that unite into four or five branches before reaching the
common trunk. The present innervation of the mandibular
line is not less misleading in its interpretation of development,
and one would hardly fancy from the distribution of the nerve
that the mandibularis primarily came in contact with its sensory
line, not near the corner of the mouth, but at the posterior
limit of the mandibular line, where this line meets the hyoman-
dibular. The point where the main nerve now forks in two
directions, once lay midway on the path of the undivided nerve,
as shown by comparison with fig. 21. In the reconstruction,
the nerves which collect the dorsal and ventral branches of the
_ hyomandibular line, and the mandibular branch of the facialis
are not united. In fact, however, the ventral branch of the hyo-
mandibularis unites with the mandibularis, and then with the
dorsal branch of the hyomandibularis, and the three nerves enter
the ventro-lateral surface of the ganglion by a common root.
From the point where the reconstruction leaves the nerves their
course is inwards, and was difficult to represent in surface view.
I have already called attention to the fact that the inner
row of mandibular sense-organs at the margin of the lower lip
is Innervated by the hyomandibularis (mandibularis internus),
thus bearing testimony to the earlier union of the two sensory
lines, hyomandibular and mandibular (fig. 21). The nerve of
the posterior half of the mandibular line anastomoses with a
branch from the hyomandibularis, so that these posterior organs
now appear to receive their innervation from both mandibular
and hyomandibular nerves.
932 JULIA B. PLATT.
Merely the trunk of the hyoid nerve is outlined, which soon
divides into three main branches that are distributed to the hyoid
muscle, and supply in general the lateral innervation of the
hyoid region. Strong (86, p. 128) says that in the tadpole the
ramus hyoideus is composed of fibres from two nerves, the
facialis and glossopharyngeus; and that “ while it is difficult
to distinguish the two sets of fibres in the ramus hyoideus,” it
is probable that the fibres from the facialis are those that
innervate the muscle, and that the cutaneous component is
derived from the glossopharyngeal vagus complex. It is
therefore of interest to note that the ramus hyoideus in Nec-
turus sends fibres to the skin when the nerve is not united
with the glossopharyngeus. Hence the difficulty which Strong
acknowledges, in distinguishing the two sets of fibres in the
tadpole, opens the way to doubt if the cutaneus fibres of the
hyoideus in Rana may not be in part derived from the facialis.
Between the mandibularis facialis and the ventral part of the
hyomandibularis a nerve is outlined which distributes its fibres
on the dorso-lateral surface of the mouth. The nerve enters the
ganglion near the root of the hyomandibularis, and I have
called it an external palatine. It may possibly be the homo-
logue of one of the palatine nerves described by v. Wijhe
(38) in the Ganoidei as belonging to the trigeminus. The
main (internal) palatine nerve is cut short in the reconstruc-
tion. It enters the ganglion at a deeper level than that at
which the hyomandibularis arises, and is distributed to the
roof of the mouth. The nerve is both larger and longer than
the external palatine.
The glossopharyngeal nerves arise from the ganglion at the
same point. The pharyngeal branch is cut short in the recon-
struction. The dorsal branch explains itself. The main
branchial nerve is post-trematic, and the distal part runs
inwards as well as forwards, ending near the axis of the
embryo. The lateral branchial nerve, which, as in the younger
embryo, extends backwards and outwards, now fuses with a
preetrematic branch from the first vagus nerve. This is the
only pretrematic branch as yet found in Necturus. The two
DIFFERENTIATIONS OF ECTODERM IN NECTURUS. 530
nerves after their fusion supply the lateral musculature, the
vascular system, and the skin, including the external gill. The
distribution of the two serially homologous branchial branches
of the first vagus arch is similar. The dorsal vagus nerve sends
branches to the upper branchial muscle, through which it
passes, before dividing into branches of the commissural and
epibranchial lines. The main branchial nerve of the posterior
vagus arch arises from the ganglion near the origin of the
nerve of the anterior arch. The two pharyngeal nerves (cut
off in the reconstruction) that arise from the vagus are dis-
tributed by numerous branches on the pharynx in their imme-
diate neighbourhood.
In regard to the dorsal and median trunk lines I have
nothing to add. From the ventral nerve of the lateral line a
few large but short motor branches arise, and a longer branch,
which supplies the ventral branchial muscles, is evidently serially
homologous with the three preceding post-trematic nerves.
In regard to the morphological value of the facial nerves
there is great difference of opinion. I believe, however, that
the development of the ramus ophthalmicus superficialis and
ramus buccalis in Necturus demonstrates that neither nerve
can be considered segmental. Of the ventral facial nerves,
v. Wijhe tells us (38, pp. 313, 314) that the ramus mandibularis
(my hyomandibularis) always divides into an external and
internal branch, and evidently does not belong to the hyoid
arch, as does the ramus hyoideus, but to an anterior visceral
arch. Therefore, if the ramus hyoideus represents a posterior
branch (post-trematic), one would be inclined to regard the
ramus mandibularis as an anterior branch; but for this it
would be necessary that its course should lie on the anterior
wall of a gill cleft, which is not the case, as the spiracle is
found anterior to the nerve. Two possibilities exist; either the
ramus mandibularis is still a ramus anterior, and the gill cleft
before which it should lie has aborted, or this is not the case,
and the nerve is a secondary outgrowth. If a gill cleft has
aborted between the ramus mandibularis and ramus hyoideus,
it probably was situated between the parts of the hyoid arch,
534: JULIA B. PLATT.
which contradicts the evidence given by Gegenbaur (15) for
viewing the hyoid as equivalent to a single visceral arch.
To the two possibilities thus suggested by v. Wijhe the
embryology of Necturus adds a third, for the hyoideus appears
in Necturus, not as a post-trematic nerve, but as a lateral
branch of the hyomandibularis similar to those supplying the
external gills and lateral walls of the following arches. The
true post-trematic nerve of the hyoid arch is the hyomandibu-
laris and its ventral continuation, the “internal mandibular.”
Nor do I regard the external mandibular nerve as the pretre-
matic nerve of the group, but because of its relation to the
mouth would homologise it also with the post-trematic nerves,
the mouth being in my opinion (81) formed by the fusion of
the ventral parts of one or more pairs of gill clefts. The
missing cleft in which, like v. Wijhe, I also believe is there-
fore to be sought in the mandibular arch rather than the
hyoid—a view that is supported by the endodermic origin of
the cells forming the mandibular musculature in Necturus
(32), and by v. Kupffer’s (24) discovery that the cavity which
gives rise to the mandibular muscles in Petromyzon is in fact
a pocket of the alimentary canal.
Of the homologies of the “ chorda tympani” I know nothing,
but was much surprised to find that Strong, in agreement with
Pollard (88), affirms that the nerve is represented by the ramus
mandibularis internus, adding that Froriep (12) may possibly
have “had the correct nerve, but was mistaken in assigning it
to the lateral line system” (p. 187). A letter from Dr. Strong
makes it probable, however, that the branch he designates
mandibularis internus corresponds to the branch I have called
“external palatine.’ This nerve is not a branch of the
hyomandibularis in Necturus, and is certainly not the homo-
logue of the nerve Froriep calls the external mandibular.
Hence the confusion.
8. Cell and Fibre.
In regard to the formation of the nerves, much that v.
Kupffer (22, 28) affirms for Petromyzon, and Dohrn (6, 8) for
DIFFERENTIATIONS OF ECTODERM IN NECTURUS. 9305
the Selachii, is equally applicable to Necturus. Nevertheless
we frequently hear that nerves are not primarily cellular in
their structure, but arise always as fibrous outgrowths from
the peripheral ganglion or central nervous system. In reply
to Dohrn, His tells us (20, p. 342) that no thoughtful investi-
gator will assume that sharks can differ from other Vertebrates
fundamentally in the manner in which their nerves are formed ;
and that when it can be shown that in higher Vertebrates, such
as Amphibia and bony fishes, the motor nerve-fibre arises as a
thread-like outgrowth from particular spinal cells, the state-
ment must (?) be valid for ray and shark.
It may, therefore, not be out of place to add a few words in
regard to the formation of the nerves in a primitive Amphibian ;
for if the Cyclostomata, Selachii, and Amphibia are shown
to agree in the cellular structure of their nerves, motor as
weli as sensory, the “ thoughtful investigator” might perhaps
conclude that the development of the human nervous system
differs from that of the shark and tadpole.
All of the nerves which I have described in Necturus,
whether motor or sensory, cranial or spinal, are formed by
the continued migration of modified ectoderm cells, either
from the neural crest, the closed medullary tube, the peripheral
ganglia, or the superficial ectoderm. The manner of their
formation, however, is not always the same, and nerves of
different orders vary greatly in the number of cellular elements
they contain.
In the development of the embryo we may distinguish two
factors, known to philosophy as efficient cause and final cause.
Efficient cause leads to that unfolding of the embryo by which
each stage grows out of the preceding one in the sequence of
its original development—its phylogeny modified by the present
changed surroundings. Final cause, however, stamps mysteri-
ously on the younger stage the image of a later form; and
thus we find throughout development rudiments of later
structures, which can be neither of immediate use to the de-
veloping embryo nor yet associated with the ancestral type its
present form repeats. To disentangle these factors is the
536 JULIA. B. PLATT.
problem of the embryologist, and we are wont to forget that in
the growth we study each cause plays its part.
Observing that the complicated nervous system of the adult
may be traced to the embryonic neural plate, we forget to
note in this the expression of final cause, and to consider that
meantime, before the neural plate enters upon its co-ordinating
functions, efficient cause must find in the egg a material
medium of co-ordination.
The living protoplasm of every cell is the first nervous
system of the egg. Step by step that specialised structure
which we recognise as a nervous system usurps the power that
belongs originally to each cell. What wonder, then, that
primitive nerves are cellular ?
In a recent publication Sedgwick (34) calls attention to the
fact that embryonic tissues are from the first connected by a
reticulum of fine protoplasmic threads. These tiny strands of
protoplasm connecting cell with cell are also evident in Necturus.
They are, as development shows, associated with the first steps
in the formation of the nervous system. When Sedgwick adds,
however, that the cell theory is a myth, and that the reticulum
forms a common medium into which the nuclei migrate, in-
stancing the formation of the neural crest, 1 cannot agree with
him. The ontogenetic changes that appear in Necturus seem
to me to refute such a supposition.
At an early period of development the superficial ectoderm
lies directly on the neural tube, and, as Sedgwick states, fine
protoplasmic threads connect the two, otherwise sharply
bounded, tissues. Later mesenchyme appears between the
skin and brain, or, as Sedgwick tells us, nuclei migrate from
below into the existing reticulum. The protoplasmic threads,
however, that at first connect the brain and superficial ectoderm
in Necturus contain no yolk granules, while the nuclei that
come from below advance in a surrounding reticulum heavy
with enormous yolk granules. Evidently they did not migrate
into the reticulum they found, but brought their own reticulum
with them. We have now a reticulum between the brain and
skin, composed in part, as before, of delicate fibres that arise
DIFFERENTIATIONS OF ECTODERM IN NECTURUS. 537
from the skin and neural tube, but chiefly formed by the
protoplasmic prolongations of the yolk-laden reticulum of the
mesenchyme. Into this reticulum the nuclei of the neural
crest, as Sedgwick tells us, migrate. But in the head of
Necturus the protoplasm of the neural crest is filled with
small yolk granules, and here again the nuclei take their
surrounding protoplasm with them. A common reticulum,
therefore, does not exist.
Is the cell, then, a separate unit, or merely the node of a
reticulum? In Necturus many cells of the neural crest mi-
grate from the dorsal wall of the brain to the branchial arches,
while cells arising in the wall of the archenteron migrate up-
wards until they lie between the dorsal wall of the brain and
the skin. I have not used microscopic methods that enable
me to state from observation that a fusion of the protoplasmic
threads passing from cell to cell does or does not take place.
But if such a fusion exists, it is continually renewed to be
immediately interrupted, since the nuclei in migrating past
one another take their “nodes” with them. That they do
this is most clearly evinced in the formation of the aortic
arches, where individual cells of endodermic origin migrate
with their large yolk granules through surrounding mesecto-
dermic tissue from which the yolk granules have nearly dis-
appeared. This constant association of a particular bit of
protoplasm with a particular nucleus makes the existence of
the separate and distinct cell highly probable, despite the
reticular structure of the mesenchyme.
I must also dissent from Sedgwick’s statement that cells of
the neural crest give rise to the walls of the vascular system
and to muscular tissues. It is, however, true that in Necturus
cells of the mesectoderm are converted into blood-
corpuscles. While the majority of the blood-corpuscles are
derived from the endoderm, the mesectoderm of the branchial
arches is also a source of their formation.
The path of the ophthalmicus profundus is originally occu-
pied by cells that, like those of the cranial ganglion, result
from the fusion of the neural crest with the ectoderm. The
538 JULIA B. PLATT.
connection of these cells with one another is at first merely by
indifferent protoplasmic prolongations, such as connect the
primitive neural crest cells with one another and with the
brain. The cells on the path of the profundus develop nerve-
fibres at about the same time as the anterior cranial ganglia.
The remaining nerves that supply the skin are formed in part by
fibrous prolongations from the cells of the ganglion with which
they are connected, in part by the migration of bipolar cells
from the ganglion, also by the distal addition of fibrous pro-
longations from the deeper layer of the superficial ectoderm,
and finally by the direct migration of cells from this layer into
the nerve. These last elements contribute also to the forma-
tion of the profundus. The difference between the formation
of this nerve and that of the remaining sensory nerves consists
chiefly in the fact that cells of the neural crest which take
part in their formation are first gathered into a mass which
we recognise as the Anlage of a ganglion, while neural crest
cells participate in the formation of the profundus throughout
its length. Even this difference is not absolute, for some of
the scattered mesectoderm cells lying near the ganglion, but
not included in its mass, contribute also to the formation of
the sensory branches of the facialis.
I speak of the rudiment of the ganglion as if already com-
posed of ganglion cells, although in fact I doubt if one com-
pletely developed ganglion cell exists in Necturus at the time
this study closes. At present the cells of the ganglion resemble
cells on the path of the nerve whose function it probably is to
contribute to co-ordination merely by conduction, and not by
modification of the impressions received.
As the nerve-cell becomes fibrillar, the entire ectoderm of
the sensory ridges of the head gives rise to a multitude of tiny
fibres which cover the inner surface of the sensory area like
fur, and appear finally to be swept together into bundles as if
by currents of conduction. Pl. 36, fig. 24, shows the tiny
threads that line the sensory ectoderm of the ventral hyoman-
dibular, inner (mandibular) line as the nerve begins to form.
Fig. 25 shows the general reticulum that connects the
DIFFERENTIATIONS OF ECTODERM IN NECTURUS. 539
skin with the underlying tissues in a region not specially
sensory.
Fig. 30 shows a later stage in the development of the hyo-
mandibular nerve, when the forest of tiny fibres seen in
fig. 24 has been swept into bundles connected with the nerve.
On the lateral lines of the trunk the fibres formed in the
deeper protoplasm of the ectoderm do not at first penetrate
the limiting membrane that bounds the inner surface of the
skin, but become gathered into a nerve before leaving the
ectoderm, thus giving rise to the nerve in the manner described
by Dohrn (8).
The branchial, pharyngeal, and palatine nerves have rela-
tively fewer cells, and are chiefly formed by outgrowths from
the ganglion, yet cells from the ganglion also migrate into
these nerves. Pl. 36, fig. 23, shows the distal part of the
glosso-pharyngeus, and, for the relative number of cells, may
be compared with the ophthalmicus superficialis (fig. 26),
the buccalis (fig. 22), or the distal hyomandibularis (PI.
37, fig. 30).
The beginning of the motor spinal nerve has been described.
The distribution of its branches to the muscle plate is peculiar.
As has been described in other Amphibia, the motor fibres of
the spinal nerves pass directly through the ganglion. Fig.
27 gives a section through the fourth spinal, second brachial,
ganglion. The muscle plate is cut above and below as it
curves over the outer surface of the ganglion, and the section
shows six motor nerves on their way to the muscle plate.
These are not the only or even the main motor nerves of the
segment, but the section serves to illustrate the irregularities
that occur in the distribution of the spinal motor nerves.
Figs. 28 and 29 show two sensory spinal nerves that underlie
the skin on the dorsal surface of the embryo. The structure
of the nerves suggests the formation of nerves in the tail of
the frog as described by Hensen (19). The nerves run longi-
tudinally, and are connected with the ganglion by a nerve that
passes through the dorsal part of the muscle plate to the sur-
face of the embryo. The cells forming these dorsal nerves do
540 JULIA B. PLATT.
not resemble the bipolar cells that migrate from a ganglion,
but look like connective-tissue cells changed in situ into
nerve cells. The cells may have migrated from the skin, but
I have no evidence that they do so, and incline to believe them
lost cells of the neural crest, since a few scattered cells of the
neural crest remain in this dorsal region. That they are in
fact nerve cells is proved by their fibrillar striation and ‘deep
stain when treated with gold chloride.
The existence of nerves such as these, where the separate
cell elements can be followed to their terminal fibrille, lends
support to the view that the cells composing any nerve are not
fused but separate, though indistinguishably so when united in
a nerve-cord wherein the fibrille lie parallel to one another
throughout their length.
The sections (figs. 28 and 29) speak more positively against
the view that each nerve-fibre extends from the ganglion to
the sensory surface, for these nerves are evidently cell chains.
The frequent use of shorter paths offered by anastomosing
branches shows, moreover, that the attachment of the super-
ficial receptive cell to one fibre of transmission is not constant.
The shorter path when offered is at once accepted.
This study, therefore, leads to the conclusion that it is of
little moment whether the motor and sensory fibres belonging
to the primitive nerves of any segment enter the brain by one
root, by two roots, or by several, the position of the nerve-
root being in great measure an expression of the co-ordinate
relations which the central nervous system subserves. The
morphological value of the nerve comes from without, and
“the metameric arrangement of the peripheral nerves is pro-
bably not primary, but occurs in adaptation to the segmenta-
tion of the structures they supply” (Froriep, 14, p. 590).
9. Summary.
(1) An early differentiation of the ectoderm into three longi-
tudinal ridges on each side of the embryo, connected by inter-
segmental transverse ridges, forms the basis from which the
lateral line system develops.
DIFFERENTIATIONS OF ECTODERM IN NEOCTURUS. 541
(2) The supra-orbital line of sense-organs and the ear form
by direct modification of the primitive ridge of the dorso-
lateral line. Sense-organs connected with the dorsal branches
of the glossopharyngeus and vagus form from ridges that
secondarily grow upwards from the epibranchial ridge after
the primitive ridges of the corresponding intersegments have
dis..ppeared in giving rise to mesectoderm. The infra-orbital
line of sense-organs, the otic sense-organ, the lower organs
supplied by the glossopharyngeal nerve, and the organs of the
epibranchial vagus line develop by the modification of the primi-
tive epibranchial ridge. The primitive intersegmental thickening
of the ectoderm where it touches the hyomandibular pocket in
the line of the gill cleft is directly modified into the hyoman-
dibular line of sense-organs. The mandibular line develops as a
secondary outgrowth from the hyomandibular line. The primi-
tive ridge of ectoderm at the third vagus cleft is the beginning
from which the ventral trunk line develops. The anterior part
of the primitive ventral longitudinal ridge is retained in the
ventral part of the hyomandibular line of sense-organs (the
internal mandibular). The lateral lines of the trunk grow
from the head backwards through indifferent ectoderm, and
do not result from the direct modification of primitive ridges.
(3) The primitive ridges differ functionally as well as struc-
turally from those of the lateral line. The former are the
source of both nervous and connective tissues, the latter of
nervous tissues alone.
(4) The primitive ridges of the trunk, like those of the head,
are sources of addition to the mesectoderm.
(5) Few of the cells that disappear from the secondary
sensory ridges of the trunk, as the sense-organs arise, enter
the lateral nerve. The majority of these cells migrate betweeu
the two layers of the ectoderm towards the terminal point of
growth in the advancing line.
(6) The third vagus cleft forms in the intersegmental plane
once occupied by the second vagus cleft, and the primitive
vagus myotome divides in the plane secondarily occupied by
the second vagus cleft, while the nerves of the second vagus
VOL. 38, PART 4,—NEW SERIES. 00
542 JULIA B. PLATT.
arch arise from the root of those of the first arch. These facts
suggest that a vagus segment is interpolated in the original
metamerism.
(7) While some fibres from the lateral line ganglion enter the
vagus root, most of the fibres enter the brain through the root
of the glosso-pharyngeus, using for this purpose the neural
crest cells which bridge the space between the vagus and
glosso-pharyngeal ganglia.
(8) Many cells of the neural crest of the trunk do not take
part in the formation of spinal ganglia, but form part of the
connective tissue at the side of the spinal cord.
(9) The motor nerves of the trunk appear before the spinal
ganglia, and are formed by the migration of bipolar cells from
the spinal cord.
(10) The nerve which underlies the infra-orbital sensory line
divides throughout its length into two nerves connected
respectively with the Gasserian and facial ganglia, thus
repeating the relation of the nerves derived from the supra-
orbital ridge, the ophthalmicus profundus, and superficialis.
(11) The ramus hyomandibularis and its direct continuation,
the mandibularis internus, appear in Necturus as the post-
trematic branch of the hyoid arch, while the hyoid nerve
resembles the lateral nerves of the posterior arches.
(12) Cells of ectodermic origin contribute to the formation
of blood-corpuscles in the branchial region.
(13) Although delicate protoplasmic prolongations connect-
ing cell with cell initiate the specialised co-ordination of the
nervous system, a common reticulum, such as Sedgwick de-
scribes, into which nuclei migrate, does not exist in Necturus.
(14) The root of the sensory nerve is no index to the seg-
mental value of the nerve.
Battimorr; April 8th, 1895.
10.
11.
12.
13.
14,
15.
16.
DIFFERENTIATIONS OF ECTODERM IN NECTURUS. 543
LITERATURE REFERENCES.
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System in Amia calva,” ‘Journal of Morphology,’ vol. ii, 1889.
. Brarp, J.—‘ On the Segmental Sense-organs of the Lateral Line, and
on the Morphology of the Vertebrate Auditory Organ,” ‘Zool.
Anzeiger,’ 1884.
. BEarp, J.—‘ The Ciliary or Motoroculi Ganglion and the Ganglion of
the Ophthalmicus Profundus in Sharks,” ‘Anat. Anzeiger,’ Bd. ii,
1887.
. Bearp, J.—‘ Morphological Studies: II. The Development of the
Peripheral Nervous System of Vertebrates,” ‘Quart. Journ. Micr.
Science,’ vol. xxix, 1888.
. Boveri, Toropor.—“ Die Nierencanalchen des Amphioxus,” ‘ Zool.
Jahrbiicher,’ Abth. f. Anat. u. Ontog. d. Thiere, Bd. v, 1892.
. Dourn, Antoy.—Studie 14: ‘“ Ueber die erste Anlage und Entwickel-
ung der motorischen Riickenmarksnerven bei den Selachiern,” ‘ Mitt-
heilungen a. d. Zoologischen Station zu Neapel,’ Bd. viii, 1888.
. Donry, Anton,—Studie 16: “‘ Ueber die erste Anlage und Entwickelung
der Augenmuskelnerven bei Selachiern und das Kinwandern von Medul-
larzellen in die motorischen Nerven,” ‘ Mittheilungen a. d. Zoologischen
Station zu Neapel,’ Bd. x, 1891.
. Dorn, ANton.—Studie 17: “ Nervenfaser und Ganglienzelle,”” ‘ Mitt-
heilungen a. d. Zoologischen Station zu Neapel,’ Bd. x, 1891.
. Ecker, A., und WIEDERSHEIM, R.—‘ Die Anatomie des Frosches,’
Braunschweig, 1881.
Ewart, J. C.—*The Lateral Sense-organs of Elasmobranchs: I. The
Sensory Canals of Lemargus,” ‘Trans. Roy. Soc. of Edinburgh,’ vol.
xxxvii, 1893.
FrorteP, A.—“‘ Ueber Anlagen von Sinnesorganen am Facialis, Glosso-
pharyngeus, und Vagus,” ‘Archiv f. Anatomie u. Entwickelungs-
geschichte,’ 1885.
Froriep, A.—‘‘ Ueber das Homologon der Chorda tympani bei niederen
Wirbelthieren,” ‘ Anat. Anzeiger,’ Bd. ii, 1887.
Froriep, A.—“ Zur Entwickelungsgeschichte der Kopfnerven,” ‘ Ver-
handlungen d. Anat. Gesellschaft,’ 1891.
Frorizr, A.—“ Entwickelungsgeschichte des Kopfes,” ‘ Ergebnisse d.
Anatomie u. Entwickelungsgeschichte,’ Bd. i, 1891.
GEcENBAUR, C.—‘ Das Kopfskelet d. Selachier,’ Leipzig, 1872.
GortTE, A.—‘ Entwickelungsgeschichte der Unke,’ Leipzig, 1875.
544, JULIA B. PLATT.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
. GoronowitscH, N.—‘‘ Die Axiale u. d. laterale Kopfmetamerie der
Vogelembryonen,” ‘ Anat. Anzeiger,’ Bd. vii, 1892.
. Goronowitscu, N.—‘ Untersuchungen iiber die Entwickelung der
sogenannten ‘ Ganglienleisten’ im Kopfe der Vogelembryonen,” ‘ Mor-
phologisches Jahrbuch,’ Bd. xx, 1893.
Hensren, V.—“ Ueber die Entwickelung des Gewebes und der Nerven
im Schwanze der Froschlarve,” ‘ Virchow’s Archiv,’ Bd. xxxi, 1864.
His, W.—“ Die Neuroblasten und deren Entstehung im Embryonalen
Mark,” ‘ Abhandl. d. math. phys. Classe d. Konig]. Sachsischen Gesell-
schaft d. Wissenschaften,’ Bd. xv, 1889.
Jounson and SHELpon.—“ Notes on the Development of the Newt,”
‘Quart. Journ. Micr. Sci.,’ vol. xxvi, 1886.
Kurrrer, C. von.—“ Die Entwickelung von Petromyzon planeri,”
© Archiv f. mikrosk. Anatomie,’ Bd, xxxv, 1890.
Kouprrer, C. von.—‘‘ Die Entwickelung der Kopfnerven der Verte-
braten,” ‘ Verhandlungen d. Anat. Gesellschaft,’ 1891.
Kurrrer, C. yon.—‘ Studien zur vergleichenden Entwickelungsgeschichte
des Kopfes der Kranioten,’ Heft 2, Munchen, 1$94.
Lez, A. B.—‘ The Microtomist’s Vade Mecum,’ London, 1893.
Leypic, F.—‘‘ Integument und Hautsinnesorgane der Knochenfische
Weitere Beitrage,” ‘Zool. Jahrbicher,’ Abt. f. Anat. u. Ontog.,
Bad. viii, 1894.
Matt, F. P.—“ Histogenesis of the Retina in Amblystoma and Nec-
turus,” ‘Journal of Morphology,’ vol. vii, 1893.
MERKEL, Fr.—Sinnesorgane,” ‘ Ergebnisse d. Anat. u. Entwickel.,’
Bd. iii, 1893.
Mrrroruanow, P.—“ Etude embryogénique sur les Sélaciens,” ‘ Archives
de zoologie expérimentale et générale,’ t. xxxi, 1893.
Pratt, Jura B.—‘‘ Contribution to the Morphology of the Vertebrate
Head,” ‘ Journal of Morphology,’ vol. v, 1891.
Pratt, Jutia B.—‘ Further Contribution to the Morphology of the
Vertebrate Head,” ‘ Anat. Anzeiger,’ Bd. vi, 1891.
Pratt, Jura B.—“ Ontogenetische Differenzirung des Ektoderms in
Necturus,” ‘ Archiv f. mikroskop. Anatomie,’ Bd. xlii, 1894,
Poriarp, H. B.—“ On the Anatomy and Phylogenetic Position of Poly-
pterus,” ‘ Zool. Jahrbiicher,’ Bd. v, 1892.
Sepewick, ApamM.—‘ On the Inadequacy of the Cellular Theory of
Development, and on the Early Development of Nerves, particularly
of the Third Nerve and of the Sympathetic in Elasmobranchii,”
‘Quart. Journ. Micro, Sci.,’ vol. xxxvii, 1894.
DIFFERENTIATIONS OF ECTODERM IN NECTURUS. 545
35. Stannius.—‘ Das peripherische Nervensystem der Fische,’ Rostock,
1849.
36. Strone, O. S.— The Cranial Nerves of Amphibia,” ‘Journal of Mor-
phology,’ vol. x, 1895.
37. Wisun, J. W. van.—<‘‘ Ueber die Mesodermsegmente und die Entwickel-
ung der Nerven des Selachierkopfes,’ Amsterdam, 1882.
38. Winx, J. W. van.—‘‘ Ueber das Visceralskelett und die Nerven des
Kopfes der Ganoiden und von Ceratodus,” ‘ Niederlandisches Archiv
fiir Zoologie,’ 1882.
39. Witson, H. V.—“The Embryology of the Sea-bass (Serranus
atrarius),” ‘ Bulletin of the U.S. Fish Commission,’ vol. ix, 1891.
EXPLANATION OF PLATES 36—38,
Illustrating Julia B. Platt’s paper, “ Ontogenetic Differen-
tiations of the Ectoderm in Necturus.”
Lettering.
a, a', b,c. Dividing cells. ar. Fore-limb. 6.4m. Branches of hyomandi-
bularis vir. 471-3. First to third external gills. duc. Ramus buccalis yit.
buc.p. Ramus buccalis profundus v. Dohrn’s “N. infra-maxillaris” in
Selachii (8, p. 267). v. Wijhe’s “ramus maxillaris superior” of Ganoidei
(88, p. 207). d'. Dorsal branch of glosso-pharyngeus. d®. Dorsal branch of
vagus. ‘d./.l, Dorsal lateral line. d.7. Dorsal longitudinal ridge. end.
Endoderm. ep. Epibranchial branch of vagus. ep.7. Epibranchial ridge.
ep.s.0. Epibranchial sense-organs. ez. d'-*, Branches of glosso-pharyngeus
and vagus supplying external gills. g/.s.o. Glosso-pharyngeal sense-organs.
hm.v. Hyomandibular ridge. m.s.o. Hyomandibular sense-organs. 70.7.
Infra-orbital ridge. io. s.0. Infra-orbital sense-organs. /. 4. Light band on
each side of the body. /./.s.0. Sense-organs of the lateral line. 7.7”. Lateral
nerve. m. Mouth. md. Ramus mandibularis v and vit. md. s.0. Mandi-
bular sense-organs. mect. Mesectoderm. m././. Median lateral line. m. x.
Motor nerve. m.7. Mandibular ridge. mz. Ramus maxillaris Vv. my!-0,
Myotomes, first to eleventh. x. Nose. z.c¢. Neural crest. oph.p. Ramus
ophthalmicus profundus v. opf.s. Ramus ophthalmicus superficialis viz. of.
Ramus oticus vi. pal.e. Ramus palatinus externus vil. pal. 7. Ramus
palatinus internus, chief palatine branch of the facialis. yp. ep.r. Primitive
epibranchial ridge. p/}-8. Rami pharyngei 1x and x. p.¢r. Posterior trans-
_verse ridge of the branchial region. so.7, supra-orbital ridge. so. s. 0. Supra-
orbital sense-organs. sp.g. Spinal ganglia. sp. ~!, *. First and second spinal
nerves. 7.7. Transverse (intersegmental) ridge. v. hm. s.o. Ventral hyo-
546 JULIA B. PLATT.
mandibular sense-organs, i.e. sense-organs of the primitive ventral longi-
tudinal ridge. v././. Ventral lateral line. v.r. Ventral longitudinal ridge.
1x, x'*. Rami post-trematici glosso-pharyngei et vagi. xg. Lateral line gan-
glion. 1X8.0.,Xs.o. Sense-organs supplied respectively by dorsal branches of
the glosso-pharyngeus and vagus. * Branch of ramus buccalis vi. ? Sense-
organs on the vagus commissural line in an unusual position. 1, 2, 3, 4.
Sense-organs supplied by buccalis vii and ophthalmicus profundus v.
PLATE 36.
Fie. 2.—a. Cross-section through the supra-orbital sensory ridge. 0.
Cross-section of the primitive supra-orbital (dorso-lateral) ridge. The planes
of sections 2, 3, 4, 5, are given in Pl. 38, fig. 1.
Fie. 3.—Cross-section through the facial part of the primitive epibranchial
ridge.
Fie. 4.—Cross-section through the primitive epibranchial ridge, as it gives
rise to the lateral ganglion.
Fic. 5.—Horizontal section through the lateral line ridge of Pl. 38, fig. 1.
Fics. 6—17 are from younger embryos than that reconstructed in Pl. 38,
fig. 1.
Figs. 6 and 7.—Cross-sections through the vagus myotome and ganglion.
Fig. 7 is posterior to Fig. 6.
Fic. 8.—Cross-section between the second and third myotomes.
Fic. 9.—Cross-section between the fourth and fifth myotomes.
Fic. 10.—Cross-section showing the first protoplasmic prolongations at the
root of the motor nerve.
Fie. 11.—Cross-section through the fifth myotome, showing the distribu-
tion of yolk granules in the tissues of the trunk at the time when the neural
crest forms.
Fic. 12.—Cross-section between the fourth and fifth myotomes in an
embryo older than that of Fig. 9.
Fie. 13.—Cross-section through the ventral root of the fifth spinal nerve.
Fies. 14 and 15.—Cross-sections through the ventral roots of the fourth
spinal nerves.
Fic. 16.—Cross-section through the root of the tenth spinal nerve.
Fic. 17.—Cross-section showing the beginning of the motor root of the
third spinal nerve.
Fie. 19.—Section through the lateral line ridge of an embryo at the stage
of development given in PI. 38, fig. 1, where the plane of the section is marked.
Fic, 20.—Section through the skin of the embryo of Fig. 18, where the
plane of the section is given.
PIFFERENTIATIONS OF ECTODERM IN NECTURUS. 547
Fie. 22.—Section through the ramus buccalis as the trigeminal and facial
parts diverge.
Fic. 23.—Section through the distal part of the post-trematic branch of the
glosso-pharyngeus.
Fic. 24.—Section through a sense-organ in the ventral part of the hyo-
mandibular line, as the nerves begin to form.
Fig. 25.—Section through the skin above the ear.
Fic. 26.—Section showing the innervation of the supra-orbital sensory
ridge by united branches from the trigeminal and facial nerves.
PLATE 37.
Fic. 27.—Section through the second brachial ganglion, showing six of the
motor nerves that supply the adjacent myotome, which the section cuts above
aud below, as it curves around the ganglion.
Fies. 28 and 29.—Sections through dorsal sensory nerves of the trunk.
The arrow in Fig. 29 shows the direction of the main stem.
Fic. 30.—Section showing the distal distribution of branches from the
ramus hyomandibularis vit.
PLATE 38.
Fic. 1.—Embryo 12 mm. long, with ectodermic ridges reconstructed, and
the position of the anterior ganglia indicated.
Fig. 18.—Embryo 13 mm. long, with the ectodermic ridges reconstructed,
and the position of the anterior ganglia and lateral line nerves indicated.
Fic. 21.—Embryo 15 mm. long, showing the ectodermic ridges and sense-
orgaus of the lateral line. Rudiments of the external gills have appeared, and
the peripheral nerves begin to develop. Facial, vagus, and lateral line nerves
are given in red; the trigeminal and glosso-pharyngeal nerves in black.
Fic. 31.—Embryo 19 mm. long, showing the anterior sense-organs of the
lateral line system, and their innervation from the ventro-lateral surface. The
external gills and fore-limb are removed. The nerves are coloured as in
Fig. 21.
Fic. 32.—Embryo at the same stage of development as in Fig. 31. The
distribution of the sense-organs on the trunk is given, and the present pig-
mentation of the embryo indicated.
Fic. 33.—Head of an embryo at the same stage as Figs. 31 and 32, showing
the distribution of sense-organs on the head, the dorsal surface of which is
now quite evenly pigmented.
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INDEX “POOL. 38;
NEW SERIES.
Amia, the early development of, by
Bashford Dean, Ph.D., 413
Amphibian embryos, ciliation of the
ectoderm of, by R. Assheton, 465
Assheton on the ciliation of the ecto-
derm of the Amphibian embryo,
465
Asterina gibbosa, development
of, by E. W. Macbride, 339
Benham on Kynotus cingulatus,
an earthworm from Madagascar,
445
Bidder on the collar-cells of Hetero-
ceela, 9
Bourne, G. C., a criticism of the
cell-theory, being an answer to
Mr. Sedgwick’s article, 137
Browne, E. T., on the variation of
Haliclystus, 1
Bury, H., on the metamorphosis of
Echinoderms, 45
Cells, iron compounds in, by A. B.
Macallum, 175
Cells, reproductive, of Elasmobranchs,
by J. E. S. Moore, 275
Cell-theory—answer to Sedgwick’s
article, by G. C. Bourne, 137
Cell-theory, further remarks on, with
a reply to Mr. Bourne, by Adam
Sedgwick, 331
VOL. 38, PART 4,—NEW SERIES.
Ciliation of the ectoderm of the
Amphibian embryo, by Richard
Assheton, 465
Collar-cells of Heteroccela, by Bidder,
9
Dean, Bashford, on the early develop-
ment of Amia, 413
Karthworm from Madagascar, by
Benham, 445
Echinoderms, the metamorphosis of,
by H. Bury, 45
Fecundation of Spherechinus and
Phallusia, by M. D. Hill, 315
Haliclystus, variation of, by E. T.
Browne, 1
Heteroccela, collar-cells of, by Bidder,
9
Hill, M. D., on the fecundation of
the egg of Spherechinus and of
Phallusia, 315
Tron compounds in cells, by A. B.
Macallum, 175
Kynotus cingulatus, an earth-
worm from Madagascar, by W. B.
Benham, 445
PoP
£50 INDEX.
Macallum, A. B., on assimilated iron _ Nervous system of Necturus, its de-
compounds in animal and vegetable velopment, by J. B. Platt, 485
cells, 175 |
Macbride, E. W., on the development | Platt on the development of the peri-
of Asterina gibbosa, 339 | pheral nervous system in Necturus,
Moore, J. E. S., on the structural 485
changes in the reproductive cells
during the spermatogenesis of Sedgwick on the cell-theory, with a
Elasmobranchs, 275 | reply to Mr, Bourne, 331
ies Spermatogenesis of Elasmobranchs,
Necturus, differentiation of the ecto- by J. H. S. Moore, 275
derm in, by Julia B. Platt, 485
PRINTED BY ADLARD AND SON,
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